Laboratory Animal Medicine, Second Edition (American College of Laboratory Animal Medicine)

Laboratory Animal Medicine 2nd edition

AMERICAN COLLEGE OF LABORATORY ANIMAL MEDICINE SERIES

Steven H. Weisbroth, Ronald E. Flatt, and Alan L. Kraus, eds.: The Biology of the Laboratory Rabbit, 1974 Joseph E. Wagner and Patrick J. Manning, eds.: The Biology of the Guinea Pig, 1976 Edwin J. Andrews, Billy C. Ward, and Norman H. Altman, eds.: Spontaneous Animal Models of Human Disease, Volume 1, 1979; Volume 2, 1979 Henry J. Baker, J. Russell Lindsey, and Steven H. Weisbroth, eds.: The Laboratory Rat, Volume 1: Biology and Diseases, 1979; Volume 2: Research Applications, 1980 Henry L. Foster, J. David Small, and James G. Fox, eds.: The Mouse in Biomedical Research, Volume 1: History, Genetics, and Wild Mice, 1981; Volume 2: Diseases, 1982; Volume 3: Normative Biology, Immunology, and Husbandry, 1983; Volume 4: Experimental Biology and Oncology, 1982 James G. Fox, Bennett J. Cohen, and Franklin M. Loew, eds.: Laboratory Animal Medicine, 1984 G. L. Van Hoosier, Jr., and Charles W. McPherson, eds.: Laboratory Hamsters, 1987 Patrick J. Manning, Daniel H. Ringler, and Christian E. Newcomer, eds." The Biology of the Laboratory Rabbit, 2nd Edition, 1994 B. Taylor Bennett, Christian R. Abee, and Roy Henrickson, eds.: Nonhuman Primates in Biomedical Research: Biology and Management, 1995 Dennis E Kohn, Sally K. Wixson, William J. White, and G. John Benson, eds.: Anesthesia and Analgesia in Laboratory Animals, 1997 B. Taylor Bennett, Christian R. Abee, and Roy Henrickson, eds.: Nonhuman Primates in Biomedical Research: Diseases, 1998 James G. Fox, Lynn C. Anderson, Franklin M. Loew, and Fred W. Quimby, eds.: Laboratory Animal Medicine, 2nd edition, 2002

La orator Anima Mec/icine 2nd edition EDITED BY

James G. Fox

Massachusetts Institute of Technology Cambridge, Massachusetts

Lynn C. Anderson Merck Research Laboratories Rahway, New Jersey

Franklin M. Loew Becker College Worcester, Massachusetts

Fred W. Quimby Laboratory Animal Research Center Rockefeller University New York, New York

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This book is printed on acid-free paper. @ Copyright 2002, 1984, Elsevier, All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science and Technology Rights Department in Oxford, UK. Phone: (44) 1865 843830, Fax: (44) 1865 853333, e-mail: permissions@elsevi'er.co.uk. You may also complete your request on-line via the Elsevier homepage: http://www.elsevier.com by selecting "CustomerSupport" and then "ObtainingPermissions". Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press Chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to Academic Press is given. Academic Press An Imprint of Elsevier 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http ://www. academicpres s.com Academic Press 32 Jamestown Road, London NW 1 7BY, UK http://www.academicpress.com Library of Congress Catalog Card Number: 2002100883 ISBN-13:978-0-12-263951-7 ISBN'10:0-12-263951-0 Printed in the United States of America 06 07 MV 9 8 7 6 5 4

Contents

B e n n e t t J. C o h e n

ix

List of C o n t r i b u t o r s

xi

Recombinant DNA Research Guidelines References B i o l o g y and D i s e a s e s of M i c e

Chapter 3

Robert O. Jacoby, James G. Fox, and Muriel Davisson

xv

Preface

I.

List of R e v i e w e r s for C h a p t e r s in This V o l u m e Chapter 1

xvii

L a b o r a t o r y A n i m a l M e d i c i n e : Historical

C ha p t e r 2

35 41 53 113

B i o l o g y and D i s e a s e s of Rats I~

II. III.

Chapter 5

Introduction Biology Diseases References

121 123 134 158

B i o l o g y and D i s e a s e s of H a m s t e r s

F. Claire Hankenson and Gerald L. Van Hoosier, Jr. Syrian Hamster

14 15 15 15 16

Laws, R e g u l a t i o n s , and Policies Affecting

Introduction II. Biology III. Diseases Introduction Biology Diseases Armenian Hamster

193

Chinese Hamster

I. II. III.

Lynn C. Anderson 19 20 29 31

168

168 173 180 190 190 190 191

I~

I. Introduction II. Biology III. Diseases

the U s e of L a b o r a t o r y A n i m a l s I. Introduction II. Animal Welfare III. Importation and Exportation of Animals and Animal Products IV. Hazardous Substances

Introduction Biology Diseases References

Dennis F Kohn and Charles B. Clifford

Franklin M. Loew and Bennett J. Cohen I. Introduction II. Origins of Animal Experimentation III. Early Veterinarians in Laboratory Animal Science and Medicine IV. The Organizations of Laboratory Animal Science V. Education and Training in Laboratory Animal Medicine VI. Impact of Laws, Regulations, and Guidelines on Laboratory Animal Medicine VII. Regulation of Animal Research in the United Kingdom and Canada VIII. Commercial and Academic Breeding of Rodents IX. Conclusion References

II. III.

Chapter 4

Perspectives

31 32

European Hamster

I. Introduction II. Biology III. Diseases Djungarian Hamster

193 194 194 194

194 194 195 196

vi

CONTENTS I. Introduction II. Biology III. Diseases References

Chapter 6

196 196 196 197

I. Introduction II. Biology III. Diseases References

203 206 212 241

Chapter 8

248 250 254 257 259 261 263 265 267 268 270 272 275 279 281 284 286 291

309 312 319 327

B i o l o g y and Diseases of Rabbits

C h a p t e r 10

329 331 339 358

M i c r o b i o l o g i c a l Q u a l i t y C o n t r o l for L a b o r a t o r y R o d e n t s and L a g o m o r p h s

William R. Shek and Diane J. Gaertner I. Overview II. Introduction III. Biosecurity

I. II. III. IV. V. VI.

C h a p t e r 13

Introduction Sources of Cats Housing Breeding Colony Management Nutrition and Feeding Infectious Disease Exclusion and Control References

365 366 366

460 462 463 466 474 475 480

B i o l o g y and Diseases of Ferrets

Robert P. Marini, Glen Otto, Susan Erdman, Lori Palley, and James G. Fox I. Introduction II. Biology III. Diseases References C h a p t e r 14

483 485 490 513

B i o l o g y and Diseases of R u m i n a n t s : Sheep, Goats, and Cattle

Margaret L. Delano, Scott A. Mischler, and Wendy J. Underwood

C h a p t e r 15

519 525 537 611

B i o l o g y and Diseases of S w i n e

Kathy E. Laber, Mark T. Whary, Sarah A. Bingel, James A. Goodrich, Alison C. Smith, and M. Michael Swindle I. Introduction II. Biology III. Diseases References

Mark A. Suckow, David W. Brammer, Howard G. Rush, and Clarence E. Chrisp I. Introduction II. Biology III. Diseases References

D o m e s t i c Cats as L a b o r a t o r y A n i m a l s

I. Introduction II. Biology III. Diseases References

Christine A. Bellezza, Patrick W. Concannon, William E. Hornbuckle, Lois Roth, and Bud C. Tennant

Chapter 9

395 397 405 454

Brenda Griffin and Henry J. Baker

W o o d c h u c k s as L a b o r a t o r y A n i m a l s

I. Introduction II. Biology III. Diseases References

372 387

B i o l o g y and Diseases of D o g s

I. Introduction II. Biology III. Diseases References C h a p t e r 12

B i o l o g y and D i s e a s e s of O t h e r R o d e n t s

Introduction Ground Squirrels or Susliks: Spermophilus Prairie Dogs: Cynomys Pocket Gophers: Geomyidae Kangaroo Rats: Dipodomys Wood Rats or Pack Rats: Neotoma Grasshopper Mice: Onychomys White-Footed Mice or Deer Mice: Peromyscus Rice Rats: Oryzomys Cane Mice: Zygodontomys Cotton Rats: Sigmodon White-Tailed Rats: Mystromys Gerbils and Jirds: Meriones Volesand Meadow Mice: Microtus Multimammate Rats: Mastomys Degus or Trumpet-Tailed Rats: Octodon Chinchillas: Chinchilla References

Microbiological (Health) Surveillance References

Robert C. Dysko, Jean A. Nemzek, Stephen L Levin, George J. DeMarco, and Maria R. Moalli

Thomas M. Donnelly and Fred W. Quimby I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII.

C h a p t e r 11

B i o l o g y and Diseases of G u i n e a Pigs

John E. Harkness, Kathleen A. Murray, and Joseph E. Wagner

Chapter 7

IV.

C h a p t e r 16

615 618 629 665

Nonhuman Primates

Bruce J. Bernacky, Susan V. Gibson, Michale E. Keeling, and Christian R. Abee I. II. III. IV. V. VI.

Introduction Taxonomy Biology Principles of Colony Management Medical Management Diseases References

676 677 680 715 724 730 777

vii

CONTENTS

Chapter 17 Biology and Diseases of Amphibians Dorcas P. O'Rourke and Terry Wayne Schultz I. II. III.

Introduction Biology Diseases References

793 801 814 823

IV. V. VI. VII.

Dogs Swine Small Ruminants Nonhuman Primates References

973 979 986 990 997

Chapter 23 Techniques of Experimentation Robert J. Adams

Chapter 18 Biology and Diseases of Reptiles Dorcas P. O'Rourke and Juergen Schumacher I. II. III.

Introduction Biology Diseases References

827 837 848 857

Chapter 19 Biology and Management of the Zebrafish Ke.ith M. Astrofsky, Robert A. Bullis, and Charles G. SagerstriJm I~

II. III. IV. V. VI. VII.

Introduction Experimental Model in Biomedical Research Health Management in the Aquatic Animal Facility Environmental Factors Important to Health Nutrition and Feeding Acquisition Infectious Diseases References

862 863 866 866 874 875 875 882

Chapter 20 Biology and Health of Laboratory Fishes Introduction Facility Design Management and Husbandry Medical Protocols Zoonotic Considerations Diseases References

886 886 893 894 898 899 907

Introduction Identification Methods Blood Collection and Intravenous Injection Vascular Cannulation Intraperitoneal Injection Subcutaneous and Intramuscular Injection Digestive System Urinary System Techniques Respiratory System Techniques Reproductive System Cardiovascular Techniques Endocrine System Techniques Orthopedic Procedures for Laboratory Animals Neurosurgical Techniques Tumor Transplantation Imaging Techniques Radiotelemetry References

1006 1006 1008 1013 1015 1015 1015 1019 1021 1023 1027 1028 1029 1029 1032 1033 1034 1034

Chapter 24 Control of Biohazards Associated with the Use of Experimental Animals Thomas E. Hamm, Jr. I.

Michael K. Stoskopf I. II. III. IV. V. VI.

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII.

II. III. IV. V. VI.

Introduction Risk Assessment Managing Facilities in Which Biohazards Are Used Other Considerations Responsibility for Reviewing and Approving Protocols Involving Biohazards Summary References

1047 1048 1048 1053 1056 1056 1056

Chapter 25 Selected Zoonoses Chapter 21 Design and Management of Animal Facilities

James G. Fox, Christian E. Newcomer, and Harry Rozmiarek I. Introduction

Jack R. Hessler and Steven L. Leary I. Introduction II. Facility Planning and Design III. Equipment IV. Commissioning and Validation V. Management VI. Conclusions References

909 910 931 946 946 947 948

Chapter 22 Preanesthesia, Anesthesia, Analgesia, and Euthanasia

Introduction Rodents Rabbits

Viral Diseases Rickettsial Diseases Chlamydial Infections Bacterial Diseases Fungal Diseases Protozoal Diseases Helminth Infections Arthropod Infestations References

1060 1060 1074 1076 1077 1088 1089 1093 1098 1098

Chapter 26 Xenozoonoses: The Risk of Infection

M. Michael Swindle, George A. Vogler, Linda K. Fulton, Robert P. Marini, and Sulli Popilskis I. II. III.

II. III. IV. V. VI. VII. VIII. IX.

after Xenotransplantation Marian G. Michaels 956 956 966

I. II. III.

Introduction Lessons from Allotransplantation Xenotransplantation References

1107 1107 1109 1113

viii

CONTENTS

C h a p t e r 27

III. History of Animal Use in Biomedical Research References

Genetic Monitoring

John J. Sharp, Evelyn E. Sargent, and Peter A. Schweitzer I. II. III. IV. v. vI.

C h a p t e r 28

Introduction The Need for Genetically Defined Animals Sources and Monitoring of Genetic Variability Colony Management Monitoring Methods Summary References

1117 1118 1119 1121 1122 1126 1127

C h a p t e r 31

I~ Introduction II. Choice of Mouse Strains for Trafisgenic Programs III. Production of Transgenic Mice: Animal Requirements IV. Management of the Transgenic Mouse Colony References

1129 1130

C h a p t e r 32

C h a p t e r 29

Henry J. Baker and J. Russell Lindsey I. II. III. IV. v. vI.

1131 1134 1140

I. II. III. IV. v. vI.

Research

Neil S. Lipman and Scott E. Perkins

C h a p t e r 30

1143 1143 1147 1165

A n i m a l M o d e l s in B i o m e d i c a l R e s e a r c h

Fred W. Quimby What Is an Animal Model? II. The Nature of Research I~

1185 1200

Introduction The Research Process Research Training Research Resources Support for Research Summary References

1228 1229 1231 1234 1235 1237 1237

Laboratory Animal Behavior

Kathryn A. L. Bayne, Bonnie V. Beaver, Joy A. Mench, and David B. Morton

Factors T h a t M a y I n f l u e n c e A n i m a l

I. Introduction II. Intrinsic Considerations III. Extrinsic Considerations References

R e s e a r c h in L a b o r a t o r y A n i m a l and Comparative Medicine

T r a n s g e n i c and K n o c k o u t M i c e

Glenn M. Monastersky and James G. Geistfeld

1206 1214

Index

Introduction Rodents Rabbits Laboratory Dogs and Cats Nonhuman Primates Farm Animals References

1240 1241 1245 1246 1248 1252 1256 1265

Bennett We would like to dedicate this book to our friend and colleague Bennett J. Cohen, D.V.M., Ph.D., who died on August 23, 1990, at the age of 65. Ben was a valued coeditor of the first edition of "Laboratory Animal Medicine." We would like to honor him with our heartfelt appreciation of his leadership, wisdom, and friendship throughout our individual careers. After graduating from Cornell University Veterinary School in 1949, Ben obtained a Ph.D. in physiology from Northwestern Medical School in 1953. He then spent several years on the faculty and was director of the vivarium at the University of California Medical School in Los Angeles. He was then recruited to the University of Michigan, where he founded the Unit for Laboratory Animal Medicine (ULAM) at the University of Michigan, and was its director for 23 years. He obtained the rank of professor of laboratory animal medicine in 1968. Dr. Cohen was a pioneer and visionary in the field of laboratory animal science for more than 40 years. His career of caring for animals used in medical research began at Northwestern University in 1949. A year later, he and veterinary colleagues in the Chicago area founded the Animal Care Panel, which later became the American Association for Laboratory Animal Science (AALAS). Ben served as the association's first secretary, as a member of the board of trustees, and later as president. Three years later Ben and a few colleagues saw the need to establish standards of training and experience for veterinarians

J.

Cohen

engaged in laboratory animal medicine. They persuaded the American Veterinary Medical Association to accept the veterinary specialty of laboratory animal medicine and establish a specialty certification board. This became the American College of Laboratory Animal Medicine (ACLAM). Today there are more than 600 board-certified veterinarians in the United States. In 1963, Dr. Cohen chaired the National Academy of Sciences committee that wrote the first edition of the document that later became "The Guide for Care and Use of Laboratory Animals." Since then, more than 400,000 copies have been distributed, and it has been accepted as a primary reference on laboratory animal care and use. The National Institutes of Health (NIH) now requires that awardee institutions comply with the provisions of the guide. Dr. Cohen was the recipient of all of the major national and international awards in laboratory animal science. In 1966, he received the Griffin Award from the American Association for Laboratory Animal Science. This, the association's highest award, was presented for "outstanding accomplishments in the improvement of care and quality of laboratory animals." In 1980, he received the Charles River Prize, the highest award of the American Veterinary Medical Association. The inscription reads, "You have been a moving force in laboratory animal science and a major figure in the founding of national

/x

X

organizations that have brought strength, cohesion, and credibility to the field." In 1990, the governing board of the International Council for Laboratory Animal Science (ICLAS) presented Dr. Cohen with the council's highest award, the Muhlbock Award, for his work in establishing high standards of laboratory animal care and use worldwide. In addition to his activity in national and international organizations, Dr. Cohen was an active clinician and scientist. He published more than 70 articles in peer-reviewed scientific journals and served on many NIH study sections and advisory boards. Dr. Cohen also established a national reputation in the field of gerontology. He originated health standards for aging animals and undertook long-term studies of rodent diseases of aging. At the University of Michigan he established the Core Facility for Aged Rodents (CFAR) in the Institute of Gerontology and the Gerontology Research and Training Center. The CFAR provides aged rodents for study by scientists campuswide.

BENNETT J. C O H E N

An additional and lasting legacy of Ben's impact on laboratory animal medicine is the stellar record he achieved in training future generations of specialists in the field. He trained 36 postdoctoral veterinary fellows from 1959 to 1985. Dr. Cohen was originally awarded the NIH training grant while at UCLA in 1960 and transferred the training grant to Ann Arbor when he relocated to the University of Michigan in 1962. This grant has been funded since its inception and is recognized internationally for its record of excellence. Dr. Cohen was a friend and an inspiration to his colleagues worldwide. He loved his work, his science, and his colleagues. He was truly a humanitarian and a lasting example--for those of us engaged in pursuits of veterinary medicine--of the values of professionalism and integrity. James G. Fox Lynn C. Anderson Franklin M. Loew Fred W. Quimby

List of Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Christian R. Abee (675), Department of Comparative Medicine, University of South Alabama, College of Medicine, Mobile, Alabama 36688 Robert J. Adams (1001), Division of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Lynn C. Anderson (19), Department of Comparative Medicine, Merck Research Laboratories, Rahway, New Jersey 07065 Keith M. Astrofsky (861), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Henry J. Baker (459, 1227), College of Veterinary Medicine, Scott-Ritchey Research Center, Auburn University, Auburn, Alabama 36849 Kathryn A. L. Bayne (1239), Association for Assessment and Accreditation of Laboratory Animal Care International, Rockville, Maryland 20852 Bonnie u Beaver (1239), Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843 Christine A. Bellezza (309), Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Bruce J. Bernacky (675), Department of Veterinary Sciences, M. D. Anderson Cancer Center, Bastrop, Texas 78602 Sarah A. Bingel (615), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 David W. Brammer (329), Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105

Robert A. Bullis (861), Center for Applied Aquaculture, The Oceanic Institute, Waimanalo, Hawaii 96795 Clarence E. Chrisp 1 (329), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Charles B. Clifford (121), Charles River Laboratories, Inc., Wilmington, Massachusetts 01887 Bennett J. Cohen 2 (1), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Patrick W. Coneannon (309), Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Muriel Davisson (35), Jackson Laboratories, Bar Harbor, Maine 04609 Margaret L. Delano (519), Animal Care, University of Massachusetts, Amherst, Massachusetts 01003 George J. DeMarco (395), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Thomas M. Donnelly 3 (247), The Kenneth S. Warren Institute, Ossining, New York 10562 Robert C. Dysko (395), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Susan Erdlnan (483), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

1Deceased February 8, 2000. 2Deceased August 23, 1990. 3present address: Post Graduate Foundation in Veterinary Science, University of Sydney, Australia.

xi

xii James G. Fox (35,483, 1059), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Linda K. Fulton (955), Godley-Snell Research Center, Clemson University, Clemson, South Carolina 29634 Diane J. Gaertner (365), Institute for Animal Studies, Albert Einstein College of Medicine, Bronx, New York 10461 James G. Geistfeld (1129), Taconic, Inc., Germantown, New York 12526 Susan V. Gibson (675), Department of Comparative Medicine, College of Medicine, University of South Alabama, Mobile, Alabama 36688 James A. Goodrich (615), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Brenda Griffin (459), College of Veterinary Medicine, ScottRitchey Research Center, Auburn University, Auburn, Alabama 36849 Thomas E. Hamm, Jr. (1047), 105 Martinique Place, Cary, North Carolina 27511 F. Claire l-lankenson (167), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 John E. l-larkness (203), College of Veterinary Medicine, Office of Research, Mississippi State University, Mississippi State, Mississippi 39762 Jack R. l-lessler (909), Washington University School of Medicine, St. Louis, Missouri, 63110 William E. l-lornbuckle (309), Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Robert O. Jacoby (35), Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut 06520 Michale E. Keeling (675), Department of Veterinary Sciences, M. D. Anderson Cancer Center, Bastrop, Texas 78602 Dennis F. Kohn (121 ), Institute of Comparative Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Kathy E. Laber (615), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Steven L. Leary (909), Washington University School of Medicine, St. Louis, Missouri 63110 Stephen I. Levin (395), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 J. Russell Lindsey (1227), Department of Comparative Medicine, University of Alabama Schools of Medicine and Dentistry, Birmingham, Alabama 35294 Neil S. Lipman (1143), Research Animal Resource Center, Memorial Sloan-Kettering Cancer Center, Weill Medical College of Cornell University, New York, New York 10021

LIST OF CONTRIBUTORS Franklin M. Loew (1), Becker College, Worcester, Massachusetts 01609 Robert P. Marini (483, 955), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Joy A. Mench (1239), Department of Animal Science, University of California, Davis, California 95616 Marian G. Michaels (1107), Division of Allergy, Immunology, and Infectious Disease, The Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 11521 Scott A. Mischler (519), Animal Care and Management, University of Vermont, Burlington, Vermont 05405 Maria R. Moalli (395), Department of Surgery and Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Glenn M. Monastersky (1129), Hoffman-LaRoche, Nutley, New Jersey 07110 David B. Morton (1239), Department of Biomedical Ethics, The Medical School Edgbaston, University of Birmingham, Birmingham, B 15 211 United Kingdom Kathleen A. Murray (203), Technical Operations, Charles River Laboratories, Wilmington, Massachusetts 01887 Jean A. Nemzek (395), Department of Pathology and Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Christian E. Newcomer 4 (1059), Division of Laboratory Animal Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 Dorcas P. O'Rourke (793, 827), Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37901 Glen Otto (483), Department of Comparative Medicine, Stanford University, Stanford, California 94305 Lori Palley (483), Astra Research Boston, Cambridge, Massachusetts 02139 Scott E. Perkins (1143), Division of Laboratory Animal Medicine, Tufts University, School of Veterinary Medicine, Boston, Massachusetts 02111 Sulli Popilskis (955), Institute of Comparative Medicine, Columbia University, New York, New York 10032 Fred W. Quimby (247, 1185), Laboratory Animal Research Center, Rockefeller University, New York, New York 10021 Lois Roth (309), Department of Pathology, MSPCA Angell Memorial, Boston, Massachusetts 02130 Harry Rozmiarek (1059), Laboratory Animal Resources, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Howard G. Rush (329), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 4presentaddress:VeterinaryResourcesProgram,NationalInstitutesof Health, Bethesda, Maryland20892.

LIST OF CONTRIBUTORS

Charles G. Sagerstriim (861), Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical Center, Worcester, Massaschusetts 01655 Evelyn E. Sargent (1117), Genetic Quality Assurance, Jackson Laboratories, Bar Harbor, Maine 04609 Terry Wayne Schultz (793), Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37901 Juergen Schumacher (827), Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37901 Peter A. Schweitzer (1117), Genetic Quality Assurance, Jackson Laboratories, Bar Harbor, Maine 04609 John J. Sharp (1117), SAIC-Frederick, Frederick, Maryland 21702 William R. Shek (365), Charles River Laboratories, Wilmington, Massachusetts, 01887 Alison C. Smith (615), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Michael K. Stoskopf (885), Environmental Medicine Consortium, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606

xiii

Mark A. Suckow (329), Friemann Life Science Center, University of Notre Dame, Notre Dame, Indiana 46556 M. Michael Swindle (615, 955), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Bud C. Tennant (309), Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Wendy J. Underwood (519), Animal Science and Veterinary Services, Eli Lilly and Company, Greenfield, Indiana 46140 Gerald L. Van Hoosier, Jr. (167), Department of Comparative Medicine, University of Washington School of Medicine, Seattle, Washington 98195 George A. Vogler (955), Department of Comparative Medicine, St. Louis University, School of Medicine, St. Louis, Missouri 63104 Joseph E. Wagner (203), College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211 Mark T. Whary (615), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

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Preface

The American College of Laboratory Animal Medicine (ACLAM) was founded in 1957 to encourage education, training, and research in laboratory animal medicine and to recognize veterinary medical specialists in the field by certification and other means. Continuing education has been an important activity in ACLAM from its inception. The second edition of this teaching text, "Laboratory Animal Medicine," reflects the College's continuing effort to foster education. It is, in part, an updated distillation for teaching purposes of a series of volumes on laboratory animals developed by ACLAM over the past two decades: "The Biology of the Laboratory Rabbit" published in 1974, with a second edition in 1994, "The Biology of the Guinea Pig" in 1976, and a two-volume work "Biology of the Laboratory Rat" in 1979 and 1980, followed by the publication "Laboratory Hamsters" in 1987. Also, in 1979 the college published a two-volume text on "Spontaneous Animal Models of Human Disease." In 1981-1983, four volumes of "The Mouse in Biomedical Research" were published. Most recently, a twovolume treatise on "Nonhuman Primates in Biomedical Research" was published in 1995 and 1998, and, finally, a text "Anesthesia and Analgesia in Laboratory Animals" in 1997. Most major advances in biology and medicine in one way or another have depended on the study of animals. During the past generation, the health, genetic integrity, and environmental surroundings of the animals have been recognized as important factors to be taken into account in planning animal studies. The ultimate responsibility for insuring the validity of scientific resuits, together with humane and scientifically appropriate animal care, resides with two categories of scientists: veterinarians responsible for the acquisition, care, nutrition, anesthesia, and other aspects of humane animal use and scientific investigators who use animals as subjects of study. This book therefore is intended for students of veterinary medicine and others in the fields of biology and medicine who utilize animals in biomed-

ical research. The editors and contributors hope it will prove useful in introducing students and scientists embarking on their careers to important concepts related to animals in research. The contents of this second edition have been greatly expanded and are presented in thirty-two chapters that provide information on the diseases and biology of the major species of laboratory animals used in biomedical research. The history of laboratory animal medicine, legislation affecting laboratory animals, experimental methods and techniques, design and management of animal facilities, zoonoses, biohazards, animal models, and genetic monitoring are also covered. Reflective of the ever increasing use of genetically engineered mice, new chapters include rodent and lagomorph surveillance and quality assurance, and transgenic and knockout mice. Also, added are chapters dealing with the emerging interest in fish biology and the use of xenotransplantation. The editors acknowledge the contributors' outstanding efforts to follow the guidelines on content and accept sole responsibility for any significant omissions. As with all volumes of the ACLAM series texts, the contributors and editors of this book have donated publication royalties to the American College of Laboratory Animal Medicine to foster continuing education in laboratory animal science. It could not have been completed without the support and resources of the editors' parent institutions. A special thanks also is extended to the reviewers of each chapter whose excellent and thoughtful suggestions helped the authors and editors present the material in a meaningful and concise manner. We acknowledge and thank Lucille Wilhelm for her excellent secretarial assistance. The assistance of the staff of Academic Press also is greatly appreciated and acknowledged. James G. Fox Lynn C. Anderson Franklin M. Loew Fred W. Quimby XI2

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List of Reviewers for Chapters in This Volume

Astrofsky, Keith Balk, Melvin Bayne, Kathryn A. Bell, Judith A. Bennett, B. Taylor Bowser, Paul Clemons, Donna Collins, Bobby R. Cullen, John DeTolla, Louis J. Erdman, Susan Faith, Robert E. Forsythe, Diane B. Geistfeld, James Herbst, Lawrence H. Huerkamp, Michael Hurley, Julie Hurley, Richard Jaax, Gerald P. James, Mary Lou Klein, Hilton Lipman, Neil S. Lohmiller, Jeffrey J. Marini, Robert P. Morrissey, J. Motzel, Sherri Noga, Edward J. Patterson, Mary Percy, Dean H. Perkins, Scott Rand, Michael S. Ringler, Daniel H. Saperstein, George Scipioni, Roberta Sive, Hazel L. Smith, Abigail Spaulding, Glen Straw, Barbara Webster, William S. White, William J.

Massachusetts Institute of Technology, Cambridge, Massachusetts Charles River Laboratories, Inc., Wilmington, Massachusetts AAALAC International, Bethesda, Maryland University of Guelph, Ontario University of Illinois, Chicago, Illinois Cornell University, Ithaca, New York Covance Laboratories, Inc., Madison, Wisconsin University of Florida, Gainesville, Florida North Carolina State University, Raleigh, North Carolina University of Maryland, Baltimore, Maryland Massachusetts Institute of Technology, Cambridge, Massachusetts Baylor College of Medicine, Houston, Texas National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina Taconic, Inc., Germantown, New York Albert Einstein College of Medicine, New York, New York Emory University, Atlanta, Georgia Consultant, Boston, Massachusetts Children's Hospital, Boston, Massachusetts Kansas State University, Manhattan, Kansas Consultant, Regulatory Compliance Merck Research Laboratories, West Point, Pennsylvania Memorial Sloan-Kettering Cancer Center, New York, New York Taconic, Inc., Germantown, New York Massachusetts Institute of Technology, Cambridge, Massachusetts The Animal Medical Center, New York, New York Merck Research Laboratories, West Point, Pennsylvania North Carolina State University, Raleigh, North Carolina Massachusetts Institute of Technology, Cambridge, Massachusetts University of Guelph, Ontario Memorial Sloan-Kettering Cancer Center, New York, New York University of Arizona, Tucson, Arizona University of Michigan, Ann Arbor, Michigan Tufts University School of Veterinary Medicine, North Grafton, Massachusetts Marshall Farms, North Rose, New York Whitehead Institute, Cambridge, Massachusetts The Jackson Laboratory, Bar Harbor, Maine Tufts University School of Veterinary Medicine, North Grafton, Massachusetts Michigan State University, East Lansing, Michigan University of Massachusetts Medical School, Worcester, Massachusetts Charles River Laboratories, Inc., Wilmington, Massachusetts xvii

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Chapter 1 Laboratory Animal Medicine: Historical Perspectives Franklin M. Loew and Bennett J. Cohen*

I. II. III. IV.

V. VI. VII. VIII. IX.

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Originsof Animal Experimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EarlyVeterinarians in Laboratory Animal Science and Medicine . . . . . . . The Organizations of Laboratory Animal Science . . . . . . . . . . . . . . . . . . . A. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The National Society for Medical Research . . . . . . . . . . . . . . . . . . . . C. The American Association for Laboratory Animal Science . . . . . . . . D. The Institute of Laboratory Animal Resources . . . . . . . . . . . . . . . . . . E. The American College of Laboratory Animal Medicine . . . . . . . . . . . Educationand Training in Laboratory Animal Medicine . . . . . . . . . . . . . . Impactof Laws, Regulations, and Guidelines on Laboratory Animal Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulationof Animal Research in the United Kingdom and Canada . . . . . Commercialand Academic Breeding of Rodents . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION

Five key terms identify the fields or activities that relate to the care and use of animals in research, education, and testing. Animal experimentation refers to the scientific study of animals, usually in a laboratory, for the purpose of gaining new biological knowledge or solving specific medical, veterinary medical, dental, or biological problems. Most commonly, such experimentation is carried out by or under the direction of persons holding research or professional degrees. Laboratory ani*Deceased. LABORATORY ANIMAL MEDICINE, 2nd edition

1

2 3 7 7 8 10 12 13 13 14 15 15 15 16

mal care is the application of veterinary medicine and animal science to the acquisition of laboratory animals and to their management, nutrition, breeding, and diseases. The term also relates to the care that is provided to animals as an aid in managing injury and pain. Laboratory animal care usually is provided in scientific institutions under veterinary supervision or guidance. Laboratory animal medicine is recognized by the American Veterinary Medical Association as the specialty field within veterinary medicine that is concerned with the diagnosis, treatment, and prevention of diseases in animals used as subjects in biomedical activities. Laboratory animal medicine also encompasses the methods of minimizing and preventing pain or discomfort in research animals and the identification of Copyright 2002, Elsevier Science (USA). All fights reserved. ISBN 0-12-263951-0

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FRANKLIN M. L O E W AND BENNETT J. COHEN

complicating factors in animal research. Comparative medicine is "the study of the nature, cause and cure of abnormal structure and function in people, animals and plants for the eventual application to and benefit of all living things" (Bustad et al., 1976). Laboratory animal science is the body of scientific and technical information, knowledge, and skills that bears on both laboratory animal care and laboratory animal medicine and that is roughly analogous to "animal science" in the agricultural sector. Laboratory animal medicine has grown rapidly because of its inherent scientific importance and because good science and the public interest require the best possible care for laboratory animals. In this chapter, we trace briefly the historical evolution of laboratory animal medicine and consider its relationship to other areas of biology and medicine.

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ON

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ORIGINS OF ANIMAL EXPERIMENTATION

The earliest references to animal experimentation are to be found in the writings of Greek philosopher-physicians of the fourth and third centuries BC. Aristotle (384-322 BC), characterized as the founder of biology, was the first to make dissections that revealed internal differences among animals (Wood, 1931). Erasistratus (304-250 BC) probably was the first to perform experiments on living animals, as we understand them today. He established in pigs that the trachea was an air tube and the lungs were pneumatic organs (Fisher, 1881). Later, Galen (AD 130-200) performed anatomical dissections of pigs, monkeys, and many other species (Cohen and Drabkin, 1948; Cohen, 1959a). Galen justified experimentation as a long, arduous path to the truth, believing that uncontrolled assertion that was not based on experimentation could not lead to scientific progress. Dogma replaced experimentation in the dark centuries following Galen's lifetime. Whereas anatomical dissection of dead animals and people had been among the earliest types of experimentation, in medieval times this practice was prohibited by ecclesiastical authorities who wanted to prevent acquisition of knowledge about the natural world that could

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be considered blasphemous. Not until the 1500s was there a reawakening of interest in science. Andreas Vesalius (15141564), the founder of modern anatomy, used dogs and pigs in public anatomical demonstrations (Saunders and O'Malley, 1950) (Fig. 1). This "vivisection" led to great leaps in the understanding of anatomy's correspondence with physiology. In 1628, Sir William Harvey published his great work on the movement of the heart and blood in animals (Singer, 1957). By the early 1700s, Stephen Hales, an English clergyman, reported the first measurement of blood pressure, using as his subject a horse "fourteen hands high, and about fourteen years of age, [and with] a fistula on her withers" (Hoff et al., 1965) (Fig. 2).

3

1. LABORATORY A N I M A L MEDICINE: H I S T O R I C A L P E R S P E C T I V E S

Fig. 3. Claude Bernard, often referred to as the founder of experimental medicine, developed and described highly sophisticated methods of animal research in his laboratory in Paris. (Photograph from Garrison, 1929.)

During the 1800s, France became a primary center of experimental biology and medicine. Scientists, such as Franqois Magendie (1783-1855) and Claude Bernard (1813-1878) (Fig. 3) in experimental physiology and Louis Pasteur (1822-1895) in microbiology, contributed enormously to the validation of the scientific method, which included the use of animals. Bernard (1865) commented: ... it is proper to choose certain animals which offer favorable anatomical arrangements or special susceptibility to certain influences. For each kind of investigation we shall be careful to point out the proper choice of animals. This is so important that the solution of a physiological or pathological problem often depends solely on the appropriate choice of the animal for the experiment so as to make the result clear and searching.

Pasteur studied infectious diseases in a variety of animals, such as silkworms ("pebrine"), dogs (rabies), and sheep (anthrax). "Pebrine" (pepper) was an economically important disease of silkworms in France when silk was a major fabric; Pasteur and others demonstrated the parasite that caused the disease (Duclaux, 1920). As pathogenic organisms were identified that could be related to specific human diseases, their animal disease counterparts also were studied. Pasteur and others perceived that the study of animal diseases benefited animals and enhanced understanding of human diseases and pathology. The extraordinary power of the experimental approach, including experiments on animals, led to what has been called the Golden Age of scientific

medicine. Despite advances in physiological and bacteriological understanding, however, criticisms of the use of animals in science began, particularly in England (Loew, 1982). The first Society for the Prevention of Cruelty to Animals (SPCA) was established in England, followed in the 1860s by an American SPCA in New York, a Philadelphia SPCA, and a Massachusetts SPCA. Objections to the use of animals in science were part of the concerns of these societies, particularly because Darwin's findings on evolution made "differences" between animals and humans less sure in many persons' minds (Loew, 1982). Most American and Canadian scientists, physicians, and veterinarians soon applied emerging scientific concepts in their research. D. E. Salmon, recipient of the first D.V.M. degree awarded in the United States (by Cornell University in 1879), studied bacterial diseases, and the genus Salmonella, a ubiquitous human and animal pathogen, was named for him. Cooper Curtice (Fig. 4), Theobald Smith, and others first demonstrated the role of arthropod victors in disease transmission in their studies of bovine Texas fever (Schwabe, 1978). The first paper published at the then fledgling Johns Hopkins Hospital and School of Medicine was by the physician William H. Welch, for whom Clostridium welchii was named, and was entitled "Preliminary Report of Investigations concerning the Causation of Hog Cholera" (Welch, 1889). Thus, it became evident that the study of the naturally occurring diseases of animals could illuminate principles applicable to both animals and mankind and lead to improved understanding of biology in general. John Call Dalton, M.D. (1825-1889), an American physiologist, spent a year in Bernard's laboratory in Paris, about 1850. He was highly impressed with Bernard's instructional methods, which included demonstrations in living animals of important physiological principles. Subsequently, Dr. Dalton included such demonstrations in his teaching at the College of Physicians and Surgeons in New York City (Mitchell, 1895), the forerunner of the "animal labs" in which generations of students in biology and medicine were once trained. When Alexis Carrel received the Nobel Prize in 1912, the citation stated in part: " . . . you have.., proved once again that the development of an applied science of surgery follows the lessons learned from animal experimentation" (Malinin, 1979). Thus, starting in ancient times and continuing to the present day, animal experimentation has been one of the fundamental approaches of the scientific method in biological and medical research and education.

llI.

EARLY VETERINARIANS IN LABORATORY ANIMAL SCIENCE AND MEDICINE

On September 15, 1915, Dr. Simon D. Brimhall (1863-1941; V.M.D., University of Pennsylvania, 1889) (Fig. 5) joined the staff of the Mayo Clinic in Rochester, Minnesota, the first

4

FRANKLIN M. LOEW AND BENNETT J. COHEN

Fig. 4. Dr. CooperCurtice, examiningticks on a cow dead of Texas fever. Curtice contributedimportantlyto the demonstrationthat arthropodscan act as carriers of mammaliandiseases. (Courtesyof The Nation's Business.)

veterinarian to fill a position in laboratory animal medicine at an American medical research institution (Cohen, 1959b; Physicians of the Mayo Clinic and the Mayo Foundation, 1937). No such field was recognized at the time, of course; but Dr. Brimhall's activities--management of the animal facilities, development of animal breeding colonies, investigation of laboratory animal diseases (Brimhall and Mann, 1917; Brimhall and Hardenbergh, 1922), and participation in collaborative and independent research (Brimhall et al., 1919-1920)mwere the prototype of the present role of "laboratory animal veterinarians" in scientific institutions throughout the world. The decision to employ a veterinarian at the Mayo Clinic in 1915 appears to have resulted from a unique juxtaposition of institutional needs and personalities. Although the Mayo Clinic was already world renowned, organized research was in only a rudimentary stage of development. About 1910, an unsuccessful effort was made to convert an old barn, belonging to the chief of surgical pathology, Dr. Louis B. Wilson, for animal experimentation (Braasch, 1969). Then, in 1914, with Dr. William J. Mayo's active encouragement, the Division of Experimental Surgery and Pathology was created, the first real research laboratory at the clinic. Dr. Frank C. Mann, a young medical scientist from Indiana, was invited to head the division, with the primary assignment of developing a first-class animal research laboratory. Dr. Brimhall's employment followed within a year and was accompanied by the planning and ultimate construction of new animal facilities (Figs. 6 and 7). Christopher Graham, M.D., then head of the Division of Medicine, greatly influenced

the decision to employ Dr. Brimhall. Perhaps the fact that Dr. Graham also was a veterinarian (V.M.D., University of Pennsylvania, 1892) provided insights into the contributions that veterinary medicine could make to experimental surgery and pathology. Certainly, the concept of mutual support among the professions was not at that time widely held; there was, in fact, relatively little interprofessional communication between medicine and veterinary medicine then. Dr. Brimhall retired in 1922 and was succeeded by Dr. John G. Hardenbergh (1892-1963; V.M.D., University of Pennsylvania, 1916). During his 5-year tenure at the Mayo Clinic, Dr. Hardenbergh was an active clinical investigator (Hardenbergh, 19261927) as well as animal facility manager. In a stout defense of animal experimentation, he also demonstrated the communication skills in the public arena that were to serve him well later in his career (1941-1958) as executive secretary of the American Veterinary Medical Association (Hardenbergh, 1923). Dr. Carl F. Schlotthauer (1893-1959; D.V.M., St. Joseph Veterinary College, 1923), who had joined the Mayo Clinic staff in 1924 as assistant in veterinary medicine, succeeded Dr. Hardenbergh in 1927. By this time, the Mayo Foundation was functioning as the graduate medical education and research arm of the Mayo Clinic and had become formally affiliated with the University of Minnesota. Dr. Schlotthauer ultimately became head of the Section of Veterinary Medicine at the Mayo Foundation (1952) and professor of veterinary medicine at the University of Minnesota Graduate School (1945). Thus, Dr. Schlotthauer was the first veterinarian to attain a full professorship

1. LABORATORY ANIMAL MEDICINE: HISTORICAL PERSPECTIVES

5

the Mayo Foundation between 1915 and 1950, Dr. Brimhall, Dr. Hardenbergh, and Dr. Schlotthauer were the ones most closely associated with activities that today are identified with laboratory animal medicine. It is noteworthy that the Mayo Clinic/Foundation has maintained a program in animal medicine continuously for more than 85 years, having initiated it long before most medical research institutions were prepared even to consider the possible value of adding veterinarians to their professional staff (P. E. Zollman, personal communication, 1982). Dr. Karl E Meyer (1884-1974; D.V.M., University of Zurich, 1924; M.D. [honorary], College of Medical Evangelists, 1936) was an internationally known epidemiologist, bacteriologist, and pathologist. Dr. Meyer was intensely interested in matters related to laboratory animals for most of his professional life. He was the author of an early review of laboratory animal diseases (Meyer, 1928), one of the first publications of its kind in the United States. Dr. Meyer was a unique personality--vigorous, dynamic, active--a world traveler on missions related to international health; a scientist who engendered in his students respect, admiration, love, and fear in varying proportions. Together with his longtime associate Bernice Eddy (Ph.D.), a bacteriologist, Dr. Meyer developed a model animal facility at the George Williams Hooper Foundation at the University of California, San Francisco, during a 30-year tenure as director (1924-1954). Dr. Meyer often was away from the laboratory, and it fell to Dr. Eddy to supervise the animal facility, which she did with great skill and dedication. Dr. Meyer foresaw the need for and was an early advocate of the participation of veterinarians in the operation of institutional laboratory animal colonies Fig. 5. SimonD. Brimhall, V.M.D., the first veterinarian in laboratoryani- (Meyer, 1958). He figured importantly in the planning that led mal medicine at an Americanmedicalresearch institution, workedat the Mayo the University of California to create the position of "statewide Clinic from 1915to 1922. (Courtesyof Universityof Minnesota Press and Dr. veterinarian" in 1953, which subsequently was superseded by Paul E. Zollman.) the appointment of veterinarians at each of the university's major campuses. Among his many honors, Dr. Meyer received the for laboratory animal medicine-related academic activities. Charles A. Griffin Award of AALAS in 1959. Dr. Charles A. Griffin (1889-1955; D.V.M., Cornell UniverDr. Schlotthauer vigorously opposed antivivisectionist attacks on animal research. He was a leader in the statewide campaign sity, 1913) was a bacteriologist at the New York State Board of that led to adoption of the Minnesota "pound law" in 1950, i.e., Health, Division of Laboratories, Albany, New York, from 1919 a law authorizing the requisitioning for research and education to 1954. Dr. Griffin pioneered the concept of the development by approved scientific institutions of impounded, but unclaimed of "disease-free" animal colonies long before gnotobiotic techdogs and cats. Dr. Schlotthauer believed that open and honest nology had evolved (Brewer, 1980). In the 1940s, Dr. Griffin communication between medical scientists and humane society utilized progeny testing to establish a rabbit colony free of pasworkers could lead to better public understanding and support teurellosis. Additionally, he showed that Salmonella spp. could of animal research. Consequently, he was also active in humane be transmitted in meat meal (Griffin, 1952). This led feed mansociety activities, serving for many years on the board of direc- ufacturers to improve the processing of laboratory animal diets tors of the Minnesota Society for the Prevention of Cruelty to so as to eliminate Salmonella contamination. The Charles A. Animals. Dr. Schlotthauer also was an important figure in the Griffin Award of AALAS was established and named in Dr. early years of the American Association for Laboratory Animal Griffin's honor. He received the award posthumously in 1955, Science (AALAS). He was a founding member of its board of the first recipient of this prestigious award. The Griffin Laboradirectors and presented a paper on animal procurement at its tory at the New York State Board of Health central facility in first meeting, in 1950 (Schlotthauer, 1950). Albany, New York, also is named in his honor. Although other veterinarians also held appointments at Dr. Nathan R. Brewer (D.V.M., Michigan State University,

6

FRANKLIN M. LOEW AND BENNETT J. COHEN

Fig. 6.

Dog breeding facility, Institute of Experimental Medicine, Mayo Clinic, constructed in the mid- 1920s. (Courtesy of Dr. Paul E. Zollman.)

Fig. 7. Interior of guinea pig breeding house, Institute of Experimental Medicine, Mayo Clinic, constructed in the early 1920s. (Courtesy of Dr. Paul E. Zollman.)

1. LABORATORY ANIMAL MEDICINE: HISTORICAL PERSPECTIVES

7

overcOme, and Dr. Brewer became supervisor of the Central Animal Quarters. Laboratory animal medicine began its modern evolution in the following years. Dr. Brewer's role was seminalmas a founder of the American Association for Laboratory Animal Science, as first president of AALAS (1950-1955), and as a "father figure" for the then youthful group of veterinarians that had been employed by other medical schools and medical research institutions in the Chicago area between 1945 and 1949. Dr. Brewer received the AALAS Griffin Award in 1962. Although retired since 1969, he remains active and attends local and national AALAS meetings. To honor his contributions to the field, AALAS instituted an annual award in 1994, the Nathan Brewer Scientific Achievement Award. Other personalities that played important roles in the early history of laboratory animal science and medicine have been characterized and their roles assessed by Brewer (1980).

IV.

THE O R G A N I Z A T I O N S OF L A B O R A T O R Y A N I M A L SCIENCE

A.

Fig. 8. Dr. NathanR. Brewer,directorof the CentralAnimalQuarters at the Universityof Chicago (1945-1969) and firstpresident of the AmericanAssociation for Laboratory Animal Science. Photograph taken in the late 1940s. (Courtesy of Dr. N. R. Brewer.)

1937; Ph.D., University of Chicago, 1936) headed the laboratory animal facilities at the University of Chicago from 1945 until his retirement in 1969 (Fig. 8). Dr. Brewer's interest in laboratory animals originated in the mid-1920s, when he started veterinary school, and continued during his graduate student years in the Department of Physiology at the University of Chicago. About 1935, Professors Anton J. Carlson (Ingle, 1979) and A. B. Luckhardt first approached Dr. Brewer about managing the University of Chicago animal facilities. They saw merit in the concept of a veterinarian, well grounded in the scientific method, as animal facility manager. They believed this arrangement would contribute to public confidence in the care and treatment of animals in research and would help to turn aside antivivisection activists. However, many investigators at the university feared that a veterinarian would dictate the conditions of care and use of animals, and they opposed the creation of this position. It was not until 1945 that this opposition was

Background

Organizations are important in scientific life as a means of implementing the content and activities of the fields they represent. Present-day students of laboratory animal science are confronted with a confusing array of organizational acronyms: AAALAC International, AALAS, ACLAM, ASLAP, CALAS, ILAR, ICLAS, NABR, and so on. It is instructive to examine why organizations such as these came into being and to evaluate their impact on laboratory animal science. Consider the milieu for research in biology and medicine in the United States about 1945. A new national policy was just being initiated to provide increased federal support of science. The use of laboratory animals began to expand rapidly as the funding of medical and biological research increased, and a host of problems as well as challenges accompanied this development. The base of knowledge about the care and diseases of laboratory animals was small. Published information was scattered and sparse. Few veterinarians were devoting themselves te "laboratory animal care," which was not yet recognized as a special field. In many institutions, animal facilities and administrative arrangements for operating them were poor. Institutions were ill prepared to accommodate increasingly large animal colonies. Simultaneously, medical scientists were under increasingly vigorous attack from antivivisectionists whose objective was to stop or limit animal research. It became essential for scientists both to confront their persistent critics and to face up to the problems they knew existed. The Chicago area was a hotbed of antivivisection activity in

8

FRANKLIN M. LOEW AND BENNETT J. COHEN

the Medical School, were notified hastily. They intercepted Mrs. McLaughlin and the dog at the entrance of the Medical School. At this point, Mrs. McLaughlin made a citizen's arrest of Dr. Ivy and Dean Miller, and the protagonists proceeded to the Chicago Avenue police station. The dog was returned to the Medical School, and the arrests subsequently were nullified. However, the incident was given wide publicity in the media, especially in the Chicago Herald-Examiner, reflecting the antivivisection sentiments of publisher William Randolph Hearst and Mr. Hearst's close friends, Mrs. McLaughlin and actress Marion Davies. This incident illustrates the flavor of the relationships between animal research scientists and their critics in the mid- and late 1940s. Without realizing it, Mrs. McLaughlin had alerted the scientific community to the significant and determined opposition it faced. An organized response was a clear necessity.

B.

Fig. 9.

Cover page of antivivisection brochure, late 1940s.

1945 (Fig. 9). The National Antivivisection Society, based in Chicago, was distributing its literature widely and working for legislative abolition of animal research in Illinois and elsewhere. Orphans of the Storm, a humane society with a strong antivivisection outlook, was headed by its founder, Irene Castle McLaughlin, a famous dancer of the World War I era. Mrs. McLaughlin had been appointed to the Animal Advisory Committee of the Arvey Ordinance. The ordinance permitted the medical schools in Chicago to obtain unclaimed dogs and cats from the public pound. On one occasion, during an inspection of the animal facilities at Northwestern University Medical School, Mrs. McLaughlin deliberately removed a dog from its cage because she felt that the animal was not receiving adequate treatment. She planned to take the dog to her shelter in Winnetka. Dr. Andrew C. Ivy, then professor and chairman of the Department of Physiology, and Dr. J. Roscoe Miller, then dean of

The National Society for Medical Research

The National Society for Medical Research (NSMR) was created in 1946 by the Association of American Medical Colleges (AAMC) and about 100 supporting groups (Grafton, 1980). AAMC had become concerned that progress in medical science could be jeopardized if antivivisectionists were successful in their numerous campaigns to prohibit or restrict animal experimentation. It was deemed essential to establish a separate organization to counter these antiscience activities and, especially, to promote better public understanding of the needs and accomplishments of animal experimentation. Public support of animal research depended upon such understanding. NSMR headquarters were established in Chicago, and Dr. Anton J. Carlson was elected the organization's first president (Fig. 10). From its inception, NSMR contributed importantly to campaigns conducted at the state, city, and county levels to win public support for the use of public pounds as a source of unclaimed dogs and cats for research (Fig. 11). Antivivisection efforts to restrict or prohibit animal experimentation were fought successfully in several states. NSMR also developed much educational material about animal research and distributed it throughout the country. In the late 1940s, NSMR provided legal counsel to several Chicago-area research scientists who had been attacked by the Hearst newspapers. Dr. Nathan Brewer was among this group. Libel suits were filed and dragged on for several years, until shortly before William Randolph Hearst's death in 1951. The suits were concluded in favor of the scientists, but without significant monetary settlement. The Hearst publications agreed to stop publishing statements tending to damage the reputations of scientists involved in animal research. The suits and Mr. Hearst's death brought to an end the extremist approach of the Hearst publications to the vivisection-antivivisection issue. In 1952, a cause celebre developed within the American Physiological Society (APS) that also involved other constitu-

9

1. LABORATORY ANIMAL MEDICINE: H I S T O R I C A L P E R S P E C T I V E S We are determining the amount of abuse that life will endure in unanaesthetized animals m i n the name of science. We are producing frustration ulcers in experimental animals under shocking conditions--in the name of science. We are observing animals for weeks, months, or even years under infamous conditions m i n the name of science. Yet it is the National Society for Medical Research and its New York satellite that are providing the means to these ends. And how is it being accomplished? By undermining one of the finest organizations of our country. THE AMERICAN H U M A N E SOCIETY.

With the aid of the halo supplied by the faith of the American people in medical science, the NSMR converts sanctuaries of mercy into animal pounds at the beck and call of experimental laboratories regardless of how the animals are to be used. What a travesty of humanity! This may well prove to be the blackest in the history of medical science.

Fig. 10. Dr. Anton J. Carlson, professor of physiology at the University of Chicago and first president of the National Society for Medical Research. (Courtesy of the University of Chicago Archives and Dr. N. R.. Brewer.)

ent societies of the Federation of American Societies for Experimental Biology (FASEB) and the NSMR. Robert Gesell, M.D., professor and chairman of the Department of Physiology at the University of Michigan from 1923 to 1954 (Fig. 12), made the following statement at the APS business meeting on April 15: The National Society for Medical Research would have us believe that there is an important issue in vivisection versus antivivisection. To a physiologist there can be no issue on vivisection per se. The real and urgent issue is humanity versus inhumanity in the use of experimental animals. But the NSMR attaches a stigma of antivivisection to any semblance of humanity. Antivivisection is their indispensable bogie which must be kept before the public at any cost. It is their only avenue towards unlimited procurement of animals for unlimited and uncontrollable experimentation. The NSMR has had but one idea since its organization, namely, to provide an inexhaustible number of animals to an ever growing crowd of career scientists with but little biological background and scant interest in the future of man. Consider what we are doing in the name of science, and the issue will be clear. We are drowning and suffocating unanaesthetized animalsmin the name of science.

Dr. Gesell had supported the formation of NSMR but subsequently took issue with Dr. Carlson on NSMR involvement in pound legislation. Dr. Gesell also was dissatisfied with what he perceived to be a lack of interest by NSMR in promulgating more detailed humane criteria for the care and use of animals than existed at that time. He knew of the formation of the American Association for Laboratory Animal Science in 1950 and of the assistance that NSMR provided to AALAS in its formative years. This did not soften his view that NSMR was not constructively dealing with the issue of humane use of animals in research. Dr. Gesell asked that his statement be made part of the minutes. Vigorous discussion followed. Dr. Ralph Gerard, APS president, explained that it was not APS policy to include all statements by APS members in the minutes. Finally, a motion by Dr. Maurice Visscher, then professor and chairman of the Department of Physiology at the University of Minnesota (later, president of NSMR), was adopted, to be included in the minutes, "that Dr. Gesell had made a statement concerning animal experimentation which criticized physiologists and the NSMR and that the statement had been challenged" (APS minutes, April 15, 1952). At a second business meeting, on April 17, 1952, APS adopted the following formal response: The American Physiological Society reaffirms its sincere belief that the moral justification for humane animal experimentation, for the purpose of furthering biological and medical knowledge, in the interest of both human and animal welfare, is completely established. The American Physiological Society rejects the sweeping allegations made by Dr. Gesell in a recent business meeting. The American Physiological Society rejects unequivocally the inference that its members are insensitive to the moral responsibilities which they have in protecting the welfare of man and animals. The American Physiological Society expresses the hope that in the future all of its members will act in unison in promoting conditions facilitating humane animal experimentation.

Despite efforts by Dr. Gesell to prevent and suppress use of his statement by antivivisection groups, it was distributed

10

Fig. 11.

FRANKLIN M. LOEW AND BENNETT J. COHEN

Insidecover and page 1 of NSMR Bulletin, May-June 1949,reporting a national opinion poll on favorable public attitudes toward animal research.

widely by these groups in their campaigns for legislative restriction of animal research. After all, it reflected the views of a respected American physiologist. The APS response was not similarly distributed by these groups. Dr. Carlson prepared a lengthy and thoughtful rebuttal of the Gesell statement for members of FASEB, but it too had only a limited distribution (A. J. Carlson, letter to FASEB members, September 17, 1952). After Dr. Gesell's death in 1954, his daughter, Christine Stevens, a founder and the president since 1950 of the Animal Welfare Institute, continued to espouse her father's views and her own strong opinion that too many scientists were insufficiently concerned about humane treatment of animals in research. These views have included critical commentary about NSMR and AALAS (Stevens, 1963, 1976, 1977). The Gesell-APS-NSMR controversy highlighted issues that, to this day, underlie the difficult relations between the scientific community and the animal welfare movement. Perhaps a positive result has been that the controversy also contributed to the climate of opinion that led additional numbers of medical research institutions to employ veterinarians to care for research animals. Ultimately, the controversy raised questions that influenced and should continue to influence all those having a constructive concern for both science and animal welfare. What, if any, are the appropriate limits on scientific freedom in

animal research? Who is best qualified to make judgments about the propriety of animal studies? Can "humaneness" be legislated? Is there not a moral imperative to conduct animal studies in the interest of human and animal welfare? How best can refinement of animal studies, reduction in the numbers of animals used, and replacement of animals, where appropriate, best be incorporated into the design of experiments (Russell and Burch, 1959)? In the 1980s, the National Society for Medical Research merged with the Association for Biomedical Research (ABR), which had been organized in 1979, to become the National Association for Biomedical Research (NABR), with Dr. Edward C. Melby as its first president. NABR works with scientists and elected officials on behalf of biomedical research. The Scientists' Center for Animal Welfare (SCAW) was formed about the same time to contribute scientific perspectives to laboratory animal welfare.

CO The A m e r i c a n Association for L a b o r a t o r y A n i m a l Science

By 1949, veterinarians were managing the laboratory animal facilities at five Chicago-area institutions: the University of

1. LABORATORY ANIMAL MEDICINE: HISTORICAL PERSPECTIVES

Fig. 12. Dr. Robert Gesell, professor and chairman, 1923-1954, Department of Physiology,Universityof Michigan. Dr. Gesell's statement at the APS business meetingin 1952became a cause celebre. Photographtaken in the late 1930s. (Courtesy of the Bentley Historical Library, Universityof Michigan.)

Chicago (Nathan R. Brewer), the University of Illinois (Elihu Bond), Northwestern University (Bennett J. Cohen), the Argonne National Laboratory (Robert J. Flynn), and the Hektoen Institute for Medical Research of Cook County Hospital (Robert J. Schroeder). The veterinarians sought one another out to exchange information and experience on the day-to-day problems they were encountering. The group met at least monthly, starting during the summer of 1949. Among the subjects reviewed at these meetings were husbandry and diseases of laboratory animals, the need to develop basic standards of animal care, and the need to counter the strident antivivisection attacks on medical science in the Chicago area. The Chicago veterinarians knew that few other veterinarians elsewhere in the country were engaged in the activity they had begun to identify as "laboratory animal care." For example, in reviewing the proceedings of a symposium on animal colony maintenance, held under the sponsorship of the New York Academy of Sciences in 1944 (Farris et al., 1945), they noted that not a single veterinarian had presented a paper. Their perception was that the problems of laboratory animal care merited organized attention, and they wondered whether others felt the same way. Special meetings were arranged when colleagues from other institutions visited Chicago. Among these colleagues were C. E Schlotthauer, D.V.M., Mayo Clinic; Charles A. Slanetz, Ph.D., director of the Central Animal Facility at the College of Physicians and Surgeons, Columbia University; Harry Herrlein, Rockland Farms, New City, New York (then a major commer-

11

cial rodent and rabbit breeding facility); C. N. W. Cumming, Carworth Farms, New City, New York (also a major rodent breeding facility at the time); and W. T. S. Thorp, D.V.M., then chief of the Laboratory Aids Branch, National Institutes of Health. These meetings were exciting, interesting, and rewarding to the participants. They demonstrated that interest in laboratory animal problems extended well beyond the Chicago area and included individuals who had a broad range of scientific, professional, and technical backgrounds. In a letter signed by the five Chicago veterinarians and sent in May 1950 to individuals in the United States and Canada thought to have an interest in the care of laboratory animals, the development of a national organization was proposed "to be open to all individuals interested in animal care work on an institutional scale" (Flynn, 1980). The response was overwhelmingly favorable, and the first meeting was convened in Chicago on November 28, 1950, with an attendance of 75. The founding members named the organization the Animal Care Panel (ACP), reflecting their broad concern with the c a r e of laboratory animals (Fig. 13). "Panel" was used in the name to emphasize the organization's purpose as a forum for the exchange of information on all aspects of animal care. Dr. Brewer was elected the first president, a post he held until 1955. During the

Fig. 13. Coverof early descriptive brochure about the Animal Care Panel, now the American Associationfor LaboratoryAnimal Science.

12

FRANKLIN M. LOEW AND BENNETT J. COHEN

early meetings of the ACP, its programs were dominated by papers on animal colony management, design of facilities and equipment, and descriptions of common diseases. This reflected the relatively underdeveloped "state of the art" with respect to the technology of animal care. ACP meetings became more sophisticated with each passing year. By the sixth meeting, in 1955, original research was being presented (Flynn, 1980). Shortly thereafter, the annual Proceedings of the Animal Care Panel was transformed into the scientific journal Laboratory Animal Care and subsequently renamed Laboratory Animal Science. The ACP grew rapidly in its institutional and individual membership and was characterized by the unique diversity of scientific,, professional, and technical backgrounds of its members. By 1960, the ACP was able to employ a full-time executive secretary, Joseph J. Garvey. He had served earlier as assistant executive secretary of NSMR and, in this position, had assisted with ACP administration, reflecting the support and encouragement the ACP received from NSMR in its formative years. From its inception, the ACP also worked to enhance the stature and training of laboratory animal technicians. This activity is exemplified in the career and contributions of George Collins (1917-1974), who served successively as supervisor of the animal facilities at the Argonne National Laboratory, Rockefeller University, and the AMA Education and Research Foundation (Brewer, 1980). He was a founding member of AALAS and, in 1963, received the AALAS Animal Technician Award. In 1967, he edited the first edition of the AALAS "Manual for Animal Laboratory Technicians," a landmark in its time (Collins, 1967). Development of standards was another early activity of the ACP. Indeed, the first edition of the "Guide for Laboratory Animal Facilities and Care" (Cohen, 1963), now known as the "Guide for the Care and Use of Laboratory Animals" (Moreland, 1978) was prepared under ACP auspices. The guide has become the basic standard regarding the use and care of animals in American research institutions. In 1967, the name of the Animal Care Panel was changed to the American Association for Laboratory Animal Science. Today, AALAS has more than 10,000 individual and institutional members and more than 48 local branches. Its annual meeting and scientific journalsmComparative Medicine (formerly Laboratory Animal Science) and a relatively new journal, Contemporary Topics in Laboratory Animal Sciencemare the principal means of scientific exchange in the field. AALAS recently published its 50-year history (McPherson and Mattingly, 1999).

D.

The Institute of Laboratory Animal Resources

Many problems of supply, standardization, and procurement of animal resources accompanied the rapid growth of medical and biological research after World War II. Concerns surfaced about these problems within the National Academy of Sciences

(NAS). These concerns developed independent of those that led to the formation of AALAS. The NAS is a private organization with a federal charter. Since 1863, it has been a principal advisor to the federal government on matters related to science and science policy (Seitz, 1967). Election to membership in NAS or its Institute of Medicine is among the highest honors a scientist can receive. It is a prestigious organization, and therefore NAS advisory groups, all of which serve without compensation, have a standing and authority they might not otherwise have. In the early 1950s, organized efforts to improve and standardize animal supply and quality had barely been initiated. Scientific standards for laboratory animal production, genetics, breeding, husbandry, and transportation did not exist. There were no good mechanisms to facilitate information exchange about laboratory animals internationally. Education and training in laboratory animal science were in an undeveloped state, and no guidelines for such training existed. Problems such as these led Dr. Paul Weiss, then chairman of the Division of Biology and Agriculture of the National Research Council (the NAS advisory arm), to appoint a Committee on Animal Resources in 1952. Dr. Weiss appointed Dr. Clarence Cook Little, the eminent geneticist and founder of the Jackson Laboratory (Bar Harbor, Maine), to be chairman. The Committee on Animal Resources recommended establishment of an Institute of Animal Resources (IAR). IAR commenced full-time operation in July 1953 (Hill, 1980). In 1956, it was renamed Institute of Laboratory Animal Resources. It was again renamed, to Institute for Laboratory Animal Research (ILAR), in the late 1990s. Historically, the ILAR office has been headed by an executive secretary (now named the director), with oversight from an advisory council and executive committee that is appointed in accordance with NAS-NRC procedures. Dr. Orson Eaton, a geneticist from the Bureau of Animal Industry, was the first executive secretary. He was succeeded by the vigorous and energetic Berton F. Hill, who also had a background in genetics. During Hill's tenure (1955-1965), ILAR became established as the major standards-development organization within laboratory animal science. In 1965, Hill was succeeded by Dr. Robert H. Yager, former director of the animal facilities at the Walter Reed Army Institute of Research. Dr. Yager was one of the "founding fathers" of ILAR, having served on the Committee on Animal Resources in 1952. Among many important ILAR accomplishments during Dr. Yager's tenure were the development of the first guidelines for education and training in laboratory animal medicine (Clarkson, 1967); the publication of an important national survey of animal facilities in the United States (Trum, 1970), following up on the first such survey, during Hill's tenure (Thorp, 1964); and enlargement of United States participation in international laboratory animal activities through support of the International Council on Laboratory Animal Science (ICLAS), known then as the Intentional Committee on Laboratory Animals (ICLA). During the formative years of ILAR and AALAS, there were

1. LABORATORY ANIMAL MEDICINE: HISTORICAL PERSPECTIVES

obvious areas of overlap. Both organizations had been involved in standards development, both were holding scientific meetings, and in many other areas their interests coincided. In 1962, the executive committees agreed on a division of responsibility that solidified ILAR's role in standards development (Garvey and Hill, 1963). This proved to be an important agreement because it enabled ILAR and AALAS to concentrate on the things each could do best. It also was important because of the position that ILAR standards subsequently achieved under the umbrella of the National Academy of Sciences.

13

sections, divisions, or departments of laboratory animal medicine or comparative medicine. With this development, laboratory animal medicine began to establish its separate identity. The committee then proceeded to seek recognition of specialization in the field by the AVMA. Late in 1956, the committee disbanded in favor of the American Board of Laboratory Animal Medicine and the new specialty was born.

V.

E D U C A T I O N AND TRAINING IN LABORATORY

ANIMAL MEDICINE E.

The American College of Laboratory Animal Medicine

Formal recognition of veterinary medical specialty fields by the American Veterinary Medical Association (AVMA) began in 1951 with the establishment of the American Board of Veterinary Public Health and the American College of Veterinary Pathologists (Grafton, 1974). In 1957, laboratory animal medicine was accorded the same recognition, when the American Board of Laboratory Animal Medicine (ABLAM) was incorporated under the laws of the state of Illinois, with 18 "Charter Fellows." In August 1961, the name was changed to American College of Laboratory Animal Medicine (ACLAM), and the designation "Fellow" was discontinued in favor of "Diplomat," a term used by other specialties. ACLAM was established to encourage education, training, and research in laboratory animal medicine, to establish standards of training and experience for qualification of specialists, and to certify specialists by examination. These objectives, which today are well understood and accepted, were but a vague concept in the early 1950s. On June 23, 1952, thirty-four veterinarians assembled in a meeting room at the Ambassador Hotel in Atlantic City, during the AVMA meeting, to consider the role of veterinarians in laboratory animal care. There was a lively discussion about this rapidly developing field, with special emphasis on defining activities that veterinarians were uniquely qualified to pursue ("News reports," 1952). Those in attendance noted that medical schools were employing an increasing number of veterinarians and that further growth seemed likely. They felt that more specific definition of this newly developing field was needed. The group organized as the Committee on the Medical Care of Laboratory Animals, with Dr. Nathan R. Brewer as chairman, Dr. Mark Morris as vice chairman, and Dr. W. T. S. Thorp as secretary. The decision was made to organize programs of special interest to laboratory animal veterinarians at future AVMA meetings. During the ensuing 4 years, the term "laboratory animal medicine" came into use to differentiate the activities of veterinarians from other professional or technical people working in the broad area of laboratory animal science. Additionally, within this period, a number of laboratory animal veterinarians were able to establish academic units in their institutions (Clarkson, 1961 a), some of which were identified as

Veterinarians entering "laboratory animal care" in the 1940s and early 1950s had to be largely self-trained. They relied on their basic education in veterinary medicine and on what they could learn from one another at AALAS and AVMA meetings (Clarkson, 1980). There were no post-D.V.M, training programs. The establishment of ACLAM in 1957, with its strong commitment to fostering education and training, stimulated more specific discussion of training needs in this new field. At this same time, NIH-supported training programs were being initiated in basic medical science and clinical fields in leading scientific institutions throughout the country. As mentioned in Section IV, E, by the late 1950s "laboratory animal medicine" was being conducted as an academic program in a few medical schools (Clarkson, 1961a). In such settings it became possible to consider establishing postdoctoral training. The animal resources program in the Division of Research Resources (DRR) at NIH had not yet matured, and the Animal Resources Branch (ARB) did not have training authority. However, with great insight about the underlying significance of laboratory animal medicine, the Physiology Training Committee, within the Division of General Medical Sciences (later to become the National Institute of General Medical Sciences [NIGMS]), decided to accept applications to establish a few research training programs in this new field. During the time this matter was under consideration, Dr. Howard Jenerick and Dr. J. H. U. Brown, both physiologists, served as secretaries of the committee. The committee chairmen were Dr. T. C. Ruch, professor and chairman of the Department of Physiology at the University of Washington, and Dr. Wallace O. Fenn, professor and chairman of the Department of Physiology at the University of Rochester. The committee's decision to sponsor such training was of paramount importance, because for the first time, training in laboratory animal medicine was placed on a par with other areas of research training in the medical and biological sciences. In the ensuing years, the specialists that now compose the academic core of present-day laboratory animal medicine were trained in such programs. The first training program was established in January 1960 at the Bowman Gray Medical School, directed by then assistant professor of laboratory animal medicine, Thomas B. Clarkson.

14

In July 1960, a second program was started at UCLA Medical School, directed by Bennett J. Cohen, then assistant professor of physiology and director of the vivarium. The program moved with Dr. Cohen to the University of Michigan in 1962. Later, programs were established at other medical schools and universities: Tulane University (1963, Dr. K. E Burns), Stanford University (1965, Dr. O. A. Soave), University of Florida (1965, Dr. A. E Moreland), Johns Hopkins University (1968, Dr. E. C. Melby), and University of Missouri (1968, Dr. C. C. Middleton). Edgewood Arsenal, Maryland, and Brooks Air Force Base, Texas, became the sites of training programs for military veterinarians. With strong encouragement from Dr. Jules S. Cass, chief veterinary medical officer at the Veterans Administration, a program was established in the mid-1960s at the Hines Veterans Administration Medical Center in the Chicago area. This program was guided initially by Dr. Robert E Locke. The "core of knowledge" comprising laboratory animal medicine was not well defined at the time these early programs were started. The curricula of the training programs simply reflected the outlook of the directors and the settings in which they were conducted. Some were formal graduate programs leading to a master of science degree. Others stressed residency training analogous to that of residency programs in the medical specialties. Thus, there were research-oriented programs and others that focused more on the clinical or managerial aspects of laboratory animal medicine (Clarkson, 1961b). By 1964, the need for better definition of the field had become apparent. An ILARsponsored workshop held in that year pointed to the need for educational guidelines to be used by all training programs (Clarkson, 1965). The first such guidelines subsequently were published (Clarkson, 1967). A formal process is now in place by ACLAM that reviews and approves training programs in laboratory animal medicine. In the mid-1960s, ARB received training authority, and the training grants in laboratory animal medicine were transferred there. Other aspects of the NIH extramural animal resources program also grew significantly, as, for example, the laboratory animal science program. Overall, the impact of these programs on laboratory animal medicine has been enormously beneficial. Some of the early training programs have been terminated, but most have continued, and several new programs have been started. These programs emphasize research training in comparative medicine and continue to be funded by NIH. Furthermore, a number of institutions have initiated residency programs independent of NIH sponsorship. Post-D.V.M. training is recognized today as necessary for a career in academic laboratory animal medicine. The American Society of Laboratory Animal Practitioners (ASLAP) was founded in 1967 to promote dissemination of knowledge about laboratory animal medicine, to foster research, and to serve as a spokesman for veterinarians in laboratory animal practice. ASLAP, together with ACLAM, has played an important role in encouraging continuing education

FRANKLIN M. LOEW AND BENNETT J. COHEN

programs in the field. Continuing education has become an important adjunct to the formal training programs in laboratory animal medicine, and such activities now are a regular component of the scientific program at AVMA and AALAS meetings.

VI. I M P A C T OF LAWS, R E G U L A T I O N S , A N D G U I D E L I N E S ON L A B O R A T O R Y A N I M A L M E D I C I N E

Prior to 1966, no federal law existed in the United States specifically regulating the acquisition or care of research animals. Pressure for federal legislation mounted steadily in the late 1950s and early 1960s. Animal welfare organizations, such as the Humane Society of the United States, the Society for Animal Protective Legislation, and the Animal Welfare Institute, argued for legislation to curb alleged "pet stealing" and abuse of animals in laboratories. They used the media effectively to generate public interest in their causes (e.g., cover headline of Life, February 4, 1966: "Concentration Camps for Lost and Stolen Pets: Your Dog Is in Cruel Danger"). Organizations of the scientific community, such as AALAS, NSMR, and FASEB, argued against the proposed legislation. Their position was that the best way to foster the humane use and care of animals was to provide better support of research and training, provide funds to upgrade animal facilities, and strengthen selfregulation through mechanisms such as the newly organized (1965) American Association for Accreditation of Laboratory Animal Care (AAALAC) and institutional committees to assess the adequacy of animal care and use programs (Galton, 1967). In the mid-1990s, AAALAC assumed a more international role and renamed itself the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). After holding hearings on a series of bills in the House of Representatives and the Senate dealing with the regulation of animal research, Congress passed the Laboratory Animal Welfare Act in 1966. The principal purposes were to regulate commercial traffic in dogs, cats, monkeys, rabbits, guinea pigs, and hamsters and to establish standards for their housing and transportation and for "adequate veterinary care." The act, administered by the U.S. Department of Agriculture (USDA), established a legal requirement for scientific institutions to provide appropriate care for research animals by or under the direction of a veterinarian. Since its initial passage, the act has been broadened and its name changed to Animal Welfare Act (see Chapter 2). According to the USDA Animal Welfare Report of Fiscal Year 2000, the number of registered research facilities totals 1,231. Thus, the act has contributed to the betterment of animal care through its requirement for participation of veterinarians in institutional animal medicine programs. The National Institutes of Health has long recognized that

1. LABORATORY ANIMAL MEDICINE: HISTORICAL PERSPECTIVES

good research requires animals that are healthy and well cared for. In 1963, NIH published the first edition of the "Guide for Laboratory Animal Facilities and Care" (Cohen, 1963), as developed by the Standards Committee of the Animal Care Panel. Revised several times since 1963 by ILAR's Committee on Revision of the Guide, it is now the "Guide for the Care and Use of Laboratory Animals." Since 1963, NIH and other granting agencies have required scientific institutions to provide assurance of compliance with the standards in the guide as a condition for receiving funds for research. The guide also is used as the basis for accreditation by AAALAC. One of the basic requirements established in the guide is for the provision of adequate veterinary medical care, a concept also expressed in the regulations of the Department of Agriculture. In 1978, the Food and Drug Administration (FDA) promulgated regulations for the conduct of animal experiments relating to new or existing pharmaceutical medicinal substances, food additives, or other chemicals. These regulations, known as the Good Laboratory Practice (GLP) regulations, also specify the need for adequate diagnosis, treatment, and control of diseases in animals used in such studies. Thus, the standards of AAALAC, NIH, USDA, and FDA all include specific references to veterinary medical participation in the care of laboratory animals. In fact, these standards provide the basis for implementation of the legal requirement that research animals receive "adequate veterinary care."

VII.

REGULATION OF ANIMAL RESEARCH IN THE UNITED K I N G D O M AND C A N A D A

Until 1986, the use of animals in the United Kingdom was governed by the Cruelty to Animals Act of 1876 (French, 1975) (see Chapter 2). The Animals (Scientific Procedures) Act 1986 is now the governing legislation in the U.K. Major debates about "vivisection" occurred in Parliament in the late 1860s and early 1870s. Finally, the act was passed with the active support of leading scientists of that time. Antivivisectionists had been working for a law that would have prohibited animal research or regulated it more strictly than called for in the act. The act requires the licensing of scientists using animals. There also is provision for certificates to be issued to scientists, depending upon the species used and the nature of the experimentation. The legal regulatory relationship is between the government and the scientist, not the government and the institution, as it is in the United States. Veterinarians, as such, do not have legal standing in the implementation of the act, although within recent years veterinarians have been added to the Home Office inspectorate. In the private sector, the British Laboratory Animals Veterinary Association was organized in the early 1970s and is affiliated with the British Veterinary Association. The

15

specialty of laboratory animal medicine does not have standing comparable to that accorded ACLAM by the AVMA in the United States. Nevertheless, laboratory animal medicine clearly is an emerging field in Great Britain, and excellent animal medicine programs are evolving under the leadership of British laboratory animal veterinarians. The use of animals in Canada is not specifically regulated by federal law. However, in 1968, the Canadian Council on Animal Care (CCAC) was established by the major agencies that fund animal research (see Chapter 2). Dr. Harry Rowsell has played an instrumental role in the founding of CCAC and in its operation over the years. The CCAC assesses animal care in Canadian research laboratories based on the standards in the "Guide to the Care and Use of Experimental Animals" (CCAC, 1980). This program has been a major factor in the elevation of animal care standards and the employment of veterinarians in Canadian research laboratories. In addition, some of the provinces have laws that apply to the requisition or use of animals in research in those provinces (e.g., Ontario and Alberta).

VIII.

C O M M E R C I A L AND ACADEMIC BREEDING OF RODENTS

The development of gnotobiology in the 1950s represented a major conceptual and technological advance in the commercial breeding of healthier rodents for research (Foster, 1958). This advance had been preceded by laboratory research (Trexler and Reynolds, 1957; Reyniers, 1957) and years of attempts to breed animals that would lead to the most unambiguous results in research as possible. With the introduction of genetically engineered mice, academic and industrial animal resource programs have increasingly been engaged in breeding and characterizing numerous lines of mice with unique genetic makeup (see Chapter 3).

IX.

CONCLUSION

A complete history of the individuals and organizations that have influenced the development of laboratory animal science and medicine would require a separate volume. Some of these topics are the history of laboratory animal science internationally; the history of the major commercial and institutional animal colonies and of the important genetic stocks and strains of laboratory animals; the evolution of animal technology, including the field of gnotobiology; the contributions of animal technicians to laboratory animal science; the origins of the NIH extramural and intramural laboratory animal science programs;

16

FRANKLIN M. LOEW AND BENNETT J. COHEN

and reviews, in historical perspective, of the major diseases of laboratory animals. Some of these topics are dealt with elsewhere (Cohen, 1979; Foster, 1980; Lindsey, 1979; McPherson, 1980; McPherson and Mattingly, 1999). Others must await documentation of the historical record. Laboratory animal science and medicine are fields of expanding horizons that provide challenging opportunities for satisfying professional careers. The following chapters clearly document the progress that has occurred while pointing to many challenges that lie ahead. It remains for each generation of laboratory animal scientists to build on the base of knowledge established by its predecessors and so determine its own future.

REFERENCES

Bernard, C. (1865). "An Introduction to the Study of Experimental Medicine" (trans. H. C. Greene, p. 123. Reprinted by Dover, New York, 1957). Braasch, W. E (1969). "Early Days of the Mayo Clinic," p. 141. Thomas, Springfield, Illinois. Brewer, N. R. (1980). Personalities in the early history of laboratory animal science and medicine. Lab. Anim. Sci. 30(4), Part 2, 741-758. Brimhall, S. D., and Hardenbergh, J. G. (1922). A study of so-called kennel lameness: Preliminary report. J. Am. Vet. Med. Assoc. 61, 145-154. Brimhall, S. D., and Mann, E C. (1917). Pathologic conditions noted in laboratory animals. J. Am. Vet. Med. Assoc. 52, 195-204. Brimhall, S. D., Mann, E C., and Foster, J. P. (1919-1920). The relation of the common bile duct to the pancreatic duct in common domestic and laboratory animals. J. Lab. Clin. Med. 5, 203-206. Bustad, L. K., Gorham, J. R., Hagreberg, G. A., and Padgett, G. A. (1976). Comparative medicine: Progress and prospects. J. Am. Vet. Med. Assoc. 169, 90-105. Canadian Council on Animal Care (CCAC) (1980). "Guide to the Care and Use of Experimental Animals," Vol. 1, 112. CCAC, Ottawa, Ontario. Clarkson, T. B. (1961a). Laboratory animal medicine and the medical schools. J. Med. Educ. 36, 1329-1330. Clarkson, T. B. (1961b). Graduate and professional training in laboratory animal medicine. Fed. Proc., Fed. Am. Soc. Exp. Biol. 20, 915-916. Clarkson, T. B. (1965). Laboratory animals. 4. Graduate education in laboratory animal medicine. Proceedings of a workshop. NAS-NRC, Publ. 1284, 33. Committee on Professional Education, Institute of Laboratory Animal Re. sources. Clarkson, T. B. (1967). A guide to postdoctoral training in laboratory animal medicine. NAS-NRC, Publ. 1483, 9. Committee on Professional Education, Institute of Laboratory Animal Resources. Clarkson, T. B. (1980). Evolution and history of training and academic programs in laboratory animal medicine. Lab. Anim. Sci. 30(4), Part 2, 790792. Cohen, B. J. (1959a). The early history of animal experimentation and animal care. 1. Antiquity. Lab. Anim. Sci. 9, 39-45. Cohen, B. J. (1959b). The evolution of laboratory animal medicine in the United States. J. Am. Vet. Med. Assoc. 135, 161-164. Cohen, B. J. (1963). "Guide for Laboratory Animal Facilities and Care." Anim. Facilities Stand. Comm., Anim. Care Panel, Public Health Serv. Publ. 1024, p. 33. U.S. Dept. of Health, Education and Welfare, Washington, D.C. Cohen, B. J. (1979). ILAR News 22(2), 26. Cohen, M. R., and Drabkin, I. E. (1948). "Sourcebook in Greek Science," p. 479. McGraw-Hill, New York.

Collins, G. R. (1967). "Manual for Laboratory Animal Technicians." Animal Technician Training Committee, American Association for Laboratory Animal Science Publ. 67-3. Joliet, Illinois. Duclaux, E. (1920). "Pasteur--The History of a Mind" (trans. E. E Smith and E Hedges), p. 363. Saunders, Philadelphia. Farris, E. J., Carnochan, E G., Cumming, C. N. W., Farber, S., Hartman, C. G., Hutt, E B., Loosli, J. K., Mills, C. A., and Ratcliffe, H. L. (1945). Animal colony maintenance. Ann. N.Y. Acad. Sci., 46, 1-126. Fisher, G. J. (1881). Historical and bibliographical notes. 12. Herophilus and Erasistratus. The Medical School of Alexandria, Bc 320-250. Ann. Anat. Surg. 4, 28-67. Flynn, R. J. (1980). The founding and early history of the American Association for Laboratory Animal Science. Lab. Anim. Sci. 30(4), Part 2, 765779. Foster, H. L, (1958). Large scale production of rats free of commonly occurring pathogens and parasites. Proc. Anim. Care Panel 8, 92-99. Foster, H. L. (1980). The history of commercial production of laboratory rodents. Lab. Anim. Sci. 30(4), Part 2, 793-798. French, R. D. (1975). "Antivivisection and Medical Science in Victorian Society," p. 425. Princeton Univ. Press, Princeton, New Jersey. Galton, L. (1967). Pain is cruel, but disease is cruel too. N.Y. Times, Sec. 6 (Magazine), February 26, p. 30. Garrison, F. H. (1929). "An Introduction to the History of Medicine," 4th ed. Saunders, Philadelphia. Garvey, J. J., and Hill, B. E (1963). Cooperation for progress. Lab. Anim. Care 13, 179-180. Grafton, T. S. (1974). The veterinary profession: A review of its progress in the United States and some indications for the future. Vet. Rec. 94, 441-443. Grafton, T. S. (1980). The founding and early history of the National Society for Medical Research. Lab. Anim. Sci. 30(4), Part 2, 759-764. Griffin, C. A. (1952). A study of prepared feeds in relation to Salmonella infection in laboratory animals. J. Am. Vet. Med. Assoc. 121, 197-200. Hales, S. (1740). "Statical Essays.'' Innys and Manby, London. Hardenbergh, J. G. (1923). The value of animal experimentation to veterinary medicine. J. Am. Vet. Med. Assoc. 62, 731-735. Hardenbergh, J. G. (1926-1927). Epidemic lymphadenitis with formation of abscess in guinea pigs due to infection with hemolytic streptococcus. J. Lab. Clin. Med. 12, 119-129. Hill, B. E (1980). The founding and early history of the Institute of Laboratory Animal Resources. Lab. Anim. Sci. 30(4), 780-785. Hoff, H. E., Geddes, L. A., and McCrady, J. D. (1965). The contributions of the horse to knowledge of the heart and circulation. Conn. Med. 29, 795-800. Ingle, D. J. (1979). Anton J. Carlson: A biographical sketch. Perspect. Biol. Med. 29, Part 2, 114-136. Lindsey, J. R. (1979). Origin of the laboratory rat. In "The Laboratory Rat" (H. G. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), Chap. 1, pp. 2-36. Academic Press, New York. Loew, E M. (1982). Animal experimentation. Bull. Hist. Med. 56, 123-126. McPherson, C. W. (1980). The origins of laboratory animal science at the National Institutes of Health. Lab. Anim. Sci. 30(4), Part 2, 786-789. McPherson, C. W., and Mattingly, S. E (1999). "Fifty Years of Laboratory Animal Science." AALAS, 9190 Crestwyn Hills Dr., Memphis TN 38125. Malinin, T. I. (1979). "Surgery and Life," p. 242. Harcourt Brace Jovanovich, New York. Meyer, K. E (1928). Communicable diseases of laboratory animals. In "The Newer Knowledge of Bacteriology and Immunity" (E. O. Jordan and I. S. Falk, eds.), pp. 607-638. Univ. of Chicago Press, Chicago. Meyer, K. E (1958). Introductory address. Lab. Anim. Sci. 8, 1-5. Mitchell, S. W. (1895). Memoir of John Call Dalton, 1825-1889. Biographical memoirs. Natl. Acad. Sci. 3, 177. Moreland, A. M. (1978). "Guide for the Care and Use of Laboratory Animals." National Academy of Sciences, National Research Council, Institute of Laboratory Animal Resources, Washington, D.C.

1. LABORATORY ANIMAL MEDICINE: HISTORICAL PERSPECTIVES News reports (1952). J. Am. Vet. Med. Assoc. 121, 257. Physicians of the Mayo Clinic and the Mayo Foundation (1937). Univ. of Minnesota Press, Minneapolis (p. 184). Reyniers, J. A. (1957). The control of contamination in colonies of laboratory animals by use of germfree techniques. Proc. Anim. Care Panel 7, 9-29. Russell, W. M. S., and Burch, R. L. (1959). "The Principles of Humane Experimental Technique," p. 238. Methuen, London. Saunders, J. B. de C. M., and O'Malley, C. D. (1950). "The Illustrations from the Works of Andreas Vesalius of Brussels," p. 128. World Publ. Co., New York. Schlotthauer, C. F. (1950). Procurement of animals. Lab. Anim. Sci. 1, 20-25. Schwabe, C. W. (1978). "Cattle, Priests, and Progress in Medicine," p. 277. Univ. of Minnesota Press, Minneapolis. Seitz, E (1967). The National Academy of Sciences. J. Wash. Acad. Sci. 57, 38-41. Singer, C. (1957). "A Short History of Anatomy and Physiology from the Greeks to Harvey," p. 209. Dover, New York. Stevens, C. (1963). Letter to the editor. Perspect. Biol. Med. 7(1) (Autumn), 129-131. Stevens, C. (1976). Humane considerations for animal-models. In "Animal

17 Models of Thrombosis and Hemorrhagic Diseases," DHEW Publ. (NIH) 76-982, pp. 151-158. U.S. Dept. of Health and Human Services, National Institutes of Health, Bethesda, Maryland. Stevens, C. (1977). Humane perspectives. In "The Future of Animals, Cells, Models, and Systems in Research, Development, Education, and Testing," pp. 16-24. National Academy of Sciences, Washington, D.C. Thorp, W. T. S. (1964). "ILAR Committee on the Animal Facilities: Survey Animal Facilities in Medical Research," Final Report and Tabular Appendix, p. 157. National Academy of Sciences, National Research Council, Institute of Laboratory Animal Resources, Washington, D.C. Trexler, P. C., and Reynolds, L. I. (1957). Flexible film apparatus for the rearing and use of germfree animals. Appl. Microbiol. 5, 406-412. Trum, B. E (1970). ILAR Committee on Laboratory Animal Facilities and Resources Survey. Laboratory animal facilities and resources supporting biomedical research. Lab. Anim. Care 20, 795-869. Welch, W. H. (1889). Preliminary report of investigations concerning the causation of hog cholera. Johns Hopkins Hosp. Bull. 1(1), 9-10. Wood, C. A., ed. (1931). "An Introduction to the Literature of Vertebrate Zoology," Chap. 4. Oxford Univ. Press, London.

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Chapter 2 Laws, Regulations, and Policies Affecting the Use of Laboratory Animals Lynn C. Anderson

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Animal Welfare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. U.S. Animal Welfare Act . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. PHS Animal Welfare Policy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. FDA Good Laboratory Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Interagency Cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. EPA Good Laboratory Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Professional and Scientific Associations . . . . . . . . . . . . . . . . . . . . . . . G. International Laws and Regulations . . . . . . . . . . . . . . . . . . . . . . . . . . III. Importation and Exportation of Animals and Animal Products . . . . . . . . . A. U.S. Department of Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. U.S. Department of Health and Human Services . . . . . . . . . . . . . . . . C. U.S. Department of the Interior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Environmental Protection Agency . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Hazardous Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Biohazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Chemical Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Radioactive Materials and Radiation-Emitting Equipment . . . . . . . . . V. Recombinant DNA Research Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix: Websites Pertaining to Laboratory Animals . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

19 20 20 25 26 27 27 28 28 29 29 30 30 30 31 31 31 31 31 32 32

INTRODUCTION

reliable data f r o m l a b o r a t o r y ani mal s has also h e l p e d assure the h u m a n e and ethical use of these animals. G o o d h u s b a n d r y practices, veterinary care, animal facility m a n a g e m e n t , and labora-

The use of animals in research, testing, and e d u c a t i o n is sub-

tory t e c h n i q u e s are all n e c e s s a r y to help assure the quality of scientific results.

ject to a m y r i a d of laws, regulations, policies, and standards. The public's interest in the t r e a t m e n t of l a b o r a t o r y ani mal s and l o b b y i n g by a n i m a l welfare and antivivisection o r g a n i z a t i o n s

ulations, and policies in the U n i t e d States that pertain to labora-

led to the p a s s a g e of m a n y of these laws and r e g u l a t i o n s during the s e c o n d half of the t w e n t i e t h century. H o w e v e r , the n e e d for

tory animal care and use. In m a n y instances, these g o v e r n i n g principles overlap, and, in s o m e cases, they p r o v i d e conflicting

LABORATORY ANIMAL MEDICINE, 2nd edition

This chapter will p r o v i d e an o v e r v i e w of the federal laws, reg-

Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

20

LYNN C. ANDERSON

minimal requirements. In addition, every state, the District of Columbia, and many cities and towns have enacted laws or ordinances pertaining to animal cruelty or the release of pound animals to research facilities. The Appendix at the end of this chapter lists websites appropriate for laboratory animal guidelines.

II.

A.

ANIMAL WELFARE

U.S. Animal Welfare Act

The first federal legislation in the United States to protect animals was the 28-Hour Law enacted in 1873. It required that farm animals be provided food, water, and rest at least once every 28 hours during transit. Animals used for research were first protected by federal legislation in 1966, with the passage of the Laboratory Animal Welfare Act (Pub. L. 89-544). To help address public concerns over "pet nabbing," this law required licensing of dealers (individuals or corporations) that bought or sold dogs or cats for research and registration of research facilities that used dogs or cats. It also mandated minimum animal care standards for dogs and cats before and after they were used for research. The standards did not apply while the animals were being used for an experimental purpose. The law authorized the U.S. Department of Agriculture (USDA) to develop and enforce these regulations. The USDA subsequently established standards for nonhuman primates, rabbits, guinea pigs, and hamsters, in addition to those for dogs and cats. Research facilities that used dogs or cats were required to observe the USDA-specified standards for all of these species. However, facilities that did not use dogs or cats were not required to comply with the regulations for the other species. The Laboratory Animal Welfare Act was amended in 1970 and renamed the Animal Welfare Act (Pub. L. 91-579). The scope of protection was broadened to include animals used for teaching, exhibitions, and the wholesale pet industry. "Animals" included dogs, cats, nonhuman primates, rabbits, guinea pigs, and hamsters and, with certain exceptions, any other warm-blooded animal designated by the Secretary of Agriculture. Institutions (except primary and secondary schools) that used these species in research, tests, or experiments were required to register as a research facility. For the first time, zoos were required to be licensed. Agricultural exhibitors and retail pet stores were specifically exempted. The definition of "dealer" was revised to include any person who bought or sold any dog or other animal designated by the USDA for use in research, teaching, or exhibition or as a pet at the wholesale level. The 1970 amendments also expanded the minimal animal care standards. These standards applied to animals during the course of research, not only before and after experimental use.

The act did not allow the Secretary of Agriculture to establish rules, regulations, or orders with regard to the design or performance of the research. However, it required that every research facility submit an annual report that provided the number of regulated species it used and assurance that it met professionally acceptable standards for the care, treatment, and use of animals, including the appropriate use of anesthetic, analgesic, and tranquilizing drugs. In 1976, the Animal Welfare Act was amended again (Pub. L. 94-279) to include regulation of common carriers and intermediate handlers and to establish transportation standards for animals. Standards were established for shipping conditions and for the containers in which animals were shipped. The amendments also prohibited interstate promotion or shipment of animals for animal fighting ventures. The Food Security Act of 1985 (Pub. L. 99-198) included provisions to amend the Animal Welfare Act, referred to as "The Improved Standards for Laboratory Animal Act." These amendments were based on the following congressional findings: 1) the use of animals is instrumental in certain research and education for advancing knowledge of cures and treatment for diseases and injuries which afflictboth humans and animals; 2) methods of testing that do not use animals are being and continue to be developed which are faster, less expensive, and more accurate than traditional animal experiments for some purposes and further opportunities exist for the development of these methods of testing; 3) measures which eliminate or minimizethe unnecessaryduplication of experiments on animalscan result in more productiveuse of federal funds; and 4) measureswhich help meet the public concernfor laboratory animalcare and treatmentare important in assuring that research will continue to progress. Until 1985, the Animal Welfare Act requirements and USDA regulations were essentially limited to animal care, housing, and transportation standards. The new amendments included specific requirements for research facilities that were related to the experimental use of animals. The law clearly states, however, that nothing in the act should be construed as authorizing the Secretary of Agriculture to promulgate rules, regulations, or orders with regard to the design or performance of research protocols. It also mandates that the USDA may not interrupt the conduct of research during inspections. The Pet Theft Act of 1990 was the fourth amendment to the Animal Welfare Act. It was incorporated in the 1990 Farm Bill and referred to as the "Protection of Pets" legislation. This amendment established a 5-day holding period for dogs and cats held at pounds and shelters (both private and public) or research facilities. This period was designed to allow pet owners and prospective owners the opportunity to claim or adopt animals before they are sold or used for research.

1. USDA Regulations The Animal Welfare Act authorizes the USDA to develop regulations based on the act. Within the USDA, the Animal Wel-

2. LAWS, REGULATIONS, AND POLICIES AFFECTING THE USE OF LABORATORY ANIMALS

fare Act is administered through the Animal and Plant Health Inspection Service (APHIS). All rules must be developed in consultation and cooperation with other federal departments and agencies and reviewed and approved by the Office of Management and Budget. The USDA is required to publish any new regulations or changes in existing regulations in the Federal Register and allow a 60-day period during which the public may comment. The final rule on the regulations is published in the Federal Register, along with an effective implementation date. The complete set of USDA regulations and standards are published in the Code of Regulations (CFR), Title 9, Animals and Animal Products, Subchapter A, Animal Welfare. Part 1 defines the terms used, Part 2 provides the regulations, Part 3 specifies the standards, and Part 4 includes the rules of practice governing proceedings under the Animal Welfare Act. In addition, the USDA has issued the "Animal Care Policy Manual" to further clarify the intent of the Animal Welfare Act. The principle components of the animal welfare regulations that pertain to research facilities are provided in Part 2, Subparts C and D, which are summarized below. a. Regulated Species. The regulated species include any live or dead dog, cat, nonhuman primate, guinea pig, hamster, rabbit, aquatic mammal, or any other warm-blooded animal that is being used or is intended for use in research, teaching, testing, experimentation, or exhibition or as a pet. Birds, rats of the genus Rattus, and mice of the genus Mus bred for use in research, teaching, or testing, and horses and farm animals intended for u s e a s food or fiber or used in studies to improve production and quality of food and fiber, are specifically excluded. b.

Licensing.

Any person operating or desiring to operate as a dealer, broker, exhibitor, or operator of an auction sale must be licensed by the USDA and pay an annual fee. A dealer is any person who, for compensation or profit of more than $500 per year, buys, sells or negotiates the purchase of, delivers for transportation, or transports a regulated animal for research, teaching, testing, experimentation, or exhibition or for use as a pet or a dog for hunting, security, or breeding purposes. Retail pet stores are exempt unless they sell to a research facility, exhibitor, or wholesale dealer. Dogs and cats acquired by a dealer or exhibitor must be held for 5 full days, not including the day of acquisition, after acquiring the animal. If the animal was acquired from a contract animal pound or shelter, the animal must be held for at least 10 full days. If the animal is then sold to another dealer, the subsequent dealer is required to hold the animal for a minimum of 24 hours. Research facilities that obtain dogs and cats from sources other than dealers, exhibitors, and exempt persons must

21

also hold the animals for 5 full days, not including the day of acquisition or time in transit, before the animals are used by the facility. c. Registration. Research facilities, intermediate handlers, and common carriers of regulated species must register with the USDA every 3 years; any revisions to the initial registration must be provided at the time of reregistration. Research facilities are defined as any institution, organization, or person that uses live animals in research, testing, or experiments; that purchases or transports live animals; or that receives federal funds for research, tests, or experiments. The Secretary of Agriculture may exempt facilities from registration if they do not use cats, dogs, or a substantial number of other regulated species. d.

IACUC Responsibilities.

To help assure humane experimental animal use, the 1985 amendments required every animal research facility to establish an Institutional Animal Committee, subsequently designated by the USDA as an Institutional Animal Care and Use Committee (IACUC). Congress mandated that the committee include at least three members appointed by the chief executive officer of the research facility. The members must possess sufficient ability to assess animal care, treatment, and practices in experimental research as determined by the needs of the research facility and shall represent society's concerns regarding the welfare of animal subjects. At a minimum, the IACUC must include one member who is a doctor of veterinary medicine and one member who is not affiliated in any way with the research facility other than as a member of the IACUC. The nonaffiliated member cannot be a member of the immediate family of a person who is affiliated with the facility and will provide representation for general community interests in the proper care and treatment of animals. In instances where the IACUC consists of more than three members, not more than three members can be from the same administrative unit of the facility. The IACUC is responsible for making recommendations to the research facility's administrative representative, designated as the Institutional Official, regarding any aspect of the research facility's animal program, facilities, or personnel training. It is required to review, at least once every 6 months, the research facility's program for humane care and use of animals, based on USDA regulations. The IACUC must also conduct an inspection of all animal study areas and animal facilities at least once every 6 months. Exceptions to the study area inspection requirement may be made by the Secretary of Agriculture if animals are studied in their natural environment or the study area is difficult to access. After each program review and inspection, the committee must file reports of its evaluations with the Institutional Official

22

of the research facility. This report must be signed by a quorum, or majority, of the committee members and must include any minority views expressed by committee members. The report must identify any violation of USDA standards, including any deficiencies in animal care or treatment and any deviations in research practices from originally IACUC-approved proposals. Significant deficiencies, defined as those that threaten animal health or safety, must be distinguished from minor deficiencies. A specific plan and reasonable opportunity for correcting problems must also be provided in the report. If, however, corrections are not implemented, the committee, in consultation with the Institutional Official, must notify the USDA and the funding federal agency of the deficiencies or deviations. The report must be maintained on file at the research facility for a minimum of 3 years and be made available during inspections by the USDA or any federal funding agency. Federal research facilities are required to have committees with the same composition and responsibilities, except that they are to report deficiencies or deviations to the head of the federal agency conducting the research. The committee is also charged with reviewing, and, if warranted, investigating concerns involving the care and use of research animals. These concerns may be raised by members of the public or laboratory or research personnel who report issues of noncompliance. The committee must also establish a mechanism for addressing such concerns. In addition, the IACUC is responsible for reviewing and approving all proposed activities or significant changes in activities related to the care and use of animals. It is authorized to require modifications in or withhold approval for these activities; it may also suspend an activity if it determines that the activity is not being conducted in accordance with the IACUC-approved procedures. Any suspended activities must be reported to the USDA. As part of its review, the IACUC must assure that the activities are in accordance with the regulations unless acceptable justification for a departure is presented in writing. A proposal to conduct an activity or to make changes in an ongoing activity involving animals must provide the following information: (1) the species and approximate number of animals to be used; (2) a rationale for involving animals and for the appropriateness of the species and numbers of animals to be used; (3) a complete description of the proposed use of the animals; (4) a description of procedures designed to assure that discomfort and pain to animals will be limited to that which is unavoidable for the conduct of scientifically valuable research, including provision for the use of pharmacologic agents to minimize animal discomfort and pain; and (5) a description of any euthanasia method to be used. Research protocols submitted to the IACUC must also provide assurance that animal discomfort, distress, or pain will be avoided or minimized. A written, narrative description of the methods and sources used to determine that alternatives are not

LYNN C. ANDERSON

available is required for procedures that might cause more than momentary or slight pain or distress. The investigator must also provide written assurance that the activities do not unnecessarily duplicate previous experiments. For potentially painful procedures, a veterinarian must be consuited. Sedatives, analgesics, or anesthetics must be provided, unless withholding them is scientifically justified in writing and approved by the IACUC. In such instances, the pain-relieving agents may be withheld only for the period of time necessary to meet research objectives. During its review, the committee must also be assured that paralytics will not be used without anesthesia. Animals that would otherwise experience severe or chronic pain or distress that cannot be relieved must be euthanatized during or after the procedure. For all research protocols, the method of euthanasia must produce rapid unconsciousness and subsequent death without evidence of pain or distress. Survival surgical procedures must be performed using aseptic technique and sterile instruments; members of the surgical team must wear gloves and masks. Appropriate preoperative and postoperative care must be provided. Major survival surgery on nonrodents may be conducted only in facilities intended for that purpose and must be maintained under aseptic conditions. An animal may not be used in more than one major operative procedure from which it is allowed to recover, unless it is scientifically justified in writing, required as a routine veterinary procedure, or required to protect the health or well-being of the animal. In other special circumstances, requests for exemptions may be made to the administrator of the USDA's Animal and Plant Health Inspection Service. The committee must also be assured that the animals' living conditions will be appropriate for their species. The housing, feeding, and nonmedical care of the animals must be directed by a veterinarian or other scientist trained and experienced in the proper care, handling, and use of the species. The IACUC must also be assured that personnel maintaining or studying animals are appropriately qualified and trained. To help protect trade secrets, the law stipulates that the research facility is not required to disclose trade secrets or commercial or financial information publicly or to the IACUC. It is unlawful for any member of an IACUC to release any confidential information of the research facility, including trade secrets, processes, operations, style of work, or apparatus. Furthermore, the law protects the identity, confidential statistical data, and amount or source of any income, profits, losses, or expenditures of the research facility. Committee members may not use or attempt to use or reveal to any other person any information that is entitled to protection as confidential information. Failure to comply with these requirements could result in a member's being removed from the committee, fined, and imprisoned. Any individual or research facility injured in its business or property by reason of a violation of the confidentiality rules may recover all actual and consequential damages.

2. LAWS, REGULATIONS, AND POLICIES AFFECTING THE USE OF LABORATORY ANIMALS

e.

Personnel Qualifications.

The 1985 amendments also, for the first time, required research facilities to ensure that all scientists, research technicians, animal technicians, and other personnel involved with animal care, treatment, and use are qualified to perform their duties. This responsibility requires each institution to provide training and instruction on the humane methods of animal maintenance and experimentation, including the basic needs and the proper handling and care of each species, preprocedural and postprocedural care of animals, and methods of aseptic surgery. Personnel must also be instructed about research or testing methods that minimize or eliminate the use of animals or limit animal pain or distress and the utilization of information services that would help them search for alternatives. In addition, they must be informed about the methods whereby deficiencies in animal care and treatment should be reported.

f.

Information Services.

To support the required training, the 1985 amendments mandated the Secretary of Agriculture to establish information services at the National Agriculture Library (NAL) to provide (1) information pertinent to employee training; (2) methods that could prevent unintended duplication of animal experimentation as determined by the needs of the research facility; (3) improved methods of animal experimentation that could reduce or replace animal use and minimize pain and distress, such as anesthetic and analgesic procedures. The Animal Welfare Information Center, NAL, meets these requirements.

g.

Attending Veterinarian.

Each research facility is required to have an attending veterinarian with training or experience in laboratory animal science and medicine who has direct or delegated program responsibility for activities involving animals at the research facility. Parttime or consulting veterinarians must provide a written program of veterinary care and regularly scheduled visits to the research facility. The veterinarian is authorized to ensure the provision of adequate veterinary care and to oversee the adequacy of animal care and use. Adequate veterinary care includes the availability of appropriate facilities, personnel, equipment, and services. It also includes the use of appropriate methods to prevent, control, diagnose, and treat diseases and injuries and the provision of emergency veterinary medical care. The veterinarian must ensure that all animals are observed at least once daily to assess their health and well-being. The veterinarian is also responsible for providing guidance to investigators and other personnel regarding the handling, immobilization, anesthesia, analgesia, tranquilization, and euthanasia of animals. Adequate preprocedural and postprocedural care must be provided in ac-

23

cordance with current established veterinary medical and nursing practices. h.

Records.

The USDA requires records to be maintained for each IACUC meeting; each proposed activity involving animals, including any significant changes; the status of IACUC approval for each activity or change; and semiannual IACUC reports and recommendations. Every research facility must also maintain records concerning any dog or cat purchased, owned, held, transported, euthanatized, or sold. These records must document the animal's source and date of acquisition, USDA-designated unique identification tag or tattoo number, species or breed, sex, date of birth or approximate age, and any distinguishing physical characteristics. The transportation, selling, or other disposition of a dog or cat must also be documented, including the name and address of the carrier (if transported) and of the new owner (if sold or donated). With the exception of the source and date of acquisition, these records must accompany any shipment of dogs or cats. A health certificate signed by a licensed veterinarian must accompany all shipments of dogs, cats, and nonhuman primates. Records that relate directly to activities approved by the IACUC must be maintained for the duration of the activity and for an additional 3 years after completion of the activity. A copy of all other records and reports must be maintained for 3 years and shall be available for inspection and copying by authorized APHIS or federal funding agency representatives. Each research facility must also submit an annual report to the USDA on or before December 1 of each calendar year to provide information relevant to the immediately preceding fiscal year (October 1-September 30). The report must assure (1) that professionally acceptable standards governing the care, treatment, and use of animals were followed; (2) that each principal investigator has considered alternatives to painful procedures; and (3) that the facility is adhering to the USDA standards and regulations, unless the IACUC has approved exceptions specified and explained by the principal investigator. A summary of any exceptions, including a brief explanation and the species and number of animals affected, must be attached to the annual report. In addition, the report must state the location of all facilities where animals were housed or used in actual research, testing, teaching, or experimentation or were held for these purposes. The common names and the numbers of animals used must be reported in one of three categories: (1) activities involving no pain, distress, or use of pain-relieving drugs; (2) experiments, teaching, research, surgery, or tests where appropriate anesthetic, analgesic, or tranquilizing drugs were used; and (3) painful activities where the use of painrelieving agents would have adversely affected the procedures, results, or interpretation of the activity. An explanation of the

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LYNN C. ANDERSON

animal procedure(s) conducted in the third categorymust be attached to the annual report. In addition, the number of animals being bred, conditioned, or held for use, but not yet used, must be listed. The USDA compiles the information contained in the reports from all registered research facilities and submits an annual summary to Congress.

2.

U S D A Standards

Dealers, exhibitors, and research facilities are required to meet minimal housing, operating, animal health, husbandry, and transportation standards. These include feeding, watering, sanitation, lighting, ventilation, shelter from extremes of weather and temperatures, adequate veterinary care, and separation by species where the Secretary of Agriculture finds it necessary for the humane handling, care, or treatment of animals. Specific standards, including minimal space requirements, are provided for dogs, cats, guinea pigs, hamsters, rabbits, nonhuman primates, and marine mammals. The specifications are similar for all species, except marine mammals, and are more detailed for dogs, cats, and nonhuman primates. General standards are also provided for other warm-blooded species, including farm animals used for biomedical research purposes or for testing and production of biologicals for humans or nonagricultural or nonproduction animals. It is beyond the scope of this chapter to provide detailed information regarding these standards. However, several of the most notable aspects, as required by the 1985 amendments to the Animal Welfare Act, are summarized below.

a.

Canine Opportunity for Exercise.

Dogs and cats must be housed in compatible groups. Dealers, exhibitors, and research facilities must develop, document, and follow an appropriate plan, approved by the attending veterinarian, to provide dogs over 12 weeks of age with the opportunity for exercise. This rule does not apply to individually housed dogs provided with at least twice the minimum floor space required and dogs that are group-housed in floor space that meets the minimum space standards for each dog. Bitches with litters and incompatible, aggressive, or vicious dogs are also exempt. The attending veterinarian may also exempt dogs from this program if participation would adversely affect the dog's health or well-being. Such exemptions made by the attending veterinarian must be documented and reviewed at least every 30 days by the veterinarian, unless the condition is permanent. The IACUC may also approve exemptions if the principal investigator determines that it is inappropriate for certain dogs to exercise or be group-housed. This exception must be reviewed annually by the IACUC. Records of these exemptions must be maintained and made available to the USDA or federal funding agency

upon request. If a dog is housed without sensory contact with another dog, it must be provided with positive contact with humans at least daily. b.

Psychological Well-being of Nonhuman Primates.

Dealers, exhibitors, and research facilities must also develop, document, and follow an appropriate plan for environmental enhancement adequate to promote the psychological well-being of nonhuman primates. The plan must be in accordance with currently accepted professional standards as cited in appropriate professional journals or reference guides and as directed by the attending veterinarian. At a minimum, the plan must address the social needs of nonhuman primate species known to exist in social groups in nature. Individual animals that are vicious, overaggressive, or debilitated should be individually housed. Nonhuman primates that are suspected of having a contagious disease must be isolated from healthy animals in the colony as determined by the attending veterinarian. Group-housed nonhuman primates must be compatible, as directed by the attending veterinarian. Individually housed nonhuman primates must be able to see and hear members of their own or compatible species unless the attending veterinarian determines that this arrangement would endanger their health or well-being. Primary enclosures must be enriched by providing means of expressing noninjurious species-specific behavior. Environmental enrichment devices may include perches, swings, mirrors, manipulanda, and foraging or task-oriented food items. Interaction with personnel is recommended, provided it is consistent with safety precautions. Special attention is required for infant and young juvenile nonhuman primates, those that exhibit signs of psychological distress, those entered in IACUC-approved research protocols that require restricted activity, and individually housed nonhuman primates without sensory contact with nonhuman primates of their own or compatible species. Great apes weighing more than 110 lb must be provided additional opportunities to express species-typical behavior. If a nonhuman primate must be maintained in a restraint device for an IACUC-approved protocol, such restraint must be for the minimum period possible. If the protocol requires more than 12 hours of continuous restraint, the nonhuman primate must be provided the daily opportunity for at least 1 continuous hour of unrestrained activity, unless the IACUC approves an exception. Such an exception must be reviewed at least annually. The attending veterinarian may also exempt an individual nonhuman primate from participation in the environmental enhancement plan in consideration of its well-being. However, such an exemption must be documented and reviewed by the attending veterinarian every 30 days. All exemptions must be available for review by the USDA and federal funding agencies upon request and reported in the annual report to the USDA.

2. LAWS,REGULATIONS,AND POLICIES AFFECTING THE USE OF LABORATORYANIMALS 3.

USDA Enforcement

The USDA is also charged with enforcement of the regulations. The Animal Care section of the USDA's Animal and Plant Health Inspection Service (APHIS) is responsible for assuring compliance of transportation, sale, and handling of animals used in laboratory research. The law requires the USDA to inspect each research facility at least once each year and, in the case of deficiencies or deviations from the standards promulgated under the act, to conduct follow-up inspections as necessary until all deficiencies or deviations are corrected. APHIS inspections and investigations may also be conducted as the result of alleged violations of the Animal Welfare Act, in response to public or internal complaints. Each research facility is required to permit APHIS officials to enter its place of business; to examine and make copies of the required records; to inspect the facilities, property, and animals; and to document, by taking photographs and other means, conditions and areas of noncompliance. Animals may be confiscated or euthanatized by the authority of an APHIS official, if the animal is suffering as a result of the research facility's failure to comply with any provision of the regulations or standards. The APHIS official must, however, give the facility the opportunity to correct the condition and provide adequate care to the animal. If deficiencies remain uncorrected, the USDA may take legal action, including the use of fines and/or suspension or revocation of registration or licenses.

B.

PHS Animal Welfare Policy

The Health Research Extension Act of 1985 (Pub. L. 99-158), Section 495, Animals in Research, mandated the Secretary of Health and Human Services, acting through the director of the National Institutes of Health, to establish guidelines for the proper care and treatment of animals used in biomedical and behavioral research. Any institution receiving support through the U.S. Public Health Service (PHS) for animal research, training, biological testing, or animal-related activities, must provide extensive written assurance of their compliance with PHS Policy on Humane Care and Use of Laboratory Animals (PHS Policy). The policy applies to all PHS-conducted or supported activities involving animals regardless of where they are conducted. Most of the federally funded animal research in the United States is channeled through the PHS, including the National Institutes of Health (NIH); Alcohol, Drug Abuse and Mental Health Administration (ADAMHA); Centers for Disease Control and Prevention (CDC); Food and Drug Administration (FDA), Health Resources and Services Administration (HRSA); and Office of the Assistant Secretary for Health. The Office of Laboratory Animal Welfare (OLAW), NIH, is responsible for the implementation, interpretation, and evaluation of compliance with the PHS Policy and for education of in-

25

stitutions and investigators receiving PHS support. No activity involving animals may be conducted or supported by the PHS unless the institution conducting the activity has an approved written Animal Welfare Assurance on file with OLAW. OLAW may approve or disapprove the assurance or may negotiate a satisfactory assurance with the institution. A new assurance must be submitted at least once every 5 years. The assurance must fully describe the institution's program for the care and use of animals in PHS-conducted or PHS-supported activities. OLAW is also responsible for conducting site visits to selected institutions and for evaluating allegations of noncompliance with PHS Policy. If significant problems are identified and not corrected within a reasonable period of time, the director of the NIH may suspend or revoke funding to an individual investigator or institution. The PHS Policy implements the "U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training," developed by the Interagency Research Animal Committee (Table I). The policy stipulates that institutions must have an Animal Welfare Assurance approved by OLAW and an Institutional Animal Care and Use Committee (IACUC) that is responsible for reviewing proposed projects, evaluating the animal care and use program, and inspecting facilities. The IACUC must maintain records of its activities and must report at least annually to OLAW. The policy also includes the information required for PHS applications or proposals that involve animal use. In addition to requiring compliance with the Animal Welfare Act, the PHS Policy requires institutions to use the "Guide for the Care and Use of Laboratory Animals" (seventh edition, 1996; or subsequent editions) as the basis for developing and implementing an institutional program for activities involving animals. The guide, written under the auspices of the Institute for Laboratory Animal Research of the National Academy of Sciences, applies to all live vertebrate animals, including traditional laboratory animals, farm animals, wildlife, and aquatic animals used in research, teaching, or testing. It emphasizes performance standards, which are less prescriptive and more flexible than engineering standards. The guide also encourages the application of professional judgment when addressing unique circumstances. Recommendations in the guide are based on published data, scientific principles, expert opinion, and experience with methods and practices that are consistent with high-quality, technically and scientifically appropriate, humane animal care and use. The guide provides recommendations for occupational health and safety programs. Numerous relevant references are provided. The PHS Policy also requires that euthanasia of animals be conducted in accordance with the "Report of the American Veterinary Medical Association (AVMA) Panel on Euthanasia." In addition, the guide recognizes accreditation by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Institutions that

26

LYNN C. ANDERSON Table I U.S. GovernmentPrinciples for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training

I. The transportation, care and use of animals should be in accordance with the Animal Welfare Act (US. Code, Vol. 7, Secs. 2131 et seq.) and other applicable Federal laws, guidelines and policies II. Procedures involving animals should be designed and performed with due consideration of their relevance to human or animal health, the advancement of knowledge or the good of society III. The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid resuits. Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered IV. Properuse of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals V. Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia. Surgical or other painful procedures should not be performed on unanesthetized animals VI. Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the procedure, or, if appropriate, during the procedure VII. The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally the housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or other scientist trained and experiencedin the proper care, handling, and use of the species being maintained or studied. In any case, veterinary care shall be provided as indicated VIII. Investigators and other personnel shall be appropriately qualified and experienced for conducting procedures on living animals. Adequate arrangements shall be made for their inservice training, including the proper and humane care and use of laboratory animals IX. Where exceptions are required in relation to the provisions of these Principles, the decisions should not rest with the investigators directly concerned but should be made, with due regard to Principle II, by an appropriate review group such as the institutional animal research committee. Such exceptions should not be made solely for the purposes of teaching or demonstration are accredited by AAALAC are assigned "Category 1" assurance status and are not required to submit their most recent semiannual report to OLAW with the assurance statement. Those that are not accredited are awarded "Category 2" assurance status and are required to submit their semiannual report to OLAW. Unlike the Animal Welfare Act, the PHS Policy requires that the IACUC consist of at least five (not three) members, including a doctor of veterinary medicine, a practicing scientist with experience in animal research, an individual whose primary concerns are in a nonscientific area, and an individual who is not affiliated with the institution in any way other than as a member of the IACUC. One person may meet more than one of these four requirements, provided there is a minimum of five IACUC members. The IACUC is responsible for reviewing all proposed research projects or proposed significant changes in ongoing re-

search projects in a manner similar to that required by the USDA animal welfare regulations. Whereas the USDA regulations require annual review of ongoing activities, PHS requires the IACUC to conduct a complete review of ongoing activities at least once every 3 years. R e c o r d s - - i n c l u d i n g the minutes of all IACUC meetings; records of applications, proposals, and proposed significant changes in the care and use of animals and their respective IACUC evaluation; IACUC semiannual program reports and recommendations; and records of accrediting body d e t e r m i n a t i o n s - - m u s t be maintained for at least 3 years. Records related to IACUC-approved activities must be held for 3 years beyond the completion of the activity. All records must be accessible for review and copying by an authorized OLAW or other PHS representative. The PHS also requires the IACUC to report to OLAW, through the Institutional Official, at least once every 12 months. This annual report must include any changes in the institution's accreditation status, program for animal care and use, or IACUC membership, as well as the dates of the IACUC's semiannual evaluations of the institution's program and facilities.

C.

FDA Good Laboratory Practices

The federal Food, Drug, and Cosmetic Act requires the Food and Drug Administration (FDA), under the Department of Health and Human Services, to ensure proper procedures for the care and use of laboratory animals, as implemented by the Good Laboratory Practice (GLP) regulations (21 CFR, Part 58) that became effective June 1979 and were amended in 1987. The regulations establish basic standards for conducting and reporting nonclinical safety testing and are intended to assure the quality and integrity of safety data submitted to the FDA in support of an application for a research or marketing permit. Such permits are required for human and animal drugs, human biological products, medical devices, diagnostic products, food and color additives, and electronic medical products. Basic research studies, clinical or field trials in animals, and human subject trials are not covered by the GLP regulations. Institutions seeking FDA approval of their products must establish written protocols and standard operating procedures (SOPs); provide adequate facilities, equipment, and animal care; properly identify test substances; and accurately record observations and report results for preclinical studies. The FDA relies heavily on documented adherence to the written protocols and SOPs in judging the acceptability of safety data submitted in support of marketing or clinical research permits. Every study conducted under GLP regulations must have a study director, who is ultimately responsible for the implementation of the protocol and conduct of the study. Each institution must also have a quality assurance unit that monitors the conduct of studies to assure that the protocol is being followed and the records are properly maintained.

2. LAWS, REGULATIONS, AND POLICIES AFFECTING THE USE OF LABORATORY ANIMALS

To help assure compliance with the GLP regulations, the FDA conducts periodic, routine surveillance inspections and data audits of public, private, and government nonclinical laboratories that may be performing tests on GLP-regulated products. They may also conduct directed inspections to verify the reliability, integrity, and compliance of important or critical safety studies being reviewed in support of pending applications for product research or premarketing approval. In addition, the FDA may conduct inspections to investigate potential noncompliance issues brought to the FDA by whistle-blowers, the news media, industry complaints, FDA reviewers, other government contacts, or other sources. Inspections of commercial laboratories are conducted without prior notification. Initial inspections of university and government laboratories are initiated only after the facility has been informed in a letter from the Bioresearch Monitoring Program coordinator, Division of Compliance Policy, Office of Enforcement, FDA, of the intent to inspect. The inspections include a review of the institution's organization and personnel, quality assurance unit, facilities, equipment, testing facility operations, reagents and solutions, test and control articles, protocols and conduct of nonclinical studies, records, and reports. In addition, the animal care program is evaluated to determine if the animal care and housing is adequate to preclude stress and uncontrolled influences that could alter the response of the test system to the test article. The inspection includes the animal housing room(s) and SOPs for the environment, housing, feeding, handling, and care of laboratory animals. Newly received animals must be appropriately isolated, identified, and evaluated for health status. Animals of different species, or animals of the same species on different projects, must be separated. Daily logs of animal health observations are randomly reviewed and treatment of animals must be authorized and documented. Cages, racks, and accessory equipment must be cleaned and sanitized, and appropriate bedding must be used. Feed and water samples must be collected at appropriate sources and analyzed periodically, and the analytical documentation must be retained. The pest control program is also reviewed. Copies of the IACUC's standard operating procedures and meeting minutes are reviewed to verify committee operation. A data audit is also conducted to compare the protocol and amendments, raw data, records, and specimens against the final safety assessment report. This audit is intended to substantiate that protocol requirements were met and that findings were fully and accurately reported. The study methods described in the final report are compared against the protocol and SOPs to confirm that the GLP requirements were met. In addition to reviewing the procedures and methods for animal housing, identification, health observations, and treatment, the audit includes review of the handling of dead or moribund animals and necropsy, histopathology, and pathology procedures. The audit also includes a detailed review of study records and raw data. These data may include animal weight records, food consump-

27

tion records, and clinical pathology analyses and ophthalmologic examinations. Inspection reports are classified according to the findings and whether or not objectionable conditions or practices were found during the inspection. If regulatory and/or administrative actions are recommended, the FDA may hold an informal conference, conduct a reinspection, or issue a warning letter. It may also reject a nonclinical study or studies, disqualify the institution, withhold or revoke a marketing permit, or terminate a permit for preclinical studies.

D.

Interagency Cooperation

As part of the Animal Welfare Act, Congress required the Secretary of Agriculture to consult and cooperate with other federal departments and agencies concerned with the welfare of animals used in research. Specifically, the Secretary of Agriculture must consult with the Secretary of Health and Human Services prior to the issuance of regulations. In 1995, authorized representatives of the USDA, NIH, and FDA signed a Memorandum of Agreement concerning Laboratory Animal Welfare. The cooperating agencies made the following agreements based on mutual concern and interest regarding animal welfare: (1) to share registries, inventories, and listings of organizations that fall under their respective authority; (2) to share significant adverse findings regarding animal care and use and the actions taken by the agency in response to those findings; (3) to share evidence of serious noncompliance with required standards or policies for the care and use of laboratory animals; (4) to inform successive evaluation teams and to avoid redundant evaluations of the same entities; (5) to consult and coordinate with each other on regulatory or policy proposals and significant policy interpretations; and (6) to provide each other with resource persons for scientific and educational seminars, speeches, and workshops related to laboratory animal welfare.

E.

EPA Good Laboratory Practices

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), enacted in 1947, authorizes the administrator of the Environmental Protection Agency (EPA) to register and control the use of pesticides. To register a new pesticide, the EPA conducts a premarket review of its potential health and environmental effects. Animal tests must be conducted, according to the EPA's Good Laboratory Practices, which differ in some respects from the FDA's Good Laboratory Practices. According to the final rules published in 1983, the Toxic Substances Control Act (TSCA) also requires the use of GLP standards for conducting chemical studies required by the TSCA.

LYNN C. ANDERSON

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F.

Professional and Scientific Associations

Many scientific and professional associations have adopted position statements regarding the care and use of laboratory animals. Several also provide resources that have made a significant impact on the generally accepted practices for animal facilities. 1.

Institute for Laboratory Animal Research

The Institute for Laboratory Animal Research (ILAR) is a division of the Commission on Life Sciences, one of eight major units with the National Research Council (NRC). The NRC is the working arm of the National Academy of Sciences, a private, nongovernmental, nonprofit organization chartered by Congress in 1863 to "investigate, examine, experiment, and report upon any subject of science or a r t . . , whenever called upon by a federal agency, a group internal to the NRC, or Congress." ILAR is governed by a 15-member council of experts in laboratory animal medicine, zoology, genetics, medicine, ethics, and related biomedical sciences. The council provides direction for ILAR's programs. Many of ILAR's reports provide a framework for governmental and institutional animal welfare policies. The most widely distributed publication from ILAR is the "Guide for the Care and Use of Laboratory Animals," which is recognized by the Public Health Service and AAALAC International as the standard reference on laboratory animal care and use programs. ILAR also published two other standard references that are used to establish and maintain optimal animal care and use programs: "Occupational Health and Safety in the Care and Use of Research Animals" and "The Psychological Well-being of Nonhuman Primates." In addition, ILAR publishes the Laboratory Animal Management Series, which provides specific recommendations for many laboratory animal species. The quarterly ILAR Journal publishes contemporary, authoritative articles relevant to laboratory animal medicine and management. ILAR also maintains a large database of commercial and investigatorheld unique animal models and an international registry of laboratory registration codes on behalf of the International Committee on Standardized Genetic Nomenclature for Mice. 2.

AAALAC International

The Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) is a nonprofit association that conducts voluntary peer review and accreditation of animal care and use programs. It is a communication-intensive program that stresses application of performance standards and professional judgment, rather than inspection and enforcement of engineering standards. AAALAC uses the "Guide for the Care and Use of Laboratory Animals" as its primary reference for program review and evaluation, but it also considers

the laws and regulations for the country in which the institution is located, and it uses other widely accepted standards and references for animal care and use, occupational health, and biosafety. The use of performance standards allows AAALAC to evaluate each program independently and to provide guidance appropriate to individual situations. This process helps institutions to maintain optimal standards for animal care and use. AAALAC accreditation is recognized by the Public Health Service. The peer review is conducted by a member of the AAALAC Council on Accreditation and at least one ad hoc consultant. Members of the council include distinguished pharmacologists, toxicologists, primatologists, animal scientists, aquatic and poultry specialists, physicians, administrators, and veterinarians. AAALAC is governed by its member organizations, including the American Association for the Advancement of Science (AAAS), the Federation of American Societies for Experimental Biology (FASEB), the American Medical Association, Society of Toxicology, and more than 40 other professional scientific organizations. More than 600 academic, government, and industrial research institutions and hospitals throughout the United States, Canada, Europe, South America, Africa, and Asia are AAALAC accredited. 3.

Federation of Animal Science Societies

To help assure the ethical and humane treatment of farm animals used in agricultural research or teaching, the agricultural community published the "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Testing" ("Ag Guide"). The first edition, published in 1988, was revised in 1999 by the Federation of Animal Science Societies. This document is intended to supplement applicable federal, state, and local laws, regulations, and policies and the "Guide for the Care and Use of Laboratory Animals." It is directed to the care and use of any warm-blooded vertebrate animal that is used to improve understanding of the animal's use in production agriculture and that may require a simulated or actual production agricultural setting consistent with consideration of the well-being of the animal. It provides standards for range or pasture production in naturalistic settings and for various degrees of confinement in relatively intensive and certain less extensive production systems. It includes guidelines for institutional policies, husbandry, veterinary care, and facility construction and maintenance. The "Ag Guide" is used by AAALAC International for relevant program assessment and accreditation purposes.

G.

International Laws and Regulations

It is not the intent of this chapter to provide detailed information on the various international laws and standards governing the care and use of laboratory animals. However, every civilized

2. LAWS, REGULATIONS, AND POLICIES AFFECTING THE USE OF LABORATORY ANIMALS

Table II CIOMS International Guiding Principles for Biomedical Research I. The advancementof biological knowledge and the developmentof improved means for the protection of the health and well-being both of man and animals require recourse to experimentationon intact live animals of a wide variety of species II. Methods such as mathematical models, computer simulation, and in vitro biological systems should be used wherever appropriate III. Animal experiments should be undertaken only after due consideration of their relevance for human or animal health and the advancementof biological knowledge IV. The animals selected for an experiment should be of an appropriate species and quality, and only the minimum number required to obtain scientifically valid results V. Investigatorsand other personnel should never fail to treat animals as sentient, and should regard their proper care and use and the avoidance or minimization of discomfort, distress, or pain as ethical imperatives VI. Investigatorsshould assume that procedures that would cause pain in human beings cause pain in other vertebrate species, although more needs to be known about the perception of pain in animals VII. Procedureswith animals that may cause more than momentary or minimal pain or distress should be performed with appropriate sedation, analgesia, or anesthesia in accordance with accepted veterinary practice. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical agents VIII. Where waivers are required in relation to the provisions of article VII, the decisions should not rest solely with the investigators directly concerned but should be made with due regard to the provisions of articles IV, V, and VI, by a suitably constituted review body. Such waivers should not be made solely for the purpose of teaching or demonstration IX. At the end of, or, when appropriate, during an experiment, animals that would otherwise suffer severe or chronic pain, distress, discomfort, or disablement that cannot be relieved should be painlessly killed. X. The best possible living conditions should be maintained for animals kept for biomedical purposes. Normally the care of animals should be under the supervision of veterinarians having experience in laboratory animal science. In any case, veterinary care should be available as required XI. It is the responsibility of the director of an institute or department using animals to ensure that investigators and personnel have appropriate qualifications or experience for conducting procedures on animals. Adequate opportunities shall be provided for in-service training, including the proper and humane concern for the animals under their care

country in the world has developed and implemented regulatory requirements for the humane and ethical treatment of research animals. Because the biomedical research community has become more global, there is increased interest in the harmonization of international standards for the care and use of laboratory animals. The member states of the European Union, in keeping with Directive 86/609, have agreed to adopt common provisions to protect animals used in experimental and other scientific procedures that may cause pain, suffering; distress, or lasting harm. This directive emphasizes the importance of seeking and en-

29

couraging the use of alternative measures with the aim of reducing the number of animals used in research. The Council for International Organizations of Medical Sciences (CIOMS) is an international, nongovernmental organization composed of medical, biomedical, and research organizations. In 1985, CIOMS developed a broad set of guidelines, the "International Guiding Principles for Biomedical Research Involving Animals" (Table II). Many countries have used these guidelines to form the basis for their regulatory requirements.

Ill.

I M P O R T A T I O N AND EXPORTATION OF ANIMALS AND A N I M A L P R O D U C T S

A.

U.S. Department of Agriculture

The U.S. Department of Agriculture (USDA), APHIS, Veterinary Services (VS), I m p o r t - E x p o r t Products Staff regulates the importation of all animals and animal-derived materials that could represent a disease risk to United States livestock. It also regulates the import and transport of infectious organisms and vectors of disease agents. This category includes not only animal products and by-products but also biological materials that contain or have been in contact with certain organisms and animal materials (including cell cultures and recombinant products). All imported materials must enter the United States through USDA-designated ports of entry. The regulations are set forth in the Code of Federal Regulations (CFR), Title 9, Chapter 1. The individual designated to receive imported material, and who will be responsible for the material, must apply for a USDA permit by submitting a complete VS application form and applicable fee. Importation of cell lines and cell culture products, such as monoclonal antibodies and recombinant proteins, requires an additional form. The information provided must be sufficient for the VS to evaluate disease risk and should include details regarding product processing, production, and nutrient factors. To protect the health of United States livestock and poultry, the USDA requires permits for importation of swine, ruminants, other hoof stock, poultry, and other birds. The USDA imposes few restrictions on the entry of small laboratory mammals. If rats, mice, guinea pigs, and hamsters have not been inoculated with or exposed to infectious agents that affect livestock or poultry, such as bovine and other transmissible spongiform encephalopathy agents, importation of these species is not restricted. However, permits are required for transgenic animals that carry receptors that enable those rodents to develop productive infection with human pathogens. At a minimum, each animal shipment should be accompanied by a health certificate endorsed by a veterinarian or person responsible for the health

LYNN C. ANDERSON

30

of laboratory animals at the facility of origin. The certificate must indicate that the animals are (1) clinically healthy, (2) have not been exposed to or inoculated with any infectious disease agents, and (3) have not originated from a facility where work with viruses affecting livestock or poultry is conducted. This certificate should be affixed to the outside of the animal transportation unit so that it is available to the United States officials at the port of arrival. A 30-day quarantine is required for poultry, ruminants, or swine, and a 60-day quarantine for horses from African horsesickness-infested regions. The importer is responsible for arranging quarantine facilities subject to the approval of the VS. The USDA, APHIS, Plant Protection and Quarantine (PPQ) service regulates the importation of plants and other vegetable matter. Feed provided to an animal during transit, such as potatoes or carrots, may be regulated. The importer must consult with the PPQ Permit Office to determine entry requirements. If it cannot be allowed entry, the prohibited vegetable matter must be removed from the cage at the point of entry by a PPQ officer.

B.

U.S. Department of Health and Human Services

Under the direction of the U.S. Department of Health and Human Services, the U.S. Public Health Service, through the Centers for Disease Control and Prevention (CDC), Office of Health and Safety, regulates the importation of nonhuman primates. Only institutions or individuals registered with the CDC may import nonhuman primates or receive them within a 31-day period of their arrival in the United States. Importers are registered for a 2-year period and must comply with CDC record-keeping and reporting requirements. The PHS is also responsible for protecting humans from zoonotic diseases and therefore regulates the importation of other animals that may be infectious to humans. Imported dogs and cats must be free of evidence of rabies, turtles must be free of salmonella, and psittacine birds must not be capable of causing psittacosis. In addition, the PHS regulates the importation or subsequent distribution of any etiologic agent or any arthropod or animal host or vector of human disease. PHS permits must be obtained for importation and distribution of these materials.

C.

U.S. Department of the Interior

The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), established in 1973 and amended in 1979, helps protect wild flora and fauna from extinction by requiring government permits for international trade in threatened wildlife and wildlife products. In the United

States, CITES is enforced by the Department of the Interior, U.S. Fish and Wildlife Service (FWS). It applies to all of the designated vertebrate and invertebrate animal or plant species, whether alive or dead, and any recognizable part of a designated animal. Protection is provided for species in two main categories: (1) those that are most endangered and (2) other species at serious risk. The most endangered species are listed in Appendix I of the CITES agreement. Appendix II includes species that are not currently threatened with extinction but may become so unless trade is subject to strict regulation. Appendix III includes all species that any country identifies as being subject to regulation within its jurisdiction for the purpose of preventing or restricting exploitation and for which the cooperation of other countries is needed. Importation or exportation of these species requires appropriate documents. The FWS is also responsible for enforcement of the Endangered Species Act, which protects threatened and endangered animal and plant species and their habitats from extinction. Protected species include birds, insects, fish, reptiles, mammals, crustaceans, flowers, grasses, and trees. The law prohibits any action, administrative or real, that results in the "taking" of a listed species or adversely affects the habitat of a listed species. It prohibits import, export, and interstate and foreign commerce of listed species. A permit issued by the Federal Wildlife Permit Office is required to use these species for scientific research. The Lacey Act was enacted in 1900 and amended several times, including substantial amendments in 1981. It authorizes the FWS to regulate the importation, exportation, transportation, sale, receipt, acquisition, or purchase of fish, wildlife, or plants that may be injurious to humans or to the interests of agriculture, horticulture, forestry, or United States wildlife resources. The Lacey Act also provides for effective enforcement of state, federal, American Indian tribal, and foreign conservation laws. In addition, the Lacey Act requires that live wildlife be transported into the United States under humane and healthful conditions and that all containers or packages containing wildlife be appropriately labeled when transported in interstate or foreign commerce.

D.

Environmental Protection Agency

The Environmental Protection Agency, which is responsible for enforcing the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), considers the potential adverse impact of pesticides on endangered species and their habitats before licensing the use of pesticides. Under FIFRA, the EPA can issue emergency suspension of certain pesticides to cancel or restrict their use if an endangered species will be adversely affected. FIFRA also provides federal control of pesticide distribution and sale and requires registration of users (farmers, utility companies, and others) and certification of applicators.

2. LAWS,REGULATIONS,AND POLICIES AFFECTING THE USE OF LABORATORYANIMALS IV.

HAZARDOUS SUBSTANCES

A.

Biohazards

The Occupational Safety and Health Act, enacted in 1970, is administered and enforced by the Department of Labor, Occupational Safety and Health Administration (OSHA). The intent is to provide workers with protection against illnesses or injury resulting from unsafe or unsanitary working conditions. The act established the National Institute for Occupational Safety and Health (NIOSH) within the Centers for Disease Control and Prevention. NIOSH plans, directs, and coordinates national programs to develop and establish recommended occupational safety and health standards and to conduct research, training, and related activities to assure safe and healthful working conditions. Chapter 24, "Control of Biohazards Associated with the Use of Experimental Animals," provides a comprehensive overview of and references for the programs, facilities, and practices necessary to help assure employee protection against biohazards. These include animal allergies, zoonoses, recombinant DNA, and infectious experimental agents.

B.

Chemical Agents

Chapter 21 also addresses the potential hazards of and precautions for working with chemical agents used in experiments. The Toxic Substances Control Act (TSCA), enacted in 1976, authorizes the Environmental Protection Agency to require testing of chemical substances entering the environment and to regulate them as necessary. Chemicals used exclusively in pesticides, food, food additives, drugs, and cosmetics are exempt from the TSCA but are regulated by other legislation. The Drug Enforcement Administration of the Department of Justice is responsible for enforcing the Drug Enforcement Act (Pub. L. 93-205). This law requires appropriate security and record management of controlled substances that are considered to be potentially addictive or habituating for human and animal use.

C. Radioactive Materials and Radiation-Emitting Equipment The Atomic Energy Act, enacted in 1954, authorizes the Nuclear Regulatory Commission (NRC) to help assure that the civilian use of radioactive materials is conducted in a manner consistent with public health and safety, environmental quality, national security, and antitrust laws. In 1974, the NRC became an independent regulatory agency under the provision of the Energy Reorganization Act. The NRC licenses individuals and

31

institutions that use radioactive material and regulates the procurement, use, storage, and disposal of these materials. The facilities, instruments, and equipment used for handling and storing radioactive materials must also meet NRC requirements. Personnel must be provided training in the safe handling and use of ionizing radiation. The Radiation Control for Health and Safety Act, enacted in 1968, authorizes the Secretary of Health and Human Services, through the Food and Drug Administration, to regulate the use of products that produce radiation, such as medical diagnostic imaging equipment, irradiators, and electron microscopes.

V.

R E C O M B I N A N T DNA R E S E A R C H GUIDELINES

The Department of Health and Human Services, National Institutes of Health, periodically publishes "Guidelines for Research Involving Recombinant DNA Molecules" in the Federal Register. Recombinant DNA molecules are defined as either (1) molecules that are constructed outside of living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate inside a living cell or (2) DNA molecules that result from the replication of these altered molecules. These guidelines apply to any recombinant DNA research project conducted at or sponsored by an institution that receives any of its support from the NIH, including intramural studies and those conducted abroad. Experiments involving recombinant DNA have been divided into four classes. The level of institutional review and containment required depends on the classification. Special care is required in the evaluation of containment levels for experiments that are likely to enhance the pathogenicity or extend the host range of viral vectors. Each institution must establish and implement policies that provide for the safe conduct of recombinant DNA research and that ensure compliance with the NIH guidelines. The institution must also appoint an Institutional Biosafety Committee (IBC) of no fewer than five members who have experience and expertise in recombinant DNA technology, biosafety, and physical containment. At least two of these members shall not be otherwise affiliated with the institution and shall represent the general interests of the surrounding community with respect to public health or the environment. The IBC acts on behalf of the institution to review all applications, proposals, and activities involving the use of recombinant DNA to assure compliance with the NIH guidelines. These responsibilities include reviewing the containment level required and assessment of the facilities, procedures and practices (including emergency spill plans) and the training and expertise of personnel conducting the research. The institution must also appoint a Biosafety Officer (BSO) if it engages in research at the Biosafety Level 3 (BSL3)or Biosafety Level 4 (BSL4) containment level.

32

LYNN C. ANDERSON Appendix: Websites Pertaining to Laboratory Animals

Resource

Homepage address

AAALAC International Animal Welfare Information Center, National Agriculture Library CITES Code of Federal Regulations Environmental Protection Agency Guidelines for Research Involving Recombinant DNA Molecules Institutional Animal Care and Use Committees Institute for Laboratory Animal Research Office of Laboratory Animal Welfare Public Health Service Policy on Humane Care and Use of Laboratory Animals USDA import permits USDA regulations and standards

http://www.aaalac.org http://www.nal.usda.gov/awic / http://international.fws.gov/cites/cites.html http://www.access.gpo.gov/nara/cfr http://www.epa.gov http ://www4.nih. gov/oba/rac/guidelines/guideline s.html http://www.iacuc.org http://www4.nas.edu/cls/ilarhome.nsf http://grants.nih.gov/grants/olaw/olaw.htm http ://grants.nih. gov/grants/olaw/references/phspol.htm http://www.aphis.usda.gov/import.html http://www.aphis.usda.gov/ac

T h e B S O is r e s p o n s i b l e for m o n i t o r i n g the facilities a n d practices to assure c o m p l i a n c e a n d for r e p o r t i n g to and s e r v i n g on the IBC. T h e p r i n c i p a l i n v e s t i g a t o r is also r e s p o n s i b l e for assuring c o m p l i a n c e w i t h the g u i d e l i n e s and m u s t r e q u e s t app r o v a l for the r e s e a r c h a n d r e p o r t a n y p r o b l e m s to the I B C .

REFERENCES

Allen, T. (1995). "Animal Welfare Legislation, Regulations, and Guidelines: January 1990-January 1995." Animal Welfare Information Center, National Agriculture Library, Beltsville, Maryland. Andrews, E. J. (1993). Report of the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc. 202, 229-249. Animal Welfare Act of 1966 (Pub. L. 89-544) and subsequent amendments (1966). US. Code, Vol. 7, Secs. 2131-2157 et seq. Barkley, W. E. (1997). "Occupational Health and Safety in the Care and Use of Research Animals." Committee on Occupational Safety and Health in Research Animal Facilities, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. National Academy Press, Washington, D.C. Bernstein, I. S. (1998). "The Psychological Well-being of Nonhuman Primates." Committee on Well-being of Nonhuman Primates, Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council. National Academy Press, Washington, D.C. Clark, J. D. (1996). "Guide for the Care and Use of Laboratory Animals." Committee to Revise the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. DHHS Publ. (NIH) 96-23. National Academy Press, Washington, D.C. Code of Federal Regulations (1999a). Title 9: Animals and Animal Products; Chap. 1: Animal and Plant Health Inspection Service, Department of Agriculture; Subchap. A: Animal Welfare; Parts 1, 2, 3, and 4. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1999b). Title 10: Energy; Chap. 1: Nuclear Regulatory Commission; Part 20: Standards for Protection against Radiation. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1998a). Title 9: Animals and Animal Products; Chap. 1: Animal and Plant Health Inspection Service, Department of Agriculture; Subchap. C: Interstate Transportation of Animals (including Poultry) and Animal Products. Office of the Federal Register, Washington, D.C.

Code of Federal Regulations (rev. 1998b). Title 9: Animals and Animal Products; Chap. 1: Animal and Plant Health Inspection Service, Department of Agriculture; Subchap. D: Exportation and Importation of Animals (Including Poultry) and Animal Products. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1998c). Title 21: Food and Drugs; Chap. 1: Food and Drug Administration, Department of Health and Human Services; Subchap. A: General; Part 58: Good Laboratory Practice for Nonclinical Laboratory Studies. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1998d). Title 29: Labor; Chap. 17: Occupational Safety and Health Administration; Part 1910: Occupational Safety and Health Standards. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1997a). Title 40: Protection of the Environment; Chap. 1: Environmental Protection Agency; Subchap. E: Pesticide Programs; Part 160: Good Laboratory Practice Standards. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1997b). Title 50: Wildlife and Fisheries; Chap. 1: U.S. Fish and Wildlife, Department of the Interior; Subchap. B: Taking, Possession, Transportation, Sale, Purchase, Barter, Exportation, and Importation of Wildlife and Plants. Office of the Federal Register, Washington, D.C. Curtis, S. E. (1994). Farm animal use in biomedical sciencemmelding the guidelines. ILAR News 36, 35-39. Dennis, M. B., Jr., and Van Hoosier, G. L., Jr. (1994). North American legislation and regulation of the use of live animals for scientific research. In "Handbook of Laboratory Animal Science," Vol. 1, "Selection and Handling of Animals in Biomedical Research" (P. Svendsen and J. Hau, eds.), pp. 23-25. CRC Press, Boca Raton, Florida. Guidelines for Research Involving Recombinant DNA Molecules (1994). Federal Register 59, 34496. Hamm, T. E., Jr., Dell, R. B., and Van Sluyters, R. C. (1995). Laboratory animal care policies and regulations: United States. ILAR J. 37, 75-78. Institutional Administrator's Manual for Laboratory Animal Care and Use (1988). Office for Protection from Research Risks, National Institutes of Health, Public Health Service. DHHS Publ. (NIH) 88-2959. U.S. Department of Health and Human Services, Washington, D.C. Institutional Animal Care and Use Committee Guidebook (1992). Office for Protection from Research Risks/Applied Research Ethics National Association, National Institutes of Health, Public Health Service. DHHS Publ. (NIH) 92-3415. U.S. Department of Health and Human Services, Washington, D.C. Interagency Research Animal Committee (1985). "U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training." Federal Register (May 20, 1985).

2. LAWS, REGULATIONS, AND POLICIES AFFECTING THE USE OF LABORATORY ANIMALS Kreger, M., Jensen, D., and Allen, T., eds. (1996). "Animal Welfare Act: Historical Perspectives and Future Directions." Working for Animals in Research, Drugs, and Surgery (WARDS). McPherson, C. W. (1984). Laws, regulations, and policies affecting the use of laboratory animals. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, F. M. Loew, eds.), pp. 19-30. Academic Press, New York. Mench, J. A. (1999). "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching." Guide Revision Committee, Federation of Animal Science Societies, Savoy, Illinois. Nomura, T. (1995). Laboratory animal care policies and regulations: Japan. ILAR J. 37, 60-61. Potkay, S., Garnett, N. L., Miller, J. G. et al. (1997). Frequently asked questions about the Public Health Service policy on humane care and use of laboratory animals. Contemp. Top. in Lab. Anim. Sci. 36, 47-50. Public Health Service Policy on Humane Care and Use of Laboratory Animals

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(1986). Office for Protection from Research Risks, National Institutes of Health, Public Health Service. (Reprinted by U.S. Department of Health and Human Services, Washington, D.C., 1996). Reid, C. S. W. (1995). Laboratory animal care policies and regulations: New Zealand. ILAR J. 37, 62-68. Richmond, J. Y., and McKinney, R. W. (1993). "Biosafety in Microbiological and Biomedical Laboratories." Centers for Disease Control and Prevention and National Institutes of Health, U.S. Public Health Service, Department of Health and Human Services. DHHS Publ. (CDC) 93-8395. U.S. Government Printing Office, Washington, D.C. Townsend, P., and Morton, D. B. (1995). Laboratory animal care policies and regulations: United Kingdom. ILAR J. 37, 68-74. Wong, J. (1995). Laboratory animal care policies and regulations: Canada. ILAR J. 37, 57-59.

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Chapter 3 Biology and Diseases of Mice Robert O. Jacoby, James G. Fox, and Muriel Davisson

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Origin and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Breeding Systems and Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . D. Housing and Husbandry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Physiology and Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Metabolic and Nutritional Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Environmental, Behavioral, and Traumatic Disorders . . . . . . . . . . . . D. Congenital, Aging-Related, and Miscellaneous Disorders . . . . . . . . . E. Neoplastic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

A.

INTRODUCTION

Origin and History

The laboratory m o u s e is assigned to the genus Mus, subfamily Murinae, family Muridae, order Rodentia. Anatomical features of the molar teeth and cranial bones help differentiate it from other murids. The house m o u s e of North A m e r i c a and Europe, Mus musculus, is the species c o m m o n l y used for biomedical research. Laboratory strains were usually derived from mice bred by mouse fanciers and their g e n o m e s are a mixture of M. musculus musculus (from eastern Europe) and M. m. domesticus (from western Europe). Since the mid-1980s, strains have LABORATORYANIMALMEDICINE,2ndedition

35 35 36 36 40 41 41 48 51 52 53 53 105 107 108 110 113

been developed from Asian mice (M. m. castaneus from Thailand and M. m. molossinus from Japan) and from M. spretus. The laboratory m o u s e was e m p l o y e d in comparative anatomical studies as early as the seventeenth century, but accelerated interest in biology during the nineteenth century, a renewed interest in M e n d e l i a n genetics, and the research requirement for a small, economical m a m m a l that was easily housed and bred were instrumental in the d e v e l o p m e n t of the "modern" laboratory mouse. These studies have grown exponentially during the current century with the recognition of the power of the m o u s e for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized m a m m a l on earth (Morse, 1979; Silver, 1995; L y o n et al., 1996). The current ability to create highly sophisticated, genetically Copyright2002,ElsevierScience(USA).Allrightsreserved. ISBN0-12-263951-0

36

ROBERT O. JACOBY,JAMES G. FOX, AND MURIEL DAVISSON

engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal.

B.

Genetics

Genetic mapping in mice began in the early 1900s. The first autosomal genes, albino and pink-eyed dilution, were linked in 1915 (Haldane et al., 1915). Extensive linkage maps and an impressive array of inbred strains are now available to expedite sophisticated genetic research. Mice have 40 chromosomes that are differentiated by the size and patterns of transverse bands. The chromosomes are designated by Arabic numbers in order of decreasing size. During the 1970s, chromosome rearrangements were used to assign known genetic linkage groups-identified by Roman numerals--to specific chromosomes and for determining locus order with respect to the centromere. Genes can now be located physically on chromosomes by fluorescent in situ hybridization (FISH), and the Genome Initiative programs are fostering development of molecular maps of mouse chromosomes. The sequence of the mouse genome is expected to be completed by 2003 (Battey et al., 1999). Inbred mice are valuable for research in virtually all fields of biomedical research, such as immunology, oncology, cardiovascular disease, metabolic disease, microbiology, biochemistry, pharmacology, physiology, anatomy, developmental biology, and radiobiology. Mice carrying spontaneous or induced mutations and strains susceptible to specific diseases provide a wide variety of mouse model systems for basic research, as well as models for biomedical research to understand specific human disorders. For example, several spontaneous mutations in genes affecting pituitary function or producing hormones provide mutant models for human dwarfing conditions. Targeted mutations in the low-density lipoprotein receptor and apolipoprotein genes provide model systems for studying cardiovascular disorders. Mice of the NOD (nonobese diabetic) strain provide a model for human insulin-dependent diabetes. Mice also provide reagents for basic research. For example, inbred histocompatible strains are used extensively as donors of plasma cell tumors to immortalize cell lines (hybridomas) that secrete highly uniform, monospecific immunoglobulins in vitro, theoretically in unlimited quantities. This technology has made a full range of functional mouse antibody molecules available for study. Development of quantitative trait loci (QTL) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (Darvasi, 1998; Frankel, 1995). One of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. Histocompatibility (H) loci control expression of cell surface molecules that modulate major immunological phenomena, such as the recognition

of foreign tissue. For example, the time, onset, and speed of skin graft rejection are controlled by two groups of H loci. The major group is located in the major histocompatibility complex (MHC, H2) on chromosome 17. These genes cause rapid rejection (10-20 days) of grafts that display foreign H2 antigens. Minor H loci groups are scattered throughout the genome and are responsible for delayed graft rejection. Genes associated with the H2 complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. Immunemediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the H2 complex. The most recent comprehensive review of the H2 complex is by Klein (1986). Because information about this subject is being published so frequently, the reader is advised to consult bibliographic indexes such as MEDLINE for recent updates. Non-MHC or minor histocompatibility systems also are under active study (Roopenian and Simpson, 2000).

C.

Breeding Systems and Nomenclature

1. Breeding Systems

Laboratory mice are identified by strain and by breeding system. A genealogy of most inbred strains is presented in the Mouse Genome Database . Table I summarizes nine breeding systems. Each requires technical skill and a firm understanding of mammalian genetics. Inbred strains were developed first in 1909 by Clarence Cook Little and offer a high degree of genetic uniformity. Mice within an inbred strain, for practical purposes, are genetically identical to other mice of the same strain and sex. They are defined as being produced by brother-sister matings for more than 20 generations. In fact, a strain should not be considered completely inbred until after 40 generations of sibling matings. Inbred strains are valuable because experimental results are reproducible with relatively small sample sizes. They are useful for genetic mapping because they are genetically well characterized, and allelic combinations can be predetermined for linkage crosses. Wild-derived inbred strains of Mus musculus castaneus and M. spretus are used extensively for mapping because of the large number of polymorphic differences from standard inbred laboratory mice. F1 hybrid mice, produced by mating mice of two inbred strains, also are genetically identical to each other and may offer a more robust animal for some studies. For example, inbred strains may differ in behavior and learning abilities, whereas hybrid mice are less likely to have learning deficits or behavioral anomalies. Mutant inbred strains carry spontaneous, targeted, or induced mutations, transgenes, or chromosome aberrations. The genetic backgrounds of such strains are homogeneous like those of regular inbred strains, but some (or all) mice of the strain carry the mutation (or chromosome aber-

37

3. BIOLOGY AND DISEASES OF MICE Table I

Kinds of Mice Used in Research

a

Definition of breeding system Random bred stock: Random mating within a large,

heterogeneous population Inbred strain: Brother- sister mating for more than 20 generations F1 hybrids: Mice from crosses between inbred strains Segregating inbred strain: Brother-sister matings system for more than 20 generations with heterozygosity for the mutations forced by (1) backcrossing, (2) intercrossing, (3)crossing and intercrossing,or (4) backcrossing and intercrossing Coisogenic inbred strains: Occurrence of a mutation within a strain

Congenic inbred strains: (A) Repeatedbackcross

of mutation-bearingmice for 10 or more generations or (B) cross-intercross system for the equivalent of 20 or more cycles with an inbred parent strain Recombinant inbred strains: Brother-sister matings for >20 generations after crossing two inbred strains and their F1 to obtain and F2 Recombinant congenic strains: Same as above except one or more backcrosses of F1 to one parent strain before beginning brother-sister matings Advanced intercross lines: Nonsibling matings from an F2 of a cross between two inbred strains a

Perpetuation of breeding system

Reference

Continue random mating, selectionpairs with random numbers method Continue brother-sister mating

Poiley (1960) Kimura and Crow (1963) Green (1981a)

Cannot be perpetuated Continue brother-sister mating with heterozygosity forced by one of the four methods at left or with homozygosity forced by intercrossinghomozygotes

Green (1981a) Green (1981a)

Perpetuate the mutation by (1) brother-sister mating within strain of origin, (2) backcross or cross-intercross system with strain of origin as parent strain, (3) brother-sister mating with heterozygosity forced by back- or intercrosses, or (4) brother-sister mating between homozygotes Perpetuate the transferred mutation by (2), (3), or (4) above. (1) may be used after 10-12 generations of backcrossing with periodic backcrosses to background strain Continue brother-sister matings

Flaherty (1981) Green (1981a)

Continue brother-sister matings

Flaherty (1981) Green (1981a) Bailey (1971) Demant and Hart (1986)

Continue nonsibling matings

Modified from Green (1981a).

ration). In segregating inbred mutant strains, mutant mice differ from their nonmutant siblings only by the mutation. Therefore, littermates can serve as controls in experiments. In homozygous mutant strains, controls must come from the same or a closely related nonmutant inbred strain. For example, C57BL/6J mice provide controls for the homozygous mutant C 5 7 B L / 6 J - m / m strain. A strain is said to be c o i s o g e n i c if the mutation of interest occurred in that strain. A strain is c o n g e n i c if the mutation or gene of interest was transferred from another strain or stock by repeated backcrossing. Detailed descriptions and diagrams of mating schemes have been provided by Green (198 l a,b), and additional contemporary information on mouse genetics and breeding can be found by consulting Table II. In contrast to inbred mice, r a n d o m bred, or outbred, mice are genetically heterogeneous and are often produced by breeding systems that intentionally minimize inbreeding. Outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. However, it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. Individual random bred mice within a colony may differ

in coat color, histocompatibility loci, enzyme and DNA polymorphisms, and other characteristics. Random breeding requires the statistically random selection of breeders by using a random numbers table or computer program. Random breeding, or outbreeding, can be achieved only in a large colony. A small breeding population or passage through the genetic "bottleneck" of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. In fact, supposedly "random bred" stocks are often genetically quite homogeneous. In a population of 25 breeding pairs, for example, heterozygosity will decrease at 1% per generation with standard randomization techniques. A random breeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. Conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. Another "circular" set of cages serves as the breeding nucleus for the n + 1 generation. Each mated pair in the nth generation contributes one female and one male to the n + 1 generation. Random breeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + 1 generation.

38

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON Table II Databases and Websites for Information about Mice Internet resource

Comprehensive database sites and mouse sources Mouse Genome Database (MGD) JAX Mice MRC Mammalian Genetics Unit, Harwell, United Kingdom The Whole Mouse Catalog ORNL Mutant Mouse Database Genetically engineered mouse sites and sources Induced Mutant Resource TBASE European Mouse Mutant Archive (EMMA) BioMedNet Mouse Knockout and Mutation Database Cre Transgenic and Floxed Gene Databases University of California Resource of Gene Trap Insertions Database of Gene Knockouts The Big Blue Web Site The Mouse Brain Library Mouse biology Mouse Tumor Biology Database (MTB) The Mammary Transgene Database Gene Expression Database (GXD) NetVet and the Electronic Zoo The Dysmorphic Human-Mouse Homology Database (DHMHD) The Mouse Atlas and Gene Expression Database Project UCD Medpath Transgenic Mouse Searcher 2.0 Mouse 2-D PAGE Database Body map Human and Mouse Gene Expression DB UNSW Embryology Mouse Development Dynamic [Embryonic] Development Zygote: A Developmental Biology Website Mouse genomics Mouse Nomenclature Guidelines and Locus Symbol Registry Trans-NIH Mouse Initiative Gene Dictionary of the Mouse Genome Genetic and Physical Maps of the Mouse Genome Mouse Backcross Service (U.K. HGMP Resource Centre) The Jackson Laboratory Mapping Panels WashU GSC Mouse EST Project Japanese Animal Genome Database NCBI LocusLink UniGene Mouse Sequences Collection TIGR Mouse Gene Index NIA/NIH Mouse Genomics Home Page WICGR Mouse RH Map Home Page Mammalian Genetics Laboratory, National Institute of Genetics (Japan) Care and use Guidelines for Ethical Conduct in the Care and Use of Animals The Ethics of Using Transgenic Animals Institute for Laboratory Animal Research Laboratory Registration Code Database Research Genetics, Genomic Tools General American Fancy Rat and Mouse Association

Web address

http://www.informatics.jax.org/ http://j axmice.jax.org/index.shtml http://www.mgu.har.mrc.ac.uk/ http://www.rodentia.com/wmc/ http://bio.lsd.ornl.gov/mouse/ http ://lena.j ax. org/resources/documents/imr/ http://tbase.j ax.org / http ://www.emma.rm. cnr.it / http://research.n.com/mkmd http://www.mshri.on.ca/nagy/cre.htm http://socrates.berkeley.edu/--~skarnes/resource.html http ://www.bioscience. org/knockout/knochome.htm http://eden.ceh.uvic.ca/bigblue.htm http://www.nervenet.org/mbl/mbl.html http ://tumor.informatics.j ax. org/cancer links.html http://bcm.tmc.edu/ermb/mtdb/mtdb.html http://www.informatics.jax.org / http://netvet.wustl.edu/vet.htm http://www.hgmp.mrc.ac.uk/dhmhd/dysmorph.html http://genex.hgu.mrc.ac.uk/ http://www-mp.ucdavis.edu/personaltgmouse 1.html http://biosun.biobase.dk/---pdi/jecelis/mouse_data_select.html http://bodymap.ims.u-tokyo.ac.jp/ http://anatomy.med.unsw.edu.au/cbl/embryo/otheremb/mouse.htm http://www.acs.ucalgary.ca/---browder/mice.html http://zygote.swarthmore.edu/info.html http://www.informatics.jax.org/mgihome/nomen/ http://www.nih.gov/science/models/mouse / http://www.nervenet.org/main/dictionary.html http://www-genome.wi.mit.edu/cgi-bin/mouse/index http://www.hgmp.mrc.ac.uk/goneaway/mbx.html http ://www.j ax.org/resources/documents/cmdata / http ://genome.wustl, edu/est/mouse_esthmpg.html http://ws4.niai.affrc.go.jp/ http://www.ncbi.nlm.nih.gov/focuslink/

http://www.ncbi.nlm.nih.gov/.unigene/mm.home.html http://www.tigr.org/tdb/mgi/index.html http://lgsun.grc.nia~nih.gov/ http://www-genome.wi.mit.edu/mouse_rh/index.html http ://www.shigen.nig.ac.jp/mouse/mouse.default.html http://www.apa.org/science/anguide.html http://oslovet.veths.no/transgenics/references.html http://www4.nationalacademies.org/cls/ilarhome.nsf http://www4.nas.edu/cls/afr.nsf/labcodesearch?openform http://www.resgen.com/index.php3 http://www.afrma.org /

3. BIOLOGY AND DISEASES OF MICE

Recombinant inbred (RI) strains are sets of inbred strains developed by single-pair random matings of mice from an F2 generation created by crossing mice of two inbred strains. Lines are propagated by brother-sister matings for more than 20 generations to obtain homozygosity. Recombinant inbred strains may take as long as 7 years to produce. RI strains are valuable for mapping phenotypic or quantitative traits that differ between the progenitor strains. Because each line is inbred, genotyping and phenotyping data are cumulative. RI sets are especially valuable for controlling for environmental variability in a trait, because several genetically identical mice from each line in a set can be typed to score the line for a trait (Bailey, 1971; Taylor, 1996). Recombinant congenic strains are sets of inbred strains derived in a manner similar to that for RI sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the F1 generation, before inbreeding is begun. The other parental strain is designated as the donor strain. The proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (Demant and Hart, 1986). Advanced intercross Lines (AILs) are a third type of RI line. AILs are made by producing an F2 generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. The purpose is to increase the possibility of recombination between tightly linked genes. For further information on this topic, consult Genetic Guidelines, Mouse Genome Database .

2.

Nomenclature

There are currently more than 1000 separate outbred stocks and inbred strains, some with multiple sublines. In addition, there are thousands of mutant strains. Therefore it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion. As an example of subline variation that makes precise nomenclature important, CBA/J carries the gene for retinal degeneration, while the CBA/CaJ subline does not. Specific nomenclatures have been developed for inbred and noninbred strains and stocks. Strains are designated by a series of letters and/or numbers, which frequently provide a shorthand description of the origin and history of the strain (Table III). For example, the inbred strain C57BL/6J originated from female 57 at the Cold Spring Harbor Laboratory (C), was the black (BL) line from this female, and is subline number 6. Sublines of an inbred strain are designated using Laboratory Registration Codes (Lab Codes), unique-2- to 4-letter codes that may be obtained from a central registry maintained at the Institute for Laboratory Animal Research in Washington, D.C. . The J in C57BL/6J means it is the subline maintained at the Jackson

39

Laboratory (J). A new type of strain designation has been created for new inbred strains made by intercrossing mice of two existing inbred strains. This is essential because many engineered mutations are made in 129-derived embryonic stem cells, recovered in C57BL/6-129 chimeras, and then maintained by brother-sister matings after the first generation. For example, an inbred strain derived by sibling matings from a C57BL/6 • 129 chimera is designated B6129. A noninbred stock that meets specific criteria is designated by placing the Lab Code before the stock symbol, separated by a full colon. For example, Hsd:ICR designates an ICR outbred stock maintained by Harlan ("International standardized nomenclature," 1972). Specific designations also distinguish coisogenic, congenic, segregating inbred, and various RI strains. The type of strain or stock often can be recognized from the correct symbol. For example,. BXD-1/Ty is line 1 in a set of RI strains derived from a C57BL/6J (B) female mated to a DBA/2J (D) male and made by Taylor (Ty). Mutant genes are designated by a brief abbreviation for the mutation (e.g., shi for shiverer). When a mutant gene is cloned, the symbol for the parent gene is used and the mutant allele is designated in superscript. For example, Mbp shi is the shiverer mutant allele in the myelin basic protein gene. Nomenclature for genetically engineered mice can be complex and may eventually require simplification. Currently, a transgenic strain is designated by a symbol for the strain followed by a symbol for the transgene. Transgene symbols take the form Tg(YYYYY) #Zzz, where Tg is the transgene, (YYYYY) is a brief description of the inserted DNA (such as a gene symbol), # is the assigned number in the series of events generated using a given construct, and Zzz is the Lab Code. When a transgene causes an insertional mutation in an endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (e.g., inrg/zz~). A targeted mutation, or knockout, is designated by the mutated gene with the identification of the mutational event as a superscript. For example, C f t r fml Unc is the first mutation in the cystic fibrosis transmembrane regulator gene created at the University of North Carolina. A gene replacement, or knockin, uses similar nomenclature; Myf5 My~ indicates that the Myf5 gene was replaced by the Myod gene. The International Committee on Standardized Genetic Nomenclature for Mice, established in the early 1950s, is responsible for genetic nomenclature rules. The rules are available online at the Mouse Genome Database (MGD) website . They are published periodically in print copy, the most recent being in Davisson (1996). The committee also maintains a list of inbred strains at the MGD site. The reader is refered to Chapters 27 and 28 for further discussion of nomenclature and to Table II for selected databases and websites relevant to mouse genetics and biology that are available as of this writing.

40

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON Table IIl Examples of Mouse Strain Nomenclature Strain name

Definition

DBA/2J

C3H/HeSn-ash/+ C57BL/6J-TyrC-2J/+ AEJ/GnJ-ae/A w-J AKR.B6-H2b

B6.CBA-D4Mit25-D4Mit80 B6.Cg m Leprdb/+ + B6.Cg-m +/+ Lepr db BXD-1/Ty CcS1 CcSI(N4) B.A-Chr 1 C57BL/6J_mt BALB/c B6; 129-Cfir tmlv"c B6.129-Myf5My~ FVB/N-TgN(MBP) 1Xxx FVB /N_mTglzzz

B6C3F1 B6EiC3-Ts65Dn

Hsd:ICR Pri:B6,D2-G#

0

1.

Inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before 20 generations of brother • sister breeding and is the subline maintained at the Jackson Laboratory (J) Coisogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on C3H/HeSn Coisogenic segregating inbred mutant strain carrying the albino 2J mutant allele of the cloned tyrosinase gene (tyr) Inbred strain segregating for two alleles at the agouti gene Congenic inbred strain in which the b haplotype at the H2 complex was transferred from C57BL/6J (B6) to the AKR background Congenic strain in which the chromosomal segment between D4Mit25 and D4Mit80 was transferred from CBA to B6 Congenic inbred strain in which the linked mutant genes misty (m) and diabetes (Lepr db) were transferred from multiple, mixed, or unknown genetic backgrounds to B6 and are carried in coupling, i.e., on the same chromosome Congenic inbred strain in which the m and Lepr db mutations are carried in repulsion Recombinant inbred (RI) strain number 1 in a set of RI strains derived from a C57BL/6J (B) female mated to a DBA/2J (D) male and made by Taylor (Ty) Recombinant congenic (RC) strain number 1 in a set made by crossing the BALB/c (C) and STS (S) strains, backcrossing 1 or 2 times to BALB/c and then inbreeding as with RI strains Recombinant congenic (RC) strain number 1 in a set made by crossing the BALB/c (C) and STS (S) strains, backcrossing N4 times to BALB/c and then inbreeding as with RI strains Chromosome substitution (CSS) or consomic strain in which Chr 1 from A/J has been transferred to the B6 background Conplastic strain with the nuclear genome of C57BL/6J, and the cytoplasmic genome of BALB/c, developed by crossing male C57BL/6J mice with BALB/c females, followed by repeated backcrossing of female offspring to male C57BL/6J First targeted mutation of the cystic fibrosis transmembrane regulator gene created at the University of North Carolina, Unc, and carried on a mixed B6 and 129 background Congenic strain carrying a replacement or "knockin" in which the Myf5 gene was replaced with the Myod gene in 129 ES cells and backcrossed onto the B6 genetic background Transgene in which the human myelin basic protein (MBP) gene is inserted into the genome of the National Institutes of Health (N) subline of the FVB strain originally maintained at the National Institutes of Health Insertional mutation caused by the TglZzz transgene made on the FVB/N genetic background F1 hybrid made by crossing a C57BL/6 female to a C3H male Strain maintained by backcrossing mice with the Ts65Dn chromosome aberration to F1 hybrid mice made by crossing females of the Eicher (Ei) subline of C57BL/6 • C3H; note that these mice are not true F1 hybrids, and the F1 designation is omitted ICR outbred stock maintained at Harlan (Hsd) Advanced intercross line (AIL) created at Princeton (Pri) from the inbred strains C57BL/6 • DBA/2; AlL are made similar to RI strains except mice are intercrossed, avoiding sibling matings, to increase the possibility of tightly linked genes recombining

H o u s i n g and H u s b a n d r y

Housing

H o u s i n g (and h u s b a n d r y ) for m i c e are often g u i d e d by microb i o l o g i c a l r e q u i r e m e n t s . A c o l o n y can be m a i n t a i n e d in a "conv e n t i o n a l " e n v i r o n m e n t or b e h i n d a barrier w h e r e the m i c e are p r o t e c t e d f r o m specific m i c r o o r g a n i s m s . E x a m p l e s of barrier h o u s i n g include positive pressure isolators and m a s s airflow racks that p ro v i d e sterile air t h r o u g h high-efficiency particulate air (HEPA) filters or individually ventilated caging. T h e integrity of the m i c r o e n v i r o n m e n t is m a i n t a i n e d by servicing and

usually m a d e of p o l y c a r b o n a t e , p o l y p r o p y l e n e , or p o l y s t y r e n e plastic (in order of d e c r e a s i n g cost and durability). M i c e are s o m e t i m e s h o u s e d in s u s p e n d e d cages with o p e n - m e s h b o t t o m s that allow e x c r e m e n t to fall t h r o u g h to a col l ect i n g pan. Susp e n d e d caging is rarely used for b r e e d i n g b e c a u s e n e o n a t a l therm o r e g u l a t i o n is difficult to m a i n t a i n w i t h o u t nest in g material. C a g e lids should be stainless steel to facilitate c l e a n i n g and inhibit rust. C a g e s should k e e p animals dry and clean, m a i n t a i n a c o m f o r t a b l e a m b i e n t temperature, allow f r e e d o m of m o v e m e n t

c h a n g i n g cages in specifically d e s i g n e d hoods. M o u s e cages vary in design, size, and c o m p o s i t i o n . T h e

and n o r m a l postural adjustments, avoid u n n e c e s s a r y p h y s ic a l restraints, provi de c o n v e n i e n t access to feed and water, and prevent overcrowdi ng. S o l i d - b o t t o m cages should contain sanitary b e d d i n g , such as

p o p u l a r s h o e b o x cage u s e d for h o u s i n g and b r e e d ing m i c e is

w o o d chips or g r o u n d corncob. Criteria for selecting b e d d i n g

3. BIOLOGY AND DISEASES OF MICE

41

Table IV

et al., 1999). However, this type of caging employs passive air exchange and is prone to accumulate CO2 and NH3, thereby requiring frequent sanitation and bedding changes (see Chapter 29). The recent large-scale introduction of individually ventilated cages obviate, to a large extent, the elevated levels of noxious gases. However, purchase of these systems requires substantial expense (see Chapter 21).

Desirable Criteria for Rodent Contact Bedding a Moisture absorbent Dust-free Does not promote microbial growth Nonstaining Atraumatic Ammonia binding Sterilizable Deleterious products not formed as a result of sterilization Easily stored Uniform from batch to batch No microbial or chemical contamination Nonpalatable Nonallergenic Nontoxic Non-enzyme-inducing Nestable Readily available Inexpensive Chemically stable during use Animal behavior is not adversely affected

aModified from Kraft (1980).

Table V Tests of Bedding Qualitya Chemical properties Pesticides and polychlorinated compounds Mycotoxins Nitrosamines Detergent residues Ether-extractable substances Heavy metals Physical properties Particle uniformity Absorbtivity Ammonia evolution Visible trauma and irritant potential Microbiological properties Standard plate count Yeasts and molds Coliforms and Salmonella Pseudomonas

aModified from Kraft (1980). vary with experimental and husbandry needs (Table IV). It is preferable to autoclave bedding prior to use, but if this is not convenient, the bedding should be used only after its origin and microbial content have been evaluated (Table V). Several caging systems with tops containing filters are routinely in use in many academic settings. Their use has been popularized because of evidence that they substantially reduce or prevent airborne transmission of microbial agents (Lipman et al., 1993) and minimize caretaker exposure to allergens (Reeb-Whitaker

2. Husbandry Nutrient requirements for the mouse are influenced by genetic background, disease status, pregnancy, and environment. The best current estimate of nutritional requirements is shown in Table VI. Nutritional requirements for laboratory mice are also published periodically by the National Research Council and have been reviewed by Knapka and coworkers (Knapka, 1983; Knapka et al., 1974). Feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. Mice consume about 3 - 5 gm of feed per day after weaning and maintain this intake throughout life. Outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (Figs. 1 and 2). Diet is often neglected as a variable in animal-related research. Diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. Therefore, diet must provide a balance of essential nutrients, and contaminants must be kept to a minimum (see also Chapter 29). Naturalproduct commercial diets for mice are satisfactory for breeding and maintenance. Fresh produce, grains, fish meal, or other supplements may expose colonies to pathogenic bacteria or harmful chemicals and should be avoided. Mice should have continuous access to potable water even if a high-moisture diet is fed. Water is needed for lubrication of dry food and for hydration. Adult mice drink 6 - 7 ml of water per day. Decreased water intake will decrease food consumption. Water imbalance may occur during disease, because sick mice commonly drink very little water. Therefore, it may be unsuitable to administer medicine orally to affected mice. However, antimicrobials can be administered in the drinking water prophylactically, a measure used commonly to prevent infection in immunodeficient mice.

II.

A.

BIOLOGY

Physiology and Anatomy

Unless otherwise indicated the information in this section is from Cook (1983) and Kaplan et al. (1983). Normative data on the mouse are presented in Table VII, and clinical chemistry reference ranges are summarized in Table VIII.

42

ROBERT O. jACOBY, JAMES G. FOX, AND MURIEL DAVISSON Naturalingredient, openformula diet e

Table VI Nutrient Requirements of Mice a Nutrient

Concentration in diet (%)

Protein (as crude protein) Fat b Fiber Carbohydrate

20-25 5-12 2.5 45-60

A (IU/kg)

Estimated Dietary Amino Acid Requirement

Amino acid

Natural -ingredient, open-formula diet (%)c

Arginine Histidine Tyrosine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

0.3 0.2 ~ 0.4 0.7 0.4 0.5 0.4 0.2 0.1 0.5

,

Purified diet (%)d

0.12 0.2 0.25 0.15 0.3 0.25 0.22 0.05 0.3

Mineral Calcium (%) Chloride (%) Magnesium (%) Phosphorus (%) Potassium (%) Sodium (%) Sulfur (%) Chromium (mg/kg) Cobalt (mg/kg) Copper (mg/kg) Fluoride (mg/kg) Iodine (mg/kg) Iron (mg/kg) Manganese (mg/kg) Molybdenum (mg/kg) Selenium (mg/kg) Vanadium (mg/kg) Zinc (mg/kg)

1.

1.23 ~ 0.18 0.99 0.85 0.36 ~ ~ 0.7 16.1 ~ 1.9 255.50 104.0 -~ -50.3

Purified dietY 0.52 0.16 0.05 0.4 0.36 0.1 __ 2.0 ~ 6.0 m 0.2 35.0 54.0 ~ 0.1 ~ 30.0

10 0.03 5000 37 3 0.2 2009 4

Purified dietg 0.81 ~ 0.073 0.42 0.89 0.39 m 1.9 ~ 4.5 ~ 36.0 299.0 50.0 ~ ~ -31.0

Chemically defined diet h 0.57 1.03 0.142 0.57 0.40 0.38 0.0023 4.0 0.2 12.9 2.3 3.8 47.6 95.2 1.55 0.076 0.25 38.0

Temperature and Water Regulation

Mice have a relatively large surface area per gram of body weight. This results in dramatic physiologic changes in response to fluctuations in the ambient temperature (TA). The

4000 7 0.01 50 0.05 0.2 1000 2 30

21 8 17

16 6 6 ,

Chemically defined diet h

Purified dietg

1100 1730 22.5 6.0 0.023 0.58 1100 1.71 32 1514 18 10.7 0.2 1 750 2375 0.45 1.43 -248 22.5 35.6

1000

82

,

Mineral and Vitamin Concentrations of Adequate Mouse Diets Naturalingredient, openformula diet e

15,000

B 6 (mg/kg) B12 (mg/kg) D (IU/kg) E (IU/kg) KI equiv. (mg/kg) Biotin (mg/kg) Choline (mg/kg) Folacin (mg/kg) Inositol (mg/kg) Niacin (mg/kg) Calcium pantothenate (mg/kg) Riboflavin (mg/kg) Thiamin (mg/kg)

,

,

Vitamin

Purified dietI

37.5 7.5 22.5

47.5 7.1 4.8

,

a Modified from Knapka (1983). bLinoleic acid: 0.6% is adequate. cJohn and Bell (1976). dTheuer (1971). eKnapka et al. (1974). IAIN (1977). gHurley and Bell (1974). hpleasants et al. (1973).

mouse responds to cold exposure, for example, by nonshivering thermogenesis. A resting mouse acclimated to cold can generate heat equivalent to about triple the basal metabolic rate, a change that is greater than for any other animal. A mouse must generate about 46 kcal/m 2 per 24 hr to maintain body temperature for each I~ drop in TA below the thermoneutral zone.

I CD | 1

4 0

O CFI| A DBA / ZN [] Cs7BI / 6

30

E 20

lO

I

21

I

28

I

35

I

42

I

49

I

56

Age (Days) Fig. 1. Growth comparison: female outbred (CD1 and CF1) and inbred mice. (Courtesy of Charles River Breeding Laboratories.)

43

3. BIOLOGY AND DISEASES OF MICE

Table VII Normative Data for the Mouse

40

30 E tO} .--

20

10

A DBA / ZN r-1 Cs~BI / 6 I 21

,

I 28

I 35

I 42

I 49

I 56

Age (Days)

Fig. 2. Growthcomparison: male outbred (CD 1 and CF1) and inbred mice. (Courtesy of Charles River Breeding Laboratories.)

Mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. Therefore, it is not advisable to conserve energy in animal quarters at night by lowering thermostats. Because of its great ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. Its biological half-time for turnover of water (1.1 days) is more rapid than for larger mammals. Water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. The conservation of water can preempt thermal stability. If the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. The mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. Mice can partially compensate for changes in TA increases from 20 ~ to 35~ It adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. Its primary means of cooling in the wild is behavioral--retreat into a burrow. In the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of 37~ or higher. Thus, the mouse is not a true endotherm. In fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before 20 days of age. The thermoneutral zone for mice varies with strain and with conditioning but is about 29.6~176 narrower than that of any other mammal thus far measured. Thermoneutrality should not be equated with comfort or physiological economy. There are repeated studies to show that mice in a TA range of 2 1 ~ 25~ grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone.

Adult weight Male Female Life span Usual Maximum reported Surface area Chromosome number (diploid) Water consumption Food consumption Body temperature Puberty Male Female Breeding season Gestation Litter size Birth weight Eyes open Weaning Heart rate Blood pressure Systolic Diastolic Blood volume Plasma Whole blood Respiration frequency Tidal volume Minute volume Stroke volume Plasma pH CO2 CO2pressure Leukocyte count Total Neutrophils Lymphocytes Monocytes Eosinophils Basophils Platelets Packed cell volume Red bl6od cells Hemoglobin Maximum volume of single bleeding Clotting time PTT Prothrombin time

2.

20-40 gm 18- 35 gm 1-3 years 4 years 0.03-0.06 cm2 40 6.7 ml/8 weeks age 5.0 gm/8 weeks age 98.8~ 99.3~ (37~ 37.2~ 28-49 days 28-49 days None 19-21 days 4-12 pups 1.0-1.5 gm 12-13 days 21 days 310- 840 beats/min 133-160 mm Hg 102-110 mm Hg 3.15 ml/100 gm 5.85 ml/100 gm 163/min 0.18 (0.09-0.38) ml 24 (11-36) ml/min 1.3-2.0 ml/beat 7.2-7.4 21.9 mEq/L 40 _ 5.4 mm Hg 8.4 (5.1-11.6) • 103/~tl 17.9 (6.7-37.2)% 69 (63-75)% 1.2 (0.7-2.6)% 2.1 (0.9-3.8)% 0.5 (0-1.5)% 600 (100-1000) • 103/ktl 44 (42-44)% 8.7-10.5 x 108/mm3 13.4 (12.2-16.2) gm/dl 5 ml/kg 2-10 min 55-110 sec 7-19 sec

Respiratory System

The respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharynx; the intermediate section consists of larynx, trachea, and

44

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON Table VIII Clinical Chemistry Reference Ranges for Adult Mice a CD-1

Analyte Serum Glucose Urea nitrogen Creatinine Sodium Potassium Chloride Calcium Phosphorus Magnesium Iron Alanine aminotransferase Aspartate aminotransferase Alkaline phosphatase Lactate dehydrogenase Protein, total Albumin Cholesterol Triglycerides Bilirubin

Units

M

F

IU/liter

99 ___86.3

49 ___22.6

41.4 ___ 16.4

29.3 +__7.1

IU/liter IU/liter IU/liter g/liter g/liter mg/dl mg/dl mg/dl

196 +_ 132.6 39 ___25.7

128 + 60.6 51 _+ 27.3

99.5 ___33.4 59 ___ 11.4

73.6 _+ 15.3 118 ___ 15.9

44 _+ 11.0

48 ___8.5

114 + 56.3 91 _+ 58.5 0.4 +__0.2

72 ___20.1 53 ___23.6 0.5 ___0.35

53.9 36.7 94.8 97

63.5 46.4 92 78

Male

Female

Female

1 0 - 40

2 0 - 40 (basal)

1500-2000 (proestrus)

8 0 - 1 2 0 (basal)

250-300 (proestrus/estrus)

Luteinizing hormone

ng/ml

Follicle stimulating hormone

ng/ml

Prolactin Growth hormone Thyroid stimulating hormone Thyroxine Cortico sterone

ng/ml ng/ml

Epinephrine Norepinephrine Progesterone

pg/dl pg/dl ng/ml

Estradiol Testosterone Urine Volume Specific gravity pH Osmolality Creatinine Glucose Protein Albumin

pg/ml ng/ml

ng/ml gg/dl gg/dl

125

<1

aSummarized from Loeb and Quimby (1999).

134.4 23.6 0.84 160.8

__+20.3 ___5.3 +_ 0.298 ___4.40

M

F

171.6 +_ 57.2

174.9 ___31.0

0.43 _+ 0.14 157.8 _+ 5.7

0.45 +_ 0.07 157 _+ 6.70

8.10 +_ 0.80 5.95 ___0.63

__+7.5 ___5.2 +_ 16.9 __+21.1

___ 8.8 ___7.0 __. 15.9 ___ 12.2

378 55.7 31.7 150.4

___269 +_ 8.9 +__4.7 ___29.9

0.7 ___0.15

35 (late proestrus, estrus)

1-5 (basal) 1.5-2.0

1.6 __+0.9 1.0341 +_ 0.005 5.011 Osm/kg 1.06 -2.63 mg/100g/24 hr 2.6 ___0.91 mg/24 hr 0.53 ___0.19 mg/24 hr 0.7 ___0.33 mg/ml 11.9 _+ 0.2

___33.2 ___3.5 ___0.08 ___8.9

10-20 1-90

300 7.4 _ 0.5 (BALB/c) 40 (middle of 9 (start of dark period) dark period) 5 (start of light period) 0-200 30-300 5 (early proestrus)

ml/16 hr

121.7 32.7 0.50 166.7

F

166 ___4.1 7.8 _ 0.75 130 ___3.9 10.30 +_ 1.58 8.00 ___ 1.85 1.38 ___0.28 473 _+ 16

8.90 8.30 3.11 474

97 ___39.9 37 ___ 16

M

BALB/cBy

__+38.1 ___20.1 +_ 0.45 ___8.6 ___0.85 +__7.2 +_ 2.06 +__ 1.46 _+ 0.37 ___44

mg/dl mg/dl mg/dl mEq/liter mEq/liter mEq/liter mg/dl mg/dl mg/dl ~tg/dl

112 38 1.10 166 8.0

C57BL/6

1.7___ 1.1

3.21 ___ 1.05 (B6C3F1)

54.6 ___8.3 39.3 • 5.4 118.2 ___36.1

3. BIOLOGYAND DISEASES OF MICE

Superior lobe

45

Left bronchus

Middle lobe

Inferior lobe

Left lung

lobe

bronchi, all of which have cartilagenous support; and the posterior portion of the respiratory tract consists of the lungs. The left lung is a single lobe. The right lung is divided into four lobes: superior, middle, inferior, and postcaval (Fig. 3). A mouse at rest uses about 3.5 ml O2/gm/hr, which is about 22 times more O2/gm/hr than is used by an elephant. To accommodate for this high metabolic rate, the mouse has a high alveolar Po2; a rapid respiratory rate; a short air passage; a moderately high erythrocyte (RBC) concentration; high RBC hemoglobin and carbonic anhydrase concentrations; a high blood 02 capacity; a slight shift in the O2-dissociation curve, enabling 02 to be unloaded in the tissue capillaries at a high Po2; a more pronounced Bohr effect, i.e., the hemoglobin affinity for 02 with changes in pH is more pronounced; a high capillary density; and a high blood sugar concentration.

3. UrinarySystem Right lung

Fig. 3. Lobesof the lung. (FromCook, 1983.)

The kidneys, ureters, urinary bladder, and urethra form the urinary system (Fig. 4). The paired kidneys lie against the dorsal body wall of the abdomen on either side of the midline. The right kidney is normally located anterior to the left kidney. Kidneys from males of many inbred strains are consistently heavier than kidneys from females. The glomeruli of mice are small, about 74 ~tm in diameter, or about half the size of glomeruli in

Left kidney

ovary Fallopian tubes Right ureter Horn of uterus

Lumen of uterus

Corpus of uterus Urinary bladder

Cervix Vaginal fornix Vagina

Urethra Clitoral gland

Fig. 4.

Femaleurogenital tract. (FromCook, 1983.)

46

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

rats. There are, however, 4.8 times as many glomeruli in the mouse, and the filtering surface per gram of tissue is twice that of the rat. Mice excrete only a drop or two of urine at a time, and it is highly concentrated (Table VIII). The high concentration is made possible by long loops of Henle and by the organization of giant vascular bundles (vasa recta) associated with the loops of Henle in the medulla. The mouse can concentrate urine to 4300 mOsm/liter, whereas the maximum permissible concentration is 1160 mOsm/liter for a human. Mice normally excrete large amounts of protein in the urine. Taurine is always present in mouse urine, whereas tryptophan is always absent. Creatinine is also excreted in mouse urine, a trait in which mice differ from other mammals. The creatinine- creatine ratio for fasting mice is about 1:1.4. Mice excrete much more allantoin than uric acid. 4.

Gastrointestinal Tract

The submaxillary salivary gland, a mixed gland in most animals, secretes only one type of saliva (seromucoid) in the mouse. The tubular portion of the gastrointestinal tract consists of esophagus, stomach, small intestine, cecum, and colon. The esophagus of the mouse is lined by a thick cornified squamous epithelium, making gavage a relatively simple procedure. The proximal portion of the stomach is also keratinized, whereas the distal part of the stomach is glandular. Gastric secretion continues whether or not food is present. The gastrointestinal flora consists of more than 100 species of bacteria that begin to colonize the alimentary canal selectively shortly after birth. The ceca of normal mice contain up to 1011 bacteria/gm of feces. The bacteria throughout the gastrointestinal tract form a complex ecosystem that provides beneficial effects, such as an increase in resistance to certain intestinal pathogens, production of essential vitamins, and homeostasis of important physiological functions (Fig. 5). Gnotobiotic animals colonized with known microbiota have been used to great advantage as models for biomedical research (Falk etal., 1998; Wostman, 1996). For certain studies, it is desirable to colonize germfree mice with a defined microbiota. In the mid-1960s, Schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice (Schaedler and Orcutt, 1983). He subsequently supplied animal breeders with this group of microorganisms. These defined bacteria included aerobic bacteria and some less oxygen-sensitive anaerobic organisms. The so-called extremely oxygen-sensitive (EOS) fusiform bacteria, which make up the majority of the normal microbiota of rodents, were not included, because of technical difficulties in isolation and cultivation. Of the defined microbiotas later used for gnotobiotic studies, the one known as the "Schaedler flora" was the most popular. In 1978, the National Cancer Institute (NCI) decided to revise the Schaedler flora, or "cocktail" consisting of eight bacteria, in order to stan-

Lactobacilli (109/g) GroupN Streptococci(109/g) Torulopsis (107/-10ag)

Lactobacilli (109/g)

GroupN Streptococci(109/g) Torulopsis (106/g) Arthromitaceae

"~#~~,/f

~ ...~~~ '~

~

Fusiform.shapedbacteria(1011/g) (GeneraFusobacterium, Clostridium and Eubacterium) Bacteroides(109-101~ Spirochetes (109/g) Catenabacterium (108-109/g) Propionibacteria (10gig) Spirullum-shapedbacteria(108109/g) Lactobacilli(109/g) GroupN Streptococci(109/g) Torulopsis (106/g)

Fig. 5. Location of bacteria composing the autochthonous microflora in the gastrointestinal tract. (From Schaedler and Orcutt, 1983.)

dardize the microbiota used to colonize germfree rodents. The new defined microbiota, now known as the "altered Schaedler flora" (ASF), consisted of four members of the original Schaedler flora (two lactobacilli, Bacteroides distasonis, and the EOS fusiform bacterium), a spiral-shaped bacterium, and three new fusiform EOS bacteria. It is difficult to monitor a gnotobiotic mouse colony with a defined microbiota. It is necessary to demonstrate that microorganisms of the specified microbiota are present and that adventitious microorganisms are absent. In the past, monitoring relied on bacterial morphology, limited evaluation of biochemical traits, and growth characteristics. Recently, the eight ASF strains were identified taxonomically by 16S rRNA sequence analysis (Dewhirst et al., 1999). Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), L. salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519), based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of L. murinis and L. animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and L. lactis. Strain ASF 519 falls into an unnamed genus containing

[Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most

3. BIOLOGYAND DISEASESOF MICE

l

I

47

(% Difference)

'1

I

I

I

I '"l

I

I

--~

~

,,

ASF 361

<

actobacillus animalis

Lactobacillus murinus - Lactobacillus mali

Lactobacillus salivarius

ASF 360

, _ !

! ' t,,

I

.......... M----

L, ,

I

t

t.......

<

ii Lactobaciilus lactis ASF 500 < r Clostridium propionicum L Clostridium neopropionicum , A S F 3 5 6 <--C/ostridiurn pi/iforme Ruminococcus gnavus

Eubacterium contortum Roseburia cecico/a

ASF 502 < II-. . . . . . Catone/la morbi F- . . . . . Acetitomaculum rumini$ q ~ ASF 492 < I Eubacterium p/exicaudatum ,

1'

{

~

d o h n s o n e l l a ignava Flexistipes sinusarabic

Deferribacter thermophilus G eo vibrio f errireducens Colobus Monkey sp. ASF 457 < Rodent sp. 1 Rodent sp. 2 = Rodent sp. 3 , ,,

~

[Bacteroides] merdae - [ B a c t e r o i d e s ] distasonis

AFS 519

<

[ B a c t e r o i d e s ] fors ythus

CDC DF-3

Fig. 6. Phylogeneticrelationships of ASF strains. (Dewhirstet al., 1999)

closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are EOS fusiform bacteria, group phylogenetically with the low-G+C content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group) (Fig. 6). The 16S rRNA sequence information determined by Dewhirst et al. (1999) should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.

accessory splenic tissue. Age, strain, sex, and health status can affect the size, shape, and appearance of the spleen. Male spleens, for example, may be 50% larger than those of females. Most lymphocytes enter and leave the spleen in the bloodstream. The so-called white pulp of the spleen is organized along the central arteriole and is subdivided into T and B cell zones. The periarteriolar sheath is composed mainly of CD4 § and CD8 § T cells, and lymph follicles, which often contain germinal centers, are located at the periphery. The red pulp consists of sinusoids and hemoreticular tissue. Cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic vessels and lymphatic ducts, which empty into the venous system. The thymus is a bilobed lymphoid organ lying in the anterior mediastinum. It reaches maximum size around the time of sexual maturity and involutes between 35 and 80 days of age. The thymus plays a major role in maturation and differentiation of T lymphocytes. This function is not complete in newborn mice. Thymectomy is routinely performed in immunological research for experimental manipulation of the immune system. Thymectomy of newborn mice causes a decrease in circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses. Thymectomy in adult mice produces no immediate effect, but several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular immune responses. The mutant athymic nude mouse is a powerful experimental tool in the study of the thymus in immune regulation (Fogh, 1982). The mucosa-associated lymph tissue (MALT) contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes. The term M A L T designates all peripheral lymphoid tissues connecting to cavities communicating with the external milieu. They include the Peyer's patches, the cecal lymphoid tissue, and the lymphoid tissue in upper and lower respiratory tract, as well as the genitourinary system. Lymphatics drain these lymphoidrich areas, thus providing a direct link with lymph nodes and the bloodstream. 6.

5.

Lymphoreticular System

The lymphatic system consists of lymph vessels, thymus, lymph nodes, spleen, solitary peripheral nodes (Fig. 7), and intestinal Peyer's patches. Mouse lymph nodes are numerous but typically are small, reaching only a few millimeters. The typical lymph node is bean-shaped and consists of a cortex and a medulla. The cortex is divided into B lymphocyte domains, called primary follicles, and T lymphocyte domains, known as the diffuse cortex. The mouse does not have palatine or pharyngeal tonsils. The spleen lies adjacent to the greater curvature of the stomach. Different strains of mice have varying degrees of

Blood and Reticuloendothelial System

Bone marrow and splenic red pulp produce erythrocytic, granulocytic, and megakaryocytic precursors over the life of the mouse. Bone marrow is located in the protected matrix of cancellous bone and is sustained by reticular tissue rich in blood vessels and adipose cells (Pastoret et al., 1998). Normal hematologic values are listed in Table VII. Bone marrow-derived mononuclear phagocytes remove particulate antigens and act as antigen-presenting cells for lymphocytes. Tissue macrophages, which often function in a similar way, are found in many tissues, including peripheral lymphoid tissues, lung, liver, intestine, and skin.

48

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

Submental Superficial - - - - ~

••D

Mandibular

cervical

Anterior

\ mediastinal - - - - - - - - -

eep cervical

------- Axillary ~-------Lateral axillary

Posterior

mediastinal

......._ Pancreatic Renal Iliac - Superficial inguinal -Hypogastric -----

Sacral Popliteal

Fig. 7. Lymph nodes. (Modified from Cook, 1983.) 7.

Cardiovascular System

The heart consists of four chambers, the thin-walled atria and the thick-walled ventricles (Fig. 8). Mice conditioned to a recording apparatus have mean systolic blood pressures ranging from 84 to 105 mm Hg. An increase in body temperature does not lead to an increase in blood pressure. Heart rate, cardiac output, and the width of cardiac myofibers are related to the size of the animal. Heart rates from 310 to 840/min have been recorded for mice, and there are wide variations in rates and blood pressure among strains. 8. Musculoskeletal System

The skeleton is composed of two parts: the axial skeleton, which consists of the skull, vertebrae, ribs, and sternum, and the appendicular skeleton, which consists of the pectoral and pelvic girdles and the paired limbs. The normal vertebral formula for the mouse is C7T13L6S4C28, with some variations among strains, especially in the thoracic and lumbar regions. Normal mouse dentition consists of an incisor and three molars in each quadrant. These develop and erupt in sequence from front to rear. The third molar is the smallest tooth in both jaws; the upper and lower third molar may be missing in wild mice and in some inbred strains. The incisors grow continuously and are worn down during mastication.

9. Nervous System

The mouse brain has a typical mammalian structure. A detailed study of the neuroanatomy of the C57BL/6J mouse was made by Sidman et al. (1971). 10. Genital System

Female reproductive organs consist of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands (Fig. 4). The clitoral glands are homologous to the male preputial glands and secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa. The female mouse normally has five pairs of mammary glands, three in the cervicothoracic region and two in the inguinoabdominal region. Detailed techniques for manipulating gametes and embryos have been developed (Daniel, 1978). The male reproductive organs consist of paired testes, urethra, penis, and associated ducts and glands (Fig. 9).

B.

Reproduction

The following section summarizes normal reproduction in the mouse. The reader is referred to more comprehensive articles for additional information (Austin and Short, 1982; Rugh,

49

3. BIOLOGYAND DISEASES OF MICE

Rt. Common Carotid

1990; Whittingham and Wood, 1983). External influences, such as noise, diet, light, and population density, play an important role in reproduction and directly or indirectly influence the hypothalamic-pituitary axis for hormonal control of ovarian and testicular function. Genotype also dramatically affects the reproductive performance of the mouse.

f f 9 Lt. Common Carotid LV'/ .~-.- Left Subclavian

Right Superior-------~ ~ Vena Cave ~ \ if/J/ Right Subclavian ................. ~:"}---~"~"~'~---

Aortic Arch

"-ight Atrium--'-"--"~.,,~rr-~ ~:::-::''-t, ................Plmuonary Artery

~':~"~I .....Left ....Atrium ......

'ght Ventricle : / L~

1.

:::::~:--'~~ ~"'' ..........Left Ventricle ~'/i~:::::---.~]..~-PulmonaryVein

/ (

Follicle-stimulating hormone promotes gametogenesis in both sexes. Luteinizing hormones promote the secretion of estrogen and progesterone in the female and androgen in the male. Prolactin promotes lactation and development of the ovary during pregnancy. These gonadal hormones also ensure proper maintenance of the reproductive tract and modulate behavior to promote successful mating. The hypophysis is usually responsive to hormonal influence by day 6 in the male and day 12 in the female. Ovarian follicle development begins at 3 weeks of age and matures by 30 days. Rising levels of gonadotropins evoke signs of sexual maturity at about the same age. in the female, estrogen-dependent changes such as cornification of vaginal epithelium at the vaginal opening can occur as early as 24-28 days. Puberty is slightly later in the male (up to 2 weeks). Sexual maturation varies among strains and stocks of mice and is subject to seasonal and environmental influences. Mating behavior and the ability to conceive and carry fetuses to parturition are under complex hormonal control mediated by the anterior pituitary.

: f'ntercoS,a'

JJ!

Superior Mesenteric-------~. L ~ ~,~l~Renal /!~----

Genital

Inferior Mesenteric Common Iliac.............

Common Iliac

Caudal Fig. 8. Heartand major vessels.(Modified from Cook, 1983.)

Seminalvesicle

Sexual Maturation

..

gl Coagulating and

, ~ ~ =,.?,,-'_C"

Ampulla

~

Caputy~s--~~

/.--"~X~_._ epididymus~ ~

[ i

..... %

~

..........! !

~,~~-. Ventral prostate Ductusdeferens--

~e'~',; ~\~ ~ Urethra' I I ~t ill ~ Bulbourethral ! I i gland.......... ~ I If--

Cauda .9 ep=dldymus ' lymu:~'-~r~

Penis

Urethral --/~F"~' .--.diverticulum

(,.%-~~

__.~.Preputialgland

Fig. 9. Malereproductivetract. (FromCook, 1983.)

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

50

Table IX Changes in the Reproductive Organs of the Mouse during the Estrous Cycle a Stage.

Smear b

Uterus

Ovary and oviduct

Proestrus

Epithelial cells to epithelial-cornified cells or epithelial-cornified cells; leukocytes to epithelial cells

Hyperemia and distension increases. Active mitoses in epithelium, few leukocytes

Estrus

Epithelial-cornified cells to cornified + cells

Distension and activity are maximal during estrus and then decrease. No leukocytes

Metestrus

Cornified + + cells, epithelial cells, leukocytes + +

Distension decreased. Leukocytes in epithelium. Walls collapsed. Epithelium degenerates. Mitoses rare

Diestrus

Epithelial cells, leukocytes, more or less

Pale in appearance, walls collapsed. Epithelium healthy but contains many leukocytes. Some secretion by uterine glands

Follicles enlarged and distended with considerable liquor folliculi. Few mitoses in germinal epithelium and in follicular cells Ovulation occurs, followed by distension of upper end of oviduct. Active mitoses in germinal epithelium and in follicular cells Follicles undergoing atresia. Growing corpora lutea. Eggs in oviduct. Few mitoses in germinal epithelium and in follicular cells Follicles begin rapid growth toward end of period

mucus

Adapted from Bronson et al. (1996). b + indicates many cells; + + indicates very many cells; - indicates transition from epithelial to cornified. The descriptions for smears are typical; there is considerable variation. a

2.

Estrous Cycle

The mouse is polyestrous and cycles every 4 - 5 days. In the first two phases (proestrus and estrus), active epithelial growth in the genital tract culminates in ovulation. Degenerative epithelial changes occur during the third phase, followed by diestrus, a period of quiescence or slow cell growth. The cycle can be followed by changes in the vaginal epithelium that are often used to determine optimum receptivity of the female for mating and fertilization (Table IX). Patency of the vaginal orifice and swelling of the vulva are useful signs of proestrus and estrus. Irregularities of the estrous cycle occur during aging. Seasonal and dietary factors, such as estrogenic substances found in a variety of feeds, and genetic backgrounds also influence estrous cycles. Estrus is routinely observed in mice at about 14-24 hr after parturition (postpartum estrus). However, cornification of the vagina is not complete, and fertile matings are not as frequent compared with normal estrus. Mice are spontaneous ovulators. Ovulation does not accompany every estrus, and estrus may not coincide with every ovulation, because estrus is dependent on gonadal hormones, whereas ovulation is responsive to gonadotropin. The cyclicity of estrus and ovulation is controlled by the diurnal rhythm of the photoperiod. Mating, estrus, and ovulation most often occur during the dark phase of the photoperiod. Reversing the timing of the light-dark cycle reverses the time of estrus, ovulation, and mating. Pheromones (Table X) and social environment also effect the estrous cycle. For example, estrus is suppressed in mice housed

in large groups because of pseudopregnancy or diestrus ("Whitten effect"). These effects can be counteracted by olfactory stimuli evoked by chemical signals (pheromones) from male mice. By contrast, pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females ("Bruce effect"). Estrus can be synchronized by group-housing females prior to pairing with males. Group housing suppresses estrus, but exposure to male pheromones restarts the cycle and leads to estrus in most females 3 days after pairing. The next estrus will occur in about 11 days.

Table X Factors Leading to Pheromone Release in Mature Micea Initiator Stressed mice Females "Foreign" females Lactating females Males "Foreign" males Males coexisting in a territory

aModified from Shorey (1976).

Effect Dispersion of other mice Stimulate the approach, and sexual and aggressive behavior of males Aggressive behavior by other females Attract preweaning young Attract females Aggression by other males "Foreign" males avoid the territory and inhibit aggressive tendencies of familiar males

51

3. BIOLOGY AND DISEASES OF MICE 3.

Mating

Mating is normally detected by formation of a vaginal plug (a mixture of the secretions of the vesicular and coagulating glands of the male) whose prevalence is highly strain dependent. The plug usually fills the vagina from cervix to vulva (Fig. 10). Plug detection is often coupled with vaginal cytology to evaluate fertility and conception. When the cervix and vagina are stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. Secretion continues for about 13 days. If fertilization has occurred, the placenta takes over progesterone production. If fertilization does not occur, a pseudopregnant period ensues, during which estrus and ovulation do not occur. Fertilization usually takes place in the ampulla or the upper portion of the oviduct. Ova can be fertilized to produce normal embryos for 10-12 hr after ovulation. 4.

5.

Postnatal Development and Weaning

Maternal care can account for about 70% of the variation in body weight of neonatal mice. Nursing females usually lactate for 3 weeks. Milk production increases up to 12 days postpartum and then declines until weaning at 21 days. Interestingly, oxytocin is required for nursing but is not essential for parturition or reproductive behavior (Nishimori et al., 1996). Some transmission of humoral immunity from dam to progeny occurs in utero, but the majority of antibody is transferred through colostrum. Transmission of passive immunity by colostral antibodies has been demonstrated to a wide variety of antigens, including viruses, bacteria, and parasites. Antibodies continue to be secreted in the milk throughout lactation. Decay of maternally acquired immunity occurs within several months after weaning. Loss of maternal immunity increases susceptibility to infection and warrants continued care of weaned mice under barrier conditions.

Gestation

Gestation is usually 19-21 days. Because of postpartum estrus, lactation and gestation can occur simultaneously. Lactation can delay gestation because of delayed implantation. This may cause prolongation of gestation for up to 12-13 days in certain inbred strains. The effective reproductive life of some inbred strains approaches 2 years where optimum environmental conditions are maintained, but litter size usually decreases as the female ages. Therefore, females are usually retired by 1 year of age. Average litter size is strain dependent and commonly ranges from 1 to 12 pups.

Fig. 10. Vaginal plug. (Courtesy of Laboratory Animal Medicine and Science AutotutorialSeries.)

C.

Behavior

Mice are social mammals in which pheromones play an important role in communication (Table X). Pheromones have been divided into two broad categories: primer pheromones and releaser pheromones (Wilson, 1970). Primer pheromones are probably detected by the vomeronasal organs, which relay messages to the central nervous system (CNS), resulting in modulated behavior. Releaser pheromones trigger a prompt CNSmediated behavioral response in a recipient. Pheromones most frequently affect developmental and reproductive processes. Primer pheromones produced by males also regulate the reproductive physiology of female mice (Keverne, 1998). More than one response can be elicited by the same pheromone. For example, small, structurally diverse ligands, which bind to the major urinary protein of male mice, demonstrate puberty-accelerated pheromone activity in recipient females. Four of these ligands have been implicated in estrus sychronization (Whitten effect). However, the same chemosensory substances now appear to be responsible for both sexual maturation and estrus cycling in female mice (Novotny et al., 1999). Pheromone communication must be accounted for in the management of mouse colonies, particularly when subtle behavioral traits or reproductive performance are critical (Ma et al., 1998). Behavior in mice also is determined by genotype and environment. Male BALB/c mice, for example, are prone to fights and have a high prevalence of bite wounds around the head, back, shoulders, perineum, and tail. Aggressive behavior can sometimes be diminished by caging together only males from one litter or males paired prior to weaning. Hair nibbling and whisker chewing are examples of social dominance (Fig. 11). These traits may be an exaggeration of inherited grooming

52

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

Fig. 11.

Barbering.(Courtesyof Dr. J. G. Fox.)

behavior. Dominant mice usually retain their whiskers--hence the name b a r b e r mouse. Hair loss from barbering must be distinguished from hair loss due to ectoparasitism, microbial dermatitides, and abrasions from improperly designed cage covers. Maintenance behavior, such as eating and drinking, is cyclic and occurs mostly during the evening or at night. Nest building is another important social behavior and can be observed by placing a nestlet cotton square or other nesting material in the mouse's cage. Mice, and particularly genetically engineered mice, are being increasingly used in behavioral research (Crawley, 1999). About 100 different genes have been studied thus far in the mouse central nervous system. They have been identified and their phenotype ascertained in transgenic and knockout mice (Bedell et al., 1997; Campbell and Gold, 1996; Nelson and Young, 1998). Initial behavioral evaluations include general health and neurological reflexes assessment. Sensory abilities and motor functions are extensively quantitated. Specific tests include observations of home cage behaviors, body weight, body temperature, appearance of the fur and whiskers, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, Digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. Hypothesis testing then focuses on at least three well-validated tasks within each relevant behavioral domain. Specific tests for mice are being utilized for the domains of learning and memory, feeding nociception, and behavior that are relevant to discrete symptoms of human anxiety, depression, schizophrenia, and drug ad-

diction (Crawley, 1999). Substantial effort also is being directed to optimize behavioral phenotyping in mice.

D.

Immunology

The mouse is the primary mammalian model for immunology research because of the extensive literature available on this species, the availability of immunological reagents, the soonto-be-published complete mouse genome, and numerous genetically defined mouse strains, as well as the ever increasing number of genetically manipulated mice with defined genetic alterations. Unless otherwise indicated, information in this section is from Pastoret et al. (1998). 1.

Immunoglobulins

The mouse has five classes of immunoglobulins, which are determined by the heavy constant region and are classified by isotype, IgM, IgD, IgG, and IgA. The IgG class is further divided into IgG1, IgG2a, IgG2b, and IgG3. The humoral immune response varies according to the type of immunogen used to evoke the response and the immunoglobulin isotypes that express the effects or functions. For example, IgM is secreted after initial exposure to an antigen, followed by IgG, the most abundant antibody in serum. In viral infections or intracellular bacterial infections, IgG2a is dominant, whereas in parasitic infection IgG~ is dominant. IgG2b and IgG3 are usually induced by T cell-independent antigens, such as carbohydrates. IgG3 also

3. BIOLOGY AND DISEASESOF MICE is important in responding to bacterial antigens, e.g., by playing a role in phagocytosis. IgE is linked to allergy, and IgA plays a pivotal role in mucosal immunity (Kramer and Cebra, 1995). Finally, IgD plasma cells are rarely found, and their exact function remains an enigma. 2.

53

turn block a given pathway despite the existence of redundant pathways.

III.

DISEASES

Cellular Immunology

Murine lymphoid cells can be phenotyped by monoclonal antibodies against surface markers and are referred to as cluster of differentiation, or CD, antigens. The nomenclature of the cells that express these antigens and other proteins that recognize them and their function have been extensively determined in the mouse, and these markers are routinely used to characterize pathological processes in mouse models. Mouse T lymphocytes can be differentiated into two primary phenotypes, based on their expression of CD molecules. CD4 § T cells (helper T cells) are MHC class II restricted and promote B lymphocyte responses essential for humoral immunity. CD8 § T cells are MHC class I restricted and serve as cytotoxic cells for cell-mediated immunity (e.g., against cells containing infectious agents) or act to suppress immune responsiveness.

Contemporary knowledge about diseases of laboratory mice has developed primarily from examining the effects of disease on traditional strains and stocks. The widespread use of genetically engineered mice is likely to modify current concepts because of novel or unpredictable interactions among genetic alterations, the genetic backgrounds on which they are expressed, and exogenous factors, such as infectious agents. Because the number of combinations is extraordinarily high, clinical and laboratory diagnosticians should be alert to the potential for altered disease expression in genetically engineered mice and not be misled by unexpected signs, lesions, and epizootiology.

A.

Infectious Diseases

Microbiological Surveillance 3.

Cytokines

Cytokines are a set of signaling molecules involved in cells communicating with one another in a complex biological system. Cytokine-mediated signaling primarily occurs after initiation and effector events of the host's immune response and in the development of hematopoietic cells. Because of their importance in modulating tissue responses to antigenic stimuli, a number of mouse cytokines have been described and are routinely used in research (Table XI). 4.

Models of Immune Dysregulation

A number of spontaneous mouse models of immune deficiency have been used extensively in research (Table XII). Their use--plus the expanding number of immune dysregulated knockout, transgenic, and dominant negative mutantsm has advanced understanding of human immune deficiency diseases as well as basic understanding of the immune system. Investigators using genetically engineered mice are constantly reminded, however, that phenotypic analysis of these animals must be done cautiously because the immune system may be profoundly affected and in ways that are not always anticipated. This may make it difficult to determine whether a given gene product is directly involved or may be secondary to a more global dysregulation of the immune system. As with other biological systems, compensation mechanisms also may mask the phenotype. Use of dominant negative mutations has reduced this possibility somewhat, because mice express transgenes encoding for abnormal, catalytically inactive proteins, which in

Many of the agents and conditions discussed under Section III,A, may interfere with mouse-based research. Housing and husbandry in microbiologically sheltered environments are designed to reduce the risks of disruptive infection, especially among immunologically dysfunctional mice, but must be accompanied by effective microbiological surveillance. Several principles are worthy of emphasis at the outset. (1) Surveillance should encomPass resident mice and mouse products (serum, cell lines, transplantable tumors) procured from external sources. Many commercial vendors currently provide sound, contemporary microbiological data on animals that they sell. Further, it is impractical, logistically and financially, to test every batch of commercially procured animals. However, thorough quarantine and testing of mice and mouse products from noncommercial sources should be mandatory. (2) Testing should be consonant with institutional needs. Therefore consideration must be given to the list of agents for which testing will be done, the minimum prevalence level for a given infection that testing is designed to detect, the frequency of testing, the number and location of animals to be tested per unit of time, sample collection strategies, and costs. Additionally, it is important to consider the impact of modern housing systems on detection strategies. Microbarrier cages, while protecting mice against infection, may also limit detection of low-level infection by impeding cage-to-cage transmission. Therefore detection should include preemptive exposure of sentinel mice to multiple bedding samples or the complementary use of molecular diagnostics to sample animals, soiled bedding, or spent air from ventilated cage racks. (3) The tests selected should provide a high degree

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ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON Table X l Major Sources, Cellular Targets, and in Vivo Effects of Select Mouse Cytokines a Cell source

Cytokine IFN-a, IFN-13 IFN-7

Macrophages, B and T cells, fibroblasts, epithelial cells T cells, NK cells

Cell targets Many cell types Macrophages, lymphocytes, NK cells Many cell types

IL-2

Macrophages, endothelial cells, keratinocytes, lymphocytes, fibroblasts, osteoblasts Activated T cells

IL-3

T cells, mast cells

IL-4

T cells, basophils, mast cells, bone marrow stromal cells

IL-5

T cells, mast cells

IL-6

B and T cells, thymocytes, hepatocytes, neurons B and T cells

IL-11 IL-12

Fibroblasts, macrophages, endothelial cells, T cells Thymic and bone marrow stromal cells Monocytes, neutrophils, fibroblasts, endothelial cells, keratinocytes, T cells T cells Macrophages, T and B cells, mast cells, keratinocytes Stromal cells T cells

IL-13

T cells

B cells

IL-14 IL-15

Endothelial cells, lymphocytes Fibroblasts, keratinocytes, endothelial cells, and macrophages

B cells T and B cells, NK cells, monocytes, eosinophils, neutrophils

IL-16

Epithelial cells, mast cells, CD4 § and CD8 § cells, eosinophils

CD4 §

IL-17

Human memory T cells, mouse tx[3TCR § CD4- CD8- thymocytes Macrophages, keratinocytes, microglial cells

Fibroblasts, keratinocytes, epithelial and endothelial cells T cells; NK cells; myeloid, monocytic, erythroid, and megakaryocytic cell lineages Hematopoietic stem cells, neutrophils, macrophages

IL-la, IL-l[3

IL-7 IL-8

IL-9 IL-10

IL-18

GM-CSF

TNF TGF-~

Macrophages, stromal cells, fibroblasts, endothelial cells, lymphocytes Macrophages, T and B cells, NK cells Platelets, macrophages, T and B cells, placenta, hepatocytes, thymocytes

Macrophages, T and B cells, NK cells Mast cells, hematopoietic progenitors B and T cells, mast cells, maerophages, hematopoietic progenitors Eosinophils, B cells

Function Antiviral, antiproliferative, stimulate NK activity and macrophage functions Proinflammatory, promotes Thl immune responses/ secretion of Thl-associated cytokines Proinflammatory, stimulates fibroblasts and bone catabolism, neuroendocrine effects (fever, sleep, anorexia, corticotropin release) T cell growth factor, stimulates NK activity Promotes proliferation and differentiation of mast cell and hematopoietic cell lineages (granulocytic, monocytic, megakaryocytic) Proliferation and differentiation of B cells (Ig switching to IgG1 and IgE) and Th2 cells (antiinflammatory by inhibiting Thl immune responses) Stimulates eosinophilia, growth and differentiation of B cells, Ig switching Differentiation of myeloid cells, induction of acute phase proteins, tropic for neurons Growth factor for B and T cells

Neutrophils, basophils, T cells

Proinflammatory, activates neutrophils, enhances keratinocyte growth

CD4 § T cells, mast cells Macrophages, T and B cells

Enhances hematopoiesis Anti-inflammatory Th2 immune responses, inhibits Thl responses Hematopoiesis Proinflammatory; promotes NK and cytotoxic lymphocyte activity; induces IFN-y, which in turn promotes Thl immune responses Activation of Ig transcription, key mediator in asthma B cell growth factor Enhances neutrophil chemokine production, cytoskeletal rearrangements, phagocytosis; delays apoptosis CD4 + T cell growth factor; proinflammatory; enhances lymphocyte chemotaxis, adhesion molecule and IL-2 receptor and HLA-DR expression Secretion of IL-6, IL-8, PGE2, MCP-1 and G-CSF, induces ICAM-1 expression, T cell proliferation Proinflammatory, induces IFN-y and other Thl cytokines, promotes Thl development and NK activity Growth and differentiation of granulocytes, macrophages

Hematopoietic progenitor cells T cells, macrophages

Many cell types Many cells types

Proinflammatory, fever, neutrophil activation, bone resorption, anticoagulant, tumor necrosis Anti-inflammatory; promotes wound healing, angiogenesis; suppresses hematopoiesis, lymphopoiesis, Ig production, NK activity; promotes Ig switching to IgA

aIFN, interferon; IL, interleukin; GM-CSF granulocyte-macrophage colony stimulating factors; NK, natural killer; TNF, tumor necrosis factor; TGF, tumor growth factor.

55

3. BIOLOGY AND DISEASES OF MICE Table XII

Common Mouse Models of Immunodeficiency ,

Model

,,,

Immunodeficiency

Phenotype

Defective transcription factor gene controlling thymic epithelial cell differentiation Defective DNA-dependent kinase that recombines gene segments coding for T (TcR) and B (Ig) cell receptors

Athymic and hairless (unrelated but linked gene defect) No T cell functions Hypoplastic lymphoid tissues No Ig or T cell responses Sensitive to ionizing radiation because of defective DNA break repair

Rag- 1 and Rag-2 mice

Defective recombinase enzymes (Rag-1 and/or Rag-2), preventing formation of functional B a (Ig) and T (TcR) cell receptors

Hypoplastic lymphoid tissues No Ig orT cell responses

XID mouse

Defect in Bruton's tyrosine kinase gene affecting signal transduction in B cells Defective phosphatase, impairing signal transduction from cell receptors

Decreased B cell numbers, low IgM Impaired response to polysaccharide antigens Deficient humoral and cellular immunity Lack cytotoxic T and NK cells Moth-eaten pelage secondary to folliculitis Autoimmune syndromes Hypergammaglobulinemia Diluted coat color Lysosomal storage disease Impaired chemotaxis, bactericidal activity of neutrophils, decreased NK activity Generalized lymphoproliferative disease (gld), autoimmunity, immunodeficiency Anemia (IL-2), wasting (IL-2, IL-10), and inflammatory bowel disease (IL-2, IL-10) when housed conventionally Lack functional response to signal of interest, variable immune compromise Inflammatory bowel disease common in TcR KO

Nude mouse SCID mouse

Moth-eaten mouse

Beige mouse

lpr and gld mice Cytokine KO mice (IL-2, IL- 10, IFN-% TNF-[3, others) Receptor KO mice (TcR, Ig, cytokine, MHC, adhesion molecules, integrins)

Mutation on chromosome 13 affects pigment granules (coat, retina) and lysosomal granules of type II pneumocytes, mast cells, and NK cells Impaired apoptosis from Fas (lpr) or Fas ligand (gld) defect Genetically engineered disruption (knockout) of cytokine gene Genetically engineered disruption (knockout) of receptor gene

Major uses Tumor and xenograft studies V (D)J recombination studies Tumor and xenograft transplantation Lymphocyte subset transfer studies Reconstitution of human hematopoietic system (Hu-PBL-SCID) V (D)J recombination studies Tumor and xenograft transplantation Lymphocyte subset transfer studies Model for human X-lined agammaglobulinemia Apoptosis studies Autoimmune syndromes

Model for Chediak-Higashi syndrome Crossed onto nude or SCID backgrounds for multiple imune deficiencies Apoptosis studies Autoimmune syndromes Physiological role of cytokines in immune response and inflammation Physiological role of receptors in immune response and inflammation

......

of sensitivity and specificity. The traditional benchmark for testing is serology, but molecular diagnostics, microscopy (especially for parasites), and isolation of the agent (especially of bacteria) may be justified for detection or verification. (4) Interpretation of test results and resulting strategies to eliminate or contain infection should be based on a thorough knowledge of the agent under consideration, its potential effects on mice knowingly or potentially exposed, and the validity of the testing and surveillance methods. Because effective surveillance strategies will vary with research needs and operating conditions, it is prudent to consult at least several literature sources about options for testing and monitoring for infectious agents and disease before launching or modifying a surveillance program (Barthold, 1998; de Souza and Smith, 1989; FELASA, 1994, 1996; Lindsey et al., 1991a;

Lussier, 1991; Nicklas et al., 1993; Rehg and Toth, 1998; Small, 1984; Waggie et al., 1994; Weisbroth et al., 1998; White et al., 1998). There are also recommendations regarding specific agents in following sections.

1.

Viral Diseases

a.

Mousepox (Fenner, 1948, 1982, 1990)

Etiology.

Mousepox is caused by ectromelia virus, an orthopoxvirus that is closely related antigenically and physicochemically to vaccinia virus. Field strains of ectromelia virus have been isolated in many countries, but several, including Hampstead (low virulence), Moscow (high virulence), and NIH-79 (high virulence), have been used extensively for laboratory

56

study. Ectromelia virus grows well on the chorioallantoic membrane of embryonated chicken eggs and can also infect HeLa cells, mouse fibroblasts (L cells), and chick embryo fibroblasts. The BS-C-1 cell line is particularly sensitive to infection. Ectromelia virus produces an envelope hemagglutinin whose detection forms the basis for hemagglutination inhibition, a historically important serological test for mousepox. Clinical signs. Mousepox usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection, or chronic infection with low mortality. The expression of clinical signs reflects an interplay among virus-related factors, including virulence and dose, and host-related factors, including age, genotype, immunological competence, and portal of entry. Acute lethal infection occurs in genetically susceptible strains (Briody, 1959). Outbreaks among susceptible mice are often volatile, with variable morbidity and high mortality. Clinical signs such as ruffled fur or prostration, may occur for only a few hours before death. This rapidly fatal form is associated with multisystemic necrosis. Mice that survive acute infection can develop chronic disease characterized by a skin rash whose severity depends on the extent of viremia secondary to infection of parenchymal organs. The rash can develop anywhere on the body and may be focal or generalized (Fig. 12). Conjunctivitis also may occur. The skin lesions usually recede within several weeks, but hairless scars can remain. Additionally, severe viral infection of the feet and tail during the rash syndrome can lead to necrosis and amputation. Epizootiology. Mousepox is not a common disease. Outbreaks occur sporadically and can usually be traced to the im-

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

portation of contaminated mice or mouse products. For example, contaminated mouse serum was responsible for recent outbreaks in the United States (Dick et al., 1996; Lipman et al., 2000). Natural exposure is thought to occur through skin abrasions, but oral inoculation can cause chronic inapparent infection of Peyer's patches accompanied by prolonged excretion of virus in feces. Mice with chronic skin disease can transmit infection by contact and also are a source of contaminated tissues. Intranasal inoculation can produce necrotizing rhinitis and pneumonia in addition to systemic disease. However, tcage-tocage transmission of infection is low and can be virtually nil if cage bonnets are employed (Bhatt and Jacoby, 1987b). Ectromelia virus is highly stable at room temperature, especially under dry conditions, leading to the potential for prolonged environmental contamination in infected colonies (Bhatt and Jacoby, 1987c). Aerogenic exposure is not a major factor in natural outbreaks, and arthropod-borne transmission does not appear to occur. However, intrauterine infection and fetal deaths have been reported. Therefore contamination of cesareanderived progeny or embryo-derived cell cultures is possible. The mouse is the only known host for ectromelia virus. Genotype can modulate the course and severity of infection. DBA/1, DBA/2, BALB/c, A, and C3H mice are among the inbred strains most susceptible to lethal infection, whereas C57BL/6 and AKR are resistant (Wallace, et al., 1985; Bhatt and Jacoby, 1987a). Immunodeficient mice also should be considered highly susceptible. The mechanisms of genetic resistance are not fully understood but appear to reflect multiple genes, some of which appear to be expressed through lymphoreticular cells, including natural killer cells (Brownstein and Gras, 1995, Jacoby et al., 1989).

Fig. 12. A hairless mouse with mousepox. (From Fenner, 1982, and with permission of the Zentral Institut fiir Veruchstiere, Hannover, Federal Republic of Germany.)

57

3. BIOLOGY AND DISEASES OF MICE

Fig. 13. Skinwith intracytoplasmictype A inclusions of ectromelia virus.

Natural transmission is facilitated by intermediately resistant mice. They frequently survive long enough to develop skin lesions that can shed virus and serve as a major reservoir for spread of infection. The risks for transmission are further increased by persistence of infectious virus in excreta and exfoliated scabs. Although virus excretion typically lasts for about 3 weeks, virus has been found in scabs and/or feces for up to 16 weeks. Resistant mouse strains also are dangerous because they can shed virus during asymptomatic infections. However, infections in resistant mice tend to be short-lived. Ironically, highly susceptible mice are a relatively small hazard for dissemination of infection, if properly discarded, because they die before virus shedding becomes prominent. Thus, the juxtaposition of enzootically infected and highly susceptible mice can provoke explosive outbreaks. Infant and aged mice are usually more susceptible to lethal infection than young adult mice. Maternal immunity among enzootically infected breeding mice may perpetuate infection by protecting young mice from death, but not from infection. Such mice may subsequently transmit infection by contact exposure. Pathology. Ectromelia virus multiplies in the cell cytoplasm and produces two types of inclusion bodies. The A type (Marchal body) is well demarcated and acidophilic in standard histological sections. It is found primarily in epithelial cells of skin or mucous membranes and can also be found in intestinal mucosa (Fig. 13). The B type of inclusion is basophilic and can be found in all ectromelia-infected cells. However, it is difficult to visualize unless cells are stained intensely with hematoxylin or, preferably, by immunohistochemistry for ectromelia virus anti-

gens on formalin-fixed, paraffin-embedded tissue sections (Jacoby and Bhatt, 1987) (Fig. 14). Following skin invasion, viral multiplication occurs in the draining lymph node and a primary viremia ensues (Fig. 14). Splenic and hepatic involvement begin within 3 - 4 days, whereupon larger quantities of virus are disseminated in blood to the skin. This sequence takes approximately 1 week and, unless mice die of acute hepatosplenic infection, ends with the development of a primary skin lesion at the original site of viral entry. The primary lesion is due to the development of antiviral cellular immunity. Severe hepatocellular necrosis occurs in susceptible mice during acute stages of mousepox. White spots indicative of necrosis develop throughout the liver. In nonfatal cases, regeneration begins at the margins of necrotic areas, but inflammation is variable. Splenic necrosis in acute disease commonly precedes hepatic necrosis but is equally or more severe (Fig. 15). Necrosis and scarring of red and white pulp can produce a macroscopic "mosaic" pattern of white and red-brown (Fig. 16). Necrosis of thymus, lymph nodes, Peyer's patches, intestinal mucosa, and genital tract also have been observed during acute infection, whereas resistant or convalescent mice can develop lymphoid hyperplasia. Severe intestinal infection may be accompanied by hemorrhage. The primary skin lesion, which occurs 6 - 1 0 days after exposure, is a localized swelling that enlarges from inflammatory edema. Necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. Secondary skin lesions (rash) develop 2 - 3 days later as the result of viremia. They are often multiple and widespread and can be associated with

58

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

Fig. 16. "Mosaicspleen"from a mousethat survivedacute mousepox.

Fig. 14. Diagramillustrating the pathogenesisof mousepox. (FromFenner, 1948.)

conjunctivitis, with blepharitis, and, in severe cases, with buccal and lingual ulcers. The skin lesions also can ulcerate and scab before scarring.

Fig. 15. Splenicnecrosis in acute mousepox.

Diagnosis. Mousepox can be diagnosed from clinical signs, lesions, serological tests, and demonstration of virus or viral antigen in tissues. Detection of characteristic intracytoplasmic eosinophilic inclusions aids detection of infection. Virus can be isolated from infected tissues by inoculation of cell cultures (BS-C-1) or embryonated eggs. Several serological tests are available to detect mousepox. Historically, the standard test was hemagglutination inhibition (HA1), using vaccinia antigen as a source of hemagglutinin. An enzyme-linked immunosorbent assay (ELISA) is more sensitive and specific and has replaced HAI for serological monitoring among nonvaccinated mice (Buller et al., 1983). Ectromelia virus infection also can be detected by an immunofluorescence assay (IFA) and a PCR (polymerase chain reaction) assay (Neubauer et al., 1997). Serological differentiation of mousepox from vaccinia infection in vaccinated mice is based on the lack of hemagglutinin in the vaccine strain of virus. Thus, serum from vaccinated mice may react by ELISA but should not react by HAI. Differential diagnosis. Mousepox must be differentiated from other infectious diseases associated with high morbidity and high mortality. These include mouse hepatitis, Tyzzer's disease, and reovirus 3 infection. Each can be expressed by acute necrosis in parenchymal organs, but they can be differentiated by morphological, serological, and virological criteria. The skin lesions of chronic mousepox must be differentiated from other skin diseases caused by opportunistic or pathogenic bacteria, acariasis, and bite wounds. Prevention and control. Mousepox is a dangerous disease because of its virulence for susceptible mice. Therefore, infected colonies should be quarantined immediately. Depopulation coupled with vaccination has been used as a primary means for control, but confirmation of infection should be obtained before exposed mice are destroyed. Tissues, supplies, instruments, or other items that have had potential contact with infected mice should be disinfected by heat or chemicals such as formalin, sodium hypochlorite, or chlorine dioxide. Materials should be autoclaved or, preferably, incinerated. Disinfected rooms should be challenged with susceptible sentinel animals that are ob-

59

3. BIOLOGY AND DISEASES OF MICE

served for clinical signs and tested for seroconversion after several weeks. Depopulation and disinfection must be done vigorously. Because modern housing and husbandry methods based on the use of microbarrier caging are effective for containing infection, testing and culling properly isolated mice is a potential alternative, especially for irreplaceable breeding mice, such as transgenic founders. Such mice can be quarantined along with cessation of breeding to permit resolution of infection (Bhatt and Jacoby, 1987b). Sequential testing with contact-exposed sentinels should be employed with this option. Additionally, maternal immunity from fully recovered dams can protect mice from infection, thereby enhancing opportunities to derive virusfree mice from previously infected dams. Vaccination can control or prevent clinically apparent mousepox. The hemagglutinin-deficient strain of vaccinia virus (IHDT) is used to scarify skin on the dorsum of the tail. "Takes" should occur in previously uninfected mice by 6 - 1 0 days, but not in infected mice (Bhatt and Jacoby, 1987d) (Fig. 17). Infected mice should be quarantined separately or eliminated. Vaccination may not prevent infection, although infection in vaccinated mice is often transient. Furthermore, vaccinia virus can be shed from scarification sites for at least several days. Therefore, other preventive measures, such as strict controls on the entry of mice or mouse products, combined with periodic serological monitoring, should not be relaxed until diagnostic testing has confirmed the elimination of vaccinia and ectromelia virus. Additionally, seroconversion evoked by

vaccination must be taken into account in serological monitoring of vaccinated colonies. Finally, vaccinia virus is a human pathogen, so vaccination procedures should include personnel protective measures to prevent exposure.

Research complications.

The primary threat from mousepox is mortality in susceptible mice. The loss of time, animals, and financial resources can be substantial.

b.

Herpesvirus Infections (Osborn, 1982, 1986)

Mice are naturally susceptible to two herpesviruses: mouse cytomegalovirus and thymic necrosis virus. They are speciesspecific viruses and antigenically distinct from each other and from other rodent herpesviruses.

i. Mouse cytomegalovirus (MCMV) infection (Lussier, 1994) Etiology. MCMV is a mouse-specific betaherpesvirus. It can, however, replicate in cell cultures from several species, including mouse (fibroblasts and 3T3 cells), hamster, rabbit, sheep, and nonhuman primate.

Clinical signs. MCMV causes asymptomatic infection in adult immunocompetent mice, but experimental inoculation of neonates has caused lethal disease due to multisystemic necrosis and inflammation.

Fig. 17. Vaccination"take" in a mouse inoculated with the IHD-T strain of vaccinia virus. (FromJacoby et al., 1983.)

60

Epizootiology. The prevalence of MCMV in laboratory mice is unresolved, because infection is clinically silent and serological surveillance is not widely practiced. Wild mice serve as a natural reservoir for infection, which implies that entry of virus into a modern vivarium is most likely to occur from contaminated animal products. Susceptibility to experimental infection varies with age, dose, route, virus strain, and host genotype. Infection can occur in young and adult mice. However, the pathogenicity of MCMV for mice decreases with age. Neonates are highly susceptible to lethal infection, but resistance to disease develops by the time mice are weaned. Immunodeficient mice, however, remain susceptible to pathogenic infection as adults. Persistence is a central feature of nonlethal infection. Persistently infected mice can excrete virus in saliva, urine, and tears for many months, resulting in horizontal transmission through mouse-to-mouse contact. Virus also can infect prostate, testicle, and pancreas, implicating other modes of excretion. Vertical transmission does not appear to be a common factor in natural infection. Further, maternal immunity protects sucklings from infection. Pathology.

Mouse cytomegalovirus can replicate in many tissues, and viremia commonly occurs. Lesions are not remarkable during natural infection and may be limited to occasional enlarged cells (megalocytosis) containing eosinophilic intranuclear and/or cytoplasmic inclusions associated with lymphoplasmacytic interstitial inflammation, especially in the cervical salivary glands. As noted above, infection in infant mice can induce multisystemic necrosis and inflammation. Persistent infection often affects the salivary glands and pancreas. The persistence of salivary gland infection appears to be dose dependent. There is experimental evidence that MCMV can produce latent infection of B cells, probably T cells as well as aforementioned tissues. Persistent infection may lead to immune complex glomerulonephritis. Latent persistent infection can be reactivated by lymphoproliferative stimuli and by immunosuppression.

Diagnosis.

MCMV antigens appear to be weak stimuli for humoral antibody production, which is consistent with the fact that cellular immunity is critical for protection against infection. Neutralizing (NT) antibody titers are low during acute infection and difficult to find during chronic infection. An ELISA has been developed for serological monitoring (Lussier et al., 1987a) and primers for PCR-based diagnosis are available, but neither is widely used because of assumptions that infection has a very low prevalence. Mouse cytomegalovirus can be grown in mouse embryo fibroblasts or 3T3 cells, but cocultivation may be required to rescue latent virus. Detection of enlarged cells with intranuclear inclusions, especially in salivary glands, are diagnostic, if they are present. In situ hybridization can be used as an adjunct to routine histopathology.

Differential diagnosis.

MCMV infection must be differentiated from infection with mouse thymic virus. The latter virus

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

can produce necrosis and atrophy of thymic and peripheral lymphoid tissue. Lytic lesions of lymphoid tissues are not a hallmark of MCMV. The viruses can also be distinguished from each other serologically. Sialoadenitis with inclusions can occur during infection with polyomavirus. Reovirus 3, mammary tumor virus, and mouse thymic virus can infect the salivary gland.

Prevention and control.

Control measures for MCMV have not been established, because it has not been considered an important infection of laboratory mice. Cage-to-cage transmission has not been demonstrated, but horizontal infection from contaminated saliva must be considered. The exclusion of wild mice is essential.

Research complications.

Mouse cytomegalovirus can suppress immune responses. Apart from the potential for interfering with immunology research, it can exacerbate the pathogenicity of opportunistic organisms such as Pseudomonas aeruginosa.

ii. Mouse thymic virus (MTV) infection (Morse, 1994) Etiology. MTV is a herpesvirus that is antigenically distinct from MCMV. No suitable in vitro method for cultivation has been developed; therefore viral propagation depends on mouse inoculation.

Clinical signs.

Natural infections are asymptomatic.

Epizootiology.

The prevalence of MTV is thought to be low. Mice can be infected at any age, although lesions develop only in mice infected perinatally. Mice infected as infants or adults can develop persistent infection of the salivary glands lasting several months or more. Excretion of virus in saliva is considered the primary factor in transmission of infection, especially to infant mice. Seroconversion occurs in adults but does not eliminate infection. Infection in neonates may not elicit seroconversion, rendering such mice serologically negative carriers. The mode of infection is obscure, but virus is excreted in saliva, suggesting that transmission from infected dams to neonatal mice occurs by ingestion. MTV also has been isolated from the mammary tissue of a lactating mouse, suggesting the potential for transmission during nursing. Prenatal transmission has not been found.

Pathology.

MTV causes severe, diffuse necrosis of the thymus in mice exposed within approximately 1 week after birth, but the severity of thymic and lymph node necrosis can be mouse strain-dependent. Grossly, the thymus is smaller than normal. Infected thymocytes display MTV-positive intranuclear herpetic inclusions. Viral antigen can be demonstrated in the thymus by immunocytochemical staining. Necrosis is followed by granulomatous inflammation and syncytium formation. Necrosis and inflammation can also occur in lymph nodes. Reconstitution of lymphoid organs takes 3 - 8 weeks.

3. BIOLOGY AND DISEASES OF MICE

Diagnosis. Thymic necrosis associated with intranuclear herpetic inclusions is the hallmark lesion. Virus also can be detected by immunohistochemistry. Seroconversion can be detected by ELISA or by IFA. Suspicion of infection in seronegative mice can be tested by inoculation of virus-free neonatal mice with homogenates of salivary gland or with saliva. Inoculated mice should be examined for typical lesions 10-14 days later. PCR methodology or the mouse antibody production (MAP) test can also be used to detect infection. Differential diagnosis. Reduction of thymus mass can occur in severe mouse coronavirus infection, during epizootic diarrhea of infant mice, or following stress. Prevention and control. Because MTV induces persistent salivary infection, rederivation or restocking should be considered if infection cannot be tolerated as a research variable. Research complications. MTV transiently suppresses cellular and humoral immune responses because of its destructive effects on neonatal T lymphocytes. Parvovirus Infections (Jacoby and Ball-Goodrich, 1995; Jacoby et al., 1996) For many years, minute virus of mice (MVM) was recognized as the sole parvovirus of laboratory mice. Recent research has confirmed natural infection with a newly recognized serogroup. The prototype isolate was initially called mouse orphan parvovirus, but has been renamed mouse parvovirus (MPV).

i. Minute virus of mice (MVM) (murine minute virus) Etiology. MVM is a small (5-kilobase) single-stranded DNA virus. The prototypic strain is designated MVM(p), An allotropic variant with immunosuppressive properties in vitro also has been identified and is named MVM(i). The genome encodes two nonstructural proteins, NS-1 and NS-2, that are highly conserved among the rodent parvoviruses and account for prominent cross-reactivity in generic serological assays. The viral capsid proteins, VP-1 and VP-2, are virus-specific and form the basis for serological differentiation of MVM from mouse parvovirus (MPV). MVM has a broad in vitro host range. It replicates in monolayer cultures of mouse fibroblasts (A9 cells), C6 rat glial cells, SV40 (simian virus 40)-transformed human newborn kidney (324K cells), T cell lymphomas (EL4), and rat or mouse embryo cells, producing cytopathic effects that can include the development of intranuclear inclusions. Clinical signs. Natural infections are asymptomatic. Neonatal mice of some inbred strains are susceptible to lethal renal and/or intestinal hemorrhage during experimental MVM(i) infection, but this syndrome has not been reported in natural outbreaks.

61

Epizootiology. MVM is perceived as a common virus of mice. Its prevalence in mouse colonies surveyed during the 1970s and 1980s was reported to be approximately 30-90%. A recent survey of major biomedical research centers revealed an overall prevalence of parvovirus infection of about 25% among specific pathogen-free mice and 40% among conventionally housed mice (Jacoby and Lindsey, 1997). However, the earlier data did not account for MPV infections, which are now known to have been present during those decades, and the recent survey did not report results for MVM and MPV separately. Therefore, the true prevalence of MVM (as opposed to MPV) is unclear. The recent development of a strain-specific ELISA should resolve this issue. MVM is highly infectious for the mouse, its only known natural host, Virus can infect the gastrointestinal track and is excreted in feces and urine. The resistance of rodent parvoviruses to environmental inactivation increases the risks of transmission after virus is excreted. Therefore, contamination of caging, bedding, food, and .clothing must be considered a risk for the spread of infection. Transmission occurs by oronasal exposure, but viral contamination of biologicals used for experimental inoculation, such as transplantable tumors, also can be a source of infection. Continuous contact exposure to infected animals or soiled bedding usually induces a humoral immune response within 3 weeks, but limited exposure may delay seroconversion. Young mice in enzootically infected colonies are protected by maternal antibody, but actively acquired immunity develops from infection sustained after the decay of maternal immunity. MVM, in contrast to MPV, is not thought to cause persistent infection; infection in immunocompetent adult mice usually lasts less than 3 weeks (Smith, 1983b; Smith and Paturzo, 1988). Infection appears to last less than 1 month even in oronasally inoculated neonatal mice. Although MVM has not been studied in immunodeficient mice, one should assume that infection will be prolonged in such mice. There is no evidence that MVM is transmitted in utero. Pathology. Natural infections or experimental inoculation of adult mice appears to be nonpathogenic, although low-level cytolysis in situ cannot be excluded. Contact-exposed neonates have been reported to develop cerebellar lesions, but these are very rare. Experimental infection of neonatal BALB/c, SWR, SJL, CBA, and C3H mice with MVM(i) can cause renal hemorrhage and infarction (Brownstein et al., 1991). DBA/2 mice also developed intestinal hemorrhages and accelerated involution of hepatic hematopoiesis. C57BL/6 neonates are resistant to vascular disease. This lesion has been attributed to viral infection of endothelium. Diagnosis. ELISA serology is the primary method to detect infection. Additionally, MVM can be differentiated from MPV using virus-specific VP-2 antigens (L. J. Ball-Goodrich, unpublished results, 2000). MVM infection also can be detected by

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PCR, in situ hybridization, and immunohistochemistry. Although the most commonly used molecular assay is PCR amplification of a conserved portion of NS-1, it does not differentiate MVM from MPV. However, virus-specific PCR assays that amplify gene segments within the capsid protein genes also are available (Besselsen, 1998). MVM can be isolated from spleen, kidney, intestine, and other tissues by inoculation of the C6 rat glial cell line. It also can be detected by the mouse antibody production test. Prevention and control. Because MVM does not persist in immunocompetent mice, control and elimination should exploit quarantine combined with thorough disinfection of the environment, because parvoviruses are resistant to environmental inactivation. However, there are no published reports confirming the success of this strategy. Additionally, reliance on quarantine presumes that MPV infection, which can be persistent in adult immunocompetent mice, has been ruled out. If the identification of the virus remains problematic, a more stringent approach such as cesarean rederivation or embryo transfer may be preferable. Prevention of MVM infection depends on strict barrier husbandry and regular surveillance of mice and mouse products destined for use in vivo. Research complications. MVM contamination of transplantable neoplasms is quite common; therefore, infection caia be introduced to a colony through inoculation of contaminated cell lines. Failures to establish long-term cell cultures from infected mice or a low incidence of tumor "takes" should alert researchers to the possibility of MVM contamination. MVM(i) has the potential to inhibit the generation of cytotoxic T cells in mixed lymphocyte cultures. ii. Mouse parvovirus (MPV) Etiology. During the mid-1980s, serological testing revealed a murine parvovirus that was antigenically distinct from MVM (McKisic et al., 1993). The virus was isolated following its detection as a lymphocytotropic contaminant in in vitro assays for cellular immunity. The virus grew lytically in a CD8 § T cell clone designated L3 and inhibited the proliferation of cloned T cells stimulated with antigen or interleukin 2 (IL-2). Additional isolates confirmed that these viruses are antigenically distinct from MVM. Thus, they constitute a second and distinct serogroup of murine parvoviruses. Molecular analysis of MPV indicates that regions encoding the NS proteins are similar to those of MVM, a finding that accounts for cross-reactivity between the viruses in generic serological tests. However, they differ significantly in regions encoding the capsid proteins, accounting for their antigenic specificity (Ball-Goodrich and Johnson, 1994). The prototype isolate was first called an "orphan" parvovirus of mice because its biology and significance were obscure, but it has subsequently been named mouse parvovirus (MPV). MPV is very difficult to grow in vitro. Immor-

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

talized T cells (L3) are the only cells found thus far to support replication of MPV. Clinical signs. MPV infection is clinically silent in infant mice and adult immunocompetent or immunodeficient mice. Epizootiology. Serologic evidence strongly suggests that MPV causes natural infection only in mice. Infection has circulated in mouse colonies in the United States for at least 30 years. Retrospective testing indicated that the prevalence of MPV approached 40% in the early 1970s, whereas only 7% of tested sera contained MVM antibody. In situ hybridization has identified the small intestine as a site of viral entry and early replication, but respiratory infection cannot be excluded. Based on these findings and initial transmission studies, MPV is thought to be transmitted primarily by fecal excretion and ingestion of contaminated material. There is no evidence of prenatal transmission. Initial studies indicated that humoral (e.g., passively or maternally acquired) immunity can protect against MPV infection. However, immunity to MVM may not confer crossimmunity to MPV (Hansen et al., 1999). MPV causes persistent infection in infant and adult mice, a property that differentiates it from MVM. In situ hybridization studies detected viral DNA in the lymph nodes of experimentally infected adult mice for at least 9 weeks. Furthermore, infection has been transmitted by adults to naive cagemates intermittently for up to 6 weeks (Smith et al., 1993). Pathology. MPV appears to enter through the intestinal mucosa, which is a site of early virus replication (Fig. 18). Acute infection is widespread but mild, involving lung, kidney, liver, and lymphoid organs. Histological lesions are not discernible, despite the potential for cytolysis during parvovirus replication. Lymphocytotropism is a characteristic of acute and persistent MPV infection in infant and adult mice. During acute infection virus is dispersed within lymph nodes, but during persistent infection virus localizes in germinal centers (Fig. 19). Diagnosis. Because infected mice do not manifest signs or lesions and the virus is very difficult to propagate in cell culture, detection and diagnosis rely on serology and molecular methods. A recently developed MPV-specific ELISA that uses MPV VP-2 as antigen is a sensitive and specific assay that differentiates MPV from MVM (L. J. Ball-Goodrich, unpublished resuits, 2001). The MAP test also can be used to detect parvovirus infections but is relatively time-consuming and expensive. As noted for MVM, a generic PCR assay for murine parvoviruses, using conserved primer sequences that are conserved among murine parvoviruses, can be used as a screening test. PCR also can be used to detect MPV-specific sequences in the VP-2 gene. Although diagnostic PCR is sensitive and specific, it is effective only in actively infected animals and requires ac-

3. BIOLOGY AND DISEASES OF MICE

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Prevention and control. The persistence of MPV in individual mice, its potential for provoking immune dysfunction, and the resistance of murine parvoviruses to environmental inactivation favor active control and prevention of MPV infection. Quarantine of infected rooms is appropriate. Elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced. For mice that are not easily replaced, virus persistence in the absence of transplacental transmission favors cesarean rederivation or embryo transfer as relatively rapid options to eliminate infection. Control of infection also should include environmental decontamination. Chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent steps. Prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice. Research complications. Murine parvoviruses can distort biological responses that depend on cell proliferation. For MPV, such effects are seen on immune function and include augmentation or suppression of humoral and cellular immune responses.

Fig. 18. Mouseparvovirus (arrows) in the intestine after oronasal inoculation of an adult mouse. In situ hybridization.

cess to tissues that are usually obtained at necropsy. Therefore, its primary value is to confirm serological results.

Differential diagnosis. MPV infection must be differentiated from MVM infection. Because both viruses are enterotropic and lymphocytotrol~ic, serology and molecular hybridization must be used to distinguish between them.

Mouse Adenovirus Infection (Richter, 1986; Porterfield and Richter, 1994a) Etiology. Adenoviruses are nonenveloped DNA viruses that produce intranuclear inclusions in vitro and in vivo. Two adenovirus strains have been associated with mice: MAdV-1 (FL) and MAdV-2 (K87). Both strains replicate in mouse kidney tissue culture but are antigenically distinct. Clinical signs. MAdV- 1 can cause severe clinical disease after experimental inoculation of infant mice. Signs include scruffiness, lethargy, stunted growth, and often death within 10 days. MAdV-2 virus is enterotropic and appears to be responsible for virtually all naturally occurring infections. Infection is usually asymptomatic in immunocompetent mice, with the possible exception of transient runting among infant mice. Epizootiology. The prevalence of adenovirus infection in mouse colonies is not well documented but appears to be low. Transmission occurs by ingestion. Adult mice experimentally infected with MAdV-1 may remain persistently infected and excrete virus in the urine for prolonged periods. Adult mice experimentally infected with MAdV-2 excrete virus in feces for at least 3 weeks but eventually recover. Athymic mice can shed MAdV-2 for at least 6 weeks and episodically for at least 6 months.

Fig. 19. Mouseparvovirus in the mesenteric lymph node of a persistently infected mouse.In situ hybridization.

Pathology. Infection with MAdV-1 causes multisystemic disease characterized by necrosis. Infant mice are especially susceptible to rapidly fatal infection characterized by necrosis of

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brown fat, myocardium, adrenal cortex, salivary gland, and kidney, including the development of intranuclear inclusions. More mature mice usually develop subclinical infection leading to seroconversion; however, athymic mice can develop intestinal hemorrhage amd wasting. Infection with MAdV-2 produces amphophilic, intranuclear inclusions in intestinal epithelium, especially in the distal small intestine and cecum (Fig. 20). Inclusions are easier to detect in infant mice than in adults.

Diagnosis. Although MAdV strains can be isolated in tissue culture, routine diagnosis depends on detection of infection by serological assay and/or demonstration of adenoviral inclusions, most commonly in the intestinal mucosa. An immunofluoresence assay and an ELISA are available for serological surveillance (Smith et al., 1986; Lussier et al., 1987b). Crossneutralization tests have revealed that antiserum to MAdV-2 neutralizes both strains, but antiserum to MAdV-1 neutralizes MAdV-2 weakly at best. Therefore, MAdV-2 antigen should be

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

used for the serological detection of adenovirus infection irrespective of the assay employed. MAdV also can be detected by PCR (S. R. Compton, personal communication, 2001).

Differential diagnosis. The intranuclear adenoviral inclusions in intestinal epithelium are pathognomonic and differentiate MAdV-2 infection from other known viral infections of mice. Prevention and control. Prevention requires serological monitoring of mice and exmination for passenger viruses of animal products such as transplantable tumors. Because MAdV-2 infection appears to be transient in individual mice, segregation of infected colonies may be effective for control. However, rederivation coupled with subsequent barrier housing is a more conservative approach. Research complications. MAdV infection is unlikely to affect research using immunocompetent mice. However, it has the potential for pathogenicity in immunodeficient mice. e.

Papovavirus Infections (Shah and Christian, 1986)

Mice can incur natural infection with two papovaviruses: polyomavirus and K virus.

i. Polyomavirus (Orcutt, 1994) Etiology. Polyomavirus is a small DNA virus. It is highly antigenic in adult mice but induces multiple types of tumors in mice infected as neonates. Its primary importance stems from use in murine models of experimental oncogenesis, with natural infection being rare. Clinical signs. Natural infections in immunocompetent mice are usually asymptomatic. However, tumor induction, neurological disease, and wasting can occur in immunodeficient mice (McCance et al., 1983; Sebesteny et al., 1980).

Fig. 20. Intranuclearadenoviralinclusions in mouseintestine. (Courtesyof Dr. S. W. Barthold.)

Epizootiology. Modern husbandry and health care have essentially eliminated natural exposure in laboratory mice. Because infection can be introduced inadvertently and is highly contagious, some additional features are highlighted here. Inoculation of neonatal mice with contaminated biologicals or cell cultures is a potential source of entry and spread. Once infection is established, virus is shed in urine, feces, and saliva, followed by intranasal exposure of other mice. Thus airborne dissemination results primarily from contaminated feed and bedding. Intrauterine infection also can occur, and persistent renal infection, contracted neonatally, can be reactivated during pregnancy. Exposure of neonatal mice also can result in viremic dissemination and high mortality. Additionally, survivors can be persistently infected and excrete virus from lung and kidney for months. Infection of breeding-age mice usually results in rapid

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3. BIOLOGY AND DISEASES OF MICE

clearance of virus and protection of offspring by maternally derived antibodies. However, PCR has revealed infection lasting up to 5 months in CBA mice inoculated with virus as adults (Berke and Dalianis, 1993). Polyomavirus infection can persist in adult immunodeficient mice.

Pathology.

Polyomavirus-induced tumors are primarily a laboratory phenomenon. Experimental inoculation of neonatal mice can produce viremia and acute, lethal disease. Tumors appear 2-12 months after inoculation of surviving mice, and in most strains the salivary glands are prevalent sites for tumor development. However, tumors can occur at other sites, especially in skin adnexa, the upper gastrointestinal tract, and the kidneys. The location of tumors varies with virus strain and, to some extent, with the route of inoculation. Athymic mice can develop cytolytic and inflammatory lesions, followed by multisystemic tumor formation. Intranuclear inclusions may be present in cytolytic lesions. Demyelinating disease has been reported in experimentally inoculated athymic mice (see references under "Clinical Signs," above), and myeloproliferative disease has been reported in experimentally inoculated SCID mice (Szomolanyi-Tsuda et al., 1994).

Diagnosis.

Virus can be isolated in mouse fibroblast cell lines, but infection is ordinarily detected serologically by ELISA (Broeders et al., 1994). Additionally, PCR and immunohistochemistry can be used (S. R. Compton, personal communication, 2001).

viremic spread. Vascular endothelium is the primary target in affected tissues, which often include lung, liver, spleen, and adrenal glands. Dyspnea occurs from pulmonary infection because of edema and hemorrhage. Infection of immunocompetent adult mice is asymptomatic and results in a vigorous immune response. However, both adults and infant mice develop persistent infection. Additionally, infection of athymic mice can lead to clinical signs and lesions akin to those described for neonatally inoculated mice. Gross lesions are limited to pulmonary hemorrhage and edema. Histologically, intranuclear inclusions, which are visualized more easily using immunohistochemistry, are present in vascular endothelium of infected tissues. Mild hepatitis with hepatocytic degeneration also may develop. Infection can be detected by ELISA serology or PCR. Prevention and control measures are similar to those described for polyomavirus, except that precautions against airborne transmission are not required.

Lactate Dehydrogenase-Elevating Virus (LD V) Infection (Brinton, 1982, 1986) Etiology.

LDV is a togavirus specific to mice that increases the concentration of several serum enzymes, most notably lactate dehydrogenase (LDH).

Clinical signs. Infection is typically asymptomatic. However, poliomyelitis has occurred in immunosuppressed C58 and AKR mice inoculated with LDV.

Differential diagnosis.

Wasting in athymic mice can be caused by other infectious agents, including coronaviruses, Sendai virus, and Pneumocystis carinii. Intranuclear inclusions can occur in infections caused by mouse adenovirus, mouse cytomegalovirus, and K virus.

Prevention and control.

Control depends on elimination of infected mice and material, together with prevention of airborne spread. Tumor and cell lines destined for mouse inoculation should be tested for polyomavirus by the mouse antibody production test or molecular diagnostics.

Epizootiology.

The prevalence of LDV infection is thought to be low. The primary mode of mouse-to-mouse transmission is mechanical transfer from aggressive behavior (e.g., bite wounds). Inoculation of mice with contaminated animal products such as cell lines, transplantable tumors, or serum is probably the most common source of induced infection. It is important to note, with respect to mechanical transmission, that infection induces lifelong viremia. Natural transmission between cagemates or between mother and young is rare even though infected mice may excrete virus in feces, urine, milk, and probably saliva.

Research complications.

Polyomavirus infection can affect experiments by inadvertent contamination of cell lines or transplantable tumors, leading to infection of inoculated mice and the potential for epizootic spread.

ii. K virus infection (Parker and Richter, 1982; Porterfield and Richter, 1994b) K virus is a papovavirus of mice that has historical importance but is of little consequence to contemporary mouse colonies. Oral inoculation of neonatal mice results in initial infection of capillary endothelium in the intestine, followed by

Pathology.

Viremia peaks within 1 day after inoculation, then persists at a diminished level. The elevation of enzyme levels in blood is thought to result primarily from viral interference with clearance functions of the reticuloendothelial system. No lesions are seen in naturally infected mice. The only significant lesion thus far associated with experimental infection is polioencephalitis in immunosuppressed C58 and AKR mice. Mild leptomeningitis and myelitis have been reported in C57BL/6 mice. T cell-dependent areas of thymus and peripheral lymphoid tissue may undergo mild necrosis early in experimental

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ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

infection. Immune complex glomerular disease is not a significant complication of LDV infection, despite the propensity of the virus to form immune complexes.

Diagnosis.

Plasma LDH levels are elevated, a response that is used to detect and titrate LDV infectivity. Of the five isoenzymes of LDH-V in mouse plasma, only LDV is elevated. SJL/J mice in particular show spectacular increases in LDH levels (15-20 times normal), a response controlled by a recessive somatic gene. LDV is detected by measuring LDH levels in mouse plasma before and 4 days after inoculation of specific pathogen-free (SPF) mice with suspect material. It is important to use nonhemolysed samples because hemolysis will produce falsely elevated readings. Plasma enzyme levels are measured in conventional units/ml, 1 conventional unit being equivalent to 0.5 International Units (IU). Normal plasma levels are 4 0 0 800 IU, whereas in LDV infection, levels as high as 7000 IU can occur. LDV also interferes with the clearance of other serum enzymes and results in their elevation in serum. Infection provokes a modest humoral antibody response, but it is difficult to detect because of formation of virus-antibody immune complexes. Molecular diagnostics also can be used to diagnose infection in mouse tissues and serum and in cell cultures. However, inhibitory factors in cells and serum may cause false negative results in PCR testing, so appropriate quality control measures are essential if this method is used (Lipman and Henderson, 2000).

Prevention and control.

Transplantable tumors have been a common source of LDV historically. Therefore, tumors or cell lines destined for mouse inoculation should be monitored for LDV contamination. Although LDV can infect tumor cells, it does not replicate in them. Therefore, one can attempt to free tumors of virus by passaging them several times in rodents nonpermissive to LDV (e.g., rats) before repassaging them in mice.

Research complications.

LDV has numerous potential effects on immunological function. It may reduce autoantibody production, cause transient thymic necrosis and lymphopenia, suppress cell-mediated immune responses, and enhance or suppress tumor growth.

g.

Lymphocytic Choriomeningitis Virus (LCMV) Infection (Lehmann-Grube, 1982; Lindsey et al., 1991b)

Etiology.

LCMV is a single-stranded, enveloped RNA virus that buds from the cell membrane without cytolysis. Its name is derived from the immune-mediated inflammation resulting from the intracerebral inoculation of virus into immunologically competent mice. Virus strains are closely related antigenically but vary in their rate of replication, tissue tropism, pathogenicity, and immunogenicity. Furthermore, these properties

can be modulated by passage in vivo or in vitro. Neurotropic and viscerotropic strains have been used extensively to develop and study mouse models of virus-induced immune injury and to reveal fundamental mechanisms of immune recognition (Doherty, 1997). LCMV can infect insect cells as well as mammalian cells and can persistently infect naturally exposed mice and cultured cells.

Clinical signs. Clinical signs of LCMV infection vary with age and strain of mouse, route of inoculation, and strain of virus. Natural infection in immunocompetent adult mice is usually self-limiting and asymptomatic. However, four basic patterns of clinical disease are recognized from study of experimentally induced infection (Fig. 21). (1) The cerebral form is characterized by illness in immunocompetent mice beginning 5 - 6 days after intracerebral inoculation of virus. Sudden death may result or subacute illness associated with one or more of the following signs may develop: ruffled fur, hunched posture, motionlessness, and neurological deficits. Mice suspended by the tail display coarse tremors of the head and extremities, culminating in clonic convulsions and tonic extension of the hindlegs. Spontaneous convulsions also can occur. Animals usually die or recover in several days. (2) A visceral form can occur in adult mice inoculated by peripheral routes with viscerotropic strains. It can be asymptomatic or lead to clinical signs, including ruffled fur, conjunctivitis, ascites, somnolescence, and death. If mice survive, recovery may take several weeks. (3) Runting and death from LCMV infection may occur in neonatally infected suckling mice and can lead to transient illness or to death. Clinical signs are nonspecific, recovery is slow, and survivors may remain runted. This early form of disease is attributed to endocrine dysfunction caused by LCMV infection. (4) Late-onset disease can occur in previously asymptomatic carrier mice that develop immune complex glomerulonephritis. It is usually the result of prenatal or neonatal infection and occurs in persistently infected mice when they are 9-12 months old. Clinical signs are nonspecific and include ruffled fur, hunched posture, weight loss, proteinuria, and ascites. Epizootiology.

LCMV is distributed widely in wild mice in North and South America and in Europe and has recently been found in Australia (Smith et al., 1993). Among common laboratory species, mice, hamsters, guinea pigs, and nonhuman primates are susceptible to infection, but only the mouse and the hamster are known to transmit virus. LCMV infection is not prevalent in laboratory mice produced and maintained in modern quarters. Infection is usually introduced through inoculation of virus-infected biologicals, such as transplantable tumors, or by feral mice. Wild mice are a natural reservoir of infection and a potential threat to research colonies if they gain entry inadvertently. Carrier mice usually develop as a result of asymptomatic prenatal or neonatal infection, which induces tolerance to the virus. Such mice can have persistently high con-

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3. BIOLOGY AND DISEASES OF MICE

Fig. 21. Basicphenomena associatedwith LCMVinfection in mice. (FromLehman-Grube, 1982.)

centrations of virus in many organs, thereby facilitating virus excretion in saliva, nasal secretions, and urine. Persistently infected neonates usually reach breeding age and can perpetuate infection in a breeding colony. Thus introduction of a single LCMV carrier mouse to a breeding colony can eventually result in a high prevalence of persistently infected mice. Infection in adult mice, by contrast, is often acute because of the onset of effective immunity, and the spread of virus is halted. Horizontal spread of infection is enhanced by close contact, but rapid horizontal spread is not characteristic. Mice can transmit LCMV to hamsters, which can remain viremic and "viruric for many months even if they contract infection as adults. Infected

hamsters can transmit virus to other hamsters and mice and are the primary source of human LCMV infection (see Chapter 5). Infected mice are not a significant source of human exposure. However, persistent infection in immunodeficient mice may carry greater risks for viral excretion and zoonotic transmission.

Pathology. LCMV disease is a prototype for virus-induced, T lymphocyte-mediated immune injury and for immune complex disease. However, lesions comparable to experimentally induced disease are rare during natural infection. Intracerebral inoculation of virus into immunocompetent adult mice can induce nonsuppurative leptomeningitis, choroiditis,

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and focal perivascular lymphocytic infiltrates. Host tissues are damaged during the course of the cellular immune response to the virus. The character of visceral lesions depends on virus strain and mouse strain; the ratio of cytolytic to proliferative responses in lymphoid organs is mouse strain-dependent. In severe infection, nonsuppurative inflammation can occur in many tissues. The severity of accompanying cytolytic lesions seems to parallel the intensity of cellular immunity. Liver lesions can include hepatocytic necrosis accompanied by nodular infiltrates of lymphoid cells and Kupffer's cells, activated sinusoidal endothelium, an occasional granulocyte or megakaryocyte, and fatty metamorphosis. Cytolysis, cell proliferation, and fibrinoid necrosis can develop in lymphoid organs. Necrosis of cortical thym0cytes can lead to thymic involution. Lesions of late-onset disease are characterized by formation of immune complexes and associated inflammation. Renal glomeruli and the choroid plexus are most severely affected, but complexes may also be trapped in synovial membranes, blood vessel walls, and skin. Lymphoid nodules can form in various organs. Lesions associated with early deaths in neonatally infected mice have not been thoroughly described but include hepatic necrosis. The lesions of acute and persistent LCMV infection reflect separate immunopathological processes. In adult mice with acute LCMV infection, virus multiplies in B cells and macrophages, whereas T cells are resistant. Internal viral epitopes induce humoral immune responses, but surface epitopes elicit cell-mediated immunity and neutralizing antibodies. Thus elimination of virus and virus-associated immunological injury are both T cell-mediated. This apparent paradox has been explained by the view that prompt cellular immunity limits viral replication and leads to host survival, whereas slower cellular immune responses permit viral spread and increase the number of virus-infected target cells subject to attack once immunity is fully developed. Antibody can be detected by 1 week after infection but does not play a significant role in eliciting acute disease. Lesions of LCMV infection appear to develop from direct T cell-mediated damage to virus-infected cells and may involve humoral factors released from immune effector T cells. LCMV also can suppress humoral and cellular immunity in acutely infected mice. Persistent infection commonly evolves from exposure early in pregnancy, and virus has been demonstrated in the ovaries of carrier mice. Prenatal or neonatal infection induces immunological tolerance to LCMV, which can then replicate to high titer in many tissues. Nevertheless, persistently infected mice develop humoral antibody to LCMV. Antibody can complex with persistent virus to elicit complement-dependent inflammation in small vessels. Immune complex glomerulonephritis exemplifies this process, as noted above. Diagnosis. LCMV infection can be diagnosed serologically by IFA or ELISA tests (Homberger et al., 1995). Whereas immunocompetent adult mice will normally seroconvert after ex-

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

posure, carrier mice may develop poor humoral immune responses. Therefore, testing must avoid false negative results. Employment of adult contact sentinel mice is a useful strategy for detecting LCMV infection by seroconversion. Alternatively, small blood samples can be collected from persistently infected live suspects, which are often viremic, and used to inoculate cultured cells or adult and neonatal mice. Intracerebral inoculation of LCMV-positive tissues should elicit neurological signs in adult mice within 10 days, whereas infant mice should remain asmptomatic. Histological examination of brains from affected adults may reveal nonsuppurative inflammation, but lesions may be minimal in mice infected with viscerotropic isolates. Immunohistochemistry can be used to detect viral antigen in brains of suckling and adult mice. Intraperitoneal inoculation of adult mice may yield short-lived infection with seroconversion, i.e., the mouse antibody protection test. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay also is available (Park et al., 1997). Virus can be grown and quantified in several continuous cell lines, including mouse neuroblastoma (N-18) cells, BHK-21 cells, and L cells. Application of immunofluorescence staining to detect LCMV antigen in inoculated cultured cells yields results more quickly than animal inoculation. Of course, all diagnostic procedures involving potential contact with live virus should be carried out under strict containment conditions to avoid infection of laboratory personnel. The use of in vitro detection has the added advantage, in this regard, of reducing biohazardous exposure and the use of live animals for testing. Differential diagnosis. Neurological signs must be differentiated from those due to mouse hepatitis virus, mouse encephalomyelitis virus, and meningoencephalitis from metastatic bacterial infection. Trauma, neoplasia, and toxicities also must be ruled out in neurological disease with low prevalence. Lateonset disease is associated with characteristic renal lesions, including deposition of viral antigen in tissues. Early-onset disease must be differentiated from other causes of early mortality, such as mouse hepatitis virus, ectromelia virus, reovirus 3 infection, Tyzzer's disease, or husbandry-related insults. Prevention and control. Adequate safeguards for procurement and testing of animals and animal products are essential to prevent entry. Because mouse-to-mouse spread is slow, selective testing and culling for seropositive or carrier mice is possible. If mice are easily replaced, however, depopulation is a safer and more reliable option. Valuable stock can be rederived, but progeny must be tested to preclude in utero transmission. Because infected hamsters can excrete large quantities of virus, exposed hamsters should be destroyed and hamsters should not be housed with mice. LCMV can be transmitted to human beings, who can contract severe CNS disease. More frequently, human infection resembles influenza or is asymptomatic. The zoonotic potential of LCMV infection makes it especially im-

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portant to detect and eliminate carrier animals and other potentially contaminated sources, such as cell cultures, transplantable neoplasms, and vaccines to prevent human exposure. Serum banking and periodic serological testing of high-risk human populations, such as those working with LCMV experimentally, is recommended.

Research complications.

LCMV may stimulate or suppress immunological responses in vivo and in vitro, and it can replicate in cells used as targets or effectors for immunological studies. Introduction of immune cells to a carrier animal may elicit an immunopathological response. Immune complex disease can complicate long-term experiments and morphological interpretations. Illness and death in mice and zoonotic risks to humans are obvious research-related hazards.

Sendai Virus Infection (Parker and Richter, 1982; Brownstein, 1986) Etiology.

Sendai virus (SV) is a paramyxovirus that is antigenically related to human parainfluenza virus 1. Viral particles are pleomorphic, contain single-stranded RNA, and have a lipid solvent-sensitive envelope that contains glycoproteins with hemagglutinating, neuraminidase, and cell fusion properties. SV grows well on embryonated hens' eggs and in several mammalian cell lines (e.g., monkey kidney, baby hamster kidney [BHK-21], and mouse fibroblast [L]). Virus replicates in the cytoplasm and by budding through cell outer membranes.

greatly in their susceptibility to lethal SV infection. For example, C57BL/6 mice are highly resistant to clinically apparent infection, whereas DBA/2 mice are highly susceptible. Aerogenic infection is promoted by high relative humidity and by low air turnover. Prenatal infection does not occur. Enzootic infection is commonly detected in postweaned mice (5-7 weeks old) and is associated with seroconversion within 7-14 days and the termination of infection. Therefore, entrenched infection is perpetuated by the introduction of susceptible animals. There is no evidence for persistent infection in imunocompetent mice, but prolonged infection is common in immunodeficient mice. Maternally acquired immunity protects young mice from infection, and actively acquired immunity is thought to be long-lived. Rats, hamsters, and guinea pigs also are susceptible to SV infection. Therefore, bidirectional cross-infection is a risk during outbreaks.

Pathology.

Viral replication during natural infection is nominally restricted to the respiratory tract and peaks by the first week after infection. Gross lesions feature partial to complete consolidation of the lungs (Fig. 22). Individual lobes are meaty and plum-colored, and the cut surface may exude a frothy serosanguinous fluid. Pleural adhesions or lung abscesses caused by secondary bacterial infection are seen occasionally, and fluid may accumulate in the pleural and pericardial cavities. SV targets airway epithelium and type II pneumocytes. Type I pneuomocytes are less severely affected. Histologically,

Clinical signs.

Susceptible adult mice often assume a hunched position and have an erect hair coat. Rapid weight loss and dyspnea occur, and there may be chattering sounds and crusting of the eyes. Although highly susceptible adults may die, lethal infection is more common in suckling mice. Sex differences in susceptibility have not been found. Genetically resistant mice usually have asymptomatic infection, especially if they are otherwise in good health. Athymic mice and immunosuppressed mice are at high risk and can develop a wasting syndrome. However, they develop illness later than their immunocompetent counterparts. Opportunistic infections can complicate the clinical presentation. For example, secondary bacterial infections of the ear can cause vestibular signs.

Epizootiology.

The prevalence of SV infection, once among the highest for infectious agents of the mouse, has decreased sharply in recent years (Jacoby and Lindsey, 1997). This is probably attributable to several factors, including improvements in housing systems, health monitoring, and production standards for mice. SV is transmitted by aerosol and is highly infectious. Morbidity in infected colonies is commonly 100%, and mortality can vary from 0 to 100%, partly because strains of mice vary

Fig. 22. Pulmonaryconsolidation resulting from Sendai viral pneumonia.

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Fig. 23. Interstitialpneumoniacausedby Sendaivirus.

the pattern of pneumonia is influenced by mouse genotype. Susceptible mice usually have significant bronchopneumonia and interstitial penumonia, whereas the interstitial component may be less prominent in resistant mice (Fig. 23). Typical changes begin with inflammatory edema of bronchial lamina propria, which may extend to alveolar ducts, alveoli, and perivascular spaces. Necrosis and exfoliation of bronchial epithelium ensue, frequently in a segmental pattern (Fig. 24). Alveolar epithelium also may desquamate, especially in severe disease, and necrotic cell debris and inflammatory cells can accumulate in airways and alveolar spaces. Alveolar septae are usually infiltrated by leukocytes to produce interstitial pneumonia. Lymphoid cells also invade epibronchial and perivascular spaces. The lymphocytic response to SV infection reflects the fact that cellular immunity contributes both to lesions and to recovery. This apparent paradox may be attributable an immunopathological

Fig. 24. Bronchopneumoniacausedby Sendaivirus.

mechanism in the development of Sendai viral pneumonia. Local immunoglobulin synthesis by infiltrating cells also occurs. The extent of inflammatory cell infiltration corresponds to the level of genetic resistance expressed by the infected host, with susceptible hosts mounting a more florid response than resistant hosts. Additionally, strain-related differences in the severity of infection may reflect differences in airway mucociliary transport. Multinucleated syncytia are occasionally seen in affected sucklings, and inclusion bodies have been reported in infected athymic mice. Regeneration and repair begin shortly after the lytic phase and are characterized by hyperplasia and squamous metaplasia of bronchial epithelium, which may extend into alveolar septae. Proliferation of cuboidal epithelium may give terminal bronchioles an adenomatoid appearance. Repair of damaged lungs is relatively complete in surviving mice, but lymphocytic infiltrates, foci of atypical epithelium, and mild scarring can persist. Acute phase lesions are prolonged in immunodeficient mice, which can lead to wasting and death. Aged mice also have a prolonged recovery phase accompanied by focal pulmonary fibrosis (Jacoby et al., 1994). Diagnosis. Clinical signs of respiratory distress are highly suggestive of SV infection, especially among infant mice or adults of genetically susceptible strains. However, ELISA or IFA serology is an effective means to detect infection in all strains of immunocompetent mice (Wan et al., 1995). Antibody can be detected by 7 days postinfection. Repeated serologic sampling over several weeks can help stage infection. An increase in titer or prevalence indicates active infection. Alternatively, sentinel animals can be added to seropositive colonies to detect active infection. Irrespective of serologic results, histopathology, immunohistochemistry (which can be performed on formalin-fixed, paraffin-embedded sections) and, where possible, virus isolation should be used to confirm infection. Virus can be isolated from the respiratory tract for up to 2 weeks, with peak titers occurring at about 9 days postinfection. Nasopharyngeal washings or lung tissue homogenates are most reliable and should be inoculated into embryonated hens' eggs or BHK-21 cell monolayer cultures. SV infection of cultured cells is noncytolytic, so erythrocyte agglutination or antigen detection methods must be used. RT-PCR also can be used to detect virus in infected lungs (Wan et al., 1995). Differential diagnosis. Respiratory infection caused by pneumonia virus of mice (PVM) is generally milder or asymptomatic. Histologically, necrosis of airway epithelium is less severe. Bacterial pneumonias of mice, including murine respiratory mycoplasmosis, are sporadic and can be differentiated morphologically and by isolation of causative organisms. Because SV pneumonia may predispose the lung to opportunistic bacterial infections, the presence of bacteria should not deter evaluation for a primary viral insult.

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Control and prevention. Sendai virus infection is self-limiting in surviving immunocompetent mice. Suckling mice from immune dams are protected from infection by maternal antibody until after weaning. Control and eradication measures must eliminate exposure of susceptible animals, so that infection can "burn out." This is most easily accomplished by a quarantine period of 4 - 6 weeks wherein no new animals are introduced either as adults or through breeding. Control also is aided by the fact that Sendai virus is highly labile. Barrier housing is preferred for prevention and for control of transmission. Vaccination with Formalin-killed virus can provide short-term protection of valuable mice but is not commonly used for prevention. Research complications. Sendai virus can cause immunosuppression and can inhibit growth of transplantable tumors. This effect has been attributed to virus-induced modification of tumor cell surface membranes. Pulmonary changes during SV pneumonia can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other bacteria. They also have been associated with breeding difficulties in mice. This sign is thought to be an indirect effect due to stress, fever, or related changes during acute infection.

Pneumonia Virus of Mice (PVM) Infection (Parker and Richter, 1982) Etiology. Pneumonia virus of mice (PVM) is an enveloped RNA virus in the genus Pneumovirus of the Paramyxoviridae. All isolates appear to have similar physicochemical, biological, and antigenic properties. The virus agglutinates erythrocytes of several rodent species, including mice. It replicates well in vitro in BHK-21 cells but, as with SV, is noncytolytic in cultured cells.

Pathology. PVM replicates exclusively in the respiratory tract and reaches peak titers in the lung 6 - 8 days after infection. Although pulmonary consolidation can occur in experimentally infected mice, gross lesions are rare during natural infection. Histological lesions can occur in the upper and lower respiratory tract. They consist of mild necrotizing rhinitis, necrotizing bronchiolitis, and interstitial pneumonia, which usually occurs within 2 weeks after exposure to virus and is largely resolved by 3 weeks. The predominant inflammatory infiltrate is compromised of mononuclear cells, but some neutrophils are usually present. Immunohistochemistry on paraffin-embedded tissues can be used to detect viral antigen in bronchial epithelium, alveolar macrophages, and possibly alveolar epithelium during acute infection. Residual lesions include nonsuppurative perivasculitis, which can persist for several weeks after acute infection has ceased. Severe pneumonia can occur in immunodeficient mice, as noted above. It is characterized by generalized pulmonary consolidation that reflects severe interstitial pneumonia with inflammatory exudates and desquamated alveolar pneumocytes filling alveolar spaces (Fig. 25). Diagnosis. Diagnosis is based primarily on serological detection that can be supplemented by histopathology, immunohistochemistry, in situ hybridization, and virus isolation. Reliable IFA and ELISA assays are available (London et al., 1983). Virus replication in B HK-21 cells is detected by immunofluorescence or other antigen detection methods. Virus also can be detected in tissues by RT-PCR (S. R. Compton, personal communication, 2001). Differential diagnosis. Because PVM is antigenically distinct from other murine viruses, serology is the most useful method to separate PVM infection from other respiratory infections of mice. However, in immunodeficient mice, where clinical signs

Clinical signs. Natural PVM infection in mice is asymptomatic. Therefore its name is clinically misleading, being derived from pneumonic illness that occurred after serial passage of the agent in mice. However, dyspnea, listlessness, and wasting may develop in immunodeficient mice infected with PVM (Weir et al., 1988). Epizootiology. PVM causes natural infections of mice, rats, hamsters, and probably other rodents and may be infectious for rabbits. Serological data indicate that PVM is prevalent in mice (Jacoby and Lindsey, 1997) and has a worldwide distribution. However, it appears to spread less rapidly than Sendai virus. Intimate contact between mice is probably required for effective transmission. This characteristic may reflect the fact that environmental inactivation of virus occurs rapidly. Infection appears to be acute and self-limiting in immunocompetent mice but may persist in immunodeficient mice.

Fig. 25. Interstitialpneumoniacaused by pneumoniavirus of mice.

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and lesions are typical, it must be differentiated from other pneumonias, especially those due to SV and Pneumocystis carinii. Additionally, PVM can coexist with and exacerbate P. carinii infection in immunodeficient mice (Bray et al., 1993). Therefore, the careful application of immunohistochemistry and argyrophilic stains is recommended for definitive diagnosis of exudative interstitial pneumonia in mice.

Prevention and control. PVM infection is acute and selflimiting in immunocompetent mice. Seropositive mice should be viewed as either immune or in the final stages of acute infection. Therefore control and prevention follows guidelines applicable to SV infection. Research complications. sis, as noted above. j.

PVM can exacerbate pneumocysto-

Reovirus Infections

Two members of the family Reoviridae infect laboratory mice: reovirus per se and murine rotavirus, also known as epizootic diarrhea of infant mice (EDIM) virus.

i. Reovirus 1, 2, or 3 (Tyler and Fields, 1986) Etiology. Reoviruses of mammals have been divided into three serotypes: reovirus 1, 2, and 3. A number of wild-type and laboratory strains have been characterized, and related viruses have been recovered from mammals, marsupials, birds, insects, and reptiles. The virion contains segmented, double-stranded RNA and is relatively heat stable. Natural infections in mice are usually not caused by pure serotypes, because reoviruses actively recombine. Reoviruses replicate well in BHK-21 cells and other continuous cell lines, as well as in primary monolayer cultures from several mammals. Clinical signs. Clinical disease is rare and age dependent. Acute disease affects sucklings at about 2 weeks of age, whereas adults usually have asymptomatic infection. Signs in sucklings include emaciation, abdominal distension, and oily, matted hair due to steatorrhea. Icterus may develop and is most easily discerned as discoloration in the feet, tail, and nose. Incoordination tremors and paralysis occur just before death. Convalescent mice are often partially alopecic and are typically runted. Alopecia, runting, and icterus may persist for several weeks, even though infectious virus can no longer be recovered. Infants born to immune dams are protected from disease by maternal immunity. Epizootiology. The prevalence of reovirus 3 infection in colonies in the United States ranges from about 5 to 20% (Jacoby and Lindsey, 1997). Reoviruses are highly infectious

among infant mice and can be transmitted by the oral-fecal or aerosol routes, but mechanical transmission by arthropods has also been documented. Additionally, virus may be carried by transplantable neoplasms and transmitted inadvertently by injection. Transmission is inefficient among adult mice. There is no evidence that vertical transmission is important or that genetic resistance or gender influence expression of disease. Infection in immunocompetent mice appears to be self-limiting, lasting up to several weeks but terminating with the development of host immunity. Nevertheless, the presence of a carrier state has not been excluded. The course of infection in immunodeficient mice should be considered prolonged, but the duration has not been determined.

Pathology. Reovirus 3 can cause severe pantropic infection in infant mice. After parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. Following ingestion, reoviruses gain entry by infecting intestinal epithelial cells (M cells) that cover Peyer's patches. Virus can be carried to the liver in leukocytes, where it is taken up by Kupffer's cells prior to infecting hepatocytes. In acute disease, livers may be large and dark, with yellow foci of necrosis. The intestine may be red and distended, and, in infants, intestinal contents may be bright yellow. Myocardial necrosis can evoke pale epicardial foci, and pulmonary hemorrhages have been reported. Myocardial edema and necrosis are especially prominent in papillary muscles of the left ventricle. The brain may be swollen and congested. Central nervous system lesions are most prevalent in the brain stem and cerebral hemispheres. Neuronal degeneration and necrosis are followed quickly by meningoencephalitis and satellitosis. Severe encephalitis may evoke focal hemorrhage. In the chronic phase, wasting, alopecia, icterus, and hepatosplenomegaly may persist. Orally infected suckling mice can develop multifocal hepatocytic necrosis, which may include the accumulation of dense eosinophilic structures resembling Councilman bodies. Hepatocytomegaly, Kupffer's cell hyperplasia, and intrasinusoidal infiltrates of mononuclear cells and neutrophilic leukocytes also can develop. In experimentally inoculated mice, necrotic foci can persist in the liver for at least 4 weeks. Chronic active hepatitis may develop after acute infection and result in biliary obstruction. Acinar cells of the pancreas and salivary glands can undergo degeneration and necrosis. Because pancreatic duct epithelium is susceptible to infection, parenchymal lesions in pancreas may be caused by obstruction rather than by viral invasion of parenchyma. Pulmonary hemorrhage and degeneration of skeletal muscles also have been observed. Both humoral and cellular immunity seem to participate in host defenses, but it is unclear how host immunity may influence the course of chronic infection. Infection with reovirus 1 results in a similar distribution of significantly milder lesions. However, reovirus 2 is highly en-

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terotropic, inducing mild enteritis without lesions in other tissues (Barthold et al., 1993a).

Diagnosis.

ELISA and IFA tests have been developed for the serological detection of reovirus 3 infection (London et al., 1983), and viral RNA can be detected by RT-PCR (Steele et al., 1995). A presumptive diagnosis of reovirus 3 infection is aided clinically by detection of the oily hair effect, accompanied by jaundice and wasting. The presence histologically of multisystemic necrosis is consistent with severe reovirus 3 infection but should be confirmed by immunohistochemistry or virus isolation.

Differential diagnosis.

Reovirus 3 infection must be differentiated from other diarrheal diseases of infant mice, including those caused by mouse coronaviruses, EDIM virus, Salmonella spp., or Clostridium piliforme.

Prevention and control.

Although surviving mice appear to recover completely from infection, the potential for a carrier state is unresolved. Therefore, it may be necessary, after adequate testing for the continued presence of virus by the use of sentinels, MAP testing, or other appropriate means, to rederive or replace infected stock. Prevention depends on adequate barrier husbandry coupled with adequate serological monitoring.

Research complications.

Reovirus 3 infection can interfere with research in several ways. Infections in breeding colonies can result in high mortality among sucklings from nonimmune dams. Virus has been commonly recovered from transmissible neoplasms and is suspected of being oncolytic. The potential exists for interference with hepatic, pancreatic, cardiovascular, or neurological research.

ii. Rotavirus (EDIM virus) (Sheridan and Vonderfecht, 1986) Etiology. Rotaviruses are double-stranded, segmented RNA viruses that have a wheel-like ultrastructural appearance. EDIM virus is a group A rotavirus that replicates in differentiated epithelial cells of the small intestine by budding into cisternae of endoplasmic reticulum. Currently, only a single antigenic strain is recognized, but antigenically distinct variants may exist. EDIM virus shares an inner capsid antigen with rotaviruses of rabbits, fowl, nonhuman primates, human beings, and domestic and companion animals. These agents tend to be speciesspecific under natural conditions and can be differentiated by serum neutralization tests. Cultivation of EDIM virus requires the presence of proteolytic enzymes to cleave an outer capsid polypeptide.

Clinical signs.

Clinical signs occur in infant mice less than 2 weeks old. This age-related susceptibility also applies to infection in immunodeficient mice. Furthermore, clinical signs

occur only in offspring of nonimmune dams, because maternal immunity protects infants until they have outgrown susceptibility to clinical disease (Rose et al., 1998). The cardinal signs are diarrhea with fecal soiling of the perineum, which may extend to the entire pelage in severe cases. Despite high morbidity, mortality is low because affected mice continue to nurse. Transient weight loss does occur, and there may be a delay in reaching adult weight. Recovery from infection usually occurs in about 2 weeks and, once weight is regained, is clinically complete.

Epizootiology.

EDIM virus appears to be infectious only for mice and occurs episodically in mouse colonies. However, infection is probably widespread geographically. Its prevalence in mouse colonies in the United States ranges between 5 and 25%, according to a recent survey (Jacoby and Lindsey, 1997). All ages and both sexes can be infected, but genetic resistance and susceptibility have not been determined. The virus is highly infectious and is transmitted by the oral-fecal route. Asymptomatically infected adult mice can shed virus in feces for at least 17 days, an interval that may be extended in immunodeficient mice (Riepenhoff-Talty et al., 1995). After oral inoculation, virus is essentially restricted to the gastrointestinal tract, particularly the small and large intestine, although small amounts of virus may be present in liver, spleen, kidney, and blood. Nursing dams can contract infection from their litters. Transplacental transmission has not been demonstrated.

Pathology.

Gross lesions occur primarily in the gastrointestinal tract, but thymic atrophy can result from infection-related stress. The intestine is often distended, flaccid, and filled with gray-green gaseous liquid or mucoid fecal material that soils the pelage. The stomach contains curdled milk, except in terminal cases with anal impaction. Virus preferentially infects terminally differentiated enterocytes in the small and large intestine, which accounts for the age-related susceptibility to disease; the number of such cells decreases as the intestinal tract matures. Characteristic histological lesions are most easily discerned in the small intestine in mice less than 2 weeks old (Little and Shadduck, 1987). They consist of increased vacuolation of villar epithelial cells with cytoplasmic swelling, which give villi a clubbed appearance (Fig. 26). The vacuoles must be differentiated from normal absorption vacuoles in nursing mice. The lamina propria may be edematous, but necrosis and inflammation are not prevalent.

Diagnosis.

EDIM virus infection is detected serologically by IFA or ELISA (Ferner et al., 1987). Clinical disease is diagnosed from signs and typical histological lesions in the intestine, which can be confirmed by immunohistochemical or ultrastructural demonstration of virus in intestine or in intestinal filtrates or smears. Rotavirus antigen can be detected in feces by

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adults while preventing access to susceptible neonates also is recommended. Alternatively, litters with diarrhea can be culled, in combination with the use of microbarrier cages. The duration of infection in immunodeficient mice has not been determined, but it is reasonable to assume that chronic infection occurs. Therefore, such animals should be eliminated. Litters from immune dams are more resistant to infection. Prevention of EDIM virus infection depends on maintenance of sanitary barrier housing with adequate serological surveillance.

Research complications.

The research complications of EDIM infection pertain to clinical illness with diarrhea and retarded growth. Transient thymic necrosis may perturb immunological responses.

Mouse Coronavirus (Mouse Hepatitis Virus [MHV]) Infection (Compton et al., 1993) Etiology.

Fig. 26. Mouserotavirus (EDIM) infection. Clubbingof intestinal villi accompanied by cytoplasmicswellingand vacuolization. ELISA, but certain dietary ingredients can cause false-positive reactions. Infection can also be diagnosed by RT-PCR (Wilde et al., 1990).

Differential diagnosis.

EDIM virus infection must be differentiated from other diarrheal diseases of suckling mice such as intestinal coronavirus (mouse hepatitis) infection, reovirus 3 infection, Tyzzer's disease, and salmonellosis. The presence of milk in the stomach can be helpful in differentiating EDIM virus infection from more severe enteric infections, such as those caused by pathogenic coronaviruses, during which cessation of nursing often occurs. The possibility of dual infections must also be considered. Thymic necrosis in EDIM virusinfected mice, although nonspecific, must be differentiated from that due to mouse thymic virus (MTV) infection.

Prevention and control.

The spread of EDIM can be controlled effectively by the use of microbarrier cages and good sanitation. Because infection appears to be acute and self-limiting, cessation of breeding for 4 - 6 weeks to allow immunity to build in

Mouse coronaviruses are large, pleomorphic, enveloped RNA viruses with radially arranged peplomers (spikes). Early clinical and laboratory investigations emphasized their potential to induce hepatitis, so their original designation, which is still used actively, is mouse hepatitis virus (MHV). Experience has shown, however, that hepatitis is not a common feature of natural infection in immunocompetent mice. Five prototype strains have received much attention from research scientists. They are: JHM (MHV4), MHV-1, MHV-3, MHV-S, and MHV-A59. However, numerous additional strains have since been identified that differ in virulence, tissue tropism, and antigenicity. This reflects the fact that mutation is common among coronaviruses, a property that increases risks for recurrent infection. As described further below, MHV isolates are often categorized according to their organotropism into two biotypes: enterotropic strains, which infect primarily the intestinal tract, and polytropic strains, which initially infect the respiratory tract but often progress to multisystemic dissemination (Homberger, 1997). However, isolates may contain features of both biotypes. Although MHV isolates and strains share internal antigens (M and N), they can be distinguished by neutralization tests that detect strain-specific spike (S) antigens. MHV shares antigens with the coronaviruses of rats, a finding that has been exploited to develop heterologous antigens for serological tests. MHV also is related to human coronavirus OC43. A number of established cell lines can be used for propagating prototype MHV strains in vitro. However, field isolates are difficult to maintain in vitro. NCTC 1469 mouse liver cells are useful for growing many polytropic strains. Enterotropic strains have been grown in CMT-93 cells derived from a rectal carcinoma in a C57BL mouse but are generally difficult to propagate in cell culture. MHV can also be grown in mouse macrophages, cells that have been used for genetic studies of resistance and

3. BIOLOGY AND DISEASES OF MICE

Fig. 27. MHV syncytiain the intestine. Immunofluorescencestain.

susceptibility to infection. Irrespective of cellular substrate used for isolation or propagation, syncytium formation is emblematic of MHV infection. (See Fig. 27 for an example of syncytia in situ. ) Clinical signs. The prevalence and severity of clinical signs depend primarily on the age, strain, and immunological status of infected mouse and strain and tropism of virus (Barthold et al., 1993b). As with many murine viruses, infection is often clinically silent among immunologically competent mature mice. Clinical morbidity is most often associated with suckling mice less than 2 weeks old or with immunodeficient mice. Suckling mice develop signs in various combinations that include diarrhea, inappetance, dehydration, weight loss, lassitude, and ruffled pelage, often terminating in death (Fig. 28). Neurotropic strains such as MHV-JHM may induce flaccid paralysis of the hindlimbs, but this sign is rarely encountered alone during natural infection. Conjunctivitis, convulsions, and circling may be seen occasionally. Mildly pathogenic strains may not cause acute disease in athymic mice but rather can cause a progressive wasting syndrome that may be accompanied by progressive paralysis.

MHV infection is, for all practical purposes, an affliction of mice and arguably the most common viral infection in this species. A recent national survey reported MHV in nearly 60% of more than 100 major vivariums, usually among conventionally housed mice but also in more than 10% of barrier-housed colonies (Jacoby and Lindsey, 1997). There are no reports of natural transmission from mice to other species, but suckling rats can develop necrotizing rhinitis after intranasal inoculation with MHV-S. Sex-related or seasonal differences in susceptibility have not been found. MHV should be considered highly contagious, with natural transmission occurring by respiratory or oral routes. Recent reports suggest that enterotropic biotypes predominate in natural infections (Hom-

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berger et al., 1998). Feces and nasopharyngeal exudates can serve as sources of infection. Natural vertical transmission has not been demonstrated. Introduction of MHV through injection of contaminated biologicals can be an important factor in epizootics, especially because some isolates infect B lymphocytes and, by implication, hybridomas nonlytically. Infection in immunocompetent mice is self-limiting. Immunemediated clearance of virus associated with seroconversion usually begins about a week after infection, and mice recover fully within 3 - 4 weeks. Humoral and cellular immunity appears to participate in host defenses to infection, and T celldependent immunity is an absolute requirement. Thus, agerelated resistance to MHV correlates with maturation of lymphoreticular tissues. Enzootic infection had been construed to include persistent infection in individual mice. Current evidence suggests, however, that enzootic infection results either from the fresh and continuous introduction of immunologically naive or deficient mice or from the recurrent infection of immune mice with MHV variants that arise by natural mutation. Mutation is favored by immune pressures in enzootically infected colonies as well as missteps during natural replication, which include copying errors and recombination. Thus, mice that have developed immunity to one strain of MHV can remain susceptible to one or more genetically and antigenically divergent strains, resulting in reinfection (Barthold and Smith, 1989a,b; Homberger et al., 1992). This caveat has practical importance for breeding colonies. Maternal immunity protects suckling mice against homologous MHV strains but not against antigenically variant strains. However, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine. Therefore, the

Epizootiology.

Fig. 28. Infant mice with enterotropic MHV infection. Upper mouse appears normal and has a milk-filled stomach. Lowermouse is runted and dehydrated and has an empty stomach. (FromBarthold et al., 1982.)

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Fig. 29. Necrosis,inflammation,and syncytiain the liver of a mousewith MHV.

susceptibility of young mice increases significantly at weaning. Strain differences in resistance and susceptibility can be inherited as an autosomal dominant trait (Barthold and Smith, 1987). For example, DBA/2 mice are highly susceptible to MHV-3 and die acutely even as adults, whereas A/J mice develop resistance to lethal infection shortly after weaning. However, genetic resistance also is virus strain-dependent. Therefore, mice resistant to one strain of MHV may be susceptible to another strain. It also is worth noting that the expanded use of genetically altered mice with novel or unanticipated deficits in antiviral responses may alter the outcome of virus-host interactions unpredictably. This pertains to MHV as well as other agents. For example, MHV infection has presented as granulomatous peritonitis and pleuritis in interferon-gamma knockout mice (France et al., 1999). Pathology. Polytropic strains replicate initially in the nasal mucosa, where necrotizing rhinitis may occur. Viremic dissemination can follow if virus gains access to regional blood vessels and lymphatics. Thus, viremia leads to secondary infection of vascular endothelium and parenchymal tissues in multiple organs including liver, brain, lymphoid organs, and other sites. Mice also may develop central nervous system disease by direct extension of infection from the olfactory mucosa along olfactory tracts. At necropsy, yellow-white foci indicative of necrosis can occur in multiple tissues, with the involvement of the liver as the classical lesion. Liver involvement may be accompanied by icterus and peritonitis. Histologically, necrosis can be focal or confluent and may be infiltrated by inflammatory cells

(Fig. 29). Syncytia commonly form at the margin of necrotic areas and, in mild infections, may develop in the absence of frank necrosis. Syncytia formation is a hallmark of infection in many tissues, including intestine (Fig. 27), lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow and in vascular endothelium in general. Although syncytia are transient in immunocompetent mice, they are a persistent feature in chronically infected, immunodeficient mice (Fig. 30). Neurotropic variants cause acute necrotizing encephalitis or meningoencephalitis in suckling mice, with demyelination in the brain stem and in peri-ependymal areas secondary to viral invasion of oligodendroglia. Convalescent mice may have residual mononuclear cell infiltrates around vessels or as focal lesions in the liver. Immunodeficient mice can develop smoldering necrotic lesions in the liver and elsewhere. Compensatory splenomegaly may occur because of expansion of hematopoietic tissue. Enterotropic strains infect primarily the intestine and associated lymphoid tissues, although some may also cause systemic lesions, especially in liver and brain. The most common sites are terminal ileum, cecum, and proximal colon. The severity of disease is age-related, with immunocompetent young infants being at highest risk for lethal infection. Pathogenic strains can cause lesions ranging from villus attentuation and atrophy to fulminant necrotizing enterotyphlocolitis, which can kill suckling mice within several days (Fig. 31). The stomach is often empty, and the intestine is filled with watery to mucoid yellowish, sometimes gaseous contents. Hemorrhage or rupture of the intestine can occur. Syncytia are a consistent feature in viable mucosa and not only are formed in intestine but also may be pres-

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Fig. 30. Hepatitisand a syncytium(center) in the liver of an athymicmouse with MHV.

ent in mesenteric lymph nodes and endothelium of mesenteric vessels. Enterocytes may contain intracytoplasmic inclusions, but they are not diagnostic. Surviving mice develop compensatory mucosal hyperplasia, which eventually recedes. Older, more resistant mice usually develop transient syncytia without necrotic lesions. The exception occurs in immunodeficient mice, such as athymic and SCID mice, which can develop chronic proliferative bowel disease of varying severity and accompanied by syncytia (Fig. 32) (Barthold et al., 1985).

Fig. 31. Typhlocolitiswith syncytiaformation and effacementof mucosal architecture in an infant mouse with enterotropic MHV infection. (Courtesyof Dr. S. W. Barthold.)

Diagnosis. Because MHV infection is usually asymptomatic, serological testing is the most reliable diagnostic tool. Many animal resources rely on sentinel mouse protocols for continuous serological surveillance, because immune dysfunction can delay or abrogate seroconversion among research mice. ELISA and IFA tests are well established, sensitive, and reliable (Smith, 1983a; Smith and Winograd, 1986). Neutralization tests are used to differentiate individual virus strains in the research laboratory but are problematic for routine use, because of cost, technical complexity, and the effects of natural antigenic mutation inherent to MHV. Additionally, strain-specific, serologic identification per se does not predict biological behavior, including virulence or tissue tropism. Serology also can be used in the context of mouse antibody production assays in which adult mice are inoculated with suspect tissues to elicit seroconversion. Molecular diagnostics are being applied more widely for the diagnosis of MHV infection. PCR protocols to detect virus in tissues or excreta are available (Casebolt et al., 1997; Homberger et al., 1991; Yamada et al., 1993), and access to testing can be obtained from specialized laboratories. Clinical signs of diarrhea, acute death, or neurological deficits are suggestive but not pathognomonic. The detection of syncytia augmented, when possible, by immunohistochemistry to detect MHV antigens (Fig. 33) is a useful and practical means to confirm infection (Brownstein and Barthold, 1982). This strategy should attempt to select mice that are in early stages of infection, because necrosis in infant mice or seroconversion in older mice may reduce the chances of detecting syncytia or viral antigens. The option of using immunodeficient mice as sentinels can be considered,

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Fig. 32. Proliferativeenteritis in an athymicmousewith MHV. because they sustain prolonged infection. However, they should be securely confined because they also amplify virus loads. If properly controlled, amplification in immunodeficient mice can, however, facilitate subsequent virus isolation in tissue culture.

Differential diagnosis. MHV infection must be differentiated from other infectious diseases that cause diarrheal illness, runt-

ing, or death in suckling mice and wasting disease in immunodeficient mice. These include EDIM, mousepox, reovirus 3 infection, Tyzzer's disease, and salmonellosis. Neurological signs or demyelinating lesions must be differentiated from mouse encephalomyelitis virus infection or noninfectious CNS lesions, such as neoplasms, including polyoma-induced tumors in athymic mice.

Fig. 33. MHV antigenin the small intestine, detectedby enzymeimmunohistochemistry.

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Prevention and control.

Control and prevention of MHV infection can be difficult because of the numerous variables that influence its expression. Perhaps the most important factor is the duration of infection in individual mice and in mouse colonies. There is evidence that infection in an individual immunocompetent mouse is acute and self-limiting. Such mice can be expected to develop immunity and eliminate virus within 30 days. Therefore, quarantine with the temporary cessation of breeding can be used effectively to eliminate infection (Weir et al., 1987). Additionally, maternally derived immunity can protect infant mice from infection until they are weaned and moved to uncontaminated quarters (Homberger, 1992; Lipman et al., 1987). Careful testing with sentinel mice should be used to assess the effectiveness of quarantine or "natural rederivation," as just described. This is especially true to account for the potential that a mutant variant could arise during quarantine and result in prolongation of colonywide infection. Immunodeficient mice, by contrast, are susceptible to chronic infection and viral excretion. Therefore, control measures must be more drastic and include depopulation. There also is an emerging gray zone, associated with the creation of genetically altered mice, in which decisions about control strategies are more problematic. It includes mice with unrecognized or unanticipated immune dysfunction or with selective immune dysfunction in which the impact of MHV infection is not known. Such colonies, which may contain highly valuable or irreplacable mice, may be rescued by cesarean rederivation or embryo transfer if vertical transmission of MHV infection is subsequently ruled out. Although rodent coronaviruses are not viable for extended periods in the environment, excreted virus may remain infectious for up to several days, so proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. The prevention of MHV requires procurement of animals from virus-free sources and maintainence under effective barrier conditions monitored by a well-designed quality assurance program. Control of feral mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbor virus (e.g., transplantable neoplasms, cell lines) are also important strategies to prevent adventitious infection.

Research complications.

Numerous research complications have been attributed to MHV (Compton et al., 1993; Homberger, 1997), and the unpredictable outcome of infection in genetically altered mice is likely to lengthen the list. For example, and apart from its clinical impact, MHV may stimulate or suppress immune responses, contaminate transplantable neoplasms, and be reactivated by treatment of asymptomatically infected animals with several classes of drugs, including immunosuppressive agents, and by intercurrent infections. It also can alter tissue enzyme levels. Additionally, the ubiquitous threat of MHV infection and uncertainty about its potential effects on a given research project provoke concerns that may ex-

ceed its true impact. For example, transient infection with a mild enterotropic strain is unlikely to disrupt systemic immune responses, whereas infection with a polytropic strain may be highly disruptive. This is not to say that asymptomatic or strictly enterotropic infection should be taken lightly but simply to caution against overreaction in assessing the impact of an outbreak.

Mouse Encephalomyelitis Virus (MEV) Infection (Downs, 1982; Lipton and Rozhon, 1986) Etiology.

MEV is a small, nonenveloped, RNA-containing cardiovirus. It was discovered by Max Theiler during experimental studies of yellow fever virus in mice, so it is also referred to as Theiler's MEV or simply TMEV. Established strains include TO (Theiler's original), FA, DA, and GD VII, the last of which is named after George Martine (George's disease), an assistant in Theiler's laboratory. The virus is rapidly destroyed by temperatures over 50~ and by alcohol but not by ether. It can be cultivated in vitro in several continuous cell lines, but B HK21 cells are routinely used for isolation and propagation. It is antigenically related to encephalomyocarditis virus, which does not infect mice by natural exposure. As with other nonenveloped viruses, MEV is resistant to environmental inactivation, a factor that must be considered in control and prevention of infection.

Clinical signs. The development of clinical disease depends on virus strain, mouse strain, and route of exposure but is exceedingly rare (estimated at 0.1-0.01% of infected mice). When clinical signs occur, they are expressed as neurological disease. The characteristic sign is flaccid posterior paralysis, which may be preceded by weakness in the forelimbs or hindlimbs, but in mice which are otherwise alert (Fig. 34). Some mice may recover, but death frequently ensues, often because of failure to obtain food or water. Furthermore, mice that recover from the paralytic syndrome are disposed to a chronic demyelinating phase, which is expressed as a gait disturbance. Epizootiology.

Infection occurs primarily in laboratory mice with the exception of the MGH strain, which has been isolated from laboratory rats and is pathogenic in mice and rats after experimental inoculation. The prevalence of MEV in mouse colonies is low, a reflection of the slow rate at which virus is transmitted from mouse to mouse. MEV infection is acquired by ingestion and replicates primarily in the intestinal mucosa. Enteric infection can persist after the development of host immunity and can result in chronic or intermittent excretion of virus in feces over several months (Brownstein et al., 1989). Mice often become infected shortly after weaning, but virus is seldom recovered in mice over 6 months of age. However, neurologic infection can persistnin the brain and spinal cordmfor at least 1 year. In contrast to MHV, immunity to one strain

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Fig. 34.

Posterior paralysis in a mouse with MEV infection.

of MEV provides cross-protection to other strains. There are no reports of differences in mice with respect to susceptibility to infection under natural conditions. Prenatal transmission has not been found.

affecting the spinal cord can also produce posterior paralysis. Polyomavirus infection in athymic mice can induce tumors or demyelination in the CNS, which may result in clinical signs resembling those of MEV infection.

Pathology.

Prevention and control.

Intestinal MEV infection does not cause lesions, but virus can be detected in enterocytes by immunohistochemistry or in situ hybridization. Poliomyelitis-like disease, the syndrome that may be encountered during natural infections, is characterized by acute necrosis of ganglion cells, neurophagia, and perivascular inflammation, which occurs particularly in the ventral horn of the spinal cord gray matter but also can involve higher centers such as the hippocampus, thalamus, and brain stem. During the subsequent demyelinating phase, mononuclear cell inflammation develops in the leptomeninges and white matter of the spinal cord, accompanied by patchy demyelination. The white-matter lesions are due to immune injury and are similar to those seen in experimental allergic encephalomyelitis (Monteyne et al., 1997). Spontaneous demyelinating myelopathy, affecting the thoracic spinal cord and associated with MEV infection, has also been reported in aged mice. Virulent strains may cause acute encephalitis after experimental inoculation, whereas less virulent isolates produce acute poliomyelitis followed by chronic demyelinating disease.

Diagnosis.

Infection is usually detected serologically by IFA or ELISA (Kraft and Meyer, 1986). A PCR assay also is available (Zoll et al., 1993). Clinical signs are striking, if they occur, but are too rare to rely on for routine diagnosis. Histological lesions in the CNS and especially the spinal cord are characteristic when present. Virus can be isolated by inoculation of B HK21 cells with intestine or CNS tissue.

Differential diagnosis.

Neurotropic variants of MHV may, on occasion, cause similar neurological signs. Injury or neoplasia

Disease-free stocks were originally developed by foster-nursing infant mice. This technique or cesarean or embryo derivation techniques can be used successfully to eliminate infection (Lipman et al., 1987). In either case, foster mothers should be surveyed in advance to ensure their MEV-free status. Selective culling can be considered as an option to eliminate infection, because infection spreads slowly. However, the virus is hardy in the environment and resists chemical inactivation, so it may be prudent to depopulate and disinfect rooms if the presence of infection is unacceptable.

Research complications.

The principal hazard from MEV for research relates to its potential effects on the CNS. 2.

Bacterial Diseases a.

Mycoplasmosis (Cassell et al., 1986; Lindsey et al., 1982, 1991i)

Several species of Mycoplasma can infect laboratory mice: M. pulmonis, M. arthriditis, M. neurolyticum, and M. collis. Antigenic cross-reactivity among these species, and especially between M. pulmonis and M. arthriditis, mandates that reliable diagnostic strategies incremental to serology be employed to distinguish potentially pathogenic infections. The following section first describes infection due to M. pulmonis, then summarizes infections associated with other murine mycoplasmas.

i. Mycoplasma pulmonis Etiology. My. pulmonis is a pleomorphic, gram-negative

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bacterium that lacks a cell wall and has a single outer limiting membrane. It causes murine respiratory mycoplasmosis (MRM).

Clinical signs. Mice are relatively resistant to florid MRM, so asymptomatic infection is common. When clinical signs occur, they reflect suppurative rhinitis, otitis media, and chronic pneumonia. Affected mice may display inactivity, weight loss, and ruffled hair coat, but the most prominent signs are "chattering" and dypsnea, due to rhinitis and purulent exudate in nasal passages. Otitis media may cause a head tilt, whereas suppurative inflammation in the brain and spinal cord, although rare, can cause flaccid paralysis. Naturally occurring disease of sites other than the respiratory tract has not been reported, but experimental infection of the genital tract can cause oophoritis, salpingitis, and metritis, which may lead to infertility or fetal deaths. Experimental inoculation of SCID mice has caused systemic infection accompanied by severe arthritis (Evengard et al., 1994). Epizootiology.

MRM used to be a common infectious disease of mice, but improved housing, husbandry, and health care have reduced its prevalence dramatically. Although a recent survey suggests that Mycoplasma infection still affects about 15% of conventionally housed mouse colonies (Jacoby and Lindsey, 1997), the data did not differentiate M. pulmonis infections from those caused by less virulent species such as M. arthritidis. Mycoplasma pulmonis infection is contracted by inhalation and can occur in suckling and adult mice. Therefore, infection should be considered highly contagious. In utero infection has been demonstrated in rats but not in mice. Concomitant viral pneumonia (Sendai virus, mouse coronavirus) or elevated environmental ammonia concentrations may increase susceptibility to MRM. Mycoplasma pulmonis also infects rats, hamsters, guinea pigs, and rabbits. Among these species, only rats are significant reservoirs of infection for mice.

Pathology.

Mycoplasma pulmonis is an extracellular organism that colonizes the apical cell membranes of respiratory epithelium. Attachment occurs anywhere from the anterior nasal passages to the alveoli and may be mediated by surface glycoproteins. The organism may injure host cells through competition for metabolites such as carbohydrates and nucleic acids or by release of toxic substances such as peroxides. Ciliostasis, reduction in the number of cilia, and ultrastructural changes leading to cell death have also been described. Detrimental effects on ciliated epithelium can lead to disrupted mucociliary transport, which exacerbates pulmonary disease. Experimental expression of MRM is dose dependent. Doses of 104 colony-forming units (CFU) or less cause mild, transient disease involving the upper respiratory tract and middle ears, whereas higher doses often lead to acute, lethal pneumonia. Additionally, Mycoplasma strains can differ in virulence. Survivors of severe infection may develop chronic broncho-

pneumonia with bronchiectasis and spread infection to other mice. Intravenous inoculation of M. pulmonis can cause arthritis in mice, but arthritis is not a significant feature of natural infection. Host genotype also is a major factor in the outcome of infection, with resistance being expressed phenotypically through the bactericidal efficiency of alveolar macrophages. Strains derived from a C57BL background appear to be resistant to pathogenic infection, whereas BALB/c, C3H, DBA/2, SWR, AKR, CBA, SJL, and others have varying degrees of increased susceptibility (Cartner et al., 1996; Lai et al., 1993). The initial lesion of MRM is suppurative rhinitis, which may involve the trachea and major airways. Early inflammatory lesions, if not quickly resolved, progress to prominent squamous metaplasia. Transient hyperplasia of submucosal glands may occur, and lymphoid infiltration of the submucosa can persist for weeks. Syncytia can sometimes be found in nasal passages, in association with purulent exudate. Affected mice also develop suppurative otitis media and chronic laryngotracheitis with mucosal hyperplasia and lymphoid cell infiltrates. Pulmonary lesions are typified by bronchopneumonia, which spreads from the hilus. Lymphoid cells and plasma cells accumulate around bronchi which often contain neutrophils in their lumena. Chronic lung disease features suppurative bronchitis, bronchiolitis, and alveolitis (Fig. 35). Chronicity also increases the prevalence of bronchiectasis and abscessation.

Diagnosis.

Accurate diagnosis should exploit the complementary use of clinical, serological, microbiological, molecular, and morphological methods. Clinical signs are variable but can be characteristic when they occur. A sensitive ELISA, a radioimmunosorbent assay, and a solid-phase radioimmunoassay are available for serological detection of infection but do not differentiate M. pulmonis infection from M. arthritidis infection (Cassell et al., 1981). Further, some mice may be poor responders and develop very low antibody titers. Therefore attempts should be made to use other methods to confirm diagnosis. Primary among these are attempts to isolate the causative organism. The upper respiratory tract should be cultured because it is a common site for natural infection. Buffered saline or Mycoplasma broth should be used to lavage the trachea, larynx, pharynx, and nasal passages. Culture from the genital tract is warranted if this site is suspected. Mycoplasma species may be difficult to grow, so it is prudent to confirm that the relevant expertise and quality control exist in the diagnostic laboratory. Speciation can be accomplished by immunofluorescence or immunoperoxidase staining or by growth inhibition. Immunohistochemistry should be considered to supplement basic histopathologic examination. Immunofluorescence and immunoperoxidase techniques are available to identify mycoplasmal antigens in tissue sections or in cytological preparations of tracheobronchial or genital tract lavages (Brunnert et al., 1994). More recently, PCR has been assessed as a diagnostic strategy

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Fig. 35. Suppurativebronchopneumoniacaused by Mycoplasmapulmonis.

(Brunnert et al., 1994; Goto et al., 1994; Harasawa et al., 1990; Kunita et al., 1990; Schoeb et al., 1997; van Kuppeveld et al., 1993). It appears capable of detecting infection rapidly, accurately, and sensitively in living mice or in paraffin-embedded tissues.

Differential diagnosis. MRM must be differentiated from bronchopneumonia associated with cilia-associated respiratory (CAR) bacillus. Silver stains may reveal CAR bacilli adherent to the respiratory epithelium. Sendai virus also can cause bronchopneumonia in mice but can be detected by serology and immunohistochemistry. Other causes of respiratory infection include pneumonia virus of mice, corynebacteriosis, and, in immunodeficient mice, Pneumocystis carinii infection. Combined infections with known pathogens or secondary opportunists also must be considered. Prevention and control. Mice mount an effective immune response to Mycoplasma pulmonis, as measured by their recovery from mild infection and their resistance to infection after active or passive immunization (Cartner et al., 1998). Antibodies of various classes are produced locally and systemically, but their role in infection is unclear. There is some evidence that antibody may facilitate phagocytosis of M. pulmonis. Classic cellular immunity, however, does not appear to play a major role in M. pulmonis infection in mice, because immunity cannot be transferred with immune cells. In addition, athymic and neonatally thymectomized mice are not more susceptible than imunocompetent mice to M. pulmonis pneumonia.

Host immunity aside, effective control and prevention of MRM depend primarily on maintenance of Mycoplasma-free colonies under barrier conditions supported by careful surveillance for infection by serology, microbiology, and histopathology. Cesarean or embryo rederivation can eliminate infection, but embryos, fetal membranes, and offspring must be tested to rule out contamination (Hill and Stalley, 1991). Treatment with tetracyclines suppresses clinical disease but does not eliminate infection. Some progress has been made in developing DNAbased vaccines against M. pulmonis, but they have not achieved clinical application (Lai et al., 1997). Research complications. Mycoplasma pulmonis can interfere with research by causing clinical disease or death. Experiments involving the respiratory tract, such as inhalation toxicology, can be compromised by chronic progressive infection. Additionally, affected mice are at greater risk during general anesthesia. Mycoplasma pulmonis may alter immunological responsiveness. For example, it is mitogenic for T and B lymphocytes and can increase natural killer cell activity. Perhaps one of the most important complications of Mycoplasma infection is contamination of cell lines and transplantable tumors.

ii. Other murine mycoplasmas Mycoplasma arthritidis is antigenically related to M. pulmonis. Therefore, serological evidence of mycoplasma infection must be supplemented by other diagnostic tests, as outlined above, to differentiate between these agents. Differentiation is important because M. arthritidis, though arthritogenic in mice after intravenous inoculation, is nonpathogenic during natural infection. Mycoplasma collis

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has been isolated from the genital tract of the mouse but does not appear to cause natural disease. Mycoplasma neurolyticum is the etiological agent of rolling disease, a rare syndrome which occurs within hours after intravenous inoculation of M. neurolytica exotoxin. Characteristic clinical signs include spasmodic hyperextension of the head and the raising of one foreleg followed by intermittent rolling on the long axis of the body. The rolling becomes more constant, but mice occasionally leap or move rapidly. After 1-2 hr of rolling, animals become comatose and usually die within 4 hr. All published reports of rolling disease are associated with experimental inoculation of organisms or exotoxin. Large numbers of organisms are needed to produce disease, and there is no indication that, under natural conditions, organisms replicate in the brain to concentrations required for the induction of these signs. Because animals are frequently inoculated with biological materials by parenteral routes, contamination with M. neurolytica may induce rolling disease inadvertently. Diagnosis can be made from the appearance of typical clinical signs, astrocytic swelling, and isolation of the causative organism. Clinical signs must be differentiated from rolling associated with Pseudomonas-caused otitis. Mycoplasma pulmonis has been recovered from the brain of mice but does not seem to cause overt neurological disease.

b.

Cilia-Associated Respiratory (CAR) Bacillus Infection

CAR bacillus is a slender, gram-negative bacillus, which, in rats, produces clinical disease and lesions that closely resemble those of MRM (see Chapter 4). Chronic respiratory disease has

been produced in mice by experimental inoculation, but natural clinical disease is rare (Griffith et al., 1988). Furthermore, putative natural cases were reported in mice that were seropositive for Sendai virus and pneumonia virus of mice. Therefore, CAR bacillus may have acted as an opportunist rather than as a primary pathogen. On balance, one can assume that mice may contract natural infection, but attributing chronic respiratory disease in mice solely to CAR bacillus is not currently warranted. An ELISA for serological detection of infection is available (Shoji et al., 1988), and PCR-based diagnostics also have been developed (Goto et al., 1995). Histologic assessment of infection requires the use of Warthin-Starry or similar stains to visualize argyrophilic bacilli adherent to the apical membranes of bronchial respiratory epithelium (Fig. 36). Alternatively, immunoperoxidase staining has also been used successfully to detect infection. There is a report that sulfamerazine (500 mg/ liter) in drinking water may be effective in eradicating infection (Matsushita and Suzuki, 1995), but this strategy has not been confirmed. Alternative approaches for eradication are similar to those described for Mycoplasma pulmonis.

Tyzzer's Disease (Fujiwara and Ganaway, 1994; Ganaway et al., 1971; Ganaway, 1982) Etiology.

Tyzzer's disease is named for Ernest Tyzzer, who first described it in a colony of Japanese Waltzing mice. The causative organism, Clostridium piliforme (formerly Bacillus piliformis), is a long, thin, gram-negative spore-forming bacterium that appears to require living cells for in vitro growth. It

Fig. 36. CAR bacilli at the ciliated borderof respiratoryepithelium (Warthin-Starry stain). The adjacentbronchial lumen contains inflammatorycells.

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has not been grown successfully on cell-free media, but it can be propagated by inoculation of susceptible vertebrates, the yolk sac of embryonated eggs, or hepatocyte cultures from mice (Ganaway et al., 1985; Kawamura et al., 1983).

Clinical signs. Clinical disease occurs as unexpected deaths that may be preceded by diarrhea and inactivity. Although outbreaks can be explosive and mortality is usually high, morbidity may be high or low. Additionally, subclinical infections can occur, accompanied by the development of antibodies to C. piliforme. Stresses, such as overcrowding, high temperature and humidity, moist food, and immunosuppression, may predispose mice to Tyzzer's disease. Susceptibility and resistance also are influenced by host genotype. It has been shown, for example, that C57BL/6 mice are more resistant than DBA/2 mice to Tyzzer's disease (Waggie et al., 1981). Resistance to severe infection appears to be due, in part, to B lymphocyte function. Athymic mice also appear to have increased susceptibility to Tyzzer's disease (Livingston et al., 1996). The role of T cells in resistance is not clear, because susceptibility among athymic mice appears to vary. However, the involvement of T cells can be inferred by the fact that several interleukins modulate resistance and susceptibility. Depletion of neutrophils or natural killer cells also increases susceptibility to infection. Epizootiology. Current prevalence rates, reservoirs of infection, carrier states, and the mechanism of spread remain speculative. Tyzzer's disease occurs in many species of laboratory animals and in domestic and free-living species, but the reservoir of infection is unknown. Some strains appear capable of crossinfecting mice, rats, and hamsters, whereas others have a more restricted host range (Franklin et al., 1994). Therefore the risks for cross-infection depend on the strain causing a given outbreak. The vegetative form of C. piliforme is unstable, but spores can retain infectivity at room temperature for at least 1 year and should be viewed as the primary means of spread. Natural infection is probably due to ingestion of organisms, which are subsequently shed in feces. Feces-contaminated food and soiled bedding are therefore the most likely sources of environmental contamination. Prenatal infection can be induced by intravenous inoculation of pregnant mice, but its importance in the natural transmission of infection has not been determined. Pathology. Infection begins in the gastrointestinal tract, followed by bacteremic spread to the liver and, to a smaller extent, the heart. The lesions are characterized by necrosis in these tissues and in the mesenteric lymph nodes. Grossly, segments of the ileum, cecum, and colon may be red and dilated, with watery, fetid contents, whereas the liver, mesenteric lymph nodes, and heart often contain gray-white foci. Histologically, intestinal lesions include necrosis of mucosal epithelium, which may be accompanied by acute inflammation and hemorrhage. In the

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

liver, foci of coagulation necrosis are generally distributed along branches of the portal vein, a finding compatible with embolic infection from the intestine. Peracute lesions are largely free of inflammation, but neutrophils and lymphocytes may infiltrate less fulminant lesions. Myocardial necrosis is sporadic in natural infection. Bundles of long, slender rods occur in the cytoplasm of viable cells bordering necrotic foci, especially in the liver (Fig. 37) and intestine. They are found more easily during early stages of infection. Organisms in tissue sections do not stain well with hematoxylin-eosin stain. Silver stains (Warthin-Starry), Giemsa stains, or periodic acid-Schiff stains are usually required.

Diagnosis. Tyzzer's disease is diagnosed most directly by the demonstration of characteristic intracellular organisms in tissue sections of liver and intestine. Supplemental procedures include inoculation of cortisonized mice or embryonated eggs with suspect material, followed by histological or immunocytochemical demonstration of organisms in tissues. Asymptomatic infection can be detected by ELISA (Waggie et al., 1987) or by PCR (Goto and Itoh, 1996). Differential diagnosis. The histological detection of organisms is essential for differentiating Tyzzer's disease from other infections that can produce similar signs and lesions, especially mousepox, coronaviral hepatitis, reoviral hepatitis, helicobacteriosis, and salmonellosis. It also is important not to misconstrue extracellular rods as Clostridium piliforme. Prevention and control. Barrier housing and husbandry that incorporate sanitation measures to avoid the introduction or buildup of spores in the environment are the bases for control or prevention of Tyzzer's disease. If infection occurs, spore formation will make control or elimination by antibiotic therapy problematic. Therefore, strict quarantine, followed by replacement of affected or exposed stock, must be considered. Rederivation by embryo transfer or cesarean section should take the potential for prenatal transmission of infection into account in housing and testing offspring. Thorough decontamination of the environment with an oxidizing disinfectant must be included in any control program. Additionally, procurement of food and bedding from suppliers with thorough quality assurance and vermin control programs is essential for both prevention and control. Husbandry supplies should be stored in verminproof quarters, and the option of heat sterilization of food and bedding should be considered. Research complications. Research complications stem from clinical morbidity and mortality. Mice with immune dysfunction are at increased risk. There is recent evidence that infection causes elevations in selected cytokines (Van Andel, et al., 2000).

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Fig. 37. Clostridiumpiliforme in the liver on a mouse with Tyzzer'sdisease (Warthin-Starry stain).

d.

Transmissible Murine Colonic Hyperplasia

Etiology. The causative agent of transmissible murine colonic hyperplasia, Citrobacter rodentium (formerly Citrobacter freundii strain 4280), is a nonmotile, gram-negative rod that ferments lactose but does not utilize citrate or does so marginally (Barthold, 1980; Schauer et al., 1995). Clinical signs. Clinically apparent infection is characterized by retarded growth, ruffled fur, soft feces or diarrhea, rectal prolapse, and moderate mortality in older suckling or recently weaned mice (Barthold et al., 1978). Epizootiology. Citrobacter rodentium is not in the gastrointestinal flora of normal mice. It is thought to be introduced by contaminated mice, food, or bedding, from which it spreads by contact or additional fecal contamination. Host genotype can influence the course and severity of disease (Barthold et al., 1977). For example, DBA, NIH Swiss, and C57BL mice are relatively resistant to mortality, whereas C3H/HeJ mice are relatively susceptible both as sucklings and as adults. Diet also can modulate infection, but specific dietary factors responsible for this effect have not been identified. Pathology. Citrobacter rodentium attaches to the mucosa of the descending colon and displaces the normal flora. Attachment is accompanied by effacement of the microvillus border and formation of pedestal-like structures (attaching and effacing lesions) (Schauer et al., 1993; Newman et al., 1999). Colo-

nization results in prominent mucosal hyperplasia, by unknown mechanisms. The characteristic gross finding is severe thickening of the descending colon, which may extend to the transverse colon and lasts for 2 - 3 weeks in surviving animals (Fig. 38) (Percy and Barthold, 2001a). Affected segments are rigid and either are empty or contain semiformed feces. Histologically, accelerated mitotic activity results in a markedly hyperplastic mucosa, which may be associated with secondary inflammation and ulceration (Fig. 39). Lesions subside after several weeks. Repair is rapid and complete in adults but slower in sucklings. Diagnosis. Diagnosis depends on clinical signs, characteristic gross and histological lesions, and isolation of C. rodentium from the gastrointestinal tract or feces. The organism is relatively easy to culture on MacConkey's agar during early phases of infection, whereas the intestine may be free of aerobic bacteria during later stages. Citrobacter rodentium also can be detected by molecular hybridization (Schauer et al., 1995). Differential diagnosis. Transmissible murine colonic hyperplasia must be differentiated from other diarrheal diseases of mice, including infections caused by coronavirus, rotavirus, adenovirus, reovirus, Salmonella, Clostridium piliforme, and Helicobacter spp. Prevention and control. Some success in curtailing epizootics has been achieved by adding antimicrobials to the drinking water (Barthold, 1980; Silverman et al., 1979). Because C.

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lance for C. rodentium should be incorporated into quality assurance programs.

Research complications. The potential effects on research of colonic hyperplasia as a clinically severe disease are obvious. Colonic hyperplasia has been shown to increase the sensitivity of colonic mucosa to chemical carcinogens and to decrease the latent period between administration of carcinogen and the appearance of focal atypical cell growth (Barthold and Beck, 1980). More recently, infection has been incriminated in immune dysfunction, poor reproductive performance, and failure to thrive in T cell receptor transgenic mice (Maggio-Price et al., 1998). It may also inhibit cytokine production by lymphoid cells. e.

Pseudomoniasis (Lindsey et al., 1991d)

Etiology. tive rod.

Fig. 38. Colonsof a normal mouse (right) and of a mouse with transmissible murine colonic hyperplasia (left). The descending colon is thickened and opaque because of mucosal hyperplasia. (FromBarthold et aI., 1978.)

rodentium may contaminate food, bedding, or water, proper disinfection of such materials is prudent before they are used for susceptible animals. Additionally, quarantine or the employment of microbarrier caging can reduce transmission. Surveil-

Pseudomonas aeruginosa is a motile, gram-nega-

Clinical signs. Pseudomonas aeruginosa infections are almost always silent, but immunologically compromised animals are prone to septicemia (Brownstein, 1978). Pseudomonas aeruginosa can, for example, cause severe or lethal infections in athymic mice. Sick mice may have equilibrium disturbances, conjunctivitis, serosanguinous nasal discharge, edema of the head, weight loss, and skin infections. Immunosuppressed mice may also develop gastrointestinal ulcers. Generalized infection is associated with severe leukopenia. Neurologic signs are rare, but there are reports of central nervous system infection.

Fig. 39. Colonichyperplasiacaused by Citrobacter rodentium.

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Chronic proliferative inflammation in the cochlea and vestibular apparatus with dissolution of surrounding bone may cause torticollis.

Epizootiology.

Pseudomonas aeruginosa is not part of the normal flora. However, it is an opportunist that inhabits moist, warm environments such as water and skin. Once established in a host, it may be found chronically in the nasopharynx, oropharynx, and gastrointestinal tract, all sites from which additional environmental contamination or direct transmission to susceptible mice can occur. Pathology.

Pathogenic infection is most common in immunodeficient mice. Organisms enter at the squamocolumnar junction of the upper respiratory tract and, in some cases, the periodontal gingiva. Bacteremia is followed by necrosis or abscess formation in liver, spleen, or other tissues. If otitis media occurs, the tympanic bullae may contain green suppurative exudate. The bowel may be distended with fluid, and gastrointestinal ulceration has been reported.

Diagnosis.

Infection is diagnosed on the basis of history (e.g., immune dysfunction), clinical signs, lesions, and isolation of P. aeruginosa from affected mice. Carrier mice can be detected either by nasal culture or by placing bottles of sterile, nonacidified, nonchlorinated water on cages for 2 4 - 4 8 hr and then culturing the sipper tubes.

Clinical signs. Many early observations concerning the pathogenicity of P. pneumotropica are questionable because they were made on colonies of mice with varying levels of bacterial and viral contamination. Infection is usually asymptomatic. Therefore, P. pneumotropica is most properly viewed as an opportunistic pathogen. Studies of experimental P. pneumotropica suggest that it may complicate pneumonias due to Mycoplasma pulmonis or Sendai virus. It also has been associated with suppurative or exudative lesions of the eye, conjunctiva, skin, mammary glands, and other tissues, especially in immunodeficient mice or in mice with a predisposing primary infection. Epizootiology.

Pasteurella pneumotropica is a ubiquitous inhabitant of the skin, upper respiratory tract, and gastrointestinal tract of mice. Litters from infected dams can become infected during the first week after birth.

Pathology.

Infections can cause suppurative inflammation, which may include abscessation. Dermatitis, conjunctivitis, dacryoadenitis, panophthalmitis, mastitis, and infections of the bulbourethral glands have been attributed to P. pneumotropica. Preputial and orbital abscesses also occur, especially in athymic mice. Its role in metritis is unclear, but it has been cultured from the uterus, and there is some evidence that it may cause abortion or infertility. Cutaneous lesions can occur without systemic disease. They include suppurative lesions of the skin and subcutaneous tissues of the shoulders and trunk.

Differential diagnosis.

Pseudomoniasis must be differentiated from other bacterial septicemias that may occur in immunodeficient mice. These include, but are not limited to, corynebacteriosis, salmonellosis, colibacillosis, staphylococcosis, and Tyzzer's disease.

Prevention and control.

Infection can be prevented by acidification or hyperchlorination of the drinking water (Homberger et al., 1993). These procedures will not, however, eliminate established infections. Entry of infected animals can be prevented by surveillance of commercially procured colonies. Maintenance of Pseudomonas-free animals usually requires barrierquality housing and husbandry.

Research complications.

Pseudomonas infection is not a substantial threat to immunocompetent mice but can complicate experimental studies by causing fatal septicemia in immunodeficient mice. Virus infections that alter host defense mechanisms, such as cytomegalovirus, may enhance susceptibility to pseudomoniasis. Pasteurella pneumotropica infection (Lindsey et al., 1991e) Etiology.

Pasteurella pneumotropica is a short, gram-

negative rod.

Diagnosis.

Diagnosis requires isolation of the organism on standard bacteriological media. However, infection can be detected serologically by ELISA (Wullenweber-Schmidt, 1988; Boot et al., 1995a,b). PCR assays also are available (Wang et al., 1996; Weigler et al., 1996).

Differential diagnosis.

Suppurative lesions in mice may be caused by other bacteria, including Staphylococcus, Streptococcus, Corynebacterium, Klebsiella, and Mycoplasma.

Treatment. Antibiotic therapy has not been highly successful, although a recent report indicates that enrofloxacin (25.5-85 mg/kg) in the drinking water for 2 weeks may be effective in eliminating infection (Goelz et al., 1996). Prevention and control.

Because P. pneumotropica is an opportunistic organism, it should be excluded from colonies containing immunodeficient mice and from breeding colonies. Achieving this goal will normally require barrier housing supported by sound microbiological monitoring. Rederivation should be considered to eliminate infection in circumstances where infection presents a potential threat to animal health or experimentation. Additionally, prophylactic administration of

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trimethoprim sulfa (50-60 mg/kg) in the drinking water has been shown to prevent infection in immunodeficient mice (J. D. Macy, personal communication, 2000).

Research complications. Clinically severe infection in immunodeficient mice is the major complication. g.

Helicobacteriosis

Helicobacteriosis appears to be a common infection of laboratory mice. It is caused by a growing list of organisms that vary in clinical, pathologic, and epidemiologic significance (Fox and Lee, 1997). Because recognition and investigation of helicobacteriosis is relatively new, many important questions about the impact on mice remain to be answered. Helicobacter hepaticus infection is emphasized here, because it is among the most prevalent causes of helicobacteriosis and has been studied more extensively than other murine enterohepatic Helicobacter spp. (Fox et al., 1994; Ward et al., 1994). However, current information about other murine helicobacters is summarized in the concluding section.

Etiology. Helicobacter is a gram-negative, microaerophilic, curved to spiral-shaped organism that has been isolated from the gastrointestinal mucosa of many mammals, including humans and mice. To date, the genus includes 20 formally named Helicobacter spp. assigned on the basis of 16S rRNA analysis complemented by biochemical and morphological characteristics (Fox, 2000). The organisms can be grown on freshly prepared antibiotic impregnated blood agar or in broth supplemented with fetal bovine serum in a microaerobic atmosphere (5% CO2, 90% N2, 5% H2) (Fox and Lee, 1997). Eight Helicobacter species have been isolated from laboratory rodents. Species isolated from mice include H. hepaticus, H. bilis (which also infects rats), H. muridarum, "H. rappini," and H. rodentium (named formally), and H. 'typhlonius' has been recently named (Fox and Lee, 1997; Franklin, et al., 2001). These organisms are most commonly urease-, catalase-, and oxidase-positive. However, H. rodentium, H. 'typhlonicus,' and another novel Helicobacter sp. are urease-negative. Clinical signs. Helicobacteriosis in adult immunocompetent mice is usually asymptomatic. Liver enzymes are elevated in H. hepaticus-infected A/J mice (Fox et al., 1996). Infection of immune-dysregulated mice with H. hepaticus can cause inflammatory bowel disease, which may present as rectal prolapse and/or diarrhea. Epizootiology. Recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barrier-maintained mouse colonies (Shames et al., 1995; Fox et al., 1998). Furthermore, H. hepaticus (and probably

other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and is readily detected in feces. These results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies.

Pathology. Helicobacter spp. colonize the crypts of the lower bowel, where, depending on host genotype, the organisms can be pathogenic or nonpathogenic. Helicobacter hepaticus, for example, can cause inflammation in the gastrointestinal tract, which is expressed as inflammatory bowel disease (IBD) in immunodeficient mice or typhlitis in A/J mice (Fig. 40) (Ward et al., 1996). Thickening of the cecum and large bowel develops because of proliferative typhlitis, colitis, and proctitis, which can occur without coincident hepatitis. Helicobacters also can cause liver disease. Bacterial translocation is thought to occur and results in colonization of the liver and progressive hepatitis (Fig. 41). It is characterized by angiocentric nonsuppurative hepatitis and hepatic necrosis. Inflammation originates in portal triads and spreads to adjacent hepatic parenchyma. Hepatic necrosis also may occur adjacent to intralobular venules, which can contain microthrombi. Additionally, phlebitis may affect central veins. This lesion has been linked to the presence of organisms by silver stains and electron microscopy. Age-related hepatocytic proliferation can develop in infected livers, a response that is more pronounced in male mice than in female mice (Fox et al., 1996). This lesion may increasesusceptibility to hepatomas and hepatocellular carcinomas among aged male A/JCr and B6C3F1 mice from infected colonies. An increased incidence of hepatic haemangiosarcomas also has been noted in H. hepaticus-infected male B6C3F1 mice. In this context, A/JCr, C3H/HeNCr, and SJL/NCr mice are susceptible to hepatitis, whereas C57BL/6 mice are resistant (Ward et al., 1994). The finding of severe liver disease and tumor induction in B6C3F1 mice infected with H. hepaticus infers that genetic susceptibility to H. hepaticus-induced neoplasia has a dominant pattern of inheritance. Recent studies with H. hepaticus in recombinant inbred mice also indicate that disease susceptibility has multigenetic properties (Hailey, 1998; Fox and Lee, 1997; Ihrig et al., 1999). Diagnosis. Rapid generic diagnosis can be accomplished by PCR detection of the highly conserved 16S rRNA region of the Helicobacter genome in feces or tissues, using suitable oligonucleotide primers (Shames et al., 1995). However, PCR does not differentiate among H. hepaticus, H. bilis, H. 'typhlonicus,' H. muridarum, and "H. rappini." Molecular speciation can be accomplished by retriction fragment length polymorphism analysis of the PCR product. This procedure requires suitable skill and experience to avoid technological pitfalls and should be performed by qualified laboratories. An IgG ELISA using outer membrane protein as the antigen shows promise for serological diagnosis. As noted above, helicobacters can be isolated

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3. BIOLOGY AND DISEASES OF MICE

Fig. 40. Inflammatorybowel disease associated with Helicobacter hepaticus in a SCID mouse reconstituted with CD45RB high CD4+ T cells.

on antibiotic-impregnated blood agar under microaerobic conditions and can then be speciated biochemically. Isolation of H. hepaticus from feces should be preceded by passing slurried samples through a 0.45 m filter before plating. If infection with larger helicobacters (H. bilis, "H. rappini") is suspected, filtration at 0.65 ~tm is preferred. Helicobacters grow slowly and re-

quire prolonged incubation of cultures (up to 3 weeks) before they can be deemed negative. Signs (rectal prolapse) and lesions (hepatitis, typhlocolitis), depending on host genotype, can be suggestive of infection. Histopathological examination should include silver stains, especially of liver, to attempt to visualize spiral or curved organisms (Fox and Lee, 1997).

Fig. 41. Hepatitiscaused by Helicobacter hepaticus in an A/JCr male mouse.

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Differential diagnos&. Clinically apparent helicobacteriosis must be differentiated from other gastrointestinal or hepatic infections of mice. Coronavirus infection, Clostridium piliforme, and Salmonella spp. can cause enterocolitis and/or hepatitis. Citrobacter rodentium also causes colonic hyperplasia, which can present as rectal prolapse. Infections caused by other helicobacters of mice. Helicobacter bilis has been isolated from the livers and intestines of aged mice and experimentally induces inflammatory bowel disease (IBD) in SCID mice as does H. hepaticus. Helicobacter muridarum colonizes the ileum, cecum, and colon. It appears to be nonpathogenic, although it can colonize the stomach of mice and induce gastritis under certain circumstances. Helicobacter "rappini" has been isolated from the feces of mice without clinical signs. Helicobacter rodentium also colonizes the intestine and may be a component of normal flora. A dual infection of H. bilis and H. rodentium was noted in a natural outbreak of IBD in immunocompromised mice (Shomer et al., 1998). A novel urease negative helicobacter, which has been named H. 'typhlonius', causes IBD in ILl0 -/- and SCID mice (Fox et al., 1999; Franklin et al., 1999, 2001). Prevention and control. Eradication of infection from small numbers of mice, such as quarantine groups, can be achieved by standard rederivation or intensive antibiotic therapy. The best results have been obtained by triple therapy with amoxicillin, metranidazole, and bismuth given for 2 weeks (Foltz et al., 1996). This strategy requires repeated daily gavage rather than administration in drinking water, but it has successfully eliminated H. hepaticus from naturally infected mice. Wide-scale, eradication of enzootic helicobacteriosis can be expensive and time-consuming, without guarantee of success. Careful husbandry procedures can limit infection within a colony (Whary et al., 2000). Therefore, strategies have to be weighed carefully against risks of enzootic infection for the health and use of mice. By contrast, infection should be avoided in immunodeficient mice, including genetically engineered mice with targeted or serendipitous immune dysfunction. Lastly, the outcome of opportunistic helicobacteriosis has not been thoroughly examined. This condition could occur during simultaneous infection with two or more Helicobacter species or during combined infection with an intestinal virus (e.g., coronavirus) and Helicobacter spp. If highly valuable animals are exposed, antibiotic therapy or rederivation may be warranted. Research complications. Chronic inflammation of the liver and or gastrointestinal tract may be injurious to health. Additionally, it may impede the development and assessment of noninfectious disease models, such as IBD models in mice with targeted deletions in T-lymphocyte receptors (Fox et al., 2000). Helicobacter hepaticus infections provoke a strong Thl pro-

ROBERT O. JACOBu JAMES G. FOX, AND MURIEL DAVISSON

inflammatory response, which may perturb other immunological responses. Helicobacter hepaticus infection also has been incriminated as a cofactor or promoter in the development of hepatic neoplasia in A/JCr and B6C3F1 mice (Hailey, et al., 1998; Fox, et al., 1998).

h.

Salmonellosis (Ganaway, 1982; Lindsey et al., 1991f)

Etiology. There are approximately 2400 known serotypes of Salmonella choleraesuis, with serotypes S. enteritidis and S. typhimurium constituting the most frequent isolates from mice. Salmonella enteritidis is a motile, gram-negative rod that rarely ferments lactose. Clinical signs. Acute infection is especially severe in young mice (Casebolt and Schoeb, 1988). It is characterized by anorexia, weight loss, lethargy, dull coat, humped posture, and occasionally conjunctivitis. Gastroenteritis is a common sign, but feces may remain formed. Subacute infection can produce distended abdomens from hepatomegaly and splenomegaly. Chronic disease is expressed as anorexia and weight loss. Enzootic salmonellosis in a breeding colony can produce epsiodic disease with alternating periods of quiescence and high mortality. The latter can be associated with diarrhea, anorexia, weight loss, roughened hair coat, and reduced production. Epizootiology. Modern production and husbandry methods have reduced the importance of salmonellosis as a natural infection of mice. However, the organisms are widespread in nature. Therefore, cross-infection from other species or from feral mice remains a potential hazard. Salmonellas are primarily intestinal microorganisms that can contaminate food and water supplies. Infection occurs primarily by ingestion. Vermin, birds, feral rodents, and human carriers are potential sources of infection. Other common laboratory species such as nonhuman primates, dogs, and cats also can serve as carriers. Conversely, murine salmonellosis presents a zoonotic hazard to people. The induction and course of infection are influencedby the virulence and dose of the organism; route of infection; host sex and genetic factors; nutrition; and intercurrent disease. Suckling and weanling mice are more susceptible to disease than mature mice. Immune deficiency, exposure to heavy metals, and environmental factors such as abnormal ambient temperatures can increase the severity of disease. Nutritional iron deficiency has an attenuating effect on Salmonella infection in mice, whereas iron overload appears to promote bacterial growth and enhance virulence. Resistance to natural infection is increased by the presence of normal gastrointestinal microflora. Resistance to infection also can be an inherited trait among inbred strains. Among the most important considerations is that mice that recover from acute infection can become asymptomatic carriers and a chronic source of contamination from fecal shedding.

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Pathology.

The virulence of S. enteritidis depends on its ability to penetrate intestinal walls, enter lymphatic tissue, multiply, and disseminate. Organisms reach Peyer's patches within 12 hr after inoculation and spread quickly to the mesenteric lymph nodes. Bacteremia results in spread to other lymph nodes, spleen, and liver within several days. In chronic infections, organisms persist in the spleen and lymph nodes as well as in the liver and gallbladder and from the latter are discharged into the intestinal contents. Bacteria reaching the intestine'can reinvade the mucosa and can be shed intermittently in the feces for months. Salmonella enteritidis infection also has been associated with chronic arthritis. Acute deaths may occur without gross lesions, but visceral hyperemia, pale livers, and catarrhal enteritis are more common. If mice survive for up to several weeks, the intestine may be distended and reddened, while the liver and spleen are enlarged and contain yellow-gray foci of necrosis. Affected lymph nodes are also enlarged, red, and focally necrotic. Focal inflammation can develop in many organs, including the myocardium (Percy and Barthold, 200 lb). Histologic lesions reflect the course of disease and the number of bacteria in affected tissues. During acute infection, necrotic foci are found in the intestine, mesenteric lymph nodes, liver, and spleen. Neutrophilic leukocytes and histiocytes accumulate in lymphoid tissues. Thrombosis from septic venous embolism may occur, especially in the liver. Granulomatous lesions are particularly characteristic of chronic salmonellosis, especially in the liver.

Diagnosis.

Diagnosis is based on isolation of salmonellas together with documentation of compatible clinical signs and lesions. In mice with systemic disease, bacteria may persist in the liver and spleen for weeks. During acute stages, bacteria can also be isolated from the blood. Asymptomatically infected animals can be detected by fecal culture using selective enrichment media, but culture of the mesenteric lymph nodes may be more reliable because fecal shedding can be intermittent. Isolates can be speciated with commercial serotyping reagents. Alternatively, isolates can be sent to a reference laboratory for confirmation. Antibodies to salmonellas can be detected in the serum of infected mice by an agglutination test. However, this method is not entirely reliable, because serological crossreactivity is common even among bacteria of different genera.

Differential diagnosis.

Salmonellosis must be differentiated from other bacterial diseases, including Tyzzer's disease, pseudomoniasis, corynebacteriosis, murine colonic hyperplasia, and pasteurellosis. Viral infections that cause enteritis or hepatitis must also be considered, especially infections caused by coronavirus, ectromelia virus, and reoviruses. Among noninfectious conditions, mesenteric lymphadenopathy is an aging-associated lesion in mice and is not indicative of chronic salmonellosis.

Prevention and control.

Salmonellosis can be prevented by proper husbandry and sanitation. Contact between mice and potential carriers, such as nonhuman primates, dogs, and cats, should be prevented. Diets should be cultured periodically to check for inadvertent contamination. Contaminated colonies should be replaced to eliminate infection and its zoonotic potential.

Research complications.

Apart from the clinical manifestations, the zoonotic potential for salmonellosis is a major concern. This includes transmission among laboratory species, but especially between mice and the people working with them.

i. Streptobacillosis (Lindsey et al., 1991g) Etiology.

Streptobacillus moniliformis is a nonmotile, gramnegative, pleomorphic rod that can exist as a nonpathogenic L phase variant in vivo. However, it can revert to the virulent bacillus form. Clinical signs. Streptobacillosis generally has an acute phase with high mortality, followed by a subacute phase and finally a chronic phase that may persist for months. Signs of acute disease include a dull, damp hair coat and keratoconjunctivitis. Variable signs include anemia, diarrhea, hemoglobinuria, cyanosis, and emaciation. Cutaneous ulceration, arthritis, and gangrenous amputation may occur during chronic infection. The arthritis can leave joints deformed and ankylosed. Hindlimb paralysis with urinary bladder distention, incontinence, kyphosis, and priapism may occur if vertebral lesions impinge on motor nerves. Breeding mice may have stillbirths or abortions.

Epizootiology.

Streptobacillosis has historical importance as a disease of mice, but modern husbandry, production, and health care strategies have reduced its impact dramatically (Wullenweber, 1995). Asymptomatic, persistently infected rats are the most likely source of dissemination to mice, but mouseto-mouse transmission can follow. Transmission may occur from aerogenic exposure, bite wounds, or contaminated equipment, feed, or bedding. Streptobacillus moniliformis also is pathogenic for humans, causing rat bite fever (Haverhill fever).

Pathology.

During acute disease, necrotic lesions develop in thoracic and abdominal viscera, especially in liver, spleen, and lymph nodes. Histological lesions include necrosis, septic thrombosis of small vessels, acute inflammation, fibrin deposition, and abscesses. Chronically infected mice may develop purulent polyarthritis because of the organism's affinity for joints.

Diagnosis.

Diagnosis depends on clinical and pathological evidence of septicemia and isolation of the organism. The organism has been recovered from joint fluid as long as 26 months

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after infection. Isolation from chronic lesions requires serumenriched medium.

Differential diagnosis. Clinical signs must be differentiated from septicemic conditions, including mousepox, Tyzzer's disease, corynebacteriosis, salmonellosis, mycoplasmosis, pseudomoniasis, and traumatic lesions. Prevention and control. Control is based on exclusion of wild rodents or carrier animals such as latently infected laboratory rats. Bacterins and antibiotic therapy are not adequately effective. The potential for cross-infection is a reason not to house rats and mice in the same room. Research complications. Infection can be disabling or lethal in mice and has zoonotic potential for humans. Corynebacteriosis (Lindsey et al., 1982; Weisbroth, 1994) Etiology. Corynebacteria are short gram-positive rods. Corynebacterium kutscheri is the cause of pseudotuberculosis in mice and rats. Corynebacterium bovis has been associated with hyperkeratosis, especially in immunodeficient mice (Clifford et al., 1995; Scanziani et al., 1998). Clinical signs. Corynebacterium kutscheri infection is often asymptomatic in otherwise healthy mice. Active disease is precipitated by immunosuppression or environmental stresses and is expressed as an acute illness with high mortality or a chronic syndrome with low mortality. Clinical signs include inappetance, emaciation, rough hair coat, hunched posture, hyperpnea, nasal and ocular discharge, cutaneous ulceration, and arthritis. Corynebacterium bovis infection causes hyperkeratotic dermatitis. It is characterized by scaly skin, which is accompanied by alopecia in haired mice. Severe infection may cause generalized weakness (Clifford et al., 1995). Corynebacterial keratoconjunctivitis has been reported in aged C57BL/6 mice (McWilliams et al., 1993). Epizootiology. The epizootiology of corynebacteriosis is unclear. Asymptomatically infected animals are presumed to harbor organisms in the upper alimentary tract, colon, and/or respiratory tract and regional lymph nodes. Other sites include the middle ears and preputial glands. Therefore, transmission by multiple routes is possible, including the fecal-oral route. Resistance to infection appears to be under genetic control in some mouse strains. Rats are susceptible to C. kutscheri, so crossinfection to mice may occur. Corynebacterium bovis infection can be transmitted from mouse to mouse by contact. Pathology. Lesions caused by C. kutscheri develop from hematogenous spread to various internal organs and appear as

gray-white nodules in kidney, liver, lung, and other sites (Percy and Barthold, 2001c). Cervical lymphadenopathy and arthritis of the carpometacarpal and tarsometatarsal joints also may occur. Septic, necrotic lesions often contain caseous material or liquefied pus. Histologic lesions are characterized by coagulative or caseous necrosis bordered by intense neutrophilic infiltration. Colonies of gram-positive organisms can usually be demonstrated in caseous lesions. Mucopurulent arthritis of carpal, metacarpal, tarsal, and metatarsal joints are related to bacterial colonization of synovium accompanied by necrosis, cartilage erosion, ulceration, and eventually ankylosing panarthritis. Corynebacterium kutscheri is not a primary skin pathogen, but skin ulcers or fistulas follow bacterial embolization and infarction of dermal vessels. Subcutaneous abscesses have also been reported. Hyperkeratotic dermatitis caused by C. bovis is characterized grossly by skin scaliness and alopecia. Microscopically, skin lesions consist of prominent acanthosis and moderate hyperkeratosis accompanied by mild nonsuppurative inflammation (Fig. 42). Hyperkeratosis is typically more severe in glabrous mice than in haired mice. Organisms can be demonstrated in hyperkeratotic layers by Gram stain.

Diagnosis. Diagnosis depends on isolation and identification of the causative bacteria. Additionally, organisms compatible with C. kutscheri are usually demonstrable with tissue Gram stains on lesions from clinically apparent cases. Agglutination serology is available, and immunofluorescence, immunodiffusion, and ELISA tests have been reported (Boot et al., 1995b). Differential diagnosis. The caseous nature of C. kutscheriinduced lesions helps separate them from necrotic changes or abscesses caused by other infectious agents of mice. Thus, they can be differentiated from streptococcosis, mycoplasmosis, and other septicemic bacterial infections in which caseous necrosis does not occur. Because mice can sustain natural infections with Mycobacterium avium, histochemical techniques for acid-fast bacilli and appropriate culture methods for mycobacteria should be considered if nodular inflammatory lesions of the lung are detected. Hyperkeratotic dermatitis caused by C. bovis must be differentiated from scaly skin caused by low humidity in glabrous mice. Prevention and control. Because clinically apparent corynebacteriosis occurs sporadically, treatment and control are difficult. Antibiotic therapy is probably inhibitory or suppressive, but not preventive or curative. Culling of clinically ill animals may be useful for conventional colonies but is of little use for immunodeficient mice or for mice that will endure significant stress during experimentation. Replacing or rederiving infected colonies in a specific pathogen-free environment can be effective in eliminating and preventing reinfection.

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3. BIOLOGY AND DISEASES OF MICE

Fig. 42. Hyperkeratosisassociatedwith Corynebacteriumbovis infection. Research complications. Corynebacteriosis is a potential threat for morbidity and mortality, especially among immunodeficient mice. Dermatologic disease in suckling mice can be fatal but is less severe and transient in weanling mice. Staphylococcosis (Besch-Williford and Wagner, 1982; Lindsey et. al., 1991h; Shimizu, 1994) Etiology. Staphylococci are gram-positive organisms that commonly infect skin and mucous membranes of mice and other animals. The two most frequently encountered species are Staphylococcus aureus, which can be highly pathogenic, and S. epidermidis, which is generally nonpathogenic. Species subtypes are identified by phage typing and biochemistry. Pathogenic staphylococci are typically coagulase-positive. Clinical signs. Staphylococcosis causes suppurative dermatitis in mice. Some evidence suggests that staphylococci can produce primary cutaneous infections, but they are more likely opportunistic organisms that induce lesions after contamination of skin wounds. Eczematous dermatitis develops primarily on the face, ears, neck, shoulders, and forelegs and can progress to ulcerative dermatitis, abscessation (including botryomycotic granulomas), and cellulitis. Because lesions are often pruritic, scratching causes additional trauma and autoinoculation. Staphylococcal infection in the genital mucosa of males may produce preputial gland abscesses. These occur as firm, raised nodules in the inguinal region or at the base of the penis and may rupture to spread infection to surrounding tissues. Male mice also

may develop septic balanoposthitis secondary to penile selfmutilation. Retrobulbar abscesses caused by S. aureus are frequently noted in athymic mice.

Epizootiology. Staphylococci are ubiquitous and can be carried on the skin and in the nasopharnx and gastrointestinal tract. They also can be cultured from cages, room surfaces, and personnel. The prevalence of staphylococcal dermatitis appears to be influenced by host genotype, the overall health of the animal, and the degree of environmental contamination with Staphylococcus spp. C57BL/6, C3H, DBA, and BALB/c mice seem to be the most susceptible strains. Age may also influence susceptibility, with young mice being more susceptible than adults. Immunodeficient mice (e.g., athymic mice) contaminated with staphylococci often develop abscesses or furunculosis (Fig. 43). As noted above, behavioral dysfunction resulting in selfmutilation, including scratching and trichotillomania, is a likely predisposing factor. Once virulent staphylococci contaminate the environment, colonization of the gastrointestinal tract can occur and produce a carrier state. Phage typing can help to determine the source of infection. Human phage types of staphylococci can infect mice, but the zoonotic importance of this connection is not clear. Pathology. Gross lesions are typified by suppurative or ulcerative dermatitis involving the head and neck but may extend to the shoulders and forelegs (Percy and Barthold, 2001d). Superficial or deep abscesses may occur in conjunction with dermatitis or separately, as, for example, in the external male

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ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

caused by [3-hemolytic organisms in Lancefield's group C, but epizootics caused by group A and group D streptococci have occurred, and group G organisms have been isolated occasionally. However, c~-hemolytic streptococci can cause systemic disease in SCID mice, and group B Streptococcus infection has been reported to cause meningoencephalitis in athymic mice (Schenkman et al., 1994). Additionally, Streptococcus equisimilis has been isolated from visceral abscesses of immunocompetent mice (Greenstein et al., 1994).

Fig. 43. Furunculosisin an athymicmouse. genitalia. Histologically, acute skin infections result in ulceration with neutrophils in the dermis and subcutis. There also may be regional lymphadenitis. Chronic lesions contain lymphocytes, macrophages, and fibroblasts. Deep infections appear as coalescing botryomycotic pyogranulomas with necrotic centers containing bacterial colonies.

Diagnosis. Diagnosis is made by documenting gross and histological lesions, including Gram staining of suspect tissues, complemented by isolation of gram-positive, coagulasepositive cocci. Differential diagnosis. Staphylococcosis must be differentiated from other suppurative infections of mice, including pasteurellosis, streptococcosis, corynebacteriosis, and pseudomoniasis. Ectoparasitism, fight wounds, and self-mutilation per se should also be considered. Prevention, control and treatment. Removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. Affected animals may be helped by nail trimming to reduce self-inflicted trauma. Conditions that facilitate aggressive or self-mutilating behavior should be avoided. Research complications. Staphylococcosis can cause illness and disfigurement in mice. Immunodeficient mice are at increased risk for these conditions. Streptococcosis (Lindsey et al., 1991j; Nakagawa and Weyant, 1994) Etiology. Streptococci are ubiquitous gram-positive organisms. Most streptococcal infections in laboratory mice are

Clinical signs and pathology. Cutaneous infections can cause ulcerative dermatitis over the trunk, which may appear gangrenous, whereas systemic infections may be expressed as conjunctivitis, rough hair coat, hyperpnea, somnolescence, and emaciation. Systemic lesions reflect hematogenous dissemination and include abscessation, endocarditis, splenomegaly, and lymphadenopathy (Percy and Barthold, 2001e). Streptococcal cervical lymphadenitis can lead to fistulous drainage to the neck complicated by ulcerative dermatitis. Infection with ahemolytic streptococci can cause inflammatory lesions affecting kidney and heart. Epizootiology. Mice can carry streptococci asymptomatically in their upper respiratory tracts. Lethal epizootics can occur, but factors leading to clinical disease are unknown, although some infections may be secondary to wound contamination. Diagnosis. Diagnosis and differential diagnosis depend on isolation of organisms from infected tissues, combined with histopathologic confirmation. Research complications. Immunodeficient mice are at increased risk for streptococcosis. m.

Colibacillosis

Escherichia coli is a small gram-negative rod that is a normal inhabitant of the mouse intestine. Infection is considered nonpathogenic in immunocompetent mice. However, hyperplastic typhlocolitis resembling transmissible murine colonic hyperplasia has been reported in SCID mice infected with a nonlactose-fermenting Escherichia coli (Waggie et al., 1988). Affected mice develop fecal staining and move slowly. Gross lesions consist of segmental thickening of the colon or cecum, which may contain blood-tinged feces. Microscopically, affected mucosa is hyperplastic and may be inflamed and eroded. Diagnosis depends on demonstrating lesions and isolating non-lactose-fermenting E. coli. This condition must be differentiated from proliferative and inflammatory intestinal disease caused by Citrobacter rodentium or by enterotropic mouse hepatitis virus, especially in immunodeficient mice. Colibacillosis provides an example of the morbidity associated with a

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3. BIOLOGY AND DISEASES OF MICE

nominally innocuous organism when it affects an immunocompomised host.

n.

Klebsiellosis

Klebsiella pneumoniae is a ubiquitous gram-negative organism that is a natural inhabitant of the mouse alimentary tract. It can be pathogenic for the respiratory and urinary tract of mice after experimental inoculation but is not a significant cause of naturally occurring disease. Klebsiella oxytoca, among other organisms, has been found in mice with suppurative endometritis, salpingitis, and oophoritis but did not elicit this condition after experimental inoculation of mice. o.

Clostridium Infection

Clostridia are large, rod-shaped, gram-positive anaerobic bacteria. Naturally occurring clostridial infection in mice is rare. Epizootics of Clostridium perfringens type D infection with high mortality have been reported in a barrier colony where heavy mortality occurred in 2- to 3-week-old suckling mice. Clinical signs included scruffy hair coats, paralysis of the hindquarters, and diarrhea or fecal impaction. However, attempts to reproduce the disease experimentally with clostridia isolated from naturally infected animals were unsuccessful. Clostridium perfringens also has been isolated from sporadic cases of necrotizing enteritis in recently weaned mice.

p.

Mycobacteriosis

Two mycobacteria are known to be pathogenic for laboratory mice: Mycobacterium avium-intracellulare and M. lepraemurium. Both are gram-positive, acid-fast, obligate intracellular bacteria. Infection with M. avium-intracellulare should be considered extremely rare, with the only published report describing an episode in a breeding colony of C57BL/6 mice (Waggie et al., 1983). The source of the outbreak was presumed to be drinking water. Clinical signs did not occur, but mice developed granulomatous pneumonia, which, in some mice, included Langhans' giant cells. Other lesions included microgranulomas in the liver and lymph nodes. Acid-fast bacilli were demonstrated in some lesions. Mycobacteria are widespread in water and soil. Their presence in laboratory mice would indicate a significant break in husbandry practices. Mycobacterium lepraemurium has been isolated from healthy laboratory mice and can persist as a latent infection, but its significance is primarily historical, as a model for human leprosy. It is highly unlikely to encounter this infection in a modern, well-managed mouse colony. If clinically apparent infection does occur, it is expressed as a chronic granulomatous disease. Clinical signs include alopecia, thickening of skin, subcutaneous swellings, and ulceration of the skin. Disease can

lead to death or clinical recovery. Gross lesions are characterized by nodules in subcutaneous tissues and in reticuloendothelial tissues and organs (lung, spleen, bone marrow, thymus, and lymph nodes). Lesions can also occur in lung, skeletal muscle, myocardium, kidneys, nerves, and adrenal glands. The histologic hallmark is perivascular granulomatosis with accumulation of large, foamy epithehoid macrophages (lepra cells) packed with acid-fast bacilli.

q.

Proteus Infection

Proteus mirabilis is a ubiquitous gram-negative organism that can remain latent in the respiratory and intestinal tracts. Clinical disease can occur following stress or induced immunosuppression. Immunodeficient mice have a heightened susceptibility to pathogenic infection. Proteus has been associated with ulcerative lesions in the gastrointestinal tract of immunodeficient mice. Infected animals lose weight, develop diarrhea, and die within several weeks. If septicemia develops, suppurative or necrotic lesions, including septic thrombi, may be found in many organs, but the kidney is commonly affected. Proteus pyelonephritis is characterized by abscessation and scarring. Ascending lesions may occur following urinary stasis, but hematogenous spread cannot be ruled out. Proteus mirabilis and Pseudomonas aeruginosa have been isolated concomitantly from cases of suppurative nephritis or pyelonephritis. Infection in immunodeficient mice is typified by splenomegaly and focal necrotizing hepatitis. Pulmonary lesions include edema and macrophage activation. Septic thrombi can occur, however, in many tissues. r.

Leptospirosis

Leptospirosis is exceedingly rare in laboratory mice. Infection with Leptospira interrogans serovar ballum has been reported on several occasions. It is a gram-negative organism that, after a septicemic phase, establishes persistent infection in the renal tubules and is excreted in the urine. Infection is asymptomatic and causes no significant lesions. Therefore, diagnosis requires isolation of organisms in kidney culture. Serological testing should be used with caution because neonatal exposure can lead to persistent infection without seroconversion. Histologic examination of kidney using silver stains can also be attempted. Persistent murine infections associated with active shedding present a zoonotic hazard for humans; therefore infected mice should be discarded. Elimination of infection from highly valuable mice requires rederivation. 3.

Rickettsial and Chlamydial Diseases (Hildebrandt, 1982)

a.

Rickettsia Infection

Etiology. Two rickettsia, Eperythrozoon coccoides and Hemobartonella muris, are known to infect mice. Eperythrozoon

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coccoides is primarily an organism of mice, whereas H. muris can infect mice but is more commonly associated with rats. Eperythrozoon occurs as a ring-shaped, coccoid, or occasionally rod-shaped organism that occurs in blood either attached to erythrocytes or free in plasma. It is enclosed by a single limiting membrane but has no cell wall and no nucleus or other membrane-bound organelles. Clinical signs. Mice infected with E. coccoides may remain clinically normal or develop febrile, hemolytic anemia and splenomegaly, which can be fatal. Hepatocellular degeneration and multifocal necrosis have been recorded in acute infections. Rickettsial infections are long-lived and are expressed clinically in one of two ways: acute febrile anemia and latent or asymptomatic infection that can be reactivated by splenectomy. The carrier state may be lifelong. Epizootiology. The primary natural vector of E. coccoides, historically, was the mouse louse, Polyplax serrata. Therefore infection was associated with primitive housing and husbandry' conditions that no longer occur in modern vivaria. Although the risks for infection have been reduced substantially by modern animal care procedures, E. coccoides can be transmitted to mice from contaminated biological products such as transplantable tumors or blood plasma. Diagnosis. Splenectomy or inoculation of test material into splenectomized mice is the most sensitive means to detect E. coccoides infection. These procedures provoke rickettsemia, usually within 2 - 4 days. Because rickettsemia may be transient, blood smears stained by the Romanowsky or indirect immunofluorescence procedure should be prepared every 6 hr beginning at 48 hr after splenectomy of index animals or inoculation of test specimens into splenectomized animals to assure that rickettsemia is not missed. Prevention and control. Treatment of E. coccoides infection is not practical. Control is based on elimination of lice and or rederivation of infected stock. If replacement animals are readily available, euthanasia is a more prudent course. Suspect biological materials destined for animal inoculation should be checked for rickettsial contamination by inoculation of splenectomized mice. Research complications. Asymptomatic infection can be reactivated by irradiation, immunosuppressive therapy, or intercurrent disease. Conversely, E. coccoides may potentiate coincident viral infections in mice. This effect has been clearly demonstrated for mouse coronavirus and has been suspected for lymphocytic choriomeningitis virus and LDV. Active infection also may suppress interferon production.

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON

b.

Chlamydia Infection

Chlamydia trachomatis is an intracellular organism that produces glycogen-positive intracytoplasmic inclusions (elementary bodies). Chlamydia trachomatis causes ocular and urogenital disease in humans. However, at least one strain, the so-called Nigg agent, is thought to be responsible for a historically noteworthy infection in mice. Natural infections are typically asymptomatic but persistent. Severe acute infection is characterized by ruffled fur, hunched posture, and labored respiration due to interstitial pneumonitis and leads to death in 24 hr. Mice dying more slowly may develop progressive emaciation and cyanosis of the ears and tail. 4.

Mycotic Diseases

a.

Pneumocystosis

Etiology. Pneumocystis carinii (Pc) is a common opportunistic organism of laboratory mice and other mammals. It had been classified initially as a protozoon, but contemporary molecular analysis of its nucleic acids and proteins places it among the fungi. Clinical signs. Infection is asymptomatic in immunocompetent mice. However, it can be clinically severe in immunodeficient mice, because an adequate complement of functional T lymphocytes is required to suppress infection (Roths et al., 1990; Shultz and Sidman, 1987; Walzer et al., 1989; Weir et aL, 1986). Infection proceeds slowly, but relentlessly in immunodeficient mice leading to clinical signs of pneumonia, usually within several months. Primary signs include dyspnea and hunched posture, which may be accompanied by wasting and scaly skin. Severe cases, such as those that occur in advanced disease in SCID mice, may be fatal. Epizootiology. Pc is a ubiquitous organism that is often present as a latent infection. Although firm prevalence data are not available, because detection methods are not simple to apply, one should assume that infection is present in mouse colonies unless ruled out by extensive surveillance. Pc isolates from different species (e.g., mice and rats) differ antigenically, but interspecies transmission can occur. Pc infection also occurs in human beings, but transmission between rodents and human beings has not been documented. Pc is transmitted aerogenically and establishes persistent, quiescent infection in the lungs of immunocompetent mice. Prenatal infection has not been demonstrated. Pathology. Pc is normally not pathogenic but can be activated by intercurrent immunosuppression. Activation fills the lung with trophic and cystic forms. Gross lesions occur in the lungs, which are often rubbery and fail to deflate (Fig. 44). Histopatho-

3. BIOLOGY AND DISEASES OF MICE

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Fig. 45. Pneumocystis pneumonia, illustrating hypercellularalveolar septa and alveoli containingproteinaceousexudate and macrophages.

that pneumonia virus of mice has been shown to accelerate the development of pneumocytosis in SCID mice (Bray et al., 1993; Roths et al., 1993).

Fig. 44. Lungfrom a mouse with Pneumocystis pneumoniathat has failed to collapse afterremoval.

Prevention and control. Pc infection is a significant disease threat to immunodeficient mice. Its widespread distribution strongly suggests that susceptible mice should be protected

logical changes are characterized by interstitial alveolitis with thickening of alveolar septa from proteinaceous exudate and infiltration with mononuclear cells (Fig. 45) (Roths et al., 1990). Alveolar spaces may contain vacuolated eosinophilic material and macrophages. Special stains are required to visualize Pc. Silver-based stains reveal round or partially flattened 3-5 mm cysts in affected parenchyma (Fig. 46). In florid cases, alveolar spaces may be filled with cysts, but cysts may be sparse in mild cases. Diagnosis. Respiratory distress in immunodeficient mice should elicit consideration of pneumocystosis. Pathologic examination of the lung, including silver methenamine staining, is essential to confirm a presumptive clinical diagnosis. Past infections of immunocompetent mice also can be detected by ELISA (Furuta et al., 1985). PCR can be used to detect active infection (Gigliotti, et al., 1993; Reddy et al., 1992) and is particularly useful for screening immunodeficient mice. Differential diagnosis. Pneumocystosis must be differentiated from viral pneumonias of mice. It is worth noting, in this regard,

Fig. 46. Pneumocystis pneumonia,illustratingPneumocystis cystsin alveoli (Gomori methenamine-silver stain).

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by microbarrier combined, where possible, with macrobarrier housing. Husbandry procedures should include proper sterilization of food, water, and housing equipment and the use of HEPA-filtered change stations. Infected colonies can be rederived by embryo transfer or cesarean methods, because infection does not appear to be transmitted in utero.

Research complications.

Pneumonia in immunodeficient mice is the major complication of Pc infection.

Dermatomycosis (Ringworm) (Besch-Williford and Wagner, 1982) Trichophyton mentagrophytes is the most common fungal agent of mice. However, infection rarely causes clinical disease. Clinical signs include sparse hair coats or well-demarcated crusty lesions, with a chalky surface on the head, tail, and legs (favus or ringworm). Skin lesions are composed of exfoliated debris, exudate, mycelia, and arthrospores with underlying dermatitis. Invasion of hair shafts is not characteristic. Diagnosis depends on effective specimen collection. Hairs should be selected from the periphery of the lesion, and hairless skin should be scraped deeply to obtain diagnostic specimens. Trichophyton mentagrophytes rarely fluoresces under ultraviolet light, and hyphae must be differentiated from bedding fibers, food particles, and epidermal debris. Histological sections should be stained with a silver stain or Schiff's reagent to reveal organisms. Trichophyton also can be cultured on Sabouraud's agar. Plates are incubated at room temperature (22~176 and growth is observed at 5-10 days. Ringworm is not easily eradicated from laboratory mice. The use of antifungal agents to treat individual mice is timeconsuming, expensive, and variably effective. Rederivation is a more prudent course. Cages and equipment should be sterilized before reuse. Concurrent infection with ectoparasites also must be considered during eradication steps. Candida albicans and other systemic mycoses are not important causes of disease in mice, but they can be opportunistic pathogens in immunodeficient mice. 5.

Parasitic Diseases

a.

Protozoal Diseases (Hsu, 1982)

i. Giardiasis Etiology. Giardia muris is a pear-shaped, flagellated organism with an anterior sucking disk. It inhabits the duodenum of young and adult mice, rats and, hamsters.

Clinical signs.

Infection is often asymptomatic, unless organisms proliferate extensively, and can cause weight loss, a rough hair coat, sluggish movement, and abdominal distension, usually without diarrhea. Additionally, immunodeficient mice may die during heavy infestation.

Epizootiology.

The contemporary prevalence of affected mouse colonies is not well documented, but surveys during the 1980s found rates exceeding 50%. Transmission occurs by the fecal-oral route. Cross-infection between mice and hamsters after experimental inoculation of organisms has been demonstrated, whereas rats were resistant to isolates from mice and hamsters (Kunstyr et al., 1992). C3H/He mice are particularly susceptible to giardiasis, whereas BALB/c and C57BL/10 mice are more resistant. Additionally, female mice appear to be more resistant to infection than male mice (Daniels and Belosevic, 1995). C57BL/6 females, for example, have lower trophozoite burdens and for a shorter interval than male mice. Females also shed cysts later than male mice. These differences may be related to a more potent humoral immune response to Giardia in female mice.

Pathology.

Gross lesions are limited to the small intestine, which may contain yellow or white watery fluid. Histopathology reveals organisms in the lumen that often adhere to microvilli of enterocytes or reside in mucosal crevices or mucus. The crypt-villus ratio may be reduced, and the lamina propria may have elevated numbers of inflammatory cells.

Diagnosis.

Diagnosis is based on detection of trophozoites in the small intestine or in wet mounts of fecal material. Organisms can be recognized in wet preparations by their characteristic rolling and tumbling movements. Ellipsoidal cysts with four nuclei also may be detected in feces. Infection also can be detected by serology (Daniels and Belosevic, 1994) and by PCR (Mahbubani et al., 1991).

Treatment, prevention, and control. Murine giardiasis can be treated by the addition of 0.1% dimetridazole to drinking water for 14 days. Prevention and control depend on proper sanitation and management, including adequate disinfection of contaminated rooms. Research complications.

Accelerated cryptal cell turnover and suppression of the immune response to sheep erythrocytes have been observed in infected mice. The potential for severe or lethal infection in immunodeficient mice was noted previously.

ii. Spironucleosis Etiology. Spironucleus muris is an elongated, pear-shaped, bilaterally symmetrical flagellated protozoan that commonly inhabits the duodenum, usually in the crypts of Lieberktihn. It is smaller than Giardia muris and lacks an anterior sucking disk.

Clinical signs. Spironucleus muris infection is usually asymptomatic in normal adult mice. It is more pathogenic, however, for young, stressed, or immunocompromised mice (Kunstyr et al., 1977). Additionally, clinical morbidity may indicate an underlying primary infection with an unrelated organism.

3. BIOLOGY AND DISEASES OF MICE

Clinically affected mice can have a poor hair coat, sluggish behavior, and weight loss. Mice at 3 - 6 weeks of age are at notably higher risk for clinically evident infection. They can develop dehydration, hunched posture, abdominal distension, and diarrhea. Severe infections can be lethal.

Epizootiology. Transmission occurs by the fecal-oral route and can occur between hamsters and mice as well as between mice. It does not appear to be transmitted between mice and rats (Schagemann et al., 1990). The most recent surveys, which are somewhat dated, indicated that prevalence rates exceeded 60% among domestic mouse colonies in the mid-1980s. There is some evidence that inbred strains vary in their susceptibility to infection and their rate of recovery (Baker et al., 1998; Brett and Cox, 1982). Pathology. Gross findings associated with infection include watery, red-brown, gaseous intestinal contents. However, it is essential to rule out primary or coinfection by other organisms before attributing such lesions to spironucleosis. Microscopically, acute disease is associated with distension of crypts and intervillous spaces by pear-shaped trophozoites and inflammatory edema of the lamina propria. Organisms can be visualized more easily with periodic acid-Schiff staining, which may reveal invasion of organisms between enterocytes and in the lamina propria. Chronic infection is associated with lymphoplasmacytic infiltration of the lamina propria and perhaps intracryptal inflammatory exudate. Diagnosis. Diagnosis is based on identification of trophozoites in the intestinal tract. They can be distinguished from Giardia muris and Tritrichomonas muris by their small size, horizontal or zigzag movements, and the absence of a sucking disk or undulating membrane. PCR-based detection also is available (Rozario et al., 1996). It is not clear whether duodenitis is a primary pathogenic effect of S. muris or represents opportunism secondary to a primary bacterial or viral enteritis. Therefore, it is prudent to search for underlying or predisposing infections.

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iii. Tritrichomoniasis Tritrichomonas muris is a nonpathogenic protozoan that occurs in the cecum, colon, and small intestine of mice, rats, and hamsters. No cysts are formed, and transmission is by ingestion of trophozoites passed in the feces. It can be detected by microscopy or by PCR (Viscogliosi et al., 1993). iv. Coccidiosis Eimeriafalciformis is a pathogenic coccidian that occurs in epithelial cells of the large intestines of mice. It was common in European mice historically but is seldom observed in the United States. Heavy infection may cause diarrhea and catarrhal enteritis. Klosiella muris causes renal coccidioisis in wild mice but is vanishingly rare in laboratory mice. Mice are infected by ingestion of sporulated sporocysts. Sporozoites released from the sporocysts enter the bloodstream and infect endothelial cells lining renal arterioles and glomerular capillaries, where schizogony occurs. Mature schizonts rupture into Bowman's capsule to release merozoites into the lumen of renal tubules. Merozoites can enter epithelial cells lining convoluted tubules, where the sexual phase of the life cycle is completed. Sporocysts form in renal tubular epithelium and eventually rupture host cells and are excreted in the urine, but oocysts are not formed. Infection is usually nonpathogenic and asymptomatic. Gray spots may occur in heavily affected kidneys and are the result of necrosis, granulomatous inflammation, and focal hyperplasia. Destruction of tubular epithelium may impair renal physiology. Diagnosis is based on detection of organisms in tissues. Prevention and control require proper sanitation and management techniques. There is no effective treatment. v. Cryptosporidiosis Cryptosporidium muris is a sporozoan that adheres to the gastric mucosa. It is uncommon in laboratory mice and is only slightly pathogenic. Cryptosporidium parvum inhabits the small intestine and is usually nonpathogenic in immunocompetent and athymic mice (Ozkul and Aydin, 1994; Taylor et al., 1999). Athymic mice may develop cholangitis and hepatitis, however, if organisms gain access to the biliary tract.

Treatment, prevention, and control. Treatment consists of adding 0.1% dimetridazole to drinking water for 14 days, as described for giardiasis. Prevention and control require good husbandry and sanitation.

vi. Entamoebiasis Entamoeba muris is found in the cecum and colon of mice, rats, and hamsters throughout the world. Organisms live in the lumen, where they feed on particles of food and bacteria. They are considered nonpathogenic.

Research complications. As with giardiasis, infection can accelerate enterocytic turnover in the small intestine. There is some evidence that infected mice may have activated macrophages that kill tumor cells nonspecifically and that infection can diminish responses to soluble and particulate antigens. Additionally, infected mice also have increased sensitivity to irradiation. Such effects should, however, be interpreted cautiously in order to rule out intercurrent viral infections.

vii. Encephalitozoonosis Encephalitozoon cuniculi is a gram-positive microsporidian that infects rabbits, mice, rats, guinea pigs, dogs, nonhuman primates, humans, and other mammals. Infection is extremely rare among laboratory mice. The life cycle of the organism is direct, and animals are infected by ingesting spores or by cannibalism. Spore cells are disseminated in the blood to the brain and other sites. Infection can last more than 1 year, and spores shed in the urine serve as a source

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of infection. Vertical transmission has not been confirmed in mice. Encephalitozoon cuniculi is an obligate intracellular parasite, but infection usually elicits no clinical signs of disease. Organisms proliferate in peritoneal macrophages by asexual binary fission. They have a capsule that accepts Giemsa and Goodpasture stains but is poorly stained by hematoxylin. Fulminating infection can cause lymphocytic meningoencephalitis and focal granulomatous hepatitis. In contrast to encephalitozoonosis in rabbits, affected mice do not develop interstitial nephritis. Infection is diagnosed by cytological examination of ascitic fluid smears, by histopathologic examination of brain tissues stained with Goodpasture stain, and ELISA serology. No effective treatment has been reported. Prevention and control require rigid testing and elimination of infected colonies and cell lines.

viii. Toxoplasmosis Toxoplasma gondii is a ubiquitous gram-negative coccidian parasite for which the mouse serves as a principal intermediate host. However, the prevalence of natural infection is negligible because laboratory mice no longer have access to sporulated cysts shed by infected cats, which were historically the major source for cross'infection. Toxoplasmosis can cause necrosis and granulomatous inflammation in the intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta, and muscles. Diagnosis is based on ELISA serology and histopathology. Control and prevention depend largely on precluding access of mice to cat feces or to materials contaminated with cat feces. Oocytes are very resistant to adverse temperatures, drying, and chemical disinfectants; therefore thorough cleaning of infected environments is required. b.

Cestodiasis (Wescott, 1982; Potkay, 1994)

Hymenolepis nana (dwarf tapeworm) infestation Etiology. Hymenolepis nana, the dwarf tapeworm, infects mice, rats, and humans. Adults are extremely small (25-40 mm) and have eggs with prominent polar filaments and rostellar hooks (Fig. 47). Clinical signs. Young adult mice are most frequently infected. Signs and lesions include weight loss and focal enteritis, but clinical disease is rare unless infestation is severe. Epizootiology. The life cycle may be direct or indirect. (Hymenolepis nana is the only cestode known that does not require an intermediate host.) The indirect cycle utilizes arthropods as intermediate hosts. Liberated oncospheres penetrate intestinal villi and develop into a cercocystis stage before reemerging into the intestinal lumen 10-12 days later. The scolex attaches to the intestinal mucosa, where the worm grows to adult size in 2 weeks. The cycle from ingestion to patency takes 2 0 - 3 0 days. Pathology. Cysticerci are found in the lamina propria of the small intestine and sporadically in the mesenteric lymph nodes, whereas adults, which have a serrated profile, are found in the lumen. Inflammation is not a feature of infection.

Fig. 47. Eggsof Hymenolepisnana. Diagnosis. Infection can be diagnosed by demonstrating eggs in fecal flotation preparations or by opening the intestine in petri dishes containing warm tap water to facilitate detection of adults. Hymenolepis nana can be differentiated from another species of rodent tapeworm, H. diminuta, by the fact that H. nana has rostellar hooks and eggs with polar filaments. However, H. diminuta requires an intermediate arthropod host, so it is rarely found in contemporary mouse colonies. Treatment, prevention, and control. Drugs recommended for treatment and elimination include praziquantel (0.05% in the diet for 5 days), albendazole, mebendazole, and thiabendazole. Although the benzimidazoles have excellent activity against cestodes and nematodes in rats, they have not been tested extensively in mice. The potential for successful treatment is high, however, because eggs do not survive well outside the host and because the prevalence of infestation is low in caged mice kept in properly sanitized facilities. Because H. nana can directly infect humans, proper precautions should be taken to avoid oral contamination during handling of rodents. Hymenolepis microstoma is found in the bile ducts of rodents and could be confused with H. nana in the mouse. However, the location of the adult as well as the large size of H. microstoma eggs compared with those of H. nana make differential diagnosis relatively simple. The mouse and the rat are intermediate hosts of the cestode Taenia taeniaformis. The definitive host is the cat. This parasite should not be found in laboratory mice housed separately from cats. c.

Nematodiasis (Wescott, 1982)

i. Syphacia obvelata (mouse pinworm) infestation Etiology. Syphacia obvelata, the common mouse pinworm, is a ubiquitous parasite of wild and laboratory mice. The rat, gerbil, and hamster are also occasionally infected. Female worms range from 3.4 to 5.8 mm in length, and male worms are smaller

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and then to a glass slide that is examined by microscopy. Aspicularis tetraptera eggs are not ordinarily found in tape preparations and are easily differentiated from eggs of S. obvelata (see below). Adult worms can be found in cecal or colonic contents diluted in a petri dish of warm tap water. They are readily observed with the naked eye or with a dissecting microscope. An ELISA also is available to detect serum antibodies to S. obvelata somatic antigens (Sato et al., 1995).

Fig. 48. Syphaciaobvelata egg. (1.1-1.5 mm). Eggs are flattened on one side and have pointed ends (Fig. 48). The nucleus fills the shell and is frequently at a larval stage when eggs are laid.

Clinical signs. Infestation is usually asymptomatic, although heavily infested mice can occasionally sustain intestinal lesions, including rectal prolapse, intussusception, enteritis, and fecal impaction. Epizootiology. Pinworm infestation is one of the most commonly encountered problems in laboratory mice. A recent national survey revealed that more than 30% of barrier colonies and about 70% of conventional colonies were affected (Jacoby and Lindsey, 1997). The epizootiological impact of pinworm infestation is increased by the airborne dissemination of eggs, which can remain infectious even after drying. The life cycle is direct and completed in 11-15 days. Females deposit their eggs on the skin and hairs of the perianal region. Ingested eggs liberate larvae in the small intestine, and they migrate to the cecum within 24 hr. Worms remain in the cecum for 10-11 days, where they mature and mate. The females then migrate to the large intestine to deposit their eggs as they leave the host. There is unconfirmed speculation that larvae may reenter the rectum. Infestation usually begins in young mice and can recur, but adult mice tend to be more resistant. Syphacia infestation often occurs in combination with Aspicularis tetraptera. Because the life cycle of Syphacia is much shorter than that of Aspicularis, the number of mice that are apt to be infected with S. obvelata is correspondingly greater. There is evidence that resistance to infestation may be mouse strain-specific (Derothe et al., 1997).

Treatment, prevention, and control. Pinworm infestation can be treated effectively by a number of regimens, which include the use of anthelmintics such as piperazine, ivermectin, and benzimidazole compounds alone or in combination (Klement et al., 1996; Le Blanc et al., 1993; Lipman et aL, 1994; Flynn et al., 1989; Wescott, 1982; Zenner, 1998). Because some of the recommended therapies have the potential for toxicity, it is prudent to keep mice under close clinical observation during treatment (Davis et al., 1999; Skopets et al., 1996; Tothet al., 2000). Prevention of reinfestation requires strict isolation because Syphacia eggs become infective as soon as 6 hr after they are laid, and they survive for weeks even in dry conditions. Strict sanitation, sterilization of feed and bedding, and periodic anthelmintic treatment are required to control infestation. The use of microbarrier cages can reduce the spread of infective eggs. Research complications. Unthriftiness and perturbation of host immune responses are the primary complications of pinworm infection. Syphacia muris is the common rat pinworm. It can potentially infest mice but is not found in well-managed colonies. It can be differentiated from S. obvelata because S. muris eggs are smaller. Treatment is the same as for pinworms of mice. ii. Aspicularis tetraptera (mouse pinworm) infection Etiology. Aspicularis tetraptera is the other major oxyurid of the mouse and may coinfest mice carrying S. obvelata. Females are 2.6-4.7 mm long, and males are slightly smaller. The eggs are ellipsoidal (Fig. 49).

Pathology. Gross lesions are not prevalent, aside from the presence of adults in the lumen of the intestine. Diagnosis. Infestation is diagnosed by demonstrating reniform-shaped eggs in the perianal area or adult worms in the cecum or large intestine. Four- to 5-week-old mice should be examined because the prevalence is higher in this age group than in older mice. Because most eggs are deposited outside the gastrointestinal tract, fecal examination is not reliable. Eggs are usually detected by pressing cellophane tape to the perineal area

Fig. 49. Aspicularistetraptera egg.

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Clinical signs. Ingested eggs hatch, and larvae reach the middle colon, where they enter crypts and remain for 4 - 5 days. They move to the proximal colon about 3 weeks after infection of the host. Because the life cycle is 10-12 days longer than in S. obvelata (see below), infestations appear in somewhat older mice; heaviest infestation is expected at 5 - 6 weeks. Infection is usually asymptomatic, but heavy loads can produce signs similar to those discussed for S. obvelata. Light to moderate loads do not produce clinical disease. Epizootiology.

As noted under S. obvelata, pinworm infestation is highly prevalent and contagious in laboratory mice. The life cycle is direct and takes approximately 23-25 days. Mature females inhabit the large intestine, where they survive from 45 to 50 days and lay their eggs. The eggs are deposited at night and are excreted in a mucous layer, covering fecal pellets. They require 6 - 7 days at 24~ to become infective and can survive for weeks outside the host.

Pathology.

See S. obvelata (Section III,A,5,c,i).

Diagnosis.

Aspicularis tetraptera eggs can be detected in the feces, and adult worms are found in the large intestine. Eggs are not deposited in the perianal area; therefore cellophane tape techniques are not useful. Treatment, prevention, and control. Measures for treatment, prevention, and control are similar to those described for S. obvelata. Because A. tetraptera takes longer to mature and because eggs are deposited in feces rather than on the host, adult

parasites are more amenable to treatment by frequent cage rotations. Immune expulsion of parasites and resistance to reinfection are hallmarks of A. tetraptera infection.

Research complications.

See S. obvelata (Section III,A,5,c,i).

d. Acariasis (Mite Infestation) (Weisbroth, 1982) Several species of mites infest laboratory mice. They include Myobia musculi, Radfordia affinis, Myocoptes musculinus and, less commonly, Psorergates simplex. The common murine mites are described below, while less frequently encountered insects are shown in Table XIII. These include the mouse mite Trichoecius romboutsi, which resembles Myocoptes and Ornithonyssus bacoti, the tropical rat mite, which can infect laboratory mice. Characteristics of specific infestations are described after a general introductory section.

Clinical signs. Mites generally favor the dorsal anterior regions of the body, particularly the top of the head, neck, and withers (areas least amenable to grooming), but in severe cases, all areas of skin can be infested (Fig. 50). Skin lesions of acariasis include pruritis, scruffiness, patchy hair loss, and, in severe cases, ulceration and pyoderma initiated or compounded by self-inflicted trauma. Epizootiology.

Ectoparasitism in mice is dominated by acariasis. A 1997 survey reported mite infestations in 15% of barrier colonies and 40% of conventional colonies (Jacoby and Lindsey, 1997). Acarids spend their entire lives on the host. Populations

Table XIIl Ectoparasites of Laboratory Mice of the Order Acarina ,

Suborder Mesostigmata

Prostigmata Family Myobiidae Subfamily Myobiinae Family Psorergatidae Family Sarcoptidae Family Demodicidae Astigmata Family Myocoptidae

Genus

Species

Common name

Ornithonyssus Ornithonyssus Liponyssoides Haemo g amasus Eulaelaps Lae laps Hae mo lae laps Haemolae laps

bacoti sylviarum sanguineus po nti g e r stabularis e c h idninus g las g o wi casalis

Tropical rat mite Northern fowl mite House mouse mite

Myobia Radfordia Psorergates Notoedres Demodex

musculi affinis simplex musculi musculi

Fur mite Fur mite Hair follicle mite

Myocoptes Trichoecius

musculinus romboutsi

Spiny rat mite

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Fig. 50. Acariasis. are limited by factors such as self-grooming, mutual grooming, the presence of hair, and immunological responses, which tend to produce hypersensitivity dermatitis. Inherited resistance and susceptibility also affect clinical expression of acariasis. Mite populations, for example, vary widely among different stocks and strains of mice housed under similar conditions.

Pathology.

Gross lesions include scaly skin, regional hair loss, abrasions, and ulcerations. Histologically, hyperkeratosis, acanthosis, and chronic dermatitis may occur. Long-standing infestation provokes chronic inflammation, fibrosis, and proliferation of granulation tissue. Ulcerative dermatitis associated with acariasis may have an allergic pathogenesis but often resuits in secondary bacterial infections. Lesions resemble allergic acariasis in other species and are associated with mast cell accumulations.

Diagnosis.

Direct observation of the hair and skin of dead or anesthetized mice is simple and straightforward. Hairs are parted with pins or sticks and examined with a dissecting microscope. Examination of young mice, prior to the onset of immune-mediated equilibrium, is likely to be more productive. Alternatively, recently euthanized mice can be placed on a black paper, and double-sided cellophane tape can be used to line the perimeter to contain the parasites. As the carcass cools, parasites will vacate the pelage and crawl onto the paper. Sealed petri dishes can also be used. Cellophane tape also can be pressed against areas of the pelt of freshly euthanatized mice and examined microscopically. Skin scrapings made with a scalpel blade can be macerated in 10% KOH/glycerin or im-

mersion oil and examined microscopically. This method has the disadvantage of missing highly motile species and low-level populations of slower-moving immature forms. It is important to remember that mite infestations may be mixed, so the identification of one species does not rule out the presence of others. Gross anatomical features facilitate differentiation of intact mites. Myocoptes has an oval profile with heavily chitinized body, pigmented third and fourth legs, and tarsal suckers (Fig. 51). Myobia and Radfordia have a similar elongated profile, with bulges between the legs. Myobia has a single tarsal claw on the second pair of legs (Fig. 52), whereas Radfordia has two claws of unequal size on the terminal tarsal structure of its second pair of legs (Fig. 53). Histopathological examination of skin is helpful for diagnosing unique forms of acariasis, such as the keratotic cysts associated with Psorergates simplex infestation.

Treatment, prevention, and control. Pharmacologic eradication with ivermectin or related compounds is costly, may cause toxic side effects, and is rarely completely effective. Therefore, this approach should be used cautiously. Because of the limitations inherent to currently available treatments, it is preferable to eliminate infestation by gnotobiotic rederivation. Control and prevention programs should be carried out on a colonywide basis, which includes thorough sanitation of housing space and equipment to remove residual eggs. Research complications.

Hypersensitivity dermatitis has the potential to confound immunological studies (Jungmann et al., 1996), especially those involving skin, and has been shown to

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neck. It is a surface dweller that feeds on superficial epidermis. Infestation can cause patchy thinning of the hair, alopecia, or erythema. Lesions can be pruritic, but ulceration has not been reported. Chronic infestations induce epidermal hyperplasia and nonsuppurative dermatitis.

Myobia musculi. This is a common mite of laboratory mice. The life cycle of Myobia can be completed in 23 days and includes an egg stage, first and second larval stage, protonymph, deutonymph, and adult. Eggs attach at the base of hair shafts and hatch in 7 - 8 days. Larval forms last about 10 days, followed by nymphal forms on day 11. Adults appear by day 15 and lay eggs within 24 hr. Myobia are thought to feed on skin secretions and interstitial fluid but not on blood. They are transmitted primarily by contact. Mite populations increase during new infestations, followed by a decrease to equilibrium in 8-10 weeks. The equilibrated population can be carried in colonies for long periods (up to years). Population fluctuations may represent waves of egg

Fig. 51. Myocoptesmusculinus male. (FromWeisbroth, 1982; courtesy of Dr. R. J. Flynn and LaboratoryAnimal Science.) elevate serum IgE (Morita et al., 1999). Heavy mite infestations can cause severe skin lesions and have been associated with weight loss, infertility, and premature deaths. Chronic acariasis also may provoke secondary amyloidosis due to long-standing dermatitis.

Additional characteristics of murine acariasis Myocoptes musculinus. This is the most common ectoparasite of the laboratory mouse but frequently occurs in conjunction with Myobia musculi. The life cycle includes egg, larva, protonymph, tridonymph, and adult stages. Eggs hatch in 5 days and are usually attached to the middle third of the hair shaft. The life cycle may range from 8 to 14 days. Transmission requires direct contact, for mice separated by wire screens do not contract infestations from infested hosts. Bedding does not seem to serve as a vector. Neonates may become infested within 4 - 5 days of birth, and parasites may live for 8 - 9 days on dead hosts. Myocoptes appears to inhabit larger areas of the body than Myobia and tends to crowd out Myobia during heavy infestations. It has some predelection for skin of the inguinal region, abdominal skin, and back, but it will also infest the head and

Fig. 52. Myobia musculi female. (From Weisbroth, 1982; courtesy of Dr. R. J. Flynn and LaboratoryAnimal Science.)

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3. BIOLOGY AND DISEASES OF MICE

by direct contact. Invasion of hair follicles leads to development of cystlike nodules, which appear as small white nodules in the subcutis. Histologically, they are invaginated sacs of squamous epithelium, excretory products, and keratinaceous debris. There is usually no inflammatory reaction, but healing may be accompanied by granulomatous inflammation. Diagnosis is made by examining the subcuticular surface of the pelt grossly or by histological examination. Sac contents also can be expressed by pressure with a scalpel blade or scraped and mounted for microscopic examination.

B. 1.

Fig. 53. Radfordiaaffiinis female. (FromWeisbroth, 1982; courtesy of Dr. R. J. Flynn and LaboratoryAnimal Science.)

hatchings. Because mites are thermotactic, they crawl to the end of hair shafts on dead hosts, where they may live for up to 4 days. Infestation may result in hypersensitivity dermatitis, to which C57BL mice are highly susceptible. Clinical signs vary from ruffled fur and alopecia to pruritic ulcerative dermatitis. Therefore, lesions can be exacerbated by self-inflicted trauma.

Radfordia ajfinis. Radfordia is thought to be common in laboratory mice, but it closely resembles Myobia and may occur as a mixed infestation. Therefore, its true prevalence is conjectural. Additionally, its life cycle has not been described. It does not appear to cause clinical morbidity. Psorergates simplex. This species has not been reported as a naturally occurring infection in well-managed colonies for several decades, but it is unique in that it inhabits hair follicles. Its life cycle is unknown, but developmental stages from egg to adult may be found in a single dermal nodule. Transmission is

Metabolic and Nutritional Diseases

Amyloidosis

Amyloidosis is caused by the deposition of insoluble (polymerized) proteins and occurs in primary and secondary forms. Primary amyloidosis is a naturally occurring disease in mice, associated with the deposition of amyloid proteins consisting primarily of immunoglobulin light chains. Secondary amyloidosis is associated with antecedent and often chronic inflammation. It results from a complex cascade of reactions involving release of multiple cytokines that stimulate amyloid synthesis in the liver (Falk and Skinner, 2000). Primary amyloidosis is common among aging mice (Lipman et al., 1993) but also may occur in young mice of highly susceptible strains such as A and SJL or somewhat later among C57BL mice. Other strains, such as BALB/c and C3H are highly resistant to amyloidosis (Dunn, 1967). Secondary amyloidosis is usually associated with chronic inflammatory lesions, including dermatitis resulting from prolonged acariasis. It can be induced experimentally, however, by injection of casein and may occur locally in association with neoplasia or in ovarian corpora lutea in the absence of other disease. Amyloidosis can shorten the life span of mice and can be accelerated by stress from intercurrent disease. Amyloid appears as interstitial deposition of a lightly eosinophilic, acellular material in tissues stained with hematoxylin and eosin. However, it is birefringent after staining with Congo red when viewed with polarized light. Deposition patterns vary with mouse strain and amyloid type. Although virtually any tissue may be affected, the following sites are common: hepatic portal triads, periarteriolar lymphoid sheaths in spleen, renal glomeruli and interstitium (which can lead to papillary necrosis), intestinal lamina propria, myocardium (and in association with atrial thrombosis), nasal submucosa, pulmonary alveolar septa, gonads, endocrine tissues, and great vessels (Fig. 54). 2.

Soft Tissue Mineralization

Naturally occurring mineralization of the myocardium and epicardium and other soft tissues is a common finding at

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Fig. 54. Renalamyloidosiswith prominent amyloiddeposition in glomeruli.

necropsy in some inbred strains of mice. It occurs in B ALB/c, C3H, and especially DBA mice (Eaton et al., 1978; Brownstein, 1983; Brunnert et al., 1999). It is found in the myocardium of the left ventricle (Fig. 55), in the intraventricular systems, and in skeletal muscle, kidneys, arteries, and lung and may be accompanied by fibrosis and mononuclear inflammatory infiltrates. DBA mice also can develop mineralization in the tongue and cornea. Dietary, environmental, disease-related, and endocrine-related factors are thought to influence the prevalence of this lesion. Although this condition is usually an incidental

finding at necropsy, interference with myocardial function cannot be ruled out if lesions are severe. 3.

A Reye's-like syndrome has been reported in BALB/cByJ mice (Brownstein et al., 1984). The etiology is unknown; however, antecedent viral infection may be involved. Affected mice rapidly become lethargic and then comatose. They also tend to hyperventilate. High mortality ensues within 6-18 hr, but some mice may recover. Lesions are characterized grossly by swollen, pale liver and kidneys. The major histopathological findings include swollen hepatocytes with fatty change and nuclear swelling among astrocytes in the brain. Hepatic lesions resembling changes in Reye's syndrome have been reported in SCID mice infected with MAdV-1 (Pirofski et al., 1991). 4.

Fig. 55. Epicardialmineralization.

Reye's-like Syndrome

Vitamin, Mineral, and Essential Fatty Acid Deficiencies (Knapka, 1983)

Vitamin deficiencies in mice have not been thoroughly described. Unfortunately, much of the information that does exist reflects work done 30-50 years ago; thus the reliability and specificity of some of these syndromes is questionable. Vitamin A deficiency may produce tremors, diarrhea, rough hair coat, keratitis, poor growth, abscesses, hemorrhages, and sterility or abortion. Vitamin E deficiency can cause convulsions and heart failure, as well as muscular dystrophy and hyaline degeneration of muscles. Deficiency of B complex vitamins produces nonspecific signs such as alopecia, decreased feed consumption,

3. BIOLOGY AND DISEASES OF MICE

poor growth, poor reproduction and lactation, as well as a variety of neurological abnormalities. Choline deficiency produces fatty livers and nodular hepatic hyperplasia, as well as myocardial lesions, decreased conception, and decreased viability of litters. Folic acid-deficient diets cause marked decreases in red and white cell blood counts and the disappearance of megakaryocytes and nucleated cells from the spleen. Pantothenic acid deficiency is characterized by nonspecific signs, such as weight loss, alopecia, achromotrichia, and posterior paralysis, as well as other neurological abnormalities. Thiamin deficiency is associated with neurological signs, such as violent convulsions, cartwheel movements, and decreased food consumption. Dietary requirements for ascorbic acid have not been shown in mice, and mouse diets are generally not fortified with ascorbic acid. Mineral deficiencies have been described only for several elements, and the consequences of the deficiencies are similar to those observed for other species. For example, iodine-deficient diets produce thyroid goiters; magnesium-deficient diets may cause fatal convulsions; manganese deficiency may cause congenital ataxia from abnormal development of the inner ear; and zinc deficiency may cause hair loss on the shoulders and neck, emaciation, decreased liver and kidney catalase activity, and immunosuppression. Chronic essential fatty acid deficiency may cause hair loss, dermatitis with scaling and crusting of the skin, and occasional diarrhea. Infertility has also been associated with this syndrome. Mice have an absolute requirement for a dietary source of linoleic and/or arachadonic acid. 5.

Alopecia and Chronic Ulcerative Dermatitis in Black Mice (Sundberg, 1994; Ward et al., 2000)

Black mice are prone to a skin condition characterized by alopecia and/or chronic ulcerative dermatitis. It is most often

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seen among C57BL/6 and C57BL/10 mice because they are used widely. Initial signs include alopecia and papular dermatitis, which usually occur over the dorsal trunk (Fig. 56). In severe cases, skin ulceration ensues, which is grounds for euthanasia, while scarring and disfigurement can occur among surviving mice. The cause is unknown. Microbiological assessments have yielded a variety of bacteria that are considered to be secondary opportunists, and acariasis has not been incriminated. Seasonal fluctuation in the incidence of disease suggests that environmental factors may play a role. The incidence appears to increase during periods of significant seasonal changes in temperature and humidity, i.e., the onset of winter and early spring. There is some evidence that incidence is related to diet, with mice on ad libitum diets being more susceptible than those on restricted diets. However, specific dietary factors have not been identified.

6.

Postpartum Ileus

Ileus associated with high mortality has been reported to occur in primiparous female mice during the second week of lactation (Kunstyr, 1986). The cause is unknown.

C.

Environmental, Behavioral, and Traumatic Disorders

Environmental variables can affect responses of mice in experimental situations. Changes in respiratory epithelial physiology and function from elevated levels of ammonia, effects of temperature and humidity on metabolism, effects of light on eye lesions and retinal function, and effects of noise on neurophysiology are examples of complications that can vary with the form of insult and the strain of mouse employed.

Fig. 56. Dorsalalopeciain a C57BL/6 mouse during an early stage of chronic uclerativedermatitis.

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1. Temperature-Related Disorders

Mice do not easily acclimatize to sudden and dramatic changes in temperature. Therefore they are susceptible to both hypothermia and hyperthermia. Mice also are susceptible to dehydration. Poorly functioning water bottles, resulting in spills (hypothermia) or obstructed sipper tubes (dehydration), are probably the major cause of these environmental insults. 2.

Ringtail

Ringtail is a condition associated with low relative humidity. Clinical signs include annular constriction of the tail and occasionally of the feet or digits, resulting in localized edema that can progress to dry gangrene (Fig. 57). It should be differentiated from dryness and gangrene that may occur in hairless mice exposed to low temperatures and perhaps other environmental or nutritional imbalances. Necrosis of legs, feet, or digits also can occur in suckling mice because of disruption of circulation by wraps of stringy nesting material such as cotton wool. 3.

Corneal Opacities

Corneal opacities can occur as a result of acute or chronic keratitis. There is some evidence that the buildup of ammonia in mouse cages may contribute to this condition, because it can be controlled by increasing the frequency of cage cleaning. 4.

Malocclusion

Malocclusion results from an inherited trait for poorly aligned incisors and results in overgrowth, especially of the lower incisors. Abscesses or necrosis may occur in the lips or oral cavity because of fighting injury or mechanical trauma from cages.

Overgrown incisor teeth can cause malocclusion, and caries can develop in molar teeth. 5.

Skin Trauma

Skin lesions can be caused by fighting, tail biting, and whisker chewing. Fighting is not limited extensively to males, but they tend to be more aggressive. Bite wounds are usually located on the head, neck, shoulders, peritoneal area, and tail. Often one animal per pen is free of lesions and is considered the aggressor or dominant animal. Removal of the unaffected male usually ends the fighting, and the wounded animals recover. However, a previously submissive male may become dominant when an aggressive male is removed, and fighting may resume. Fighting has some strain predilection, and is especially notorious among BALB/c males, but tail lesions resembling bite wounds have been reported in other strains. Hair nibbling or whisker chewing (barbering) is also a manifestation of social dominance. Dominant animals retain whiskers, whereas cagemates have "shaved faces" (Fig. 11). Chronic hair chewing can produce histological abnormalities such as poorly formed or pigmented club hairs. Once chewing has ceased, many mice regrow previously lost hair in several weeks. Both sexes may engage in this activity, and sometimes females may be dominant. Regional alopecia, especially around the muzzle, may result from abrasion against cage surfaces. Improperly diluted disinfectants may also cause regional hair loss. Metal tags used for animal identification may cause pruritis and self-induced trauma. Clipping prior to application of experimental compounds to the skin may cause pruritic responses and can augment lesions that interfere with test results. Dermatophytosis, ectoparasitism, or idiopathic hair loss must be considered in the differential diagnoses for muzzle or body alopecia.

D. Congenital, Aging-Related, and Miscellaneous Disorders (Burek et al. 1982; Percy and Barthold, 2001f) 1.

Cardiovascular System

Atrial thrombosis appears to be strain-related, with a high prevalence in RFM mice. It also is more common in aged mice. It usually involves the left atrium and auricle and may be accompanied by amyloidosis. Affected mice may display signs of heart failure, particularly severe dyspnea. Myocardial and epicardial mineralization is described above, in Section III,B,2. Periarteritis occurs in aged mice. 2.

Fig. 57. Ringtail.

Respiratory Tract

Hyperplasia of alveolar or bronchial epithelium occurs in old mice and must be differentiated from pulmonary tumors. Pulmonary histiocytosis is an incidental finding in selected strains of mice, including C57BL/6, and the incidence increases with

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aging. Histologically, alveoli are filled with macrophages containing eosinophilic crystalline material. 3.

Alimentary Tract a.

Stomach

Gastric lesions include crypt dilatation, submucosal fibrosis, adenomatous gastric hyperplasia, mineralization, and erosion or ulceration. Gastric ulcers may be stress-related, especially in mice with prolonged illness. Germfree mice may have reduced muscle tone in the intestinal tract. Cecal volvulus is a common finding in germfree mice and is caused by rotation of the large cecum. b.

Pancreas

Exocrine pancreatic insufficiency has been reported in CBA/J mice. Acinar cell atrophy is common but is strain- and sex-dependent. 4.

5.

Musculoskeletal System

Age-associated osteoporosis or senile osteodystrophy can occur in some mice. It is not associated with severe renal disease or parathyroid hyperplasia. Nearly all strains of mice develop some form of osteoarthrosis. It is generally noninflammatory, affects articulating surfaces, and results in secondary bone degeneration.

Liver

Age-associated lesions are common in the livers of mice. Cellular and nuclear pleomorphism, including binucleated and multinucleated cells, are detectable by 6 months. Mild focal necrosis occurs with or without inflammation, but an association of mild focal hepatitis with a specific infectious disease is often hard to confirm. Other geriatric hepatic lesions include biliary hyperplasia with varying degrees of portal hepatitis, hepatocellular vacuolization, amyloid deposition (especially in periportal areas), strangulated or herniated lobes, hemosiderosis, lipofuscinosis, and fibrosis. Extramedullary hematopoiesis occurs in young mice and in response to anemia. c.

bined immunodeficient (SCID) mice, which lack both T and B lymphocytes, are used widely used and are highly susceptible to opportunistic agents such as Pneumocystis carinii. Specific immune deficits have become excellent models for studying the ontogeny and mechanisms of immune responsiveness (Table XII).

Lymphoreticular System

Blood-filled mesenteric lymph nodes may occur in aged mice, especially C3H mice. This condition is an incidental finding and should not be confused with infectious lymphadenopathy such as that associated with salmonellosis. Aggregates, or nodules of mononuclear cells, are found in many tissues of aged mice, including the salivary gland, thymus, ovary, uterus, mesentery and mediastinum, urinary bladder, and gastrointestinal tract. These nodules should not be mistaken for lymphosarcomas. The spleen is subject to amyloidosis and hemosiderin deposition. Lipofuscin deposition is common, especially in older mice. The thymus undergoes age-associated atrophy. A variety of genetic immunodeficiencies have been described in mice, many of which increase susceptibility to infectious diseases. Perhaps the most widely known of these is the athymic nude mouse that lacks a significant hair coat and, more important, fails to develop a thymus and thus has a severe deficit of T cell-mediated immune function. Additionally, severe com-

6.

Urinary Tract

Glomerulonephritis is a common kidney lesion of mice. It is more often associated with persistent viral infections or immune disorders rather than with bacterial infections. Its prevalence in some strains approaches 100%. NZB and NZB X NZW F1 hybrid mice, for example, develop immune complex glomerulonephritis as an autoimmune disease resembling human lupus erythematosus, whereas glomerular disease is relatively mild in NZB mice (NZB mice have a high incidence of autoimmune hemolytic anemia). Renal changes occur as early as 4 months of age, but clinical signs and severe disease are not present until 6 - 9 months. The disease is associated with wasting and proteinurea, and lesions progress until death intervenes. Histologically, glomeruli have proteinaceous deposits in the capillaries and mesangium. Later, tubular atrophy and proteinaceous casts occur throughout the kidney. Immunofluorescence studies show deposits of immunoglobulin and the third component of complement, which lodge as immune complexes with nuclear antigens and antigens of murine leukemia virus in glomerular capillary loops. Mice infected with LCMV or with retroviruses can also develop immune complex glomerulonephritis. Mice also can develop chronic glomerulopathy characterized by progressive thickening of glomerular basement membrane by PAS-positive material that does not stain for amyloid. This lesion can be accompanied by proliferation of mesangial cells; local, regional, or diffuse mononuclear cell infiltration; and fibrosis. Advanced cases may lead to renal insufficiency or failure. Interstitial nephritis can be caused by bacterial or viral infections but may also be idiopathic. Typical lesions include focal, regional, or diffuse interstitial infiltration of tubular parenchyma by mononuclear cells, but glomerular regions also may be involved. Severe lesions can be accompanied by fibrosis, distortion of renal parenchyma, and intratubular casts, but not by mineralization. If renal insufficiency or failure ensues, it can lead to ascites.

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Some strains of mice, such as BALB/c, can develop polycystic kidney disease, which, if severe, can compromise normal renal function. Urinary tract obstruction occurs as an acute or chronic condition in male mice. Clinical signs usually include wetting of the perineum from incontinence. In severe or chronic cases, wetting predisposes to cellulitis and ulceration. At necropsy, the bladder is distended, and proteinaceous plugs are often found in the neck of the bladder and proximal urethra. In chronic cases the urine may be cloudy, and calculi may develop in the bladder. Additionally, cystitis, urethritis, prostatitis, balanoposthitis, and hydronephrosis may develop. This condition must be differentiated from infectious cystitis or pyelonephritis and from the agonal release of secretions from accessory sex glands, which is not associated with an inflammatory response. Hydronephrosis per se also may occur without urinary tract obstruction. Ascending pyelitis occurs in mice secondary to urinary tract infection. 7. Genital Tract a.

9. Nervous System Symmetrical mineral deposits commonly occur in the thalamus of aged mice. They may also be found in the midbrain, cerebellum, and cerebrum and are particularly common in A/J mice. Lipofuscin accumulates in the neurons of old mice. Ageassociated peripheral neuropathy with demyelination can be found in the nerves of the hindlimbs in C57BL/6 mice. Deposits of melanin pigment occur in heavily pigmented strains, especially in the frontal lobe. A number of neurologically mutant mice have been described. They commonly have correlative anatomical malformations or inborn errors of metabolism.

Female

Parvovarian cysts are observed frequently and may be related to the fact that mouse ovaries are enclosed in membranous pouches. Amyloidosis is also common in the ovaries of old mice. Cystic endometrial hyperplasia may develop unilaterally or bilaterally and may be segmental. In some strains, the prevalence in mice older than 18 months is 100%. Endometrial hyperplasia is often associated with ovarian atrophy. Mucometra is relatively common in adult female mice. The primary clinical sign is abdominal distension resembling pregnancy among mice that do not whelp. b.

posited in the thyroid and parathyroid glands as well as in the adrenal glands. Spontaneous diabetes mellitius occurs in genetic variants of several strains such as nonobese diabetic (NOD) mice. High levels of estrogen in pregnancy may influence postpartum hair shedding. Various endocrine effects on hair growth have also been described. Abdominal and thoracic alopecia have been reported in B6C3F1 mice.

Male

Testicular atrophy, sperm granulomas, and tubular mineralization occur with varying incidence. Inflammation of accessory sex glands may occur. Preputial glands, especially of immunodeficient mice, can become infected with opportunistic or pathogenic bacteria. 8. Endocrine System Accessory adrenal cortical nodules are found in periadrenal and perirenal fat, especially in females. These nodules have little functional significance other than their potential effect on failures of surgical adrenalectomy. Lipofuscinosis, subcapsular spindle cell hyperplasia, and cystic dilatation of cortical sinusoids are found in the adrenal cortices of aged mice. Some inbred strains have deficiencies of thyrotropic hormone, resulting in thyroid atrophy. Thyroid cysts lined by stratified squamous epithelium and generally of ultimobranchial origin may be seen in old mice. Amyloid can be de-

10. Organs of Special Sense a.

Eye

Retinal degeneration can occur as either an environmental or a genetic disorder in mice. Nonpigmented mice, both inbred and outbred, can develop retinal degeneration from exposure to light, with the progression of blindness being related to light intensity and duration of exposure. Other strains such as C3H and CBA are genetically predisposed to retinal degeneration. C3H/He mice express the rd gene, which leads to retinal degeneration within the first few weeks of life and has been used extensively as a model for retinitis pigmentosa (Farber and Danciger, 1994). Blindness does not interfere with health or reproduction and blind mice cannot be distinguished from nonblind mice housed in standard caging. Cataracts can occur in old mice and have a higher prevalence in certain mutant strains. b.

Ear

Vestibular syndrome associated with head tilt, circling or imbalance can result from infectious otitis or from necrotizing vasculitis of unknown etiology affecting small and mediumsized arteries in the vicinity of the middle and inner ear.

E. Neoplastic Diseases (Jones et al., 1983-1993; Maronpot et al., 1999; Percy and Barthold, 2001g) 1. Lymphoreticular and Hematopoietic Systems Neoplasms of lymphoid and hematopoietic tissues are estimated to have a spontaneous prevalence of 1-2%. There are,

3. BIOLOGY AND DISEASES OF MICE

however, some strains of mice that have been specifically inbred and selected for susceptibility to spontaneous tumors. Leukemogenesis in mice may involve viruses and chemical or physical agents. Viruses associated with lymphopoietic and hematopoietic neoplasia belong to the family Retroviridae (type C oncornaviruses) and contain RNA-dependent DNA polymerase (reverse transcriptase). These viruses are generally noncytopathogenic for infected cells, and mice appear to harbor them as normal components of their genetic apparatus. Although they may be involved in spontaneous leukemia, they are not consistently expressed in this disease. Recombinant viruses have recently been discovered that can infect mouse cells and heterologous cells and are associated with spontaneous leukemia development in high leukemia strains such as AKR mice. Their phenotypic expression is controlled by mouse genotype. Endogenous retroviruses are transmitted vertically through the germ line. Horizontal transmission is inefficient but can occur by interuterine infection or through saliva, sputum, urine, feces, or milk. The leukemia induced by a given endogeous virus is usually of a single histopathological type. Chemical carcinogens, such as polycyclic hydrocarbons, nitrosoureas, and nitrosamines and physical agents such as X-irradiation can also induce hematological malignancies in mice.

Lymphoblastic Lymphoma (Thymic Lymphoma, B-Cell Lymphoma) The most common hematopoietic malignancy in the mouse is lymphocytic leukemia that originates in the thymus. Disease begins with unilateral atrophy and then enlargement of one lobe of thymus as tumor cells proliferate. Cells can spread to the other lobe and then to other hematopoietic organs, such as the spleen, bone marrow, liver, and peripheral lymph nodes. Clinical signs include dyspnea and ocular protrusion. The latter sign is due to compression of venous blood returning from the head. Tumor cells spill into the circulation late in disease. Most of these tumors originate from T lymphocytes or lymphocblasts, but there are leukemias of B lymphocyte or null cell lineage. In the last two syndromes, the lymph nodes and spleen are often involved, but the thymus is generally normal.

Reticulum Cell Sarcoma (Histiocytic Lymphoma, Follicular Center Cell Lymphoma) Reticulum cell sarcomas are common in older mice, especially in inbred strains such as C57BL/6 and SJL. Primary tumor cell types have been divided into several categories based on morphological and immunohistochemical features. Histiocytic sarcomas correspond to the older Dunn classification as type A sarcomas and are composed primarily of reticulum cells. The tumor typically causes splenomegaly and nodular lesions in other organs, including liver, lung, kidney, and the female reproductive tract. Follicular center cell lymphomas correspond to Dunn type B sarcomas. They originate from B cell regions

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(germinal centers) of peripheral lymphoid tissues, including spleen, lymph nodes, and Peyer's patches. Typical tumor cells have large vesiculated, folded, or cleaved nuclei and ill-defined cytoplasmic borders. Tumors also often contain small lymphocytes. Type C reticulum cell tumors often involve one or several lymph nodes rather than assuming a wide distribution. They consist of reticulum cells with a prominent component of welldifferentiated lymphocytes.

c.

Myelogenous Leukemia

Myelogenous leukemia is uncommon in mice and is associated with retrovirus infection. Disease begins in the spleen, resuiting in marked splenomegaly, but leukemic spread results in involvement of many tissues including liver, lung, and bone marrow. Leukemic cells in various stages of differentiation can be found in peripheral blood. In older animals, affected organs may appear green because of myeloperoxidase activity, giving rise to the term chloroleukemia. The green hue fades on contact with air. Affected mice are often clinically anemic and dyspneic.

d.

Erythroleukemia

Erythroleukemia is rare in mice. The major lesion is massive splenomegaly, which is accompanied by anemia and polycythemia. Hepatomegaly can follow, but there is little change in the thymus or lymph nodes.

e.

Mast Cell Tumors

Mast cell tumors are also very rare in mice. They are found almost exclusively in old mice and grow slowly. They should not be confused with mast cell hyperplasia observed in the skin following painting with carcinogens or X-irradiation.

f.

Plasma Cell Tumors

Natural plasma cell tumors are infrequent in the mouse. They can, however, be induced by intraperitoneal inoculation of granulomatogenic agents such as plastic filters, plastic shavings, or a variety of oils. 2.

M a m m a r y Gland

Mammary tumors can be induced or modulated by a variety of factors, including viruses, chemical carcinogens, radiation, hormones, genetic background, diet, and immune status. Certain inbred strains of mice, such as C3H, A, and DBA/2, have a high natural prevalence of mammary tumors. Other strains, such as BALB/c, C57BL, and AKR, have a low prevalence. Among the most important factors contributing to the development of mammary tumors are mammary tumor viruses. Several major variants are known. The primary tumor virus

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MMTV-S (Bittner virus) is highly oncogenic and is transmitted through the milk of nursing females. Infected mice typically develop a precursor lesion, the hyperplastic alveolar nodule, which can be serially transplanted. Spontaneous mammary tumors metastasize with high frequency, but this property is somewhat mouse strain dependent. Metastases go primarily to the lung. Some mammary tumors are hormone dependent, some are ovary dependent, and others are pregnancy dependent. Ovary-dependent tumors contain estrogen and progesterone receptors, whereas pregnancy-dependent tumors have prolactin receptors. Ovariectomy will dramatically reduce the incidence of mammary tumors in C3H mice. If surgery is done in adult mice 2 - 5 months of age, mammary tumors will develop, but at a later age than normal. Grossly, mammary tumors may occur anywhere in the mammary chain. They present as one or more firm, well-delineated masses, which are often lobular and maybe cystic (Fig. 58). Histologically, mammary tumors have been categorized into three major groups; carcinomas, carcinomas with squamous cell differentiation, and carcinosarcomas. The carcinomas are divided into adenocarinoma types A, B, C, Y, L, and E Most tumors are type A or B. Type A consists of adenomas, tubular carcinomas, and alveolar carcinomas. Type B tumors have a variable pattern with both well-differentiated and poorly differentiated regions. They may consist of regular cords or sheets of cells or papillomatous areas. These two types are locally invasive and may metastasize to the lungs. Type C tumors are rare and are characterized by multiple cysts lined by low cuboidal to squamous epithelial cells, and they have abundant stroma. Type Y tumors, which are also rare, are characterized by tubular branching of cuboidal epithelium and abundant stroma. Adenocarcinomas

Fig. 58.

with a lacelike morphology (types L and P) are hormone dependent and have a branching tubular structure. The control or prevention of mammary neoplasms depends on the fact that some strains of mammary tumor virus are transmitted horizontally whereas others are transmitted vertically. Although one can rid mice of horizontally transmitted virus such as MMTV-S by cesarean rederivation or by foster nursing, endogenous strains of tumor virus may remain. Fortunately, these latter tumor viruses have generally low oncogenicity relative to the Bittner virus.

3. Liver Mice develop an assortment of liver changes as they age, including proliferative lesions. The latter can range from hyperplastic foci to hepatomas to hepatocellular carcinomas. Almost all strains of mice have a significant prevalence of hepatic tumors, some of which appear to result from dietary contamination or deficiency. The prevalence of spontaneous liver tumors in B6C3F 1 hybrids is increased by feeding choline-deficient diets. Tumors also can develop in mice exposed to environmental chemicals, many of which are carcinogenic or potentially carcinogenic. Spontaneous liver tumors in mice occur grossly as gray to tan nodules or large, poorly demarcated dark red masses. They are usually derived from hepatocytes, whereas cholangiocellular tumors are rare. Hepatomas are well circumscribed and well differentiated, but they compress adjacent liver tissue as they develop. Hepatocellular carcinomas are usually invasive and display histopathological patterns ranging from medullary to

Mammarytumorsin a C3H mouse.

3. BIOLOGY AND DISEASES OF MICE trabecular. Large carcinomas also may contain h e m o r r h a g e and necrosis. Carcinomas also may metastasize to the lungs.

4.

Lung

P r i m a r y respiratory tumors of mice occur in relatively high frequency. It has been estimated that more than 95% of these tumors are p u l m o n a r y a d e n o m a s that arise either from type 2 p n e u m o c y t e s or from Clara cells lining terminal bronchioles. P u l m o n a r y adenomas usually appear as distinct whitish nodules that are easily detected by examination of the lung surface. Malignant alveologenic tumors are infrequent and consist of adenocarcinomas and squamous cell carcinomas. They invade p u l m o n a r y p a r e n c h y m a and are prone to metastasize. The prevalence of spontaneous respiratory tumors is m o u s e straindependent. For example, the prevalence is high in aging A strain mice but low in aging C 5 7 B L mice. The n u m b e r of tumors per lung is also higher in susceptible mice. P u l m o n a r y tumors often occur as well-defined gray nodules. Microscopically, a d e n o m a s of alveolar origin consist of dense ribbons of cuboidal to c o l u m n a r cells with sparse stroma. A d e n o m a s of Clara cell origin are usually associated with bronchioles. They have a tubular to papillary architecture consisting of c o l u m n a r cells with basal nuclei. P u l m o n a r y adenocarcinomas, though comparatively rare, are locally invasive. They often form papillary structures and have considerable cellular pleomorphism.

5.

Neoplasms of Other Organ Systems

N e o p l a s m s of other organ systems are described in Jones 1 9 8 3 - 1 9 9 3 ; M a r o n p o t et al., 1999; and Percy and Barthold, 1993g. et al.,

REFERENCES

American Institute of Nutrition (AIN) (1977). Ad Hoc Committee on Standards for Nutritional Studies. Report of the Committee. J. Nutr. 107, 13401348. Austin, C. R., and Short, R. V., eds. (1982). "Reproduction in Mammals," 2nd ed., Vols. 1-5. Cambridge Univ. Press, London. Bailey, D. W. (1971). Recombinant inbred strains. Transplantation 11, 325327. Baker, D. G., Malieni, S., and Taylor, H. W. (1998). Experimental infection of inbred mouse strains with Spironucleus muris. Vet. Parasitol. 77, 305-310. Ball-Goodrich, L. J., and E. A. Johnson. (1994). Molecular characterization of a newly recognized mouse parvovirus. J. Virol. 68, 6476-6486. Barthold, S. W. (1980). The microbiology of transmissible murine colonic hyperplasia. Lab. Anita. Sci. 30, 167-173. Barthold, S. W. (1998). Opportunistic infections in research rodents: The challenges are great and the hour is late. ILAR J. 39, 316-321. Barthold, S. W., and Beck, D. (1980). Modification of early dimethylhydrazine carcinogenesis by colonic mucosal hyperplasia. Cancer Res. 40, 44514455.

113 Barthold, S. W., and Smith, A. L. (1987). Response of genetically resistant and susceptible mice to intranasal inoculation with mouse hepatitis virus JHM. Virus Res. 7, 225-239. Barthold, S. W., and Smith, A. L. (1989a). Duration of challenge immunity to coronavirus JHM in mice. Arch Virol. 107, 171-177. Barthold, S. W., and Smith, A. L. (1989b). Virus strain specificity of challenge immunity to coronavirus. Arch. Virol. 104, 187-196. Barthold, S. W., Osbaldiston, G. W., and Jonas, A. M. (1977). Dietary, bacterial, and host genetic interactions in the pathogenesis of transmissible murine colonic hyperplasia. Lab. Anim. Sci. 27, 938-945. Barthold, S. W., Coleman, G. L., Jacoby, R. O., Livstone, E. M., and Jonas, A. M. (1978). Transmissible murine colonic hyperplasia. Vet. Pathol. 15, 223-236. Barthold, S. W., Smith, A. L., Lord, P. S., Bhatt, P. N., Jacoby, R. O., and Main, A. J. (1982). Epizootic coronaviral typhlitis in suckling mice. Lab. Anita. Sci. 32, 376-383. Barthold, S. W., Smith, A. L., and Povar, M. L. (1985). Enterotropic mouse hepatitis virus infection in nude mice. Lab. Anim. Sci. 35, 613-618. Barthold, S. W., Smith, A. L., and Bhatt, P. N. (1993). Infectivity, disease patterns, and serologic profiles of reovirus serotypes 1, 2, and 3 in infant and weanling mice. Lab. Anim. Sci. 43, 425-430. Barthold, S. W., Beck, D. S., and Smith, A. L. (1993). Enterotropic coronavirus (mouse hepatitis virus) in mice: Influence of host age and strain on infection and disease. Lab. Anim. Sci. 43, 276-284. Battey, J., Jordan, E., Cox, D., and Dove, W. (1999). An action plan for modse genomics. Nat. Genet. 21, 73-75. Bedell, M. A., Jenkins, N. A., and Copeland, N. G. (1997). Mouse models of human disease. Part 1: Techniques and resources for genetic analysis in mice. Genes Dev. 11, 1-43. Berke, Z., and Dalianis, T. (1993). Persistence of polyomavirus in mice infected as adults differs from that observed in mice infected as newborns. J. Virol. 67, 4369-4371. Besch-Williford, C., and Wagner, J. E. (1982). Bacterial and mycotic diseases of the integumentary system. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 55-76. Academic Press, New York. Besselsen, D. G. (1998). Detection of rodent parvoviruses by PCR. Methods Mol. Biol. 92, 31-37. Bhatt, P. N., and Jacoby, R. O. (1987a). Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. 1. Clinical responses. Lab. Anim. Sci. 37, 11-15. Bhatt, P. N., and Jacoby, R. O. (1987b). Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. 3. Experimental transmission of infection and derivation of virus-free progeny from previously infected dams. Lab. Anim. Sci. 37, 23-28. Bhatt, P. N., and Jacoby, R. O. (1987c). Stability of ectromelia virus strain NIH79 under various laboratory conditions. Lab. Anita. Sci. 37, 33-35. Bhatt, P. N., and Jacoby, R. O. (1987d). Effect of vaccination on the clinical response, pathogenesis, and transmission of mousepox. Lab. Anita. Sci. 37, 610-614. Boot, R., Thuis, H. C., Veenema, J. L., and Bakker, R. G. (1995a). An enzymelinked immunosorbent assay (ELISA) for monitoring rodent colonies for Pasteurella pneumotropica. Lab. Anita. 29, 307-313. Boot, R., Thuis, H., Bakker, R. G., and Veenema, J. L. (1995b). Serological studies of Corynebacterium kutscheri and coryneform bacteria using an enzyme-linked immunosorbent assay (ELISA). Lab. Anita. 29, 294-299. Bray, M. V., Barthold, S. W., Sidman, C. L., Roths, J., and Smith, A. L. (1993). Exacerbation of Pneumocystis carinii pneumonia in immunodeficient (SCID) mice by concurrent infection with a pneumovirus. Infect. Immun. 61, 1586-1588. Brett, S. J., and Cox, E E. (1982). Immunological aspects of Giardia muris and Spironucleus muris infections in inbred and outbred strains of laboratory mice: A comparative study. Parasitology 85, 85-99.

114 Brinton, M. A. (1982). Lactate dehydrogenase-elevating virus. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 194-208. Academic Press, New York. Brinton, M. A. (1986). Lactate dehydrogenase-elevating virus. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 389-420. Academic Press, Orlando, Florida. Briody, B. A. (1959). Response of mice to ectromelia and vaccinia viruses. Bacteriol. Rev. 23, 61-95. Broeders, H. W., Groen, J., van Steenis, G., and Osterhaus, A. D. (1994). An enzyme-linked immunosorbent assay for the detection of mouse polyomavirus-specific antibodies in laboratory mice. Lab. Anim. 28, 257-261. Bronson, E H., Dagg, C. P., and Snell, G. D. (1966). Reproduction. In "Biology of the Laboratory Mouse" (E. L. Green, ed.), 2nd ed., pp. 187-204. McGraw-Hill, New York. Brownstein, D. G. (1978). Pathogenesis of bacteremia due to Pseudomonas aeruginosa in cyclophosphamide-treated mice and potentiation of virulence of endogenous streptococci. J. Infect. Dis. 137, 795-801. Brownstein, D. G. (1983). Genetics of dystrophic epicardial mineralization in DBA/2 mice. Lab. Anim. Sci. 33, 247-248. Brownstein, D. G. (1986). Sendai virus. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 37-61. Academic Press, Orlando, Florida. Brownstein, D. G., and Barthold, S. W. (1982). Mouse hepatitis virus immunofluorescence in formalin- or Bovin's-fixed tissues using trypsin digestion. Lab. Anim. Sci. 32, 37-39. Brownstein, D. G., and Gras, L. (1995). Chromosome mapping of Rmp-4, a gonad-dependent gene encoding host resistance to mousepox. J. Virol. 69, 6958-6964. Brownstein, D. G., Johnson, E. A., and Smith, A. L. (1984). Spontaneous Reye's-like syndrome in BALB/cByJ mice. Lab. Invest. 51, 386-395. Brownstein, D. G., Bhatt, P. N., Ardito, R. B., Paturzo, E X., and Johnson, E. A. (1989). Duration and patterns of transmission of Theiler's mouse encephalomyelitis virus infection. Lab. Anim. Sci. 39, 299-301. Brownstein, D. G., Smith, A. L., Jacoby, R. O., Johnson, E. A., Hansen, G., and Tattersall, P. (1991). Pathogenesis of infection with a virulent allotropic variant of minute virus of mice and regulation by host genotype. Lab. Invest. 65, 357-364. Brunnert, S. R., Dai, Y., and Kohn, D. E (1994). Comparison of polymerase chain reaction and immunohistochemistry for the detection of Mycoplasma pulmonis in paraffin-embedded tissue. Lab. Anim. Sci. 44, 257-260. Brunnert, S. R., Shi, S., and Chang, B. (1999). Chromosomal localization of the loci responsible for distrophic cardiac calcinosis in DBA/2 mice. Genomics 59, 105-107. Buller, R. M., Bhatt, P. N., and Wallace, G. D. (1983). Evaluation of an enzymelinked immunosorbent assay for the detection of ectromelia (mousepox) antibody. J. Clin. Microbiol. 18, 1220-1225. Burek, J. D., Molello, J. A., and Warner, S. D. (1982). Selected non-neoplastic diseases. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 425-440. Academic Press, New York. Campbell, I. L., and Gold, L. H. (1996). Transgenic modeling of neuropsychiatric disorders. Mol. Psychiatry 1, 105-120. Cartner, S. C., Simecka, J. W., Briels, D. E., Cassell, G. H., and Lindsey, J. R. (1996). Resistance to mycoplasmal lung disease in mice is a complex genetic trait. Infect. Immun. 64, 5326-5331. Cartner, S. C., Lindsey, J. R., Gibbs-Erwin, J. Cassell, G. H., and Simecka, J. W. (1998). Roles of innate and adaptive immunity in respiratory mycoplasmosis. Infect. lmmun. 66, 3485-3491. Casebolt, D. B., and Schoeb, T. R. (1988). An outbreak in mice of salmonellosis caused by Salmonella enteritidis serotype enteritidis. Lab. Anim. Sci. 38, 190-192. Casebolt, D. B., Qian, B., and Stephensen, C. B. (1997). Detection of en-

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON terotropic mouse hepatitis virus fecal excretion by polymerase chain reaction. Lab. Anim. Sci. 47, 6-10. Cassell, G. H., Lindsey, J. R., Davis, J. K., Davidson, M. K., Brown, M. B., and Mayo, J. G. (1981). Detection of natural Mycoplasma pulmonis infection in rats and mice by an enzyme linked immunosorbent assay (ELISA). Lab. Anim. Sci. 31, 676-682. Cassell, G. H., Davis, J. K., Simecka, J. W., Lindsey, J. R., Cox, N. R., Ross, S., and Fallon, M. (1986). Mycoplasmal infections: Disease pathogenesis, implications for biomedical research and control. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E New, eds.), pp. 87-130. Academic Press, Orlando, Florida. Clifford, C. B., Walton, B. J., Reed, T. H., Coyle, M. B., White, W. J., and Amyx, H. L. (1995). Hyperkeratosis in athymic nude mice caused by a coryneform bacterium: Microbiology, transmission, clinical signs, and pathology. Lab. Anim. Sci. 45, 131-139. Compton, S. R., Barthold, S. W., and Smith, A. L. (1993). The cellular and molecular pathogenesis of coronaviruses. Lab. Anim. Sci. 43, 15-28. Cook, M. J. (1983). Anatomy. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 102-120. Academic Press, New York. Crawley, J. N. (1999). Behavioral phenotyping of transgenic and knockout mice: Experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Res. 835, 18-26. Daniel, J. C. Jr., ed. (1978). "Methods in Mammalian Reproduction." Academic Press, New York. Daniels, C. W., and Belosevic, M. (1994). Serum antibody responses by male and female C57BL/6 mice infected with Giardia muris. Clin. Exp. Immunol. 97, 424-429. Daniels, C. W., and Belosevic, M. (1995). Comparison of the course of infection with Giardia muris in male and female mice. Int. J. Parasitol. 25, 131135. Darvasi, A. (1998). Experimental strategies for the dissection of complex traits in animal models. Nat. Genet. 18, 19-24. Davis, J. A., Paylor, R., McDonald, M. P., Libbey, M., Ligler, A., Bryant, K., and Crawley, J. N. (1999). Behavioral effects of ivermectin in mice. Lab. Anim. Sci. 49, 288-296. Davisson, M. T. (1996). Rules for nomenclature of inbred strains. In "Genetic Variants and Strains of Laboratory Mouse" (M. E Lyon, S. Rastan, and S. D. M. Brown, eds.), 3rd ed., Vol. 2, pp. 1532-1536. Oxford University Press, Oxford. Demant, P., and Hart, A. A. M. (1986). Recombinant congenic strainsma new tool for analyzing genetic traits determined by more than one gene. Immunogenetics 24, 416- 422. Derothe, J. M., Loubes, C., Orth, A., Renaud, E, and Moulia, C. (1997). Comparison between patterns of pinworm infection (Aspicularis tetraptera) in wild and laboratory strains of mice, Mus musculus. Int. J. Parasitol. 27, 645-651. de Souza, M., and Smith, A. L. (1989). Comparison of isolation in cell culture with conventional and modified mouse antibody producton tests for detection of murine viruses. J. Clin. Microbiol. 27, 185-187. Dewhirst, E E., Chien, C. C., Paster, B. J., Ericson, R. L., Orcutt, R. P., Schauer, D. B., and Fox, J. G. (1999). Phylogeny of the defined murine microbiota: Altered Schaedler flora. Appl. Environ. Microbiol. 65, 3287-3292. Dick, E. J., Kittell, C. L., Meyer, H., Farrar, P. L., Ropp, S. L., Esposito, J. J., Buller, M. L., Neubauer, H., Kang, Y. H., and McKee, A. E. (1996). Mousepox outbreak in a laboratory mouse colony. Lab. Anim. Sci. 46, 602611. Doherty, P. C. (1997). The Nobel lectures in immunology. Cell-mediated immunity in virus infections. Scand. J. Immunol. 46, 527-540. Downs, W. G. (1982). Mouse encephalomyelitis virus. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 341-352. Academic Press, New York.

3. BIOLOGY AND DISEASES OF MICE

Dunn, T. B. (1967). Amyloidosis in mice. In "Pathology of Laboratory Rats and Mice" (E. Cotchin and E J. C. Roe, eds.), pp. 181-212. Blackwell Scientific, Oxford, United Kingdom. Eaton, G. J., Custer, R. P., Johnson, E N., and Stabenow, K. T. (1978). Dystrophic cardiac calcinosis in mice: Genetic, hormonal, and dietary influences. Am. J. Pathol. 90, 173-186. Evengard, B., Sandstedt, K., Bolske, G., Feinstein, R., Reisenfelt-Orn, I., and Smith, C. I. (1994). Intranasal inoculation of Mycoplasma pulmonis in mice with severe combined immunodeficiency (SCID) causes a wasting disease with grave arthritis. Clin. Exp. ImmunoL 98, 388-394. Falk, P. G., et al. (1998). Creating and maintaining the gastrointestinal ecosystem: What we know and need to know from gnotobiology. MicrobioL Mol. Biol. Rev. 62, 1157-1170. Falk, R. H., and Skinner, M. (2000). The systemic amyloidoses: An overview. Adv. Intern. Med. 45, 107-137. Farber, D. B., and Danciger, M. (1994). Inherited retinal degenerations in the mouse. In "Molecular Genetics of Inherited Eye Disorders" (A. E Wright and B. Jay, eds.), pp. 123-152. Harwood, Chur, Switzerland. FELASA (1994). Recommendations for the health monitoring of mouse, rat, hamster, guinea pig, and rabbit breeding colonies. Report of the Federation of European Laboratory Animal Science Associations (FELASA) Working Group on Animal Health. Lab. Anim. 28, 1-12. FELASA (1996). Recommendations for the health monitoring of mouse, rat, hamster, guinea pig, and rabbit experimental units. Report of the Federation of European Laboratory Animal Science Associations (FELASA) Working Group on Animal Health. Lab. Anim. 30, 193-208. Fenner, E (1948). The pathogenesis of the acute exanthems. An interpretation based on experimental investigations with mouse-pox (infectious ectromelia of mice). Lancet 2, 915-920. Fenner, E (1982). Mousepox. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 209-230. Academic Press, New York. Fenner, E (1990). Wallace P. Rowe lecture. Poxviruses of laboratory animals. Lab. Anim. Sci. 40, 469-480. Ferner, W. T., Miskuff, R. L., Yolken, R. H., and Vonderfecht, S. L. (1987). Comparison of methods of detection of serum antibody to murine rotavirus. J. Clin. Microbiol. 25, 1364-1369. Flaherty, L. (1981). Congenic strains. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 1, p. 215. Academic Press, New York. Flynn, B. M., Brown, E A., Eckstein, J. M., and Strong, D. (1989). Treatment of Syphacia obvelata in mice using ivermectin. Lab. Anita. Sci. 39, 461-463. Fogh, J., ed. (1982). 'q'he Nude Mouse in Experimental and Clinical Research." Academic Press, New York. Foltz, C. J., Fox, J. G., Yan, L., and Shames, B. (1996). Evaluation of various oral antimicrobial formulations for the eradication of Helicobacter hepaticus. Lab. Anita. Sci. 46, 193-197. Fox, J. G. (2000). Helicobacter species other than H. pylori: Emerging pathogens in humans and animals. In "Emerging Infections" (W. M. Scheld, W. A. Craig, and J. M. Hughes, eds.), pp. 121-135. Washington, DC: ASM Press, in press. Fox, J. G., and Lee, A. (1997). The role of Helicobacter species in newly recognized gastrointestinal diseases of animals. Lab. Anita. Sci. 47, 222255. Fox, J. G., Dewhirst, E E., Tully, J. G., Paster, B. J., Yan, L., Taylor, N. S., Collins, M. J., Gorelick, E L., and Ward, J. M. (1994). Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J. Clin. Microbiol. 32, 12381245. Fox, J. G., Li, X., Yan, L., Cahill, R. J., Hurley, R., Lewis, R., and Murphy, J. C. (1996). Chronic proliferative hepatitis in A/JCr mice associated with persistent H. hepaticus infection: A model of Helicobacter induced carcinogenesis. Infect. Immun. 64, 1548-1558.

115 Fox, J. G., MacGregor, J., Shen, Z., Li, X., Lewis, R., and Dangler, C. A. (1998). Comparison of methods to identify Helicobacter hepaticus in B6C3F1 mice used in a carcinogenesis bioassay. J. Clin. Microbiol. 36, 1382-1387. Fox, J. G., Gorelick, P. L., Kullberg, M. C., Ge, Z., Dewhirst, E E., and Ward, J. M. (1999). A novel urease-negative Helicobacter species associated with colitis and typhlitis in IL- 10-deficient mice. Infect. Immun. 67, 1757-1762. Fox, J. G., Dangler, C. A., and Schauer, D. B. (2000). Inflammatory bowel disease in mouse models: Role of gastrointestinal microbiota as proinflammatory modulators." In "Pathology of Genetically Engineered,~clice" (J. M. Ward, J. Mahler, R. R. Maronpot, J. P. Sundberg, and R. Frederickson, eds.), pp. 297-313. Iowa State University Press, Ames. France, M. P., Smith, A. L., Stevenson, R., and Barthold, S. W. (1999). Granulomatous peritonitis in interferon-gamma gene knockout mice naturally infected with mouse hepatitis virus. Aust. Vet. J. 77, 600-604. Frankel, W. N. (1995). Taking stock of complex genetic traits in mice. Trends Genet. 11, 471-477. Franklin, C. L., Motzel, S. L., Besch-Williford, C. L., Hook, R. R., and Riley, L. K. (1994). Tyzzer's infection: Host specificity of Clostridium piliforme isolates. Lab. Anim. Sci. 44, 568-572. Franklin, C. L., Riley, L. K., Livingston, R. S., Beckwith, C. S., Hook, R. R., Jr., Besch-Williford, C. L., Hunziker, R., and Gorelick, P. L. (1999). Enteric lesions in SCID mice infected with "Helicobacter typhlonicus," a novel urease-negative Helicobacter species. Lab. Anim. Sci. 49, 496-505. Franklin, C. L., Gorelick, P. L., Riley, L. K., Dewhirst, F. E., Livingston, R. S., Ward, J. M., Beckwith, C. S., Fox, J. G. (2001). Helicobacter typhlonius sp. nov., a novel murine urease-negative Helicobacter species. J. Clin. MicrobioL 39, 3920-3926. Fujiwara, K., and Ganaway, J. R. (1994). Bacillus piliformis (1994). In "Manual of Microbiological Monitoring of Laboratory Animals" (K. Waggie, N. Kagayima, A. M. Allen, and T. Nomura, eds.), 2nd ed., pp. 117-120. National Institutes of Health Publication 94-2498, Bethesda, Maryland. Furuta, T., Fujiwara, K., Yamanuochi, K., and Ueda, K. (1985). Detection of antibodies to Pneumocystis carinii by enzyme-linked immunosorbent assay in experimentally infected mice. J. Parasitol. 71, 533-523. Ganaway, J. R. (1982). Bacterial and mycotic diseases of the digestive system. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 7-20. Academic Press, New York. Ganaway, J. R., Allen, A. M., and Moore, T. D. (1971). Tyzzer's disease. Am. J. Pathol. 64, 717-732. Ganaway, J. R., Spencer, T. H., and Waggie, K. S. (1985). Propagation of the etiologic agent of Tyzzer's disease (Bacillus piliformis) in cell culture. In "Contribution of Laboratory Animal Science to the Welfare of Man and Animals: Past, Present, and Future," Eighth Symposium of ICLAS/ CALAS (1983), pp. 59-70. Gustav Fischer Verlag, Stuttgart. Gigliotti, E, Haidaris, P. J., Haidaris, C. G., Wright, T. W., and Van der Meid, K. R. (1993). Further evidence of host species-specific variation in antigens of Pneumocystis carinii using the polymerase chain reaction. J. Infect. Dis. 168, 191-194. Goelz, M. F., Thigpen, J. E., Mahler, J., Rogers, W. P., Locklear, J., Weigler, B. J., and Forsythe, D. B. (1996). Efficacy of various therapeutic regimens in eliminating Pasteurella pneumotropica from the mouse. Lab. Anim. Sci. 46, 280-285. Goto, K., and Itoh, H. (1996). Detection of Clostridium piliforme by enzymatic assay of amplified cDNA segment in microtitration plates. Lab. Anim. Sci. 46, 493-496. Goto, K., Kunita, S., Terada, E., and Itoh, T. (1994). Comparison of polymerase chain reaction and culture methods for detection of Mycoplasma pulmonis from nasal, tracheal, and oral swab samples of rats. Jikken Dobutsu 43, 413-415. Goto, K., Nozu, R., Takakura, A., Matshushita, S., and Itoh, T. (1995). Detection of cilia-associated respiratory bacillus in experimentally and naturally infected mice and rats by the polymerase chain reaction. Exp. Anim. 44, 333-336.

116 Green, E. L. (1981a). "Genetics and Probability in Animal Breeding Experiments." Macmillan, New York. Green, E. L. (1981b). Breeding systems. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 1, pp. 89-104. Academic Press, New York. Greenstein, G., Drozdowicz, C. K., Nebair, E, and Bozik, R. (1994). Isolation of Streptococcus equisimilis from abscesses detected in specific pathogenfree mice. Lab. Anim. Sci. 44, 374-376. Griffith, J. W., White, W. J., Danneman, P. J., and Lang, C. M. (1988). Cilia-associated respiratory bacillus infection of obese mice. Vet. Pathol. 25, 72-76. Hailey, J. R., Haseman, J. K., Bucher, J. R., Radovsky, A. E., Malarkey, D. E., Miller, R. T., Nyska, A., and Maronpot, R. R. (1998). Impact of Helicobacter hepaticus infection in B6C3F1 mice from twelve National Toxicology Program two-year carcinogenesis studies. Toxicol. Pathol. 26, 602-611. Haldane, J. B. S., Sprunt, A. D., and Haldane, N. M. (1915). Reduplication in mice. J. Genet. 5, 133-135. Hansen, G. M., Patruzo, F. X., and Smith, A. L. (1999). Humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus. Lab. Anim. Sci. 49, 380-384. Harasawa, R., Koshimizu, K., Uemori, T., Takedo, O., and Asada, K. (1990). The polymerase chain reaction for Mycoplasma pulmonis. MicrobioL Immunol. 34, 393-395. Hildebrandt, P. K. (1982). Rickettsial and chlamydial diseases. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 99-109. Academic Press, New York. Hill, A. C., and Stalley, G. P. (1991). Mycoplasma pulmonis infection with regard to embryo freezing and hysterectomy derivation. Lab. Anim. 41, 563566. Homberger, E R. (1992). Maternally-derived passive immunity to enterotropic mouse hepatitis virus. Arch. Virol. 122, 133-141. Homberger, E R. (1997). Enterotropic mouse hepatitis virus. Lab. Anim. 31, 97-115. Homberger, E R., Smith, A. L., and Barthold, S. W. (1991). Detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction. J. Clin. Microbiol. 29, 2789-2793. Homberger, E R., Barthold, S. W., and Smith, A. L. (1992). Duration and strain specificity of immunity to enterotropic mouse hepatitis virus. Lab. Anim. Sci. 42, 347-351. Homberger, E R., Pataki, Z., and Thomann, P. E. (1993). Control of Pseudomonas aeruginosa infection in mice by chlorine treatment of drinking water. Lab. Anim. Sci. 43, 635-637. Homberger, E R., Romano, T. P., Seiler, P., Hansen, G. M., and Smith, A. L. (1995). Enyzme-linked immunosorbent assay for detection of antibody to lymphocytic choriomeningitis virus in mouse sera, with recombinant nucleoprotein as antigen. Lab. Anim. Sci. 45, 493-496. Homberger, E R., Zhang, L., and Barthold, S. W. (1998). Prevalence of enterotropic and polytropic mouse hepatitis virus in enzootically infected mouse colonies. Lab. Anim. Sci. 48, 50-54. Hsu, C.-K. (1982). Protozoa. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 359-372. Academic Press, New York. Hurley, L. S., and Bell, L. T. (1974). Genetic influence on response to dietary manganese deficiency. J. Nutr. 104, 133-137. Ihrig, M., Schrenzel, M., and Fox, J. G. (1999). Differential susceptibility to hepatic inflammation and proliferation in AXB recombinant inbred mice chronically infected with Helicobacter hepaticus. Am. J. Pathol. 155, 571582. International standardized nomenclature for outbred stocks of laboratory animals (1972). ICLA Bull. 30, 4-17. Institute of Laboratory Animal Resources, National Research Council, 2101 Constitution Avenue, Washington, DC 20418. Jacoby, R. O., and Ball-Goodrich, L. J. (1995). Parvovirus infections of mice and rats. Semin. Virol. 6, 329-333.

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON Jacoby, R. O., and Bhatt, E N. (1987). Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. 2. Pathogenesis. Lab. Anim. Sci. 37, 16-22. Jacoby, R. O., and Lindsey, J. R. (1997). Health care for research animals is essential and affordable. FASEB J. 11, 609-614. Jacoby, R. O., Bhatt, P. N., and Johnson, E. A. (1983). The pathogenesis of vaccinia (IHD-T) virus infection in BALB/cAnN mice. Lab. Anim. Sci. 33, 435-441. Jacoby, R. O., Bhatt, P. N., and Brownstein, D. G. (1989). Evidence that NK cells and interferon are required for genetic resistance to infection with ectromelia virus. Arch. Virol. 108, 49-58. Jacoby, R. O., Bhatt, P. N., Barthold, S. W., and Brownstein, D. G. (1994). Sendai virus pneumonia in aged BALB/c mice. Exp. Gerontol. 29, 89-100. Jacoby, R. O., Ball-Goodrich, L. J., Besselsen, D. G., McKisic, M. D., Riley, L. K., and Smith, A. L. (1996). Rodent parvovirus infections. Lab. Anim. Sci. 46, 370-380. John, A. M., and Bell, J. M. (1976). Amino acid requirements of the growing mouse. J. Nutr. 106, 1361-1367. Jones, T. C., Mohr, U., and Hunt, R. D., eds. (1983-1993). "Monographs on Pathology of Laboratory Animals." Springer-Verlag, Berlin. Jungmann, P., Freitas, A., Bandiera, A., Nobrega, A., Coutinho, A., Marcos, M. A., and Minoprio, P. (1996). Murine acariasis. 2. Immunologic dysfunction and evidence for chronic activation of Th-2 lymphocytes. Scand. J. Immunol. 43, 604-612. Kaplan, H. M., Brewer, N. R., and Blair, W. H. (1983). Physiology. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 248-292. Academic Press, New York. Kawamura, S., Taguchi, E, Ishida, T., Nakayama, M., and Fujiwara. K. (1983). Growth of Tyzzer's organism in primary monolayer cultures of mouse hepatocytes. J. Gen. Microbiol. 129, 277-283. Keverne, E. B. (1998). Vomeronasal/accessory olfactory system and pheromonal recognition. Chem. Senses 23, 491-494. Kimura, M., and Crow, J. E (1963). On maximum avoidance of inbreeding. Genet. Res. 4, 399-415. Klein, G. (1986). "The Natural History of the Major Histocompatibility Complex." Wiley and Sons, New York. Klement, P., Augustine, J. M., Delaney, K. H., Klement, G., and Weitz, J. I. (1996). An oral ivermectin regimen that eradicates pinworms (Syphacia spp.) in laboratory rats and mice. Lab. Anim. Sci. 46, 286-290. Knapka, J. J. (1983). Nutrition. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 52-67. Academic Press, New York. Knapka, J. J., Smith, K. P., and Judge, E J. (1974). Effect of open and closed formula rations on the performance of three strains of laboratory mice. Lab. Anim. Sci. 24, 480-487. Kraft, L. M. (1980). The manufacture, shipping, receiving, and quality control of rodent bedding materials. Lab. Anim. Sci. 30, 366-377. Kraft, V., and Meyer, B. (1986). Diagnosis of murine infections in relation to test methods employed. Lab. Anim. Sci. 36, 271-276. Kramer, D. R., and Cebra, J. J. (1995). Role of maternal antibody in the induction of virus specific and bystander IgA responses in Peyer's patches of suckling mice. Int. Immunol. 7, 911-918. Kunita, S., Terada, E., Goto, K., and Kagiyama, N. (1990). Sensitive detection of Mycoplasma pulmonis by using the polymerase chain reaction. Jikken Dobutsu 39, 103-107. Kunstyr, I. (1986). Paresis of peristalsis and ileus lead to death in lactating mice. Lab. Anim. 20, 32-35. Kunstyr, I., Ammerpohl, E., and Meyer, B. (1977). Experimental spironucleosis (hexamitiasis) in the nude mouse as a model for immunologic and pharmacologic studies. Lab. Anim. Sci. 27, 782-788. Kunstyr, I., Schoeneberg, U., and Friedhoff, K. T. (1992). Host specificity of Giardia muris isolates from mouse and golden hamster. Parasitol. Res. 78, 621-622.

3. BIOLOGY AND DISEASES OF MICE Lai, W. C., Linton, G., Bennett, M., and Pakes, S. P. (1993). Genetic control of resistance to Mycoplasma pulmonis infection in mice. Infect. Immun. 61, 4615-4621. Lai, W. C., Pakes, S. P., Ren, K., Lu, Y. S., and Bennett, M. (1997). Therapeutic effect of DNA immunization of genetically susceptible mice infected with virulent Mycoplasma pulmonis. J. Immunol. 158, 2513-2516. Le Blanc, S. A., Faith, R. E., and Montgomery, C. A. (1993). Use of topical ivermectin treatment for Syphacia obvelata in mice. Lab. Anim. Sci. 43, 526528. Lehmann-Grube, E (1982). Lymphocytic choriomeningitis virus. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 231-266. Academic Press, New York. Lindsey, J. R., Cassell, G. H., and Davidson, M. K. (1982). Mycoplasma and other bacterial diseases of the respiratory system. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 21-41. Academic Press, New York. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991a). Health surveillance programs. In "Infectious Diseases of Mice and Rats," pp. 21-27. National Research Council, National Academy Press, Washington, D.C. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991b). Lymphocytic choriomeningitis. In "Infectious Diseases of Mice and Rats," pp. 199-205. National Research Council, National Academy Press, Washington, D.C. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991c). Mycoplasma pulmonis. In "Infectious Diseases of Mice and Rats," pp. 42-48. National Research Council, National Academy Press, Washington, D.C. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991d). Pseudomonas aeruginosa. In "Infectious Diseases of Mice and Rats," pp. 141-145. National Research Council, National Academy Press, Washington, D.C. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991 e). Pasteurella pneumotropica. In "Infectious Diseases of Mice and Rats," pp. 187-190. National Research Council, National Academy Press, Washington, D.C. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991f). Salmonella enteritidis. In "Infectious Diseases of Mice and Rats," pp. 132-139. National Research Council, National Academy Press, Washington, D.C. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991g). Streptobacillus moniliformis. In "Infectious Diseases of Mice and Rats," pp. 176-179. National Research Council, National Academy Press, Washington, D.C. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991h). Staphylococcus aureus. In "Infectious Diseases of Mice and Rats," pp. 182-185. National Research Council, National Academy Press, Washington, D.C. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991i). Mycoplasma pulmonis. Mycoplasma neurolyticum. Mycoplasma collis. Mycoplasma arthritidis. In "Infectious Diseases of Mice and Rats," pp. 42-48, 77-79, 80-81, 173-176. National Research Council, National Academy Press, Washington, D.C. Lindsey, J. R., Boorman, G. A., Collins, M. J., Hsu, C.-K., Van Hoosier, G. L., and Wagner, J. E. (1991j). Streptococcus pneumoniae. In "Infectious Diseases of Mice and Rats," pp. 51-54. National Research Council, National Academy Press, Washington, D.C. Lipman, N. S., and Henderson, K. (2000). False negative results using RT-PCR for detection of lactate dehydrogenase-elevating virus in a tumor cell line. Comp. Med. 50, 255-256. Lipman, N. S., Newcomer, C. E., and Fox, J. G. (1987). Rederivation of MHV and MEV antibody positive mice by cross-fostering and the use of the microisolator caging system. Lab. Anim. Sci. 37, 195-199.

117 Lipman, N. S., Dalton, S. D., Stuart, A. R., and Arruda, K. (1994), Eradication of pinworms (Syphacia obvelata) from a large mouse breeding colony by combination oral anthelminthic therapy. Lab. Anim. Sci. 44, 517-520. Lipman, N. S., Perkins, S., Nguyen, H., Pfeffer, M., and Meyer, H. (2000). Mousepox resulting from the use of ectromelia virus-contaminated, imported mouse serum. Comp. Med. 50, 426-435. Lipman, R. D., Gaillard, E. T., Harrison, D. E., and Bronson, R. T. (1993). Husbandry factors and the prevalence of age-related amyloidosis in mice. Lab. Anim. Sci. 43, 439-444. Lipton, H. L., and Rozhon, E. J. (1986). The Theiler's murine encephalitis viruses. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E New, eds.), pp. 253-275. Academic Press, Orlando, Florida. Little, L. M., and Shadduck, J. A. (1987). Pathogenesis of rotavirus infection in mice. J. Virol. 61, 3345-3348. Livingston, R. S., Franklin, C. L., Besch-Williford, C. L., Hook, R. R., and Riley, L. K. (1996). A novel presentation of Clostridium piliforme infection (Tyzzer's disease) in nude mice. Lab. Anim. Sci. 46, 21-25. Loeb, W, and Quimby, F., eds. (1999). "The Clinical Chemistry of Laboratory Animals." Taylor and Francis, Philadelphia. London, B. A., Kern, J., and Gehle, W. D. (1983). Detection of murine virus antibody by ELISA. Lab. Anim. 12, 40-47. Lussier, G., ed. (1991), Detection methods for the identification of rodent viral and mycoplasmal infections. Lab. Anim. Sci. 41, 199-225. Lussier, G. (1994). Murine cytomegalovirus. In "Manual of Microbiological Monitoring of Laboratory Animals," 2nd ed., pp. 69-74. National Institutes of Health Publication 94-2498, Bethesda, Maryland. Lussier, G., Guenette, D., and Descoteaux, J. P. (1987a). Comparison of serological tests for the detection of natural and experimental murine cytomegalovirus. Can. J. Vet. Res. 51, 249-252. Lussier, G., Smith, A. L., Guenette, D., and Descouteaux, J.-P. (1987b). Serological relationship between mouse adenovirus strains FL and K87. Lab. Anim. Sci. 37, 55-57. Lyon, M. E, Rastan, S., and Brown, S. D. M., eds. (1996). "Genetic Variants and Strains of the Laboratory Mouse." Oxford Univ. Press, Oxford, United Kingdom. Ma, W., Miao, Z., and Novotny, M. V. (1998). Role of the adrenal gland and adrenal-mediated chemosignals in suppression of estrus in the house mouse: The Lee-Boot effect revisited. Biol. Reprod. 59, 1317-1320. Maggio-Price, L., Nicholson, K. L., Kline, K. M., Birkebak, T., Suzuki, I., Wilson, D. L., Schauer, D. L., and Fink, P. J. (1998). Diminished reproduction, failure to thrive, and altered immunologic function in a colony of T-cell receptor transgenic mice: Possible role of Citrobacter rodentium. Lab. Anim. Sci. 48, 145-155. Mahbubani, M. H., Bej, A. K., Perlin, M., Schaeffer, E W., Jakubowski, W., and Atlas, R. M. (1991). Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts. Appl. Environ. Microbiol. 57, 3456-3461. Maronpot, R. R., Boorman, G. A., and Gaul, B. (1999). "Pathology of the Mouse." Cache River Press, Vienna, Illinois. Matsushita, S., and Suzuki, E. (1995). Prevention and treatment of cilia-associated respiratory bacillus in mice by use of antibiotics. Lab. Anim. Sci. 45, 503 -507. McCance, D. J., Sebesteny, A., Griffin, B. E., Balkwill, F., Tilly, R., and Gregson, N. A. (1983). A paralytic disease in nude mice associated with polyomavirus infection. J. Gen. Virol. 64, 57-67. McKisic, M. D., Lancki, D. W., Otto, G., Padrid, R., Snook, S., Cronin, D. C., Lohmar, P. D., Wong, T., and Fitch, E W. (1993). Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells. J. Immunol. 150, 419-428. McWilliams, T. S., Waggie, K. S., Luzarraga, M. B., French, A. W., and Adeams, R. J. (1993). Corynebacterium species-associated keratoconjunctivitis in aged C57BL/6J mice. Lab. Anim. Sci. 43, 509-512.

118 Monteyne, P., Bureau, J. E, and Brahic, M. (1997). The infection of mouse by Theiler's virus: From genetics to immunology. Immunol. Rev. 159,163-176. Morita, E., Kaneko, S., Hiragun, T., Shindo, H., Tanaka, T., Furukawa, T., Nobukiyo, A., and Yamamoto, S. (1999). Fur mites induce dermatitis associated with IgE hyperproduction in an inbred strain of mice, NC/Kuj. J. Dermatol. Sci. 19, 37-43. Morse, H. C., III, ed. (1979). "Origins of Inbred Mice." Academic Press, New York. Morse, S. S. (1994). Mouse thymic virus. In "Manual of Microbiological Monitoring of Laboratory Animals" (K. Waggie, N. Kagiyama, A. M. Allen, and T. Nomura, eds.), 2nd ed., pp. 63-67. National Institutes of Health Publication 94-2498, Bethesda, Maryland. Nakagawa, M., and Weyant, R. S. (1994). Streptococcus pneumoniae. In "Manual of Microbiological Monitoring of Laboratory Animals," K. Waggie, N. Kagiyama, A. M. Allen, and T. Nomura, ed.), 2nd ed., pp. 165-169. National Institutes of Health, Bethesda, Maryland. Nelson, R. J., and Young, K. A. (1998). Behavior in mice with targeted disruption of single genes. Neurosci. Biobehav. Rev. 22, 453-462. Neubauer, H., Pfeffer, M., and Meyer, H. (1997). Specific detection of mousepox virus by polymerase chain reaction. Lab. Anim. 31, 201-205. Newman, J. V., Zabel, B. A., Jha, S. S., and Schauer, D. B. (1999). Citrobacter rodentium espB is necessary for signal transduction and for infection of laboratory mice. Infect. lmmun. 67(11), 6019-6025. Nicklas, W., Kraft, V., and Meyer, B. (1993). Contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses. Lab. Anim. Sci. 43, 296-300. Nishimori, K., Young, L. J., Guo, Q., Wang, Z., Insel, T. R., and Matzuk, M. M. (1996). Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc. Natl. Acad. Sci. U.S.A. 93, 11699-11704. Novotny, M. V., Ma, W., Wiesler, D., and Zidek, L. (1999). Positive identification of the puberty-accelerating pheromone of the house mouse: The volatile ligands associating with the major urinary protein. Proc. R. Soc. Lond. B Biol. Sci. 266, 2017-2022. Orcutt, R. (1994). Polyoma virus. In "Manual of Microbiological Monitoring of Laboratory Animals" (K. Waggie, N. Kagiyama, A. M. Allen, and T. Nomura, eds.), 2nd ed., pp. 79-86. National Institutes of Health, Bethesda, Maryland. Osborn, J. E. (1982). Cytomegalovirus and other herpesviruses. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 267-292. Academic Press, New York. Osborn, J. E. (1986). Cytomegalovirus and other herpesviruses of mice and rats. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E New, eds.), pp. 421-450. Academic Press, Orlando, Florida. Ozkul, I. A., and Aydin, Y. (1994). Natural Cryptosporidium muris infection of the stomach in laboratory mice. Vet. Parasitol. 55, 129-132. Park, J. Y., Peters, C. J., Rollin, P. E., Ksiazek, T. G., Gray, B., Waites, K. B., and Stephensen, C. B. (1997). Development of a reverse-transcriptasepolmerase chain reaction assay for diagnosis of lymphocytic choriomeningitis virus infection and its use in a prospective surveillance study. J. Med. Virol. 51, 107-114. Parker, J. C., and Richter, C. B. (1982). Viral diseases of the respiratory system. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 110-158. Academic Press, New York. Pastoret, P. P., Brazin, P. G. H., and Govaerts, A., eds. (1998). "Handbook of Vertebrate Immunology." Academic Press, Boston. Percy, D. H., and Barthold, S. W. (2001a). Colonic hyperplasia (Citrobacterfreundii infection). In "Pathology of Laboratory Rodents and Rabbits," 2nd ed., pp. 38-40. Iowa State University Press, Ames. Percy, D. H., and Barthold, S. W. (2001b). Salmonellosis. In "Pathology of Laboratory Rodents and Rabbits," 2nd ed., pp. 33-34. Iowa State University Press, Ames. Percy, D. H., and Barthold, S. W. (2001c). Pseudotuberculosis (Corynebac-

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON terium kutscheri infection). In "Pathology of Laboratory Rodents and Rabbits," 2nd ed., pp. 62-63. Iowa State University Press, Ames. Percy, D. H., and Barthold, S. W. (2001d). Staphylococcal infections. In "Pathology of Laboratory Rodents and Rabbits," 2nd ed., pp. 64-65. Iowa State University Press, Ames. Percy, D. H., and Barthold, S. W. (2001e). Streptococcal infections. In "Pathology of Laboratory Rodents and Rabbits," 2nd ed., pp. 65-66. Iowa State University Press, Ames. Percy, D. H., and Barthold, S. W. (2001f). Aging, degenerative, and miscellaneous disorders. In "Pathology of Laboratory Rodents and Rabbits," 2nd ed., pp. 88-92. Iowa State University Press, Ames. Percy, D. H., and Barthold, S. W. (2001g). Neoplasms. In "Pathology of Laboratory Rodents and Rabbits," 2nd ed., pp. 95-106. Iowa State University Press, Ames. Pirofski, L., Howitz, M. S., Scharff, M. D., and Factor, S. M. (1991). Murine adenovirus infection of SCID mice induces hepatic lesions that resemble human Reye syndrome. Proc. Natl. Acad. Sci. U.S.A. 88, 4358-4362. Pleasants, J. R., Wostmann, B. S., and Reddy, B. S. (1973). Improved lactation in germfree mice following changes in the amino acid and fat components of a chemically defined diet. In "Germfree Research" (J. B. Heneghan, ed.), p. 245. Academic Press, New York. Poiley, S. M. (1960). A systematic method of breeder rotation for non-inbred laboratory animal colonies. Proc. Anim. Care Panel 10, 159-166. Porterfield, P. D., and Richter, C. B. (1994a). Mouse adenovirus. In "Manual of Microbiological Monitoring of Laboratory Animals" (K. Waggie, N. Kagiyama, A. M. Allen, and T. Nomura, eds.), 2nd ed., pp. 47-51. National Institutes of Health, Bethesda, Maryland. Porterfield, P. D., and Richter, C. B. (1994b). K virus. In "Manual of Microbiological Monitoring of Laboratory Animals" (K. Waggie, N. Kagiyama, A. M. Allen, and T. Nomura, eds.), 2nd ed., pp. 25-29. National Institutes of Health, Bethesda, Maryland. Potkay, S. (1994). Hymenolepis spp. In "Manual of Microbiological Monitoring of Laboratory Animals" (K. Waggie, N. Kagiyama, A. M. Allen, and T. Nomura, eds.), 2nd ed., pp. 191-195. National Institutes of Health, Bethesda, Maryland. Reddy, L. V., Zammit, C., Schuman, P., and Crane, L. R. (1992). Detection of Pneumocystis carinii in a rat model by polymerase chain reaction. Mol. Cell. Probes 6, 137-143. Reeb-Whitaker, C. K., Harrsion, D. J., Jones, R. B., Kacergis, J. B., Meyers, D. D., and Paigen, B. (1999). Control strategies for aeroallergens in an animal facility. J. Allergy Clin. Immunol. 103, 139-146. Rehg, J. E., and Toth, L. A. (1998). Rodent quarantine programs: Purpose, principles, and practice. Lab. Anim. Sci. 48, 438-447. Richter, C. B. (1986). Mouse adenovirus. K virus, pneumonia virus of mice. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E New, eds.), pp. 137-192. Academic Press, Orlando, Florida. Riepenhoff-Talty, M., Dharakul, T., Kowalski, E., Michalak, S., and Ogra, P. L. (1995). Persistent rotavirus infection in mice with severe combined immunodeficiency. Virology 211, 474-480. Roopenian, D. C., and Simpson, E., eds. (2000). "Minor Histocompatibility Antigens: From the Laboratory to the Clinic." Landes Bioscience, Austin, Texas. Rose, J., Franco, M., and Greenberg, H. (1998). The immunology of rotavirus infection in the mouse. Adv. Virus Res. 51, 203-235. Roths, J. B., Smith, A. L., and Sidman, C. L. (1993). Lethal exacerbation of Pneumocystis carinii pneumonia in severe combined immunodeficient mice after infection with pneumonia virus of mice. J. Exp. Med. 177, 11931198. Roths, J. B., Marshall, J. D., Allen, R. D., Carlson, G. A., and Sidman, C. L. (1990). Spontaneous Pneumocystis carinii pneumonia in immunodeficient mutant SCID mice. Natural history and pathobiology. Am. J. Pathol. 136, 1173-1186.

3. BIOLOGY AND DISEASES OF MICE Rozario, C., Morin, L., Roger, A. J., Smith, M. W., and Muller, M. (1996). Primary structure and phylogenetic relationships of glyceraldehyde-3-phosphate dehydrogenase genes of free-living and parasitic diplomonad flagellates. J. Eukaryot. Microbiol. 43, 330-340. Rugh, R. (1990). "The Mouse: Its Reproduction and Development." Oxford Univ. Press, New York. Scanziani, E., Gobbi, A., Crippa, L., Guisti, A. M., Pesenti, E., Cavaletti, E., and Luini, M. (1998). Hyperkeratosis-associated coryneform infection in severe combined immunodeficient mice. Lab. Anim. 32, 330-336. Sato, Y., Ooi, H. K., Nonaka, N., Oku, Y., and Kamiya, M. (1995). Antibody production in Syphacia obvelata infected mice. J. ParasitoL 81, 559-562. Schaedler, R. W., and Orcutt, R. P. (1983). Gastrointestinal microflora. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 327-345. Academic Press, New York. Schagemann, G., Bohnet, W., Kunstyr, I., and Friedhoff, K. T. (1990). Host specificity of cloned Spironucleus muris in laboratory rodents. Lab. Anim. 24, 234-239. Schauer, D. B., Zabel, B. A., Pedraza, I. E, O'Hara, C. M., Steigerwalt, A. G., Brenner, D. J. (1995). Genetic and biochemical characterization of Citrobacter rodentium sp. nov. J. Clin. Microbiol. 33(8), 2064-2068. Schauer, D. B., and Falkow, S. (1993). The eae gene of Citrobacterfreundii biotype 4280 is necessary for colonization in transmissible murine colonic hyperplasia. Infect. Immun. 61(11), 4654-4661. Schenkman, D. I., Rahija, R. J., Klingenberger, K. L., Elliot, J. A., and Richter, C. B. (1994). Outbreak of group B streptococcal meningoencephalitis in athymic mice. Lab. Anim. Sci. 44, 639-641. Schoeb, T. R., Dybvig, K., Keisling, K. E, Davidson, M. K., and Davis, J. K. (1997). Detection of Mycoplasma pulmonis in cilia-associated respiratory bacillus isolates and in respiratory tracts of rats by nested PCR. J. Clin. Microbiol. 35, 1667-1670. Sebesteny, A., Tilly, R., Balkwill, E, and Trevan, D. (1980). Demyelination and wasting associated with polyomavirus infection in nude (nu/nu) mice. Lab. Anim. Sci. 14, 337-345. Shah, K. V., and Christian, C. (1986). Mouse polyoma and other papovaviruses. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 505-528. Academic Press, Orlando, Florida. Shames, B., Fox, J. G., Dewhirst, E E., Yan, L., Shen, Z., and Taylor, N. S. (1995). Identification of widespread H. hepaticus infection in feces in commercial mouse colonies by culture and PCR assay. J. Clin. Microbiol. 33, 2968-2972. Sheridan, J. E, and Vonderfecht, S. L. (1986). Mouse rotavirus. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 217-244. Academic Press, Orlando, Florida. Shimizu, A. (1994). Staphylococcus aureus. In "Manual of Microbiological Monitoring of Laboratory Animals" (K. Waggie, N. Kagayima, A. M. Allen, and T. Nomura, eds.), 2nd ed., pp. 159-164. National Institutes of Health Publication 94-2498, Bethesda, Maryland. Shoji, Y., Itoh, T., and Kagiyama, N. (1988). Enzyme-linked immunosorbent assay for detection of serum antibody to CAR bacillus. Exp. Anim. 37, 67-72. Shomer, N. H, Dangler, C. A, Marini, R. P, and Fox, J. G. (1998). Helicobacter bilis/Helicobacter rodentium co-infection associated with diarrhea in a colony of SCID mice. Lab. Anim. Sci. 48, 455-459. Shorey, H. H. (1976). "Animal Communication by Pheromones." Academic Press, New York. Shultz, L. D., and Sidman, C. L. (1987). Genetically determined models of immunodeficiency. Annu. Rev. Immunol. 5, 367-403. Sidman, R. L., Angevine, J. B., and Taber-Pierce, E. ( 1971). "Atlas of the Mouse Brain and Spinal Cord." Harvard Univ. Press, Cambridge, Massachusetts. Silver, L. M. (1995). "Mouse Genetics: Concepts and Applications." Oxford Univ. Press, New York. Silverman, J., Chavannes, J. M., Rigotty, J., and Ornaf, M. (1979). A natural out-

119 break of transmissible murine colonic hyperplasia in A/J mice. Lab. Anim. Sci. 29, 209-213. Skopets, B., Wilson, R. P., Griffith, J. W., and Lang, C. M. (1996). Ivermectin toxicity in mice. Lab. Anim. Sci. 46, 111-112. Small, J. D. (1984). Rodent and lagomorph health surveillance-quality assurance. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.) 1st ed., pp. 709-724. Academic Press, Orlando, Florida. Smith, A. L. (1983a). An immunofluorescence test for detection of serum antibody to rodent coronaviruses. Lab. Anim. Sci. 33, 157-160. Smith, A. L. (1983b). Response of weanling random-bred mice to inoculation with minute virus of mice. Lab. Anim. Sci. 33, 37-39. Smith, A. L., and Paturzo, E X. (1988). Explant cultures for detection of minute virus of mice in infected mouse tissue. J. Tissue Cult. Methods 11, 45-47. Smith, A. L., and Winograd, D. E (1986). Two enzyme immunoassays for the detection of antibody to rodent coronaviruses. J. Virol. Methods 14, 335343. Smith, A. L., Winograd, D. E, and Burrage, T. G. (1986). Comparative biological characterization of mouse adenovirus strains FL and K87 and seroprevalence in laboratory rodents. Arch. Virol. 91, 233-246. Smith, A. L., Jacoby, R. O., Johnson, E. A., et al. (1993). In vivo studies with an "orphan" parvovirus of mice. Lab. Anim. Sci. 43, 175-182. Steele, M. I., Marshall, C. M., Lloyd, R. E., and Randolph, V. E. (1995). Reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease. Hepatology 21, 697-702. Sundberg, J. P. (1994). Chronic ulcerative dermatitis in black mice. In "Handbook of Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools" (J. P. Sundberg, ed.), pp. 485-492. CRC Press, Boca Raton, Florida. Szomolanyi-Tsuda, E., Dundon, P. L., Joris, I., Shultz, L. D., Woda, B. A., and Welsh, R. M. (1994). Acute, lethal, natural killer cell-resistant myeloproliferative disease induced by polyomavirus in severe combined immunodeficient mice. Am. J. Pathol. 144, 359-371. Taylor, B. A. (1996). Recombinant inbred strains. In "Genetic Variants and Strains of the Laboratory Mouse" (M. E Lyon, S. Rastan, and S. D. M. Brown, eds.), 3rd ed., pp. 1597-1659. Oxford Univ. Press, Oxford, United Kingdom. Taylor, M. A., Marshall, R. N., Green, J. A., and Catchpole, J. (1999). The pathogenesis of experimental infections of Cryptosporidium muris (strain RN 66) in outbred nude mice. Vet. Parasitol. 86, 41-48. Theuer, R. C. (1971). Effect of essential amino acid restriction on the growth of female CS7BL mice and their implanted BW 10232 adenocarcinomas. J. Nutr. 101, 223-232. Toth, L. A, Oberbeck, C., Straign, C. M., Frazier, S., and Rehg, J. E. (2000). Toxicity evaluation of prophylactic treatments for mites and pinworms in mice. Contemp. Top. Lab. Anim. Sci. 39, 18-21. Tyler, K. L., and Fields, B. N. (1986). Reovirus infection in laboratory rodents. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 277-304. Academic Press, Orlando, Florida. Van Andel, R. A., Franklin, C. L., Besch-Williford, C. L., Hook, R. R., and Riley, L. K. (2000). Prolonged perturbations of tumor necrosis factor-alpha and interferon gamma in mice inoculated with Clostridium piliforme. J. Med. Microbiol. 49, 557-563. van Kuppeveld, E J., Melchers, W. J., Willemse, H. E, Kissing, J., Galama, J. M., and van der Logt, J. T. (1993). Detection of Mycoplasma pulmonis in experimentally infected laboratory rats by 16S rRNA amplification. J. Clin. Microbiol. 31, 524-527. Viscogliosi, E., Philippe, H., Baroin, A., Perasso, R., and Brugerolle, G. (1993). Phylogeny of trichomonads based on partial sequences of large subunit rRNA and on cladistic analysis of morphological data. J. Eukaryot. Microbiol. 40, 411-421. Waggie, K. S., Hansen, C. T., Ganaway, J. R., and Spencer, T. S. (1981). A study

120 of mouse strain susceptibility to Bacillus piliformis (Tyzzer's disease): The association of B-cell function and resistance. Lab. Anim. Sci. 31, 139-142. Waggie, K. S., Wagner, J. E., and Lentsch, R. H. (1983). A naturally occurring outbreak of Mycobacterium avium-intracellulare infections in C57BL/6N mice. Lab. Anim. Sci. 33, 249-253. Waggie, K. S., Spencer, T. H., and Ganaway, J. R. (1987). An enzyme-linked immunosorbent assay for detection of anti Bacillus piliformis serum antibody in rabbits. Lab. Anim. Sci. 37, 176-179. Waggie, K. S., Hansen, C. T., Moore, T. D., Bukowski, M. A., and Allen, A. M. (1988). Cecocolitis in immunodeficient mice associated with an enteroinvasive lactose negative E. coli. Lab. Anim. Sci. 38, 389-393. Waggie, K., Kagayima, N., Allen, A. M., and Nomura, T., eds. (1994). "Manual of Microbiological Monitoring of Laboratory Animals," 2nd ed. National Institutes of Health Publication 94-2498, Bethesda, Maryland. Wallace, G. D., Buller, R. M., and Norse, H. C. III (1985). Genetic determinants of resistance to ectromelia (mousepox) virus-induced mortality. J. Virol. 55, 890-891. Walzer, P. D., Kim, C. K., Linke, M. J., Pogue, C. L., Huerkamp, M. J., Chrisp, C. E., Lerro, A. V., Wixson, S. K., Hall, E., and Shultz, L. D. (1989). Outbreaks of Pneumocystis carinii pneumonia infection colonies of immunodeficient mice. Infect. ImmUn. 57, 62-70. Wan, C. H., Riley, M. I., Hook, R. R., Franklin, C. L,, Besch-Williford, C. L., and Riley, L. K. (1995). Expression of Sendai virus nucleocapsid protein in a baculovirus expression system and application to diagnostic assays for Sendai virus. J. Clin. Microbiol. 33, 2007-2011. Wang, R.-E, Campbell, W., Cao, W.-W., Summage, C., Steele, R. S., and Cerniglia, C. E. (1996). Detection of Pasteurella pneumotropica in laboratory mice and rats by polymerase chain reaction. Lab. Anim. Sci. 46, 8185. Ward, J. M., Fox, J. G., Anver, M. R., Haines, D. C., George, C. V., Collins, M. J., Jr, Gorelick, P. L., Nagashima, K., Gonda, M. A., Gilden, R. V., Tully, J. G., Russell, R. J., Benveniste, R. E., Paster, B. J., Dewhirst, E E., Donovan, J. C., Anderson, L. M., and Rice, J. M. (1994). Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J. Natl. Cancer Inst. 86, 1222-1227. Ward, J. M., Anver, M. R., Haines, D. C., Melhorn, J. M., Gorelick, P., Yan, L., and Fox, J. G. (1996). Inflammatory large bowel disease in immunodeficient mice naturally infected with Helicobacter hepaticus. Lab. Anim. Sci. 46, 15-20. Ward, J. M., Mahler, J. E, Maronpot, R. R., and Sundberg, J. P. (2000). "Pathology of Genetically Engineered Mice." Iowa State University Press, Ames. Weigler, B. J., Thigpen, J. E., Goelz, M. E, Babineau, C. A., and Forsythe, D. B. (1996). Randomly amplified polymorphic DNA polymerase chain reaction assay for molecular epidemiologic investigation of Pasteurella pneumotropica in laboratory rodent colonies. Lab. Anim. Sci. 46, 386-392. Weir, E. C., Brownstein, D. G., and Barthold, S. W. (1986). Spontaneous wasting disease in nude mice associated with Pneumocystis carinii infection. Lab. Anim. Sci. 36, 140-144. Weir, E. C., Barthold, S. W., Cameron, G. A., and Simack, E A. (1987). Elimination of mouse hepatitis from a breeding colony by temporary cessation of breeding. Lab. Anita. Sci. 37, 455-458.

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON Weir, E. C., Brownstein, D. G., Smith, A. L., and Johnson, E. A. (1988). Respiratory disease and wasting in athymic mice infected with pneumonia virus of mice. Lab. Anim. Sci. 38, 133-137. Weisbroth, S. H. (1982). Arthropods. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 385-402. Academic Press, New York. weisbroth, S. H. (1994). Corynebacterium kutscheri. In "Manual of Microbiological Monitoring of Laboratory Animals" (K. Waggie, N. Kagayima, A. M. Allen, and T. Nomura, eds.), 2nd ed., pp. 129-133. National Institutes of Health Publication 94-2498, Bethesda, Maryland. Weisbroth, S. H., Peters, R., Riley, L. K., and Shek, W. (1998). Microbiological assessment of laboratory rats and mice. ILAR J. 39, 272-290. Wescott, R. B. (1982). Helminths. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, p. 373. Academic Press, New York. Whary, M. T., Cline, J. H., King, A. E., Hewes, K. M., Chojnacky, D., Salvarrey, A., and Fox, J. G. (2000). Monitoring sentinel mice for Helicobacter hepaticus, H. rodentium, and H. bilis infection by PCR and serology. Comp. Med. 50, 436-443. White, W. J., Anderson, L. C., Geistfeld, J., and Martin, D. G. (1998). Current strategies for controlling/eliminating opportunistic microorganisms. ILAR J. 39, 291- 305. Whittingham, D. G., and Wood, M. J. (1983). Reproductive physiology. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 137-164. Academic Press, New York. Wilde, J., Eiden, J., and Yolken, R. (1990). Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J. Clin. Microbiol. 28, 1300-1307. Wilson, E. O. (1970). Chemical communication within animal species. In "Chemical Ecology" (E. Sondheimer and J. B. Simeone, eds.), pp. 133155. Academic Press, New York. Wostman, B. S. (1996). Germfree and gnotobiotic animal models. CRC Press, Boca Raton, Florida. Wullenweber, M. (1995). Streptobacillus moniliformisma zoonotic pathogen. Taxonomic considerations, host species, diagnosis, therapy, and geographical distribution. Lab. Anim. 29, 1-15. Wullenweber-Schmidt, M. (1988). An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Pasteurella pneumotropica in murine colonies. Lab. Anim. Sci. 38, 37-41. Yamada, Y. K., Yabe, M., Yamada, A., and Taguchi, E (1993). Detection of mouse hepatitis virus by the polymerase chain reaction and its application to the rapid diagnosis of infection. Lab. Anim. Sci. 43, 285-290. Zenner, L. (1998). Effective eradication of pinworms (Syphacia muris, Syphacia obvelata, and Aspicularis tetraptera) from a rodent breeding colony by oral anthelminthic therapy. Lab. Anim. 32, 337-342. Zoll, J., Galama, J., and Melchers, W. (1993). Molecular identification of cardioviruses by enzymatic amplification. MoL Cell Probes 7, 447-452.

Chapter 4 Biology and Diseases of Rats Dennis F Kohn and Charles B. Clifford

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. MajorTaxonomic and Historical Considerations . . . . . . . . . . . . . . . . B. Usesin Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Summaryof Laboratory Managementand Husbandry . . . . . . . . . . . . II. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Morphophysiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. NormalPhysiological Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Behavior. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. InfectiousDiseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Noninfectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Traumaticand Iatrogenic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . D. NeoplasticDiseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. MiscellaneousConditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

A.

INTRODUCTION

Major Taxonomic and Historical Considerations

The laboratory rat, Rattus norvegicus, is within the order Rodentia and family Muridae. The genus Rattus contains more than 130 species; however, the Norway rat, R. norvegicus, and the black rat, R. rattus, are the 2 species most commonly associated with the genus. Rattus rattus preceded R. norvegicus in migration from Asia to Europe and the Americas by several hundred years. The former species reached Europe in the twelfth century, and the Americas in the sixteenth century; LABORATORY ANIMAL MEDICINE, 2nd edition

121 121 122 122 122 123 123 126 126 127 133 134 134 152

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whereas R. norvegicus emerged in the eighteenth century in Europe and in the nineteenth century in the Western Hemisphere. Globally, the Norway rat has largely displaced the black rat, probably because of the Norway rat's larger size and aggressiveness. The domestication and introduction of the albino R. norvegicus is rooted by its use in Europe and America in the 1800s as prey for a sport (rat baiting) in which individuals would wager on which terrier dog would most swiftly kill the largest number of rats confined to a pit. Because of the need for large numbers of rats for this sport, wild rats were purposebred, and albinos were selected out by some people as a hobby (Robinson, 1965). The first reported use of the rat in an experiment, in 1856, has Copyright 2002, Elsevier Science (USA). All fights reserved. ISBN 0-12-263951-0

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been credited to J. M. Philipeaux, who studied the effects of adrenalectomy in albino rats (Richter, 1954). Rats were used in experiments only sporadically in Europe and the United States until about 1890. Pivotal to the development of the rat for use in research was H. H. Donaldson, who at the Wistar Institute did much to produce and define early stocks of laboratory rats (Lindsey, 1979).

B.

Uses in Research

The rat is second only to the mouse as the most frequently used mammal in biomedical and behavioral research. Characteristics such as a short gestation and a relatively short life span, a docile behavior, and ready availability of animals with welldefined health and genetic backgrounds are responsible for the importance of the rat as a laboratory animal. The rat is a standard species for toxicological, teratological, and carcinogenesis testing by the pharmaceutical industry and governmental agencies. Its early use in behavioral, neurological, nutritional, and endocrinology studies continues today. The size of the rat enables it to be used for surgical procedures, varying from organ transplantation to vascular techniques. Although the number of commonly used inbred strains is dwarfed by those of the mouse,

Table I

Commonly Used Rat Strains Inbred strains

Usefulness as models

ACI

Congenital genitourinary anomalies,prostatic adenocarcinomas

BB/Wor

Juvenile insulin-dependent diabetes mellitus Inducible, transplantable myeloid leukemia,

BN (Brown Norway) BUF (Buffalo) COP (Copenhagen) F-344 (Fischer 344)

LEW (Lewis) LOU/C

SHR (spontaneous hypertensive rat) WF (Wistar-Furth) Zucker Mutant strains Brattleboro

Gunn Nude Obese SHR

hydronephrosis, bladder carcinoma Spontaneous autoimmunethyroiditis, host for transplantable Morris hepatoma Prostate adenocarcinoma Inbred rat model for National ToxicologyProgram's Carcinogen Bioassay Program and the National Institute on Aging Multiple sclerosis, various experimentallyinduced autoimmune diseases Myeloma,production of IgG autoantibody Hypertension, cardiovascular research Mononuclear cell leukemia

Obesity Characteristics Diabetes insipidus (autosomalrecessive) Jaundice, kernicterus (autosomalrecessive) T cell deficient (autosomal recessive) Type 4 hypeflipoproteinemia(autosomalrecessive)

inbred rat strains do represent an important repertoire of disease models (Table I).

C.

Sources

Commercial sources for outbred and inbred rats have been significantly consolidated during the past decade or two, resulting in a high percentage of rats being sold in the United States by several companies. Many of the small firms that had regional niches have been acquired by companies that have multiple divisions within the United States and internationally. Concomitantly, there has been a raising of the bar regarding the health status and genetic integrity of laboratory rats available to investigators.

D. Summary of Laboratory Management and Husbandry 1.

Secondary Enclosures

Rooms in which rats are to be housed should meet the guidelines of the "Guide for the Care and Use of Laboratory Animals" (National Research Council, 1996a). Wall, ceiling, and floor surfaces should be made of materials that allow for effective sanitation and that are resistant to damage from normal use and manipulation of equipment. The environment of the room should be well controlled, to ensure animal well-being and to help limit variables to those of the experimental design. Although rats, like most other species, can adapt to changes in temperature and humidity, room temperatures within a range of 700-76 ~F and with a relative humidity of 3 0 - 7 0 % are typically accepted as being appropriate. Twenty-four-hour temperature/ humidity recorders, either located in animal rooms or as a component of an electronic environmental management system, are useful in detecting changes in environmental conditions. Practice over many years has shown that, in general, ventilation rates of 10-15 air changes/hr of fresh air are sufficient to compensate for heat load and the generation of NH3 and CO2 from animals. A stable photoperiod is necessary to avoid changes in reproductive behavior, food intake, and weight gain. A cycle of 12 to 14 hr light and 10 to 12 hr dark is typically used for rats. Rats are particularly susceptible to phototoxic retinopathy. There is sufficient evidence to indicate that light intensity at cage level should be between 130 and 300 lux to prevent retinopathy (National Research Council, 1996a,b). Prevention and control of infectious diseases are partially a function of the location, size, and environmental conditions of a rat housing room. Strategies for limiting the transfer of pathogens will vary according to the potential impact that infectious agents may have on a particular group of rats and the study in

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which they are being used. For instance, an appropriate-sized room or cubicle may reflect the space necessary to separate rats by such criteria as pathogen status, immunological status, vendor, protocol, or investigator. Modifications such as the incorporation of Class 100 flexible wall enclosures may be useful to help ensure the specific pathogen status of rats over an extended period of time. Because of stress produced by noise, rat rooms should be located distant from mechanical rooms, cage washing centers, and species that are apt to produce noise (National Research Council, 1996b). 2.

Primary Enclosures

The amount of cage space needed for rats, whether group or individually housed, is a function of animal weight and the specific physiological or protocol requirements of the animal(s) (National Research Council, 1996a). Unless there is an experimental need, rats should be housed in solid-bottom rather than in wire-bottom cages. This will help prevent pododermatitis and injuries that are more frequently associated with wire floors (National Research Council, 1996b), and bedding within solidbottom cages provides environmental enrichment. The most frequently used materials for solid-bottom cages are polycarbonate and polypropylene. The former plastic is often preferred because it may be repeatedly autoclaved without damage and because its translucency allows for observation of animals. Various contact bedding materials are appropriate for rats (e.g., hardwood chips, ground corncob, cellulose sheets). 3.

Sanitation

Solid-bottom cages should typically be sanitized at a frequency of 1-2 times per week. A less frequent cycle may be appropriate if cage density is very low, if there are perinatal considerations, or if ventilated cages are used; and a more frequent cycle, if cage density is high or if pathophysiological considerations exist (e.g., diabetes). A detailed description of appropriate sanitation for rodent housing is given in "Laboratory Animal Management--Rodents" (National Research Council, 1996b).

lI.

A.

BIOLOGY

Morphophysiology

This section provides a summary of some of the morphophysiological characteristics of the rat that may be useful to the reader. For more comprehensive descriptions, see the references cited in this section.

1.

General Appearance

The Norway rat has small, thick ears and a tail that is about 85% of the length of the body (in contrast, R. rattus has larger ears and a tail that is distinctly longer than the body). The hair coat is composed of two classes--long and short hair shafts, with the former being more sparse. Hair growth in the young rat is cyclic, with the resting period and the growing period each being 17 days. In the female, there are usually 12 teats, with 3 pairs in the pectoral and 3 pairs in the abdominal region (Greene, 1963). Body weights and growth rates are dependent on the stock, strain, and source of rats. Of the two most commonly used outbred stocks, the Sprague-Dawley is larger than the Wistar, and the inbred Fischer 344 rat is smaller than either of the outbreds. 2.

Sensory Organs

Rat eyes are exophthalmic, which increases the risk of injury from trauma and drying during anesthesia. The eyelids are well developed, and only the cornea is visible. The Harderian gland, located medially to the orbit, and the lacrimal glands moisten the cornea. The Harderian gland secretes porphyrin in excessive amounts, termed chromodacryorrhea, when the animal is stressed (e.g., because of malnutrition, dehydration, disease, or environmental factors). Accordingly, a reddish secretion or crust located periorbitally and at the nares may be a useful indicator of illness or a husbandry problem (Moore, 1995). The orbital venous sinus beneath the medial aspect of the orbit is a useful site for blood collection in the anesthetized animal. It has been accepted that rats lack color vision; however, one recent study suggests that rats may have dichromatic color vision (Koolhass, 1999). Olfactory signals are strong determinants for behavior in the rat. Male rats recognize the social status of other males, females in estrus, and kinship by olfactory cues. Rats also detect alarm pheromones from other rats (Koolhass, 1999). The hearing frequency range for rats at 70 dB is 250 Hz to 7 0 - 8 0 kHz, with 8 kHz to 32 kHz being the most sensitive range. Except for the rat's high-frequency sensitivity, its hearing capability corresponds closely to that of other mammals (Kelly and Masterson, 1977). This high-frequency sensitivity corresponds to the 2 2 - 8 0 kHz vocalizations emitted by pups left alone by their dam, or by adults during sexual and aggressive behavior (Koolhass, 1999). 3.

Skeleton

The skull is composed of the following bones: paired nasal, premaxillary, maxillary, zygoma, palatine, lacrimal, frontal, parietal, squamosal, periotic capsule, tympanic bulla, and mandible; 6 auditory ossicles; 4 turbinates; and single vomer,

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Fig. 1. Salivaryglands, cervical lymph nodes, and adipose tissue. (From Greene, 1963.)

ethmoid, basisphenoid, presphenoid, occipital, interparietal, and hyoid bones. The vertebral column consists of 7 cervical, 13 thoracic, 6 lumbar, 4 sacral, and 27-.30 caudal vertebrae. The ribs consist of ventral calcified and dorsal ossified segments without true costal cartilages. The humerus, ulna, and radius are similar to those of other mammalian species. The carpus consists of 9 bones. The pelvis is formed by 2 ossa coxae, which articulate with the first 2 sacral vertebrae. The bones of the hindlimb are the femur, the tibia, and the fibula, which articulates proximally with the tibia but is fused distally. The tarsus is composed of 8 bones (Greene, 1963). 4.

Digestive System

The dental formula of the rat is 2(1 1/1, C 0/0, PM 0/0, M 3/3). Incisors grow continuously. If the incisors are not worn evenly or are misaligned due to gingivitis or congenital defects, the resuiting malocclusion may lead to nonfunctional, spiral elonga-

tion of the incisors, injury to the palate, and reduced food intake. The salivary glands are paired and consist of the parotid, the submandibular, and the smaller sublingual glands. The parotid gland is serous and consists of three or four lobes located ventrolaterally from the caudal border of the mandible to the clavicle. The submandibular glands are mixed glands situated ventrally between the caudal border of the mandibles and the thoracic inlet. The sublingual glands are mucous and located at the rostral pole of the submandibular glands. Multilocular adipose tissue, referred to as brown fat or the hibernating gland, is located in the ventral and lateral portions of the neck and can be confused with salivary glands. Figure 1 depicts the location of the salivary glands, cervical lymph nodes, and adipose tissue (Greene, 1963). The stomach is divided into two parts: the forestomach, or cardiac portion, which is nonglandular; and the corpus, or pyloric portion, which is glandular. A ridge separates the two portions, with the esophagus entering at the lesser curvature of the stomach through a fold of the ridge. This fold is responsible for the inability of the rat to vomit. The small intestine consists of the duodenum (8 cm), jejunum (80 cm), and ileum (3 cm). The comma-shaped cecum is thinwalled, with a prominent mass of lymphoid tissue in its apical portion. The colon consists of the ascending colon, with prominent oblique mucosal ridges, and the transverse and descending portions, with longitudinal mucosa folds. The liver has four lobes: the median, which has a deep fissure for the hepatic ligament; the right lateral, which is partially divided; the left, which is large; and the caudate, which is small and surrounds the esophagus. The rat does not have a gallbladder, and bile ducts from each lobe form the common bile duct, which enters the duodenum about 25 mm from the pyloric sphincter. The pancreas is a very diffuse and lobulated organ that can be differentiated from adjacent adipose tissue by its darker color and firm consistency. Numerous excretory ducts fuse into 2 - 8 large ducts, which empty into the common bile duct (Bivin et al., 1979). Figure 2 depicts the abdominal and thoracic viscera in situ (Greene, 1963). 5.

Respiratory System

The rat has a maxillary recess (sinus) located between the maxillary bone and the lateral lamina of the ethmoid bone. The recess contains the lateral nasal gland (Steno's gland), which secretes a watery product discharged at the rostral end of the nasal turbinate. It has been postulated that this secretion may act to regulate the viscosity of the mucous layer overlying the nasal epithelium. The lungs consist of the left lung, which is single-lobed, and the right lung, which is divided into the cranial, middle, accessory, and caudal lobes. The pulmonary vein has cardiac striated muscle fibers within its wall that are contiguous with those

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4. BIOLOGY AND DISEASES OF RATS

Fig. 2.

Abdominal and thoracic viscera in situ. (From Greene, 1963.)

in the heart. The rat does not have an adrenergic nerve supply to the bronchial musculature, and bronchoconstriction is controlled by vagal tone (Bivin et al., 1979).

6. GenitourinarySystem The male rat has a number of highly developed accessory sex glands (Fig. 3). The paired bulbourethral glands (Cowper's glands) at the base of the penis open into the dorsal surface of the urethral flexure. Within the abdominal cavity and surrounding the bladder are the large seminal vesicles and the pros-

tate gland, which is composed of the dorsocranial (coagulation gland), ventral, and dorsolateral lobes. The female rat has a bicornate uterus, and although the uterine horns appear fused distally, there are two distinct ossa uteri and cervices. The right kidney is more craniad than the left and has its cranial edge at the L1 vertebra and its caudal edge at the level of L3. Like the kidneys of other rodents, the rat kidney is unipapillate, making the rat useful for studies in which cannulization of the kidney is done. The rat is also widely used as a model for investigating nephron transport in an in vivo micropuncture system, because of the presence of superficial nephrons in the renal cortex.

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DENNIS F. KOHN AND CHARLES B. CLIFFORD

Fig. 3.

7.

Maleurogenital system. (FromGreene, 1963.)

Central Nervous System

The brain is characterized by large olfactory bulbs, a smooth cerebrum, and the two parafloccular lobes of the cerebellum, which lie in deep sockets of the periotic capsule of the skull. The hypophysis lies behind the optic chiasma and is attached to the base of the brain by a thin hollow stalk, the infundibulum. The ventricular system is similar to that of other animals, but the rat lacks a foramen of Magendie. The spinal cord ends at the fourth lumbar vertebra, with the ilium terminale ending at the level of the tail beyond the third caudal nerves (Greene, 1949).

B.

N o r m a l Physiological Values

Many of the normal values determined for a specific group of rats may be accurate for only that source and stock/strain. Other factors such as age, pathogen status, sample collection methods, and husbandry conditions of the colony are also important variables (Suber and Kodell, 1985; Dameron et al., 1992; Perez et al., 1997). Selected physiological, clinical chemistry, and hematological values are listed in Tables II, III, and IV.

C. 8.

Cardiovascular System

The heart is located on a midline in the thorax, with its apex near the diaphragm and its lateral aspects bounded mainly by the lungs. The heart is exposed to the left thoracic wall between the third and fifth ribs, making it a useful site for cardiac blood collection. The blood supply to the atria of the rat, unlike that of higher mammals, is largely extracoronary from branches of the internal mammary and subclavian arteries.

Nutrition

Nutritionally adequate diets for rats are readily available from commercial sources. However, the refinement of ingredients within diet formulations may vary according to classifications of commercially available products. The three classifications of diets are (1) natural-ingredient, (2) purified, and (3) chemically defined. The most commonly used type for most research applications is the natural-ingredient diet, composed of agricultural products and by-products. This class of diet can be either

127

4. BIOLOGY AND DISEASES OF RATS

an open-formula diet, in which the information on the amount of each ingredient is available, or a closed-formula diet, in which such information is held confidential by the producer. The nutrient composition of ingredients in natural-ingredient diets varies from batch to batch because of various factors (e.g., relative costs of grains, weather conditions, harvesting and storage conditions, and concentrations of contaminants). Certified, natural-ingredient diets are used for toxicological and other Good Laboratory Practice (GLP) studies because each lot is assayed and certified not to exceed established maximum concentrations of a set list of contaminants (e.g., pesticides, heavy metals, mycotoxins, and estrogens) that could influence study results. The nutrient concentrations in purified diets are less variable because defined ingredients, each composed of a single nutrient or nutrient class (e.g., casein, sugar, starch, vegetable oil, cellulose), are used in their formulation. A frequently used purified diet for rats is AIN-76. The downside of this class of diets is that Table II Selected Normative Data for the Rat a Adult weight Male Female Life span Body temperature Basal metabolism rate (400 gm rat) Chromosome number (diploid) Puberty Gestation Litter size Birth weight Eyes open Weaning Food consumption/24 hr Water consumption/24 hr Cardiovascular Arterial blood pressure Mean systolic Mean diastolic Heart rate Cardiac output Blood volume Respiratory Respirations/min Tidal volume Alveolar surface area (400 gm rat) Renal Urine volume/24 hr Na + excretion/24 hr K § excretion/24 hr Urine osmolarity Urine pH Urine specific gravity

300 -500 gm 250 - 300 gm 2.5-3 years 37.5~ 35 kcal/24 hr 42 50 _ 10 days 21-23 days 8-14 5 - 6 gm 10-12 days 21 days 5 gm/100 gm body weight 8-11 ml/100 gm body weight

ll6mmHg 90 mm Hg 300-500 beats/min 50ml/min 6 ml/100 gm body weight 85 1.5 ml 7.5 m 2 5.5 ml/100 gm body weight 1.63 mEq/100 gm body weight 0.83 mEq/100 gm body weight 1659 mOsm/kg of H20 7.3-8.5 1.04-1.07

aData from Baker (1979) and Bivin et al. (1979).

they are more expensive and often less palatable. Chemically defined diets are formulated with very basically defined ingredients (e.g., specific amino acids, sugars, triglycerides, and essential fatty acids). These diets are costly and tend to lack palatability (National Research Council, 1996b). Table V summarizes the nutrient requirements of rats (National Research Council, 1995). In most instances, rats are fed ad libitum. However, there are numerous reports that demonstrate that unlimited feeding of rats on long-term carcinogenesis and toxicological studies reduces longevity and increases the incidence of neoplasia relative to rats fed at 7 0 - 8 0 % of the ad libitum food amount. These effects have been found in Sprague-Dawley, Wistar, and F-344 rats and have caused increased variability among 2-year carcinogenicity and safety assessment studies, compromising the usefulness of bioassays in risk assessment (Keenan et al., 1996). For instance, Wistar rats fed 80% of ad libitum beginning at 16 weeks of age had very significant reductions in the incidence of lung, mammary, pancreatic islet cell, and pituitary tumors relative to controls fed ad libitum. The overall incidence of malignant tumors was 16% in the feed-limited group and 37% in the ad libitum group, even though the feed-limited group had a greater longevity. There was also a reduction in chronic inflammation and fibrosis of the heart, acute inflammation of the prostate, radiculoneuropathy, and acinal hyperplasia of the mammary gland in feed-limited animals (Roe et al., 1995).

D. 1.

Reproduction

Reproductive Physiology

In the rat, the vagina is closed at birth by compact epithelium, referred to as the vaginal plate (Del Vecchio, 1992). This begins to degenerate and cornify at 20-35 days of age and is completely open between 40 and 80 days of age. Persistence of the vaginal plate is an occasional cause of infertility. Puberty is defined as the onset of sexual maturity, the ability to bear viable young, and occurs before full body size and weight are attained. As in most species, puberty occurs in females earlier than in males and also varies with stock or strain. Puberty most often occurs at 2 - 3 months of age in the rat (Fox and Laird, 1970), although considerable variation exists in reported values. Kohn and Barthold (1984) report 4 0 - 6 0 days, and Bennett and Vickery (1970) report 5 0 - 7 2 days. More recently, Ayala et al. (1998) report 4 5 - 4 7 days. Estrus, which should be distinguished from puberty, begins before full reproductive competency is reached and has been reported to occur at 36 days in the Wistar rat (Eckstein et al., 1973). However, some authors report successfully breeding Wistar B H rats at 35 days of age (Rosen et al., 1987). The estrous cycle of rats is most often 4 - 5 days in length and

128

DENNIS F. KOHN AND CHARLES B. CLIFFORD Table III Clinical Chemistry Reference Ranges for Adult Rats a Sprague-Dawley b Analyte

Serum Glucose Urea nitrogen Creatinine Sodium Potassium Chloride Calcium Phosphorus Magnesium Iron

M

Units

mg/dl mg/dl mg/dl mEq/liter mEq/liter mEq/liter mg/dl mg/dl Ixg/dl

115 19 0.70 150 7.00 103.0 12.0 7.30 3.12 152

Fisher 344 a

+- 16.9 +- 2.2 +- 0.11 ___ 3.4 +_ 0.65 ___ 1.90 +- 0.94 ___ 1.5 +- 0.41 f ___70

111 +- 17.2 21 ___3.4 0.70 ___0.13 148 ___3.5 6.1 __+0.67 104.0 ___2.4 12.1 ___0.71 5.80 ___ 1.10 2.60 +- 0.21 f 220 ___ 130 (19-21 weeks)

Total iron binding capacity Alanine aminotransferase Aspartate aminotransferase Alkaline phosphatase Lactate dehydrogenase

Ixg/dl IU/liter IU/liter IU/liter IU/liter

368 r 49 +- 24.1 95 +- 31.7 130 +- 43.7 275 ___ 112

Sorbitol dehydrogenase

IU/liter

~/-Glutamyl transpeptidase Creatinine kinase Protein, total Albumin Cholesterol Triglycerides Bilirubin Bile acids

IU/liter IU/liter gm/liter gm/liter mg/dl mg/dl mg/dl Ixmol/liter

Uric acid

mg/dl

20 +- 5 ~ (32 weeks) 2.5 ___ 1.25 c 275 +- 112.5 ~ 70 ___5.0 34 ___2.0 119 ___51.3 266 ___ 121.4 0.3 +- 0.16 40 +- 10 c (20 weeks) 1.52 ___0.30

1.25 +- 0.36

ml/16hr rnl/22 hr

15.7 +_ 6.7

11.0 ___5.0

943 7.8 148 31 121 142 136

995 +- 367 7.7 "--0.5 151 +- 51 50 +- 22 110+- 45 156 +- 62 116 ___41

Urine Volume Specific gravity Osmolality pH Chloride Sodium Potassium Phosphorus Creatinine Glucose Protein Alkaline pho sphatase Lactate dehydrogenase N-Acetyl-13-glucosaminidase Aspartate aminotransferase ~/-Glutamyl transpeptidase Insulin clearance Glomerular filtration rate Urine flow

mOsm/kg mmol/liter mmol/liter mmol/liter mg/dl mg/dl mg/dl mg/dl IU/liter IU/liter IU/liter IU/liter ixl/min/100 gm lxl/min lxl/min/100gm

+- 327 __. 0.5 +- 36 +- 11 +- 31 +- 34 +- 40

M

F

69 ___44.9 99 ___54.5 117 +- 41.7

F

115 +- 12.5 c 15 ___2.5 c 149 4.80 106 10.5

___3.0 c + 0.35 c +- 3.0 ~ +__0.50 c

78--- 11 49.5 +- 9.25 c 650 ___75

49"--8

650 +__75 (20 weeks)

20.0 ___7.5 c

75- - - 5 40 __. 2.5 119 ___29.0 249 +_ 159.7 0.4 +- 0.27

400 +- 50 c 70.5 +__4.75 c 42.5 +- 3.75 c 96.5 ___ 14.25 122 +- 21.25 0.3 +__0.1c 30"-- 10 c

130 __. 10.0 62.5 ___ 11.25

9.5 ___4.0

9.3 +- 5.6

1.022 ___0.007

1.017 +- 0.007

6 . 0 - 6.5 c

80 __. 28 9.9 --- 3.9 98.8 _ 54.4 152 +_ 61 28___ 15 12.2 __+7.8 14.4 +_ 6.5 4964 +__780

54 __. 25 5.5 +_ 2.3 11.2 _ 5.5 73 ___ 37 16+--8 5.9 ___2.7 3.6 + 2.5 1873 +_ 215

857 +- 178 275 +- 33 c 5.2 ___2.0

occurs throughout the year, as well as postpartum. Seasonal variation is not observed in laboratory colonies. For a 4-day estrous cycle, approximately 1 day is spent in each of the four

stages: estrus, metestrus, diestrus, and proestrus. However, cycles of up to 6 days are not uncommon, with the additional time in diestrus or proestrus (Peluso, 1992). In proestrus, the uterus

129

4. BIOLOGY AND DISEASES OF RATS

Table III (Continued) Analyte Hormonee Luteinizing hormone

Units

Male

ng/ml

0.16-0.64

Follicle stimulating hormone

ng/ml

Prolactin

ng/ml

Growth hormone Thyroid stimulating hormone

ng/ml ng/ml

5.56-11.1 (light period) 11.1-20.0 (dark period) 28.6 (dark period) 1.8-10.7 (light period) 71.4 (after coitus) 0.4-80 c 2.27-3.4

Adrenocorticotropic hormone Vasopressin Oxytocin Thyroxine (T4) Triiodothyronine (T3) Free T4 Free T3 Calcitonin Parathyroid hormone 1,25-Dihydroxy vitamin D Corticosterone

pg/ml pg/ml pg/ml ~g/dl ng/dl ng/dl pg/dl pg/ml pg/ml pg/ml Ixg/dl

Aldosterone

ng/dl

Epinephrine

pg/dl

Norepinephrine

pg/dl

Progesterone

ng/ml

Estradiol

pg/ml

Testosterone

ng/ml

Female

0.32-0.64 (basal) 24.6-32.8 (late proestrus) 2.22-4.44 (basal) 8.85-13.3 (preovulatory, estrus) 5.4-10.7 (basal) 71.4 -107 (late proestrus) 0.57 (basal) 1.14 (early light period)

30-100 1-8 c 4-10.5 c 5.1 ___0.4 66 ___3.5 2.212 _+ 0.055c 208.49 + 8.55c 200-1000 c (F-344/9 months) 140-180 120 _+ 24 15-23 (late light period) 1-6 (late dark period) 12-35 (late light period) 4-11 (middle light period) 253 ___30c (awake, undisturbed; Sprague-Dawley) 460 + 60c (handled) 180 ___24c (asleep) 710 _+ 110c (awake, undisturbed; Sprague-Dawley) 830 ___130c (handled) 460 ___80c (asleep)

4.9 _+ 0.1 (Long-Evans) 83 ___3 (Long-Evans)

<50-400 (Sprague-Dawley) 96 ___17 (Wistar) 70 (late light period) 17 (late dark period) 25-35 (9-10 AM)

1-5 (early proestrus) 40-50 (estrus) 20-30 (first diestrus day) < 10 (basal) 20-30 (2nd diestrus day) 40-50 (proestrus)

3 (1330-1600 hr) <1 (2130hr)

pg/ml

500-600 (proestrus) 1O0 (estrus)

a Values are for 12-month-old animals, unless noted otherwise, as summarized from Loeb and Quimby (1999). b6-- 18 months old. cGender not specified. d58--112 weeks old. Species not specified. YWistar strain. e

can appear "ballooned" with fluid, especially in a peripubertal rat; this condition should not be mistaken for hydrometra. Ovulation occurs approximately 8 - 1 1 hr after the onset of estrus, usually between midnight and 2 A.M. (Peluso, 1992), although this would obviously d e p e n d on timing of the light cycle. Ova remain viable for approximately 1 0 - 1 2 hr (Fox and Laird, 1970). Testes descend from the a b d o m e n into the scrotum at approx-

imately 15 days of age (Russell, 1992). S p e r m are first produced at about 4 5 - 4 6 days of age, but fertility (puberty) does not occur until approximately 6 2 - 6 5 days of age, and sperm production is not m a x i m a l until 75 days of age (Russell, 1992). Interestingly, on histologic examination of young rat testes, more degenerate g e r m cells are noted prior to 75 days of age than afterward, indicative of the poor efficiency of spermatogenesis at early ages in the rat (Russell et al., 1987). Male sexual behavior

130

DENNIS F. KOHN AND CHARLES B. CLIFFORD Table IV

Hematological Parameters in the Rat a Erythrocyte parameters (mean values) Erythrocyte

PCV (%)b

(X 106mm 3)

Stock/strain Cfl:CD(SD)BR Cfl:(WI)BR

CDF(F-344)CRIBR

Hsd:SD Hsd:WI F-344/NHsd Tac:N(SD)fBR Tac:Sim(LE)

Hemoglobin (gm/dl)

MCV (fl)~

MCHC (%)0

Age (weeks)

M

F

M

F

M

F

M

F

M

F

8 12 6-8 19-21 32-34 6- 8 19-21 32-34 11-12 11-12 11-12 6 10-12 11

7.98 8.32 6.46 8.31 8.4 5.38 7.62 5.51 7.05 7.85 8.61 7.1 7.82 8.0

7.44 7.6 6.92 7.81 7.8 5.35 7.0 5.05 6.68 7.06 7.32 6.9 8.1 7.3

43 44.5 36 41 42 43 46 49 41.7 43.8 44.9

41 41.5 38 40 40.7 40 50 42 39.9 39.5 40.1

55 55 55 50 52 76 72 83 59.7 56.0 49.8

35 34 37 38 37 35 33 34 36.3 37.2 37.4

35 35 36 39 40 35 36 36 37.8 37.1 36.7

40.7

14.2 14.5 14.1 15.6 16.3 14.0 16.4 15.0 15.0 14.7 14.7 14.6 16.65 13.4

54 53 56 53 50 79 61 89 59.2 55.8 52

45.2

14.9 15.3 13.5 16.0 15.5 14.9 15.3 16.6 15.1 16.2 16.7 15.5 16.4 14.5

56

56

32.0

32.9

Leukocyte parameters (mean values)

Stock/strain Crl:CD(SD)BR Crl:(WI)BR CDF(F-344)CrlBR Hsd:SD Hsd:WI F-344:NHsd Tac:N(SD)fBR Tac:Sim(LE)

Age (weeks ) 8 12 6-8 19-21 32-34 6-8 19-21 32-34 11-12 11-12 11-12 6 10-12 11

WBC ( • 103)

Neutrophil

Lymphocyte

M

F

M

F

M

F

11.82 11.25 8.66 9.37 7.8 6.4 8.2 4.7 4.5 15.2 5.8 6.8 5.24 9.3

9.28 8.79 6.96 8.43 6.0 5.5 8.3 6.9 4.9 7.7 5.4 4.8 6.71 7.4

10 11 12 15 18 17 30 52 29 12 17 8.2 11.33 17.2

10 9 13 17 23 14 43 38 30 12 15 9.7 13.88 21.8

84 83 85 82 80 78 68 46 65 87 81 91.6 88.11 80.8

86 85 84 80 75 83 52 56 64 83 79 89.8 85.75 75.9

Derived from vendor data. bpcv, packed cell volume a

is, in part, dependent on age and experience. Prepubertal males have no preference for females in estrus, and female-oriented sexual behavior is reported to decrease after 150 days of age ( M a t u s z c z y k et al., 1994; Smith et al., 1992). D e c r e a s e d s e r u m testosterone m a y be partially responsible for age-related decreases in mating behavior, but this does not appear to completely explain the p h e n o m e n o n . Coitus occurs m o r e frequently during dark periods than light periods, and m o r e frequently during the latter portion of the dark cycle than during the early portion (Mercier et al., 1987). Multiple intromissions ( 5 - 1 5 ) , each lasting 0 . 3 - 0 . 6 seconds and with 2 - 9 pelvic thrusts (Bennett and Vickery, 1970), precede the first ejaculation, which lasts about 1 second. This first

Monocyte (differential %) M 2.6 2.6 2 2 0 4 1 2 <3 <1 <2 0 0 1.2

F 2.3 2.3 2 2 0 3 5 4 <3 <4 <4 0 0 1.2

Eosinophil

Basophil

M

F

M

F

1 1 1 1 2 1 1 1 <2 <1 <1 0 0 0.9

1 1 1 1 2 1 1 1 <2 <1 <2 0 0 1.1

<1 <1 0 0 0 0 0 0 <3 <1 <1 0 0 0

<1 <1 0 0 0 0 0 0 <2 <1 <1 0 0 0

cMCV, mean corpuscular volume dMCHC, mean corpuscular hemoglobin contact series of intromissions is called the ejaculatory latency and lasts about 10 minutes, followed by a refractory period (Dewsbury, 1970). A single ejaculation with fewer intromissions is less likely to impregnate the female. M u l t i p l e series of intromissions and ejaculations occur, usually about seven, with increasing refractory periods b e t w e e n successive episodes. Coitus can be confirmed in rats by detection of s p e r m a t o z o a on a vaginal smear, by observation of a vaginal plug (the pale c o a g u l u m f o r m e d by seminiferous fluid visible in the vagina), or by direct observation of sexual behavior. Implantation of the blastocyst into the e n d o m e t r i u m occurs b e t w e e n 4 and 7 days after fertilization and actually represents a process that requires 1 2 - 2 4 hours for c o m p l e t i o n (Peluso,

131

4. BIOLOGY AND DISEASES OF RATS

Table V Recommended Composition of Diet for Growing Rats a Main components and amino acids Digestible energy (kJ/gm) Fat (gm/kg) Fiber (gm/kg) Protein (gm/kg) Arginine (gm/kg) Asparagine (gm/kg) Glutamic acid (gm/kg) Histidine (gm/kg) Isoleucine (gm/kg) Leucine (gm/kg) Lysine (gm/kg) Methionine + cystine (gm/kg) Phenylalanine + tyrosine (gm/kg) Proline (gm/kg) Threonine (gm/kg) Tryptophan (gm/kg) Valine (gm/kg) Glycine (gm/kg)

16.0 50 r.u. b 120 6 4 40 3 5 7.5 7 6 8 4 5 1.5 6 s.u. c

Minerals and trace elements Calcium (gm/kg) Chloride (gm/kg) Magnesium (gm/kg) Phosphorus (gm/kg) Potassium (gm/kg) Sodium (gm/kg) Sulfur (gm/kg) Chromium (mg/kg) Copper (mg/kg) Fluoride (mg/kg) Iodine (mg/kg) Iron (mg/kg) Manganese (mg/kg) Selenium (mg/kg) Zinc (mg/kg)

5 0.5 0.4 4 3.6 0.5 0.3 0.3 5 1 0.15 35 50 0.1 12

Vitamins Retinol (mg/kg) Cholecalciferol (Ixg/kg) DL-oL-Tocopherylacetate (mg/kg) Menadione (mg/kg) Thiamin (mg/kg) Riboflavin (mg/kg) Pyridoxine (mg/kg) Cyanocobalamin (l~g/kg) Nicotinic acid (mg/kg) Folic acid (mg/kg) Biotin (mg/kg) Pantothenic acid (mg/kg) Choline (mg/kg) Inositol (mg/kg) Ascorbic acid (mg/kg)

1.2 25 27 0.05 4 3 6 50 20 1 s.u. c 8 1000 n.r. d n.r. d

From National Research Council (1995). b r.u., required but degree unknown. cs.u., status unknown. dn.r., not required.

a

1992; Enders, 1970; Garside et al., 1996). Parturition occurs 21-23 days after the time of coitus, although it can occur as early as 19 days (Bennett and Vickery, 1970; Baker, 1979). Pregnant rats whose time of coitus is known are therefore of known gestational length and are called timed pregnant.

10 days, careful palpation can detect the developing fetuses; this is especially accurate after 12 days of gestation. By 14 days of gestation, mammary gland and nipple development are evident (Bennett and Vickery, 1970). 3.

2.

Detection of Estrus and Pregnancy

Several methods can be used to determine if a rat is in estrus, which is useful in production of timed pregnant rats. Rats in estrus often exhibit specific behavior, including ear quivering when the back or head are stroked, and lordosis ("swayback" posture) when the pelvic area is stimulated (Blandau et al., 1941). Additionally, the vulva becomes swollen, and the vaginal wall appears dry in estrus, instead of the moist pink appearance during metestrus or diestrus. This is due to cornification of the vaginal epithelium during estrus (Baker, 1979). These changes in the vaginal epithelium can be assessed by cytologic examination of vaginal smears (Montes and Luque, 1988). In estrus, 25-100% of the epithelial cells are cornified (Bennett and Vickery, 1970). Changes in vaginal fluids and cytology also lead to changes in the electrical impedance in the vagina during estrus. This has been widely exploited as a more precise method of estrus detection (Koto et al., 1987a, b), using a device referred to as an impedance meter in which an electrical probe is inserted into the vagina. Pregnancy is difficult to detect early in gestation, but conception rates of 85% or more are often observed for outbred rat stocks, somewhat less in inbred strains. After approximately

Husbandry Needs

Inbred rats are generally bred monogamously or in trios, with one male and two females in each cage. Outbred rats may also be bred monogamously but are more often bred polygamously by commercial breeders for reasons of economy. Pregnant females are removed to separate cages a few days before parturition to minimize cannibalism or abandonment of litters. A number of variables have been identified that may influence the husbandry requirements of a reproducing population of laboratory rats. Despite the lack of seasonal variation in estrous cycles in the rat, both ovarian function and the estrous cycle are influenced by light cycles. Continuous light has been reported to cause persistent estrus and cystic follicles in the ovaries, without formation of corpora lutea (Fox and Laird, 1970). Chronic exposure to even low intensity light during the dark cycle has been reported to result in earlier vaginal opening and ovarian atrophy (Beys et al., 1995; Fox and Laird, 1970). Caloric restriction, a 15-30% decrease from ad libitum caloric intake, may cause cessation of estrous cycles and delayed sexual maturation (Fox and Laird, 1970). High ambient temperatures can result in male infertility (Pucak et al., 1977) by causing irreversible degeneration of the seminiferous epithelium. Significantly, the damage may occur

132

DENNIS F. KOHN AND CHARLES B. CLIFFORD

in rats as young as 4 days and in rats with prolonged exposure to temperatures as low as 26.6 ~C. 4.

Parturition

Female rats increase nest-building activity approximately 5 days prepartum and continue through lactation (Bennett and Vickery, 1970). Females will use any material available, but it is not necessary to add material for this purpose to the cages. Approximately 1.5-4 hours before the first pup is born, clear mucoid fluid discharges from the vagina. In early labor, the female walks about the cage and stretches. This behavior becomes more exaggerated as events progress; then the female will lie on her abdomen with rear legs extended off the cage floor. As pups are born, the female pulls the placenta from the birth canal and eats it. Parturition averages 1-3.5 hours, varying with litter size. Nursing usually begins only after all pups are born. Litter size varies with stock, strain, source, and maternal age. The following are examples of the effect of maternal age. Wistar BH rats had an average of 1.69 more pups (11.80 vs. 10.11) when first mated at 105 days than when first mated at 35 days of age (Rosen et al., 1987). The second litter is usually the largest (Bennett and Vickery, 1970). After 9 months of age, litter size is further decreased, and the pregnancy rate declines after 12 months of age (Niggeschulze and Kast, 1994). Loss of fetuses, termed pregnancy wastage, occurs as a function of age (Mattheij and Swarts, 1991), with less than 5% wastage in 4-month-old rats, 30% in 9-month-old rats, and 65% at 11 months of age. Wastage is primarily due to preimplantation and early postimplantation mortality. In contrast to the decremental effects of aging on litter size, maternal behavior in virgin rats is enhanced at 19-20 months, when compared with those at 3 4 months of age (Gonzalez and Deis, 1990). In addition, at least some maternal stressors can lead to fetal wastage. An increase in fetal wastage was reported to be due to an earthquake that occurred when the dams were at 7, 8, or 9 days of gestation, although no difference was noted in the number of live births, fetal weight, or incidence of runts (Fujinaga et al., 1992). This raises the possibility of similar fetal loss when rats are shipped at this stage of gestation, although such has not been documented in the peer-reviewed literature. Strenuous maternal exercise, i.e., running on a treadmill--has also been reported to result in decreased litter size and decreased fetal weight (Mottola et al., 1992). Dystocia is rare in rats. Cannibalism is not frequently encountered and is an indicator of maternal stress. 5.

Early Development of the Newborn

Rat pups are altricial and nidicolous; they are hairless and blind, with poorly developed limbs, short tails, and closed ear canals (Baker, 1979). There is an inverse relationship between fetal or birth weight and litter size (Romero et al., 1992). This phenomenon is significant for the reproductive toxicologist because the tendency of a test substance to cause decreased fetal

weight may be masked if it also causes fetal loss. Other factors also influence birth weight and weaning weight, including the age of the dams. Pups of dams mated at 105 days of age weighed more at weaning than pups of dams mated at 35 or 70 days (Rosen et al., 1987). The external acoustic meati open between 2.5 and 3.5 days of age. Internally, the cochlea and organ of Corti are immature at birth but develop rapidly to approximately adult morphology by the time of weaning. Rats appear to first be able to hear at about 9 days of age, although they are able to vocalize from the time of birth (Feldman, 1992). Incisors erupt at 6 - 8 days of age, although molars do not erupt until 16 (molar 1), 18 (molar 2), and 32-34 days of age (molar 3) (Brown and Leininger, 1992). The retina is poorly developed at birth, equivalent to a human fetus of 4 - 5 months. The eyelids open at about 14-17 days of age, although the retina does not fully mature until 3 0 - 4 0 days of age, and the final components in the angle of the anterior chamber are not fully formed until 60 days of age (Weisse, 1992). Some hairs may be present on the trunk at birth, usually associated with touch domes, indicating that they are guard hairs (English and Munger, 1992). Pups are considered fully haired at about 7-10 days of age. Maternal antibody is transferred passively across the yolk sac in utero (Laliberte et al., 1984). Antibody can also be transferred across the intestinal mucosa from maternal colostrum and milk in the suckling rat. This transfer occurs at low rates shortly after birth, reaches maximal rates at day 14, and ceases by the 21 days, when gut closure is said to be complete (Martin et al., 1997). 6.

Sexing

Sex is readily determined in mature rats by direct observation of the perineal region. Males have a distinct scrotum located between the anus and the preputial opening. The penis is often visible and is larger than the urethral papilla of the female. In addition the distance between the anus and the genital opening, called the anogenital distance, is greater in the male than in the female. Sex discrimination is more difficult in prepubertal rats but is possible even in neonates. Comparative evaluation will reveal that neonatal males have a greater anogenital distance than their female littermates, although the distinction is more subtle than in adults. More recently, a technique for sex determination of preblastocyst embryos has been described (Utsumi et al., 1991). Male embryos ceased development in the presence of antibody to the HY antigen, and resumed development only after the antibody was washed off. In contrast, 80% of the embryos that developed into blastocysts in the presence of the HY antibody produced female pups after the blastocysts were implanted. 7.

Weaning

Rats are weaned at 20-21 days of age, although they may be weaned successfully as early as 17 days. Prior to 17 days, the pups may not be fully capable of urination without maternal

133

4. BIOLOGY AND DISEASES OF RATS

stimulation, and weaning may result in obstructive urinary tract disease. 8.

Synchronization of Estrus

Synchronization of estrus in the rat can be accomplished by administration of 40 mg methoxyprogesterone in the drinking water for 6 days (in 200 ml ethanol/liter water, prepared fresh daily), followed by intramuscular injection with 1 IU of pregnant mare's serum (Baker, 1979). Although synchronization of estrus may be useful in the production of large numbers of timed pregnant rats, use of the impedance meter, as described above, may be more practical in most circumstances. More recently, synchronization of estrus to prepare recipients in embryo transfer has been reported. Mature females were administered 40 ~tg of luteinizing hormone releasing hormone agonist (Rouleau et al., 1993). Five days later estrus was confirmed by vaginal cytology. 9.

Artificial Insemination and Embryo Transfer

Artificial insemination (AI) in rats is complicated by the rapid coagulation of semen, especially when the semen is obtained by electroejaculation, due to the contributions of the coagulating glands and seminal vesicles (Bennett and Vickery, 1970). These glands may be surgically removed without compromising fertility rates from AI. Alternatively, sperm may also be collected by stripping directly from the epididymis, although probably not more than twice from each male rat. Sperm from the proximal portion of the cauda epididymidis are reported to have greater fertility than sperm for the middle or caudal portions (Moore and Akhondi, 1996). Once collected, sperm may be surgically introduced directly into the uterus of estrous females (Orihuela et al., 1999). An essential step in assuring the success of AI is the induction of pseudopregnancy in the recipient female by prior mating with a vasectomized male, by mechanical stimulation of the vagina, or by electrical stimulation of the cervix (Bennett and Vickery, 1970; Rouleau et al., 1993). Embryo transfer in rats is becoming more widely used as an alternative to cesarean rederivation in order to eliminate pathogens from breeding lines. Embryo transfer can also be used to investigate whether specific characteristics are due to, or modified by, the uterine environment, in contrast to being solely determined by genetic factors (Kubisch and Gomez-Sanchez, 1999; Rouleau et al., 1993). Additionally, embryo collection is the first step in cryopreservation. In embryo transfer, embryos are collected 2 - 4 days after the females are bred. Embryos are usually washed in phosphate-buffered saline (PBS) and bovine serum albumin (BSA), with or without added trypsin. Trypsin may more effectively remove pathogens than PBS alone. Embryos are then suspended in PBS with BSA and fetal calf serum and surgically transferred into the uterus or oviduct of the pseudopregnant recipient (Kubisch and Gomez-Sanchez, 1999; Rouleau et al., 1993). Nonsurgical implantation of embryos

through the cervix, using an otoscope, has also been reported (Bennett and Vickery, 1970) but has not found wide use. In vitro fertilization (IVF) is performed in the rat but is used primarily as a research tool for events in fertilization and early development rather than as a colony management tool (Gaddum-Rosse et al., 1984; Vanderhyden et al., 1986). One form of IVF, the microinjection of spermatids into individual oocytes, is, however, used in mice to rescue or maintain strains that do not produce motile spermatozoa (Tanemura et al., 1997; Songsasen and Leibo, 1998). The same techniques will probably become more common in rats.

10.

Cryopreservation

Cryopreservation has not been performed in rats as often as it has in mice, but the technique is becoming more widespread, for the same reasons that it is used widely in mice (Tada et al., 1995). Cryopreservation can be an efficient method of maintaining the potential of raising live mice of the thousands of genetically modified genotypes currently available (Songsasen and Leibo, 1998). It can serve as a fail-safe measure, should a strain become genetically contaminated. In addition to being used for murine reproductive purposes, frozen embryos are also used to test culture reagents and environments for human IVF (Meyer et al., 1997). Although embryos, two-cell through morula, are most frequently cryopreserved, the techniques for cryopreservation of mouse sperm have recently been developed (Songsasen and Leibo, 1998; Tanemura et al., 1997). Cryopreservation of sperm has not yet been reported for rats.

E.

Behavior

Relatively little space in many laboratory animal medicine texts, including the previous edition of this volume, has been devoted to the behavior of laboratory rats, especially as it relates to experimental design or disease status. Although this unfortunate, perhaps unavoidable, lack may lead some readers to conclude that rat behavior is not an important aspect of laboratory animal science, quite the contrary is actually true. Many aspects of the rat's normal behavior may affect scientific use of rats in biomedical research and should be investigated by researchers prior to initiating studies in those specific areas. Aspects of rat behavior relevant to experimental design and disease status may be considered in two broad and overlapping categories: normal behavior, and stressors and stress responses. Only a few examples will be cited here. Laboratory rats of all stocks and strains have been selected for many years for a variety of traits, among which is docility. Nonetheless, strain differences exist. Sprague-Dawley rats, such as the CD and SD, and Lewis rats are generally more docile than Brown Norway or F-344 rats. Frequent gentle handling will increase docility, whereas infrequent or rough handling will

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evoke fear responses. Gentle handling not only reduces the likelihood of occupational injury for animal workers but also avoids stress for the rats. Handling-induced stress can lead to altered responses in behavioral studies (Hirsjarvi and Valiaho, 1995; Shalev et al., 1998). Handling also leads to vocalization, much of which is ultrasonic, in the range of 22 kHz (Brudzynski et al., 1993; Brudzynski and Chiu, 1995; Brudzynski and Ociepa, 1992). Stress-induced vocalization can make handling more difficult for other rats within hearing range. An additional interesting fact regarding rat vocalization, illustrative of its importance in rat behavior, is that rat pups vocalize in the ultrasonic range, probably to signal their mothers, even before their ears are sufficiently developed for them to be capable of hearing (Feldman, 1992). Rats are most active at night but will also move and feed some during the day; they are also more active in the mornings than in afternoons (Saibaba et al., 1996). This circadian rhythm is relevant to a broad range of behavioral measurements. For example, pain threshold is often determined in a tail flick test. Female rats have shorter tail flick response times in the middle of the dark period, as well as during estrus and metestrus (Martinez-Gomez et al., 1994). Rats, as are other rodents, are coprophagic and vary considerably between individuals in the percentage of feces consumed. This may be of significance when measuring fecal output volume or intestinal absorption of some agents. However, it appears to have no effect on iron absorption (Tidehag et al., 1988). Rats may be housed singly or in groups. In general, males are less likely to fight when housed together than are male mice, but they also do well when housed singly, as is the norm in many toxicology and safety assessment studies. Temporary single housing of female Wistar:Han rats accustomed to group housing resulted in elevated glucose levels, although the same was not observed in males (Perez et al., 1997). It is not clear if the change in glucose levels was due to stress of being alone, was just a generic response to any change in environment, or was a result of the higher food consumption recorded in the singly housed females. Also, given a report that transportation stress reduced blood glucose in Wistar:WU rats (Van Ruiven et al., 1988), it is not clear whether the higher or lower blood glucose levels are more "normal." When afforded the choice, rats have shown preferences for solid flooring, bedding consisting of large particles of aspen wood chips, and nest boxes (Manser et al., 1995a,b; Blom et al., 1995; Manser et al., 1998a,b), although the consequences of being deprived of the preferred housing factors have not been reported. When provided with objects as part of an environmental enrichment program, rats will chew on inanimate objects such as wooden blocks and nylon bones and balls (Watson, 1993; Chmiel and Noonan, 1996). No deleterious effects of these objects have been found; neither have benefits been measured.

DENNIS F. KOHN AND CHARLES B. CLIFFORD

III.

A. 1.

DISEASES

Infectious Diseases

Bacterial, Mycoplasmal, and Rickettsial Infections

a.

Streptococcosis

Streptococcosis is disease caused by infection with Streptococcus spp: Several species of Streptococcus are opportunistic pathogens in rats (i.e., they can cause clinical disease under at least some circumstances). Streptococcus pneumoniae, which is a-hemolytic, is the Streptococcus species of most historic concern in the rat, although various members of the [3-hemolytic group also occasionally cause opportunistic infection. In addition, Enterococcus spp., which are not truly streptococci, are often considered together with Streptococcus spp. Pneumonia caused by S. pneumoniae has historically been referred to as streptococcosis. However, because the term streptococcosis could be used to describe any streptococcal infection, it is inherently nonspecific and should be avoided. Streptococcus pneumoniae is rarely present today in commercially obtained rats and is now considered to be a pathogen of low significance in laboratory animals (National Research Council, 1991). Humans are the natural host of S. pneumoniae, with both adults and children frequently colonized. Streptococcus pneumoniae is transmitted primarily via aerosol, although fomites may play a minor role. Disease due to S. pneumoniae has been infrequently reported in rats, but infection is usually asymptomatic. In asymptomatic rats the organism colonizes the nasopharynx. Numerous serotypes of S. pneumoniae exist; disease is predominantly associated with infection by more pathogenic serotypes, especially 2, 3, 8, 16, and 19 (Fallon et al., 1988). Infection in rats resembles that in both human and nonhuman primates, characterized by suppurative inflammation in the upper respiratory tract, which spreads to the lung to cause bronchopneumonia (Kohn and Barthold, 1984) and sometimes fibrinosuppurative pleuritis. Affected rats may become bacteremic and may develop fibrinopurulent inflammation of other serous surfaces (e.g., peritoneum, synovium) and other tissues. Monitoring for S. pneumoniae infection is conducted by na.sopharyngeal culture onto blood agar. Differentiation of S. pneumoniae from other a-hemolytic streptococci is most often performed by the optochin inhibition test. Optochin inhibition is greater for most S. pneumoniae strains than for other a-hemolytic streptococci. However, because of the occurrence of nonpathogenic isolates (Fallon et al., 1988), isolation of S. pneumoniae from rats, even if a respiratory problem is present in the colony, does not necessarily provide a diagnosis, nor does isolation of S. pneumoniae from asymptomatic rats necessarily indicate a colony health threat. Action to eliminate

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4. BIOLOGY AND DISEASES OF RATS

S. pneumoniae is indicated in the presence of characteristic lesions or detection of known pathogenic serotypes. 13-Hemolytic streptococci are also present in many rats but rarely cause disease. 13-Hemolytic streptococci are divided into groups based on Lancefield antigens, with Lancefield groups B and G most commonly isolated from rats. Infrequently, they may be isolated from abscesses, but exclusion from most colonies is neither necessary nor practical, for humans are often carriers. So-called streptococcal enteropathy is actually due to nonhemolytic (T-hemolytic) Lancefield group D enterococci, including Enterococcus hirae, E. faecium-durans 2, and E. faecalis 2 (Barthold, 1997). Streptococcal enteropathy is a disease that affects only suckling rats, not postweaning animals. Affected litters develop diarrhea or soft stool, with bright yellow pasty feces. Mortality can be high. Microscopically, the villi of the small intestine are carpeted with gram-positive cocci. Disease is clearly associated with some strains of enterococci and not with others, but the factors determining the pathogenic potential have not been elucidated. They may, however, involve the ability of pathogenic isolates to adhere to the surface of the microvilli. Control of Streptococcus spp. and Enterococcus spp. is problematic, because the organisms are virtually ubiquitous, including being present in a high percentage of the human population (Del Vecchio, 1992). Some Enterococcus spp. have even been considered autochthonous flora of the rat (Savage, 1971). Streptococci can be excluded by aseptic microisolator technique or by use of isolators (Pleasants, 1974), yet the low incidence of disease may not warrant the additional time, expense, or other resources that such housing techniques would require. b.

Pseudotuberculosis

Pseudotuberculosis is caused by Corynebacterium kutscheri, which can infect rats, mice, guinea pigs, and hamsters, although in the last two there is only bacteriological evidence. Infections with C. kutscheri are usually clinically silent (Suzuki et al., 1988; Amao et al., 1995). Nonspecific clinical signs may be observed, such as ruffled fur, hunched posture, dyspnea and rales, porphyria, mucopurulent ocular and nasal discharges, lethargy, and lameness. These are usually followed by death in 1 to 7 days. In infected colonies, C. kutscheri will typically cause latent infections and may be cultured from submaxillary (cervical) lymph nodes, oropharynx and nasopharynx, middle ears, and preputial glands. Latent infections may be triggered to become clinical by a variety of stressors that can cause immunosuppression in the host. These include poor husbandry, overcrowding, shipping, malnutrition, intercurrent infections, irradiation, and treatment with immunosuppressive drugs (Barthold and Brownstein, 1988). As with other persistent infections, such as mycoplasmosis, disease is more frequent in older animals.

Transmission is probably through direct contact or oronasal exposure. Lesions are due to septic emboli becoming trapped in organs or tissues with an extensive capillary network, such as lung, liver, kidney, and synovium. Although any organs and tissues may be involved, the lung is the organ most frequently involved in the rat. Gross lesions of C. kutscheri infection consist of solitary or multiple randomly distributed abscesses in the lung, liver, kidney, skin, and joints. Suppurative inflammation may also be found in the preputial gland and tympanic bullae. Histopathologically, the lesions are generally as expected from the gross findings. Interstitial inflammation in the lung is due to the hematogenous seeding of the lung with bacteria, although bronchi and bronchioles may also contain suppurative exudate. Caseous necrosis is often prominent, and epithelioid macrophages and multinucleated giant cells may be present in areas of abscessation. Large areas of caseous necrosis may also be present in the liver. Septic embolic glomerulitis may be present in the kidneys, as may abscesses with or without pyelonephritis. Abscesses and caseous necrosis may also be observed in virtually any tissue. Definitive diagnosis is accomplished by bacteriologic culture (Fox et al., 1987). The best site, other than lesions, to culture is probably the submandibular (cervical) lymph nodes. The oral cavity, cecum, colon, and rectum may also harbor the organism. Microscopic evaluation may reveal the characteristic irregularly branching arrays of gram-positive rods in tissue sections (Brown and Brenn stain) or impression smears (Gram stain). However, if possible, histopathology should always be confirmed by bacteriology. Disease provocation tests, often called stress tests, have also been employed to activate latent infections with C. kutscheri, as with Pneumocystis carinii and Clostridium piliforme. Adequate culture techniques should obviate the need for stress tests. Serology has also been widely employed for detection of C. kutscheri infection in immunocompetent rats (Fox et al., 1987). As with other serologic assays, especially serologic assays for agents more antigenically complex than viruses, false positives and false negatives occasionally occur, so positive resuits should always be confirmed by culture. Differential diagnosis for the presence of multiple abscesses in rats should include streptococcosis, streptobacillosis, mycoplasmosis (pulmonary abscesses), CAR bacillus infection (pulmonary abscesses), or other miscellaneous bacteria. Of these, only mycoplasmosis and CAR bacillus infection would be found predominantly in older animals.

c.

Tyzzer's Disease

Tyzzer's disease, first discovered by Tyzzer in Japanese Waltzing mice (Tyzzer, 1917), is caused by Clostridium piliforme (Duncan et al., 1993), formerly known as Bacillus piliformis. The host range is protean among mammals, including

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rat, a distended abdomen has been observed in weanlings with Tyzzer's disease, albeit at a very low incidence (Hansen et al., 1992b). Clostridium piliforme is transmitted horizontally in rats by spores through fecal-oral contamination. The spores are highly resistant to desiccation and some disinfectants (Ganaway, 1980). The delicate vegetative form, however, survives only inside of cells. After being ingested, C. piliforme spores produce a vegetative form, which is actively phagocytosed by mucosal epithelial cells covering the gut-associated lymphoid tissue, or Peyer's patches (Tyzzer, 1917; Duncan et al., 1993). Multiple, pale foci, pinpoint or larger, of necrosis are often visible on the surface of and within the liver. Megaloileitis--a greatly dilated, flaccid, and hyperemic ileummmay be present (Fig. 4). Hyperemia, edema, hemorrhage, and ulceration may affect any part of the intestine, especially the terminal ileum, cecum, and colon. Secondary to intestinal involvement, mesenteric lymph nodes may be enlarged, hyperemic, and edematous. In the heart, pale circumscribed areas may be visible on the epicardium. Myocardial necrosis due to Tyzzer's disease may also appear as pale linear streaks or areas in the heart, especially near the apex. Histopathologically, characteristic lesions may be observed in the liver, ileum, cecum, and colon, and, less frequently, the heart. In the intestinal tract, there may be necrotizing enteritis, typhlitis, and colitis. Coagulative necrosis in the liver is the hallmark lesion and is often accompanied by a moderate leukocytic infiltrate, usually neutrophils and mononuclear cells, at the periphery of the lesions. Acute lesions may be hemorrhagic, and mineralization may occur with time. In the heart, myocardial degeneration and necrosis occurs in a minority of cases, often with a mixed leukocytic infiltrate and dystrophic calcification. Histopathologic evaluation is diagnostic if the characteristic bacilli are observed (Tyzzer, 1917; Duncan et al., 1993). The vegetative form of the organism is a filamentous bacillus, 8 20 ~tm long and 0.3-0.5 ~tm wide (Fig. 5). Bacilli are intracellular, are often numerous, and may appear as either a jumbled array (pickup stick) or parallel arrangement, as dictated by the shape of the cell. The vegetative form may rarely be visible in hepatocytes in tissue sections stained with hematoxFig. 4. Tyzzer'sdiseasein an adolescentrat with the skin reflected. Note en- ylin and eosin, but usually special stains are necessary, includlargement of ileal loops in situ. (Courtesyof Dr. StevenWeisbroth.) ing Warthin-Starry silver (best), Giemsa, and methylene blue stains. Although gram-negative, C. piliforme stains very poorly with gram stains. In the liver, the organisms are most often obnumerous rodent species, rabbits, carnivores, horses, and both served in surviving hepatocytes at the periphery or within lenonhuman and human primates (DeLong and Manning, 1994; sions. In the intestine, normal gut flora within mucosal crypts Skelton et al., 1995). Clostridium piliforme infection is usually clinically silent and superimposed upon the mucosal epithelial cells may com(Motzel and Riley, 1992; Hansen et al., 1992a). Overt disease in plicate evaluation. Organisms may also occasionally be obrats, as in other species, is most likely to be observed in young, served in cardiac myocytes or myocytes of the tunica muscurecently weaned animals. In these, the clinical signs are non- laris of the intestine. Differential diagnoses for necrotizing hepatitis in the rat specific (anorexia, lethargy, emaciation, ruffled fur) and may include acute death without clinical signs. Diarrhea may be should include other bacterial septicemias, such as Corynebacnoted and may contain mucus and blood. Particularly in the terium kutscheri, as well as infection with rat virus. Diagnosis

4. BIOLOGY AND DISEASES OF RATS

137

Fig. 5. Tyzzer's disease in rat ileum. Clostridium piliforme in villous submucosa at base of crypt. Giemsa stain. Magnification: X800. (Courtesy of Dr. Steven Weisbroth.)

of clinical disease depends on demonstration of the organism in tissue. Tissue smears may facilitate rapid diagnosis; Giemsastained smears of suspicious liver lesions are especially useful (Percy and Barthold, 1993a). Colony screening for latent infection is problematic. Serologic screening is rapid and technically simple (Motzel and Riley, 1991) but is subject to false positives, yielding results that can be difficult to put into context. Disease provocation tests, or stress tests, to exacerbate latent infections are widely used and are recommended as a follow-up test when serologic positive results are obtained. However, there is some doubt as to efficacy of stress tests that rely on chemical immunosuppression, usually with cyclophosphamide (Boivin et al., 1990), followed by histopathologic evaluation. The doubt arises because test animals may have already cleared the C. piliforme infection and therefore may no longer be susceptible to activation of "latent" infection. Alternatively, sentinel animals can be placed on soiled bedding, but this may require sentinels to be of the same species (to avoid species specificity's causing false negatives), for not even gerbils are susceptible to all strains of C. piliforme (Motzel and Riley, 1992; Franklin et al., 1994). Interference of Clostridium piliforme with research has primarily been attributed to the morbidity and mortality, although effects on coagulation and leukokines have also been reported (Van Andel et al., 1996).

d.

Pasteurellosis

Pasteurella pneumotropica is a gram-negative coccobacillus. It grows aerobically on sheep blood agar without producing hemolysis, but producing smooth, gray translucent colonies (Car-

ter, 1984). It has been isolated from numerous mammalian species, including humans, and is generally considered to be of low significance in immunocompetent rats (National Research Council, 199 ld). Pasteurella pneumotropica has a high prevalence in infected colonies and is most often isolated from the nasopharynx, cecum, vagina, uterus, and conjunctiva during routine monitoring (National Research Council, 1991). The vast majority of animals are asymptomatic, with only rare instances of conjunctivitis, metritis, and mastitis (Percy and Barthold, 1993b). Histologically, lesions are characterized by necrotizing, suppurative inflammation. Control of the agent may not be necessary in immunocompetent animals, because of the rarity of P. pneumotropica-induced disease. However, treatment with enrofloxacin has been described (Goelz et al., 1997). Rederivation by either cesarean section or embryo transfer will also eliminate the agent (SultanDosa et al., 1983). Antibiotic treatment of infected dams prior to cesarean section has been recommended by at least one major rodent vendor (C. Clifford, unpublished observations, 1998), because P. pneumotropica can be present in the uterus. The probability of successful elimination of P. pneumotropica by cesarean section can be further increased by culturing all uteri after the pups are removed and eliminating any offspring from a culture-positive uterus. Offspring should also be held in strict isolation--i.e., not mixed in with a breeding colony-until repeatedly cultured negative for P. pneumotropica. Pasteurella pneumotropica is not transmitted to a significant degree by fomites, does not persist or multiply in the environment, and only rarely colonizes humans. Therefore, once a colony is free

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of the agent, there is relatively little risk of reinfection except through introduction or incursion of infected animals. e.

Salmonellosis

Salmonellosis is the disease caused by bacteria of the genus Salmonella. The taxonomic classification and subdivision of the genus are controversial and, like much taxonomy, subject to change (see Chapter 3). However, for clarity in communication, it is useful to refer to all salmonellas that one is likely to encounter in rats as belonging to S. enteritidis (Percy and Barthold, 1993a; Le Minor, 1984). Salmonella enteritidis is composed of more than 2400 serovars. These vary greatly in pathogenicity and geographic distribution, which makes serovar classification of epizootiologic significance. However, this discussion will treat S. enteritidis as a single entity. Salmonellosis may be virtually nonexistent in laboratory rats in the United States, but because infection is thought to be prevalent among many other species of vertebrates, including wild rodents, the potential for introduction remains. In rats, as in most species, clinical signs of infection with S. enteritidis are rare but may include a hunched posture, ruffled fur, lethargy, weight loss, and conjunctivitis. Soft stools and diarrhea may also be observed, usually in less than 20% of animals. Salmonella enteritidis is transmitted by ingestion of contaminated materials, including feed, bedding, or water. Incursion of wild or feral rodents into a laboratory facility poses a further risk. In addition, salmonellosis is an anthropozoonosis (Wray, 1994); humans not only are at risk of infection from rodents but also may serve as a source of the agent. In rats with subclinical infections, gross and microscopic lesions will usually be absent. Rats with clinical disease may have evidence of gastrointestinal involvement and septicemia, including mural thickening and mucosal ulcers in the cecum and ileum, as well as splenomegaly. Microscopically, enteric lesions are characterized by edema of the lamina propria, leukocyte infiltration in areas of ulceration, and reactive hyperplasia of crypt epithelial cells. Lymphoid hyperplasia, with focal necrosis and neutrophil infiltration, may be observed in Peyer's patches, as well as in the spleen and mesenteric lymph nodes. Septicemic rats will have necrosis in the spleen and liver, with emboli composed of fibrin, bacteria, and debris present in liver, spleen, and lymph nodes (Percy and Barthold, 1993b). Salmonellosis is most often diagnosed by culture of feces, mesenteric lymph nodes, liver, spleen, or blood. Material is placed in enrichment broth and then inoculated onto selective growth medium. Although symptomatic animals should be culture-positive, an infected colony may have only a low incidence of asymptomatic carriers, perhaps less than 5%. Detection of S. enteritidis in these colonies may require repeated testing of significant numbers of samples. Using a probability formula (National Research Council, 1976), 58 animals would have to

be tested to provide a 95% confidence of finding at least 1 positive if the true prevalence of positive samples was 5%. Differential diagnoses for diarrheal disease in rats include Tyzzer's disease, rotavirus infection, enterococcal enteropathy, cryptosporidiosis, and problems with feed and/or water. Salmonellosis is prevented by rigorous pest control and by ensuring that food and bedding are not contaminated. Good personal hygiene of employees will prevent them from serving as a source of Salmonella or other enteric pathogens to the colony. Once S. enteritidis is detected in a colony, all animals are usually destroyed, and all surfaces and materials either sterilized or safely discarded. Strict quarantine of a small group of animals may be practical in some situations, prior to rederivation by embryo transfer or cesarian section. This may be most feasible in a flexible film or semirigid isolator. Treatment is not recommended, because a chronic carrier state may result and there is the potential for zoonotic disease. Rats infected with S. enteritidis should not be used in research, because of the zoonotic potential and the risk they animals pose to other animals. Research complications from salmonellosis have primarily been reported in mice (National Research Council, 1991c). f

Pseudomoniasis

Pseudomoniasis refers to clinical disease caused by Pseudomonas aeruginosa, a gram-negative bacillus of the order Eubacteriales, family Pseudomonadaceae. Pseudomonas aeruginosa is motile, aerobic, oxidase-positive, and widely distributed in water, soil, sewage, and the skin and gastrointestinal tract of many animals. It is considered as part of the common commensal flora of humans, domestic animals, and laboratory rodents and is more frequently isolated from animals and humans receiving antibiotics (Kiska and Gilligan, 1999). Despite its near ubiquity, P. aeruginosa is rarely implicated in disease except in mammals with specific and severe host defense deficits, particularly hosts or tissues deficient in functional phagocytes (i.e., macrophages and neutrophils, and their serum opsonins). Thus, athymic nude mice are not subject to a high incidence of pseudomoniasis unless irradiated or treated with myelosuppressive agents. In general, pseudomoniasis is considered to be of low significance in rats (National Research Council, 199 l a) but should be suspected when rats that are irradiated or treated with radiomimetic agents die earlier than expected (Percy and Barthold, 1993b). In particular, pseudomoniasis has been reported as a consequence of infection of indwelling jugular catheters (Wyand and Jonas, 1967). Signs were those of septicemia. Necropsy findings included vegetative valvular endocarditis and multifocal hemorrhagic pneumonia. Histologically, fibrin emboli, leukocytes, and gram-negative bacteria were observed in the heart, lung, and occasionally other organs.

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4. BIOLOGY AND DISEASES OF RATS

Pseudomoniasis is diagnosed by cultural identification of the organism. However, caution should be exercised in attributing observed morbidity and mortality to an organism as nearly ubiquitous as P. aeruginosa. Although Pseudomonas will grow on blood agar, isolation is enhanced by the use of selective media, such as Pseudomonas isolation agar, or Pseudomonas P agar. The use of selective media is particularly recommended when screening clinically healthy animals, because only low numbers of organisms may be harbored in the common sites for isolation, the cecum, and nasopharynx. Exclusion of P. aeruginosa is rarely justified in a research setting (National Research Council, 1991c). Where appropriate, exclusion requires gnotobiotic methods and sterilization of all water reaching the animals, as well as sterilization of cages, feed, and bedding. Animals must be maintained in isolators or microisolators and must be routinely monitored. All possible sources of contamination from human skin or any wet surface must be strictly prohibited. Control of P. aeruginosa infection often begins with the watering system. Pseudomonas aeruginosa is one of a group of organisms that forms biofilms, layers of bacteria, usually with reduced metabolic activity, embedded in a dense glycocalyx. Bacteria in a biofilm are extraordinarily resistant to chlorine (150-3000 times more resistant than free-floating bacteria) and monochloramine (2- to 100-fold) (LeChevallier et al., 1988) and may be inaccessible to antibiotics. Nonetheless, chlorination (10-13 ppm) or acidification (pH 2.5-3.0) can significantly reduce the colonization of mice with P. aeruginosa but will not eliminate infection. Rederivation by cesarian section or embryo transfer is required to eliminate P. aeruginosa from an infected colony. Treatment with gentamycin in the animal drinking water, at 1 gm liter, has been reported to eliminate the infection in mice but is probably not practical for large groups of rats (Urano et aL, 1977). g.

Streptobacillosis

One cause of rat-bite fever, Streptobacillus moniliformis is primarily of historic interest. This zoonotic agent is virtually nonexistent in modern laboratory animals but nonetheless bears brief mention because of the potentially serious consequences of infection (Anderson et al., 1983; Wullenweber, 1995). The agent is a gram-negative pleomorphic bacillus, which will grow nonhemolytically on sheep blood agar, although trypticase soy agar enriched with 20% horse serum is preferred (Weisbroth, 1982; Savage, 1984). Streptobacillus moniliformis is commensal in wild rats, inhabiting the nasopharynx, middle ear, and respiratory tract. It is present in blood and urine of infected rats and is transmitted to humans by bite wounds, aerosols, and fomites (Will, 1994). The organism is nonpathogenic in rats. Clinical signs in humans follow a 3- to 10-day incubation period and include fever, vomit-

ing, arthralgia, and rash. Disease is treated with antibiotics, and mortality is low. Colonies of laboratory rats are monitored by culture of blood and nasopharyngeal swabs for Streptobacillus moniliformis, and any colony in which the organism is confirmed should immediately be terminated. Because wild rats are the reservoir for S. moniliformis, its detection in a laboratory rat colony would indicate exposure to infected wild rats. h.

Helicobacteriosis

Several Helicobacter spp. have been found as natural infections of rats in the last few years. Helicobacter muridarum, one of the first helicobacters identified in rodents, was first reported in 1992 (Lee et al., 1992). More recently, H. trogontum has been identified as a naturally occurring intestinal helicobacter (Mendes et al., 1996), and H. bilis has been reported from the large bowel of immunodeficient rats (Haines et al., 1998). Organisms with the ultrastructural morphologic appearance of "H. heilmannii" (H. bizzozeroni) have also been found in the stomach of wild rats (Giusti et al., 1998). All currently identified helicobacters of laboratory rodents are microaerophilic, gram-negative flagellated bacteria that may be spiral, slightly curved, or straight. Coccoid forms have also been described for H. bilis (and H. pylori) (Fox et al., 1995). Although the pathogenic potential of H. pylori in human gastritis and ulcers is widely accepted, less work has been completed to confirm suspicions of similar pathogenicity of rodent helicobacters. Koch's postulates have been fulfilled for H. hepaticus as a causative agent of enterocolitis and hepatitis in mice (Fox et al., 1996b), but no experimental reproduction of disease by natural routes of infection has been performed for Helicobacter spp. in rats. Lesions reported in athymic nude rats infected with H. bilis are similar to those reported in immunodeficient mice inoculated with H. bilis or H. hepaticus and include proliferative and ulcerative typhlitis, colitis, and proctitis (Haines et al., 1998), although no causal role was confirmed. No lesions have been reported in immunocompetent laboratory rats from any naturally occurring Helicobacter species. No studies have been published on the transmission of naturally occurring Helicobacter infections in rats. In mice, horizontal transmission by soiled bedding, probably fecal-oral transmission, has been demonstrated (Livingston et al., 1998). Fecal-oral transmission is also probable in rats. The host range of rat Helicobacter species is not fully elucidated. Clearly, H. bilis has been found in rats and mice, and there is also an additional report of it in a dog (Eaton et al., 1996). Helicobacter muridarum has been reported in rats and mice. No host range has been reported for H. trogontum. Many other Helicobacter species, however, are able to colonize a phylogenetically wide range of mammalian hosts. The only lesions in rats reported due to natural Helicobacter infection are in a small series of 11 male athymic nude rats,

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5 - 8 months of age, infected with H. bilis (Haines et al., 1998). Helicobacter infection in rats is nonetheless expected to be conIn these rats, gross lesions consisted of focal or diffuse thicken- taminated rodents or other laboratory animals. No indication ing of the cecal wall, with normal-appearing colon and rectum. of transmission by feed, bedding, water, or aerosols has been Cystic mesenteric lymph nodes were also noted in 8 of the 11 reported. rats. Histologically, all 11 rats had proliferative typhlitis, with Once a colony is infected, treatment of small groups of ani8 of the animals also having similar lesions in the colon and mals may be possible. Several antibiotic regimens have been rectum. Crypt epithelium was hyperplastic, with cytoplasmic reported to be successful for mice and might similarly be atbasophilia, increased mitoses, and fewer goblet cells than nor- tempted in rats. These have primarily involved oral dosing by mally observed. The lamina propria was infiltrated by lympho- gavage several times each day or incorporation of antibiotics cytes, plasma cells, and a few eosinophils. Mucosal erosion and into the diet. A triple therapy combination of amoxicillin or ulceration were observed in the cecum of the most severely af- tetracycline, with metronidazole and bismuth, administered by fected rats. The authors experimentally reproduced many as- oral gavage 3 times each day for 2 weeks has been the most pects of the disease by intraperitoneal injection of approxi- effective (Orcutt, 1980). Elimination of infection from large mately 5 x 108 H. bilis bacteria in phosphate-buffered saline. groups of rats would be less likely to be 100% effective, even if Once established, infection by any Helicobacter species is practical obstacles of dosing could be overcome, because even typically lifelong. Infection, or colonization, should be distin- a single rat retaining any viable Helicobacter could lead to reguished from disease. Helicobacter muridarum and H. trogon- infection of the entire colony. turn may be nonpathogenic in rats, although H. muridarum has Research complications due to infection by Helicobacter spp. been reported to cause lymphocytic gastritis in aged mice, pos- in rats have not been reported. sibly associated with a loss of parietal cell mass leading to increased gastric pH. Key pathogenic factors for H. pylori include i. Cilia-Associated Respiratory Bacillus urease, a vacuolating cytotoxin (vacA), and the presence of a Usually referred to as CAR bacillus, the cilia-associated respathogenicity island. All three Helicobacter spp. currently piratory bacillus is not taxonomically classified in the genus identified in rats--H, bilis, H. trogontum, and H. muridarum m are urease-positive (Fox and Lee, 1997). Other virulence factors Bacillus. Rather, it has recently been tentatively placed in a group of bacteria known as "gliding bacteria," based on the fact have not been reported. Helicobacter infection should be a prime differential diagno- that they are motile but without visible means for such motility, sis when proliferative lesions of the large bowel are observed in and may be related to Flavobacterium or Flexispira, based on rats. Spontaneous chronic ulcerative colitis has also been re- 16S rRNA sequencing (Cundiff et al., 1995a). Final identiported in athymic nude rats (Thomas and Pass, 1997). The au- fication, however, is still pending. CAR bacillus has been identified in rats, mice, and rabbits thors were unable to culture Helicobacter spp. from affected anamong common laboratory animals (Van Zwieten et al., 1980; imals, although no molecular techniques were employed. Diagnosis of Helicobacter infection in laboratory rats is best MacKenzie et al., 1981; Waggie et al., 1987; Griffith et al., 1988). In rats, infection is usually asymptomatic, although nonaccomplished by polymerase chain reaction (PCR) (Riley et al., 1996; Fox and Lee, 1997). A common approach is to use genus- specific clinical signs such as weight loss and dyspnea may specific primers capable of detecting any Helicobacter spp., as o c c u r . Transmission is primarily via direct contact with infected anwell as primers specific for a single species, such as H. bilis, or to follow the initial amplification with restriction endonuclease imals. Fomites probably do not play a significant role in natural digestion and gel separation to identify digestion product band- transmission of CAR bacillus, and bedding does not transmit ing patterns characteristic of various species. Samples are most the infection well (Matsushita et al., 1989). Airborne exposure often fecal pellets, although cecal mucosal scrapings or tissue is not an important means of transmission (Itoh et al., 1987). CAR bacillus infection may not always present gross lesions, may also be used. Helicobacter spp may also be cultured from a variety of although translucent gray cystic lesions, representing dilated, sources. Culture from contents of the large intestine is greatly mucus-filled airways may be visible on the pleural surface complicated, however, by the rich flora of the site. Fox et al. (Itoh et al., 1987). Coinfection with Mycoplasma pulmonis (1997) recommend passage of cecal contents through a 0.65-ktm or other pathogens may occur, resulting in suppurative bronchiofilter, then culture on Brucella agar with antibiotics (trimetho- pneumonia. Histopathologically, hyperplastic peribronchial and peribronprim, vancomycin, polymyxin) to suppress growth of unwanted organisms that are not removed by the filter (Fox and Lee, 1997). chiolar mononuclear cell cuffs are observed in the lungs (Itoh In general, it is easy to exclude from animal colonies those ro- et al., 1987; Matsushita and Joshima, 1989). A thin basophilic dent-specific organisms that do not persist or multiply in the en- layer may be observed on the surface of the airway epithelium vironment. However, given the uncertainty as to the full host in hematoxylin and eosin-stained sections, giving the impresrange of rat helicobacters, the possibility of transmission by hu- sion that the cilia are more basophilic than normal, but this is mans is difficult to exclude. The usual source of infection for not specific and should not be used as a definitive diagnostic

4. BIOLOGY AND DISEASES OF RATS

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Fig. 6. Warthin-Starry silver stain of rat bronchus. Innumerable filamentousbacteria are densely clustered at the ciliated surface of the columnar epithelium. Note the polymorphonuclearcell exudate in the bronchial lumen. Magnification: X100. feature. With Warthin-Starry or methenamine silver stain, filamentous bacilli are readily observed among cilia of respiratory epithelium from the nasal cavity to the bronchioles (Fig. 6). The upper respiratory tract is involved earlier in the course of infection than the lower tract and should be included in histologic examinations for CAR bacillus infection. CAR bacillus infection should be distinguished from murine respiratory mycoplasmosis, pneumonia due to other bacteria

(i.e., Streptococcus pneumoniae, Corynebacterium kutscheri, etc.), and viruses. Detection of CAR bacillus infection should also raise the suspicion of coinfection with other pathogens (Van Zwieten et al., 1980; MacKenzie et al., 1981) (Fig. 7). Colonies are best screened for CAR bacillus infection by serologic techniques, such as enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assay (IFA) (Matsushita et al., 1987; Shoji et al., 1988; Lukas et al., 1987). Because

Fig. 7. Electronmicrograph showing filamentous CAR bacillus organisms (large arrow) at the surface of a ciliated bronchial epithelial cell. Note coinfection with Mycoplasma pulmonis (small arrow). Bar:1 ~tm.

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false-positive reactions can occur (Hook et al., 1998), any positive results should be confirmed by a Steiner stain of tracheal mucosal scraping or histopathology with use of special stains, or by PCR. Interestingly, because infection is not readily transmitted by soiled bedding (Cundiff et al., 1995b), many sentinel programs may fail to detect CAR bacillus infection. Infection is lifelong, and the organisms are readily retrievable by tracheal lavage or scraping (Medina et al., 1998). Therefore, CAR bacillus may be readily detected by PCR (Cundiff et al., 1994), which may serve as an important confirmatory test to follow positive serologic results. PCR may be positive prior to serologic conversion, and samples for PCR may be collected as nasal swabs as a nonterminal procedure (Franklin et al., 1999). CAR bacillus infection is prevented by exclusion of infected animals. No effective treatment has been described. As an alternative to elimination and rederivation of entire infected colonies, the requirement for direct contact for transmission may possibly be exploited to advantage. If individual animals or cages are monitored by serology, and then negative individuals are monitored by PCR, all rats that are positive (or all cages that have a positive rat) by either test may be eliminated or quarantined. Because the infection is not transmitted well by aerosol or fomites, it may be possible to control the spread of infection. However, the expense, the labor, and the consequences of possible failure would have to be weighed against the value of possibly saving some of the rats. The interference of CAR bacillus with research is unknown. Interference with ciliary function has been suspected but not measured. Effects of CAR bacillus on other respiratory functions and on the immune response have also been postulated but not documented in the scientific literature.

j.

et aL, 1985); host nutritional deficiencies such as vitamin A and E deficiencies may exacerbate disease (Tvedten et al., 1973). Mycoplasma isolates may also vary in virulence (Davidson et al., 1988). Environmental factors may include intracage ammonia, temperature, humidity, etc. (Schoeb et al., 1982). Mycoplasma pulmonis possibly damages host cells by causing dysfunction and/or loss of cilia (Kohn, 1971) (Fig. 8), which is a likely cause of the accumulation of exudate, opportunistic bacterial infections, and impaired transport of ova (infertility). Mycoplasma pulmonis competes for the host cell nutrients and metabolites (Cassell et al., 1986) and may also produce toxic metabolites, such as peroxides and nonspecific mitogens (Naot et al., 1979a,b). The latter may cause proliferation of autoreactive clones of lymphocytes, leading to the host's becoming a victim of its own immune system. Mycoplasma pulmonis successfully evades the host's immune defenses, so infection and some lesions (especially those in the upper respiratory tract) are persistent and often progressive. The exact mechanism by which M. pulmonis evades the host immune system, however, is unknown.

Mycoplasmosis

Murine respiratory mycoplasmosis (MRM), also known as chronic respiratory disease, is caused by Mycoplasma pulmonis (Kohn and Kirk, 1969; Lindsey et al., 1971). Clinical signs are usually observed only in older animals; M. pulmonis infection is clinically silent in young animals. Clinical signs are nonspecific, referable to the respiratory and auditory involvement, and include rales and dyspnea, snuffling and chattering, and ocular and nasal discharges, as well as chromodacryorrhea, rubbing of eyes, and head tilt. Rats with severe middle ear involvement may spin when held up by the tail. Decreased reproductive efficiency has also been reported in rats (Leader et al., 1970). Mycoplasma pulmonis is transmitted horizontally by direct contact and aerosol and vertically by in utero transmission (Lindsey et al., 1982). Venereal transmission may also be possible. The disease outcome depends on a complex interaction of factors relating to host, pathogen, and environment (Lindsey et al., 1985). Host factors include age, strain (Davis and Cassell, 1982), immune status and lymphoreticular function, and the presence of intercurrent infections such as Sendai virus (Schoeb

Fig. 8. Electronmicrographof bronchial epithelial cell. Note the numerous M. pulmonis organisms cytadsorbed to the cell surface and the associated cytopathology.

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4. BIOLOGY AND DISEASES OF RATS

Gross lesions of MRM (Percy and Barthold, 1993b), listed in decreasing order of frequency, include suppurative rhinitis, otitis media, laryngitis, and tracheitis in the upper respiratory tract. In the lung, suppurative bronchopneumonia with or without atelectasis, bronchiectasis, and abscesses may occur; widespread bronchiectatic abscesses lead to the appearance referred to as "cobblestone" lung, primarily seen in adults with endstage disease. This classic lesion of MRM is rare in recent years. Arthritis may rarely be observed. No genital tract lesions are usually observed, but occasionally partially resorbed fetuses and suppurative salpingitis may be found. Histopathologically (Van Andel et al., 1996), airway lesions in the respiratory tract are usually characterized by suppurative exudate, hyperplasia (squamous metaplasia) of mucosal epithelium, and often striking hyperplasia of the bronchial-associated lymphoid tissue (BALT). Other respiratory tract lesions include pseudoglandular hyperplasia of the nasal epithelium in chronic cases, and hyperplasia of peribronchial alveolar type II pneumocytes. CAR bacillus and/or secondary bacterial pneumonias also frequently accompany MRM. Lesions in the female genital tract of rats with mycoplasmosis may include suppurative oophoritis and salpingitis, or hydrosalpingitis, and chronic suppurative endometritis or pyometra. Differential diagnoses for MRM include other bacterial pneumonias, such as Corynebacterium kutscheri infection, streptococcosis, cilia-associated respiratory (CAR) bacillus infection, and (rarely) mycotic pneumonia. Viral infections are less likely to be mistaken for MRM, but intercurrent infections are common, including Sendai virus, pneumonia virus of mice, and others. Diagnosis of mycoplasmosis in an individual rat is usually based on cultural isolation (especially exudate in the upper respiratory tract and middle ears). Surveillance of infections in colonies, however, is most effectively accomplished by ELISA (Cassell et al., 1981; Lussier, 1991). Pathology, including gross examination, and histopathology should not be considered diagnostic by themselves but may provide guidance in selecting more definitive tests. Mycoplasma pulmonis interferes with research by its effects on the immune system, the respiratory system, and the reproductive system and by being a primary cause of early mortality in infected colonies (Cassell et al., 1986; Swing et al., 1995; Lindsey et al., 1971, 1982).

k.

Hemobartonellosis

Haemobartonella muris is a gram-negative bacterium, order Rickettsiales, family Anaplasmataceae, that parasitizes erythrocytes of rats (Ristic and Kreier, 1984). Like other members of the family, it is an obligate parasite and cannot be grown in vitro. Clinical signs are typically observed only if the normally latent infection is activated by immunosuppression or splenec-

tomy (National Research Council, 1991e). Signs are due to erythrocyte destruction and may include weight loss, hemoglobinuria, pallor, and dyspnea. Clinical pathology demonstrates anemia, reticulocytosis, increased coagulation times, decreased plasma proteins, and increased serum immunoglobulins (IgG and IgM). Because H. muris is transmitted by the spiny rat louse, Polyplax spinulosa, which is very rare in modern laboratory animal facilities, hemobartonellosis is also correspondingly rare (National Research Council, 199 lb). However, the potential exists for infection of biological materials, which would provide a route of introduction into rat colonies. In addition, both the agent and the vector are still extant in North America and presumably elsewhere, indicating a continuing, albeit low-level, threat. Necropsy of rats with hemobartonellosis is unrewarding except in the case of active infections, when anemia, hemoglobinuria, and splenomegaly may be observed. Blood films are likely to show parasitemia only in active infections. Hemobartonellosis should be suspected whenever lice are found in a rat colony or whenever anemia and hemoglobinuria are observed. Diagnosis should be based on detection of the organisms on erythrocytes, where they appear as round (coccoid), elongate (rod), or dumbbell-shaped densities on the erythrocyte surface. Hemobartonellosis is readily prevented by excluding Polyplax spinulosa and controlling biologic materials being introduced into a colony. Once the disease is confirmed in a colony, rederivation by embryo transfer or cesarian section is warranted, although treatment with antirickettsial compounds such as tetracyclines or arsenicals may be appropriate for small groups of rats (Ristic and Kreier, 1984). Hemobartonellosis exerts its effects on research by virtue of its parasitism of erythrocytes. It reduces the half-life of erythrocytes, can alter function of the mononuclear phagocyte system, and can increase rejection of transplantable tumors, as well as interfering with research in other blood-borne parasitic diseases such as malaria and trypanosomiasis. 2.

Viral Infections

a.

Sendai Virus Infection

Sendai virus is an RNA virus (genus Paramyxovirus, species parainfluenza 1) comprised of strains that are antigenically homologous (National Research Council, 1991a). Unlike Sendai virus-induced disease in mice, an asymptomatic and selflimiting disease is usually induced by Sendai virus in rats. Clinical signs associated with the virus may include reduced production and litter sizes, as well as retarded growth of young within breeding colonies. Infrequently, clinical respiratory signs occur (Makino et al., 1972). It has been shown in Lewis rats, inoculated intranasally with

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Sendai virus, that draining lymph nodes of the upper respiratory tract are the initial and major site of antibody production. Development of serum immunoglobulin G (IgG) antibodies coincides with clearance of respiratory tract infection and recovery from viral infection (Liang et al., 1999). Coinfection with other respiratory pathogens such as Mycoplasma pulmonis, Flexibacter sp. (CAR bacillus), Pasteurella pneumotropica, and pneumonia virus of mice (PVM) increases the severity of clinical disease and pulmonary lesions (BeschWilliford et al., 1987; Carthew and Aldred, 1988). Although Sendai virus was once very prevalent in commercial sources of mice and rats, today it rarely occurs in barrier-maintained commercial sources. However, commercial and institutional sources that are conventionally maintained may still be sources for introduction of the virus to naive colonies of rats or mice. Sendai virus is highly contagious, with transmission occurring through the respiratory tract either by aerosol or direct contact. After exposure, the initial tropism in the upper respiratory tract induces a rhinitis characterized by focal to diffuse necrosis of the epithelial cells, and a leukocytic infiltrate composed of neutrophils, lymphocytes, and plasma cells. Within the lungs there is a hyperplastic to suppurative bronchitis and focal alveolitis. Alveolar septa are hypercellular, with infiltrates of alveolar macrophages, neutrophils, and lymphocytes. Viral replication occurs in bronchial epithelial cells, type I and type II pneumocytes, and alveolar macrophages. Later, there is pronounced perivascular and peribronchial cuffing with a lymphocytic and plasmacytic infiltrate that may remain 7 months after the acute phase of the infection (Burek et al., 1977; Percy and Barthold, 1993b). Based upon experimental infection, lesion severity has been shown to be more severe in Brown Norway and LEW rats than in F-344 rats (Sorden and Castleman, 1991; Liang et al., 1995). Due to the low prevalence of clinical signs, diagnosis is best achieved by detection of antibodies to the virus and demonstration of typical lesions in the respiratory tract. The ELISA is the test of choice for diagnosis of Sendai virus infections in rats. Comparison with Complement fixation (CF) and hemagglutination inhibition (HAI)) tests indicates that ELISA is the most sensitive, particularly in detection of early antibody to Sendai virus and detection of small amounts of antibody (Rottinghaus et al., 1986). Prevention of Sendai virus introduction into an existing colony requires knowledge of the pathogen status of the source and, in some cases, quarantine with serological testing of incoming rats and mice. Regular and periodic serologic testing within colonies of rats and mice should be done to help prevent and control infection within rodent housing facilities. Mouse and rat antibody production (MAP, RAP) testing should be done on all transplantable tumors, cell lines, and other biological materials to prevent transmission of Sendai virus from infected materials to recipient animals. Recently, the use of PCR testing

DENNIS F. KOHN AND CHARLES B. CLIFFORD

for the presence of Sendai virus (and other viral pathogens) in tumors and cell lines has been shown to be more sensitive than MAP testing (Riley et al., 1999). If Sendai virus is introduced into a rat colony of immunocompetent rats, neutralizing antibody in infected rats renders the infection self-limiting. Accordingly, if antibody-naive rats are not introduced and if pregnant and preweanling rats are killed and breeding is halted, the virus will be eliminated from the colony within 4 - 8 weeks (Jacoby et al., 1979). In addition to research complications associated with the respiratory tract tropisms of the virus, it may modulate some immunological responses, e.g., reducing the severity of adjuvant arthritis (Garlinghouse and Van Hoosier, 1978) and depressing T cell and thymocytotoxic autoantibody (Takeichi et al., 1988). b.

Rat Coronavirus Infection

The two prototype coronaviruses in rats are Parker's rat coronavirus (RCV-P) and sialodacryoadenitis virus (RCV-SDA). In addition to these two coronavirus strains, there are others that have been isolated and found to differ antigenically from either RCV-P or RCV-SDA. Historically, RCV-P and RCV-SDA were considered to induce two rather distinct sets of clinical signs and types of lesions in rats (Jacoby et al., 1979). More recently, however, the clinical signs, pathogenicity, and histological lesions are considered to be variable but similar for both RCV-P and RCV-SDA, and defining the neutralization group of a new RCV isolate is not useful in predicting its pathogenic potential (Compton et al., 1999). Accordingly, infection with either RCV-SDA or RCV-P cannot be differentiated by comparison of clinical signs or lesions. The antigenic differences between RCV-P and RCV-SDA are significant enough to allow crossinfection with either virus. Probably the most important point to be made from a clinical perspective is that neutralizing antibodies to one virus prototype will not offer significant cross protection from the other virus strain, thus allowing viral shedding and recurrence of clinical signs and lesions, albeit diminished (Percy and Barthold, 1993b; Jacoby, 1986; Bihun and Percy, 1994; Kojima and Okaniwa, 1991; Weir et al., 1990). Rat coronaviruses may induce either asymptomatic infections or transient clinical infections (sialodacryoadenitis) associated with tissue tropisms for the salivary glands, lacrimal glands, Harderian glands, and respiratory epithelium. There are two distinctive types of clinical disease associated with the virus. The first is associated with breeding colonies in which the virus is endemic with mature rats immune to infection, and in which clinical disease is primarily associated with preweanling, nonimmune animals that display ocular signs associated with conjunctivitis. These signs are transient, lasting for a week or less. The second type of clinical picture is associated with a sudden onset of clinical signs in naive postweanling-to-adult rats that have been exposed to infected rats. Signs include cervical

4. BIOLOGY AND DISEASES OF RATS

Fig. 9. Edematouspale submaxillary gland (arrow) and moderately enlarged cervical lymphnodes. (Courtesyof Dr. Robert Jacoby.)

swelling due to inflammation and edema of submaxillary salivary glands (Fig. 9), nasal and ocular discharges that are usually porphyrin stained, photophobia, corneal opacities, and corneal ulcers. In most animals the signs last for less than 2 weeks. However, in some animals a chronic keratitis and megaloglobus may persist (Jacoby et al., 1979). Rat coronaviruses are very contagious, with transfer to susceptible rats by direct contact with infected rats, and indirectly by aerosol and fomites (La Regina et al., 1992). Virus is present in target tissues for about 1 week after exposure, at which time heightened antibody levels render the infection self-limiting. However, immunity is not lifelong. Under experimental conditions, it has been shown that rats are susceptible to reinfection as early as 6 months after initial infection and that such rats are able to transfer infection to naive rats by cage contact. However, the severity of lesions in reinfected rats is minimal compared

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with those associated with primary infections (Percy et al., 1990; Weir et al., 1990). Differences in pathogenicity have been reported among a few rat strains (Jacoby et al., 1979; Carthew and Slinger, 1981). Histological changes associated with sialodacryoadenitis (SDA) in affected salivary and lacrimal glands include coagulation necrosis of ductal and acinar epithelial cells during the acute stages of the disease, followed by squamous metaplasia during the reparative period that begins 7-10 days after infection. There is a mixed leukocyte infiltrate. Regeneration of the epithelial cells occurs in about 4 weeks postinfection. However, focal lesions may persist an additional several weeks in the Harderian glands. The microscopic changes associated with rhinitis, tracheitis, and focal bronchitis during the acute stage of the disease include a mononuclear and polymorphonuclear cell infiltration, hyperplastic respiratory epithelia with loss of ciliated surfaces, and focal alveolitis. The lesions within the lower respiratory tract abate in about 7-10 days, and those in the nasopharynx remain somewhat longer (Percy and Barthold, 1993b). Diagnosis of SDA is best achieved by serological means using the ELISA method and histological examination of the Harderian glands and the submaxillary and parotid salivary glands (National Research Council, 1991). Because the disease is often subclinical, typical signs associated with salivary gland and Harderian gland tropisms may not be useful. Differential diagnoses include M y c o p l a s m a , Sendai virus, and pneumonia virus of mice (PVM) infections, and stress-associated factors that induce chromodacryorrhea (Percy and Barthold, 1993). Preventing transfer of this highly contagious coronavirus to naive colonies is predicated upon preventing entry of infected rats into a facility through knowledge of the pathogen status of vendor colonies and an effective quarantine program. Control of infection within a colony or facility is based upon the fact that rats shed the virus for only about 1 week, after which they are immune and not latently infected. The virus is not transmitted vertically. Eliminating rat coronavirus from a colony is achieved by allowing the virus to spread quickly to all animals, preventing entry of susceptible rats to the room, and suspension of breeding and removal of preweanlings. The rapidity in which all animals will seroconvert and no longer shed the virus will determine the period of time needed before susceptible animals can be safely introduced or breeding resumed. In most instances, a 6- to 8-week period should be allowed (Jacoby et al., 1979). Alternatively, if suspension of breeding cannot be done, a method to continue breeding and eliminate SDA is to define a subset of the breeding colony that is seropositive and to relocate these breeding animals to a separate room, allow litters to be born in the original colony until the relocated breeders are in late gestation, and then kill all animals in the original colony (Brammer et al., 1993). Research complications associated with SDA reflect tropisms

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for the lacrimal and salivary glands, the eye, vomeronasal organ, and respiratory epithelium (Percy and Barthold, 1993b). Except for long-term ocular lesions, research complications would be expected to be linked to the period of active infection and the 2- to 3-week reparative period. During this period, food intake frequently decreases if cervical swelling occurs. c.

Rat Parvovirus Infection

Parvoviruses are single-stranded DNA viruses that have a predilection for mitotically active host cells. Parvoviruses that infect rats include (Kilham's) rat virus (RV), (Toolan's) H-1 virus, and rat parvovirus (RPV). The first two viruses were initially isolated from a transplantable tumor (RV) and a tumor cell line passed in rats (H-1). In the 1980s, testing of rat sera indicated the presence a parvovirus that was neither RV nor H-1. This virus, which was initially referred to as rat orphan parvovirus (OPV), is now designated rat parvovirus (RPV) (Jacoby et al., 1996). Clinical signs associated with RV infection occur very sporadically in colonies showing serological evidence of infection and are usually seen only in preweanling animals. In such colonies, reduced litter size, runted litters, and fetal and neonatal death may be observed. Although subclinical infections in postweanling rats are the rule, an outbreak characterized by hemorrhage and necrosis of the brain, testes, and epididymides has been reported in young adult rats (Coleman et al., 1983). The ability of RV to cross the placenta appears to depend on the virus strain, dose, and time of gestation. Resistance to lethal infection develops during the first postpartum week (Jacoby et al., 1988; Gaertner et al., 1996). Serological surveys of rat colonies have indicated a rather high prevalence of antibody to H- 1 virus; however, clinical disease is not associated with this virus (National Research Council, 1991 b). Although the pathogenesis of RPV needs further definition, it appears that RPV infections are characterized as being subclinical (Weisbroth et al., 1998). Rat virus is excreted in urine and milk and is transmitted by aerosol through direct contact or fomites (Jacoby et al., 1996). RV-contaminated bedding, stored at room temperature for up to 5 weeks, is capable of inducing seroconversion of rats for up to 5 weeks (Yang et al., 1995). Rats may harbor and transmit RV long after seroconversion occurs, with the frequency of persistent infection during natural outbreaks being RV straindependent (Gaertner et al., 1996). After experimental inoculation of RV into neonatal rats, the virus persists in tissues for up to 14 weeks, and the duration of infectivity to cage contacts up to 10 weeks. If weanling rats are inoculated, the duration of viral recovery and infectivity is decreased to 7 and 3 weeks, respectively (Paturzo et al., 1987; Jacoby et al., 1988). In another study, RV was recoverable for up to 6 months from tissue explant cultures derived from newborn rats (Jacoby et al., 1991). In persistent infections, DNA and antigenic evidence of RV is most likely to be observed in lymphoid tissues, endothelium,

vascular muscle tunics, and renal tubular epithelium (Gaertner et al., 1996; Jacoby et al., 1991). The correlation of age and RV pathogenicity is thought to be due to the decreased complement of target cells in the S phase of division needed for productive infection. The immune status of the host is also significant to the outcome of RV infection. Rat virus in athymic rats induces a more severe and persistent infection than in euthymic rats (Gaertner et al., 1995; Gaertner et al., 1989). Diagnosis of RV, RPV, and H-1 virus can be accomplished by testing sera by ELISA or IFA, using RV, H-l, or recombinant NS1 as the antigen. A positive response does not delineate which rat parvovirus antibody exists but only indicates that antibody to a rodent parvovirus is present. Positive ELISA or IFA sera are then tested by hemagglutination inhibition (HAI) for RV and H-1. Samples negative by HAI tests are interpreted to be positive for RPV (Weisbroth et al., 1998). PCR assays for RV, H- 1, and RPV have been developed that provide a rapid, specific and sensitive means for detecting viral DNA in tissue (Besselsen et al., 1995a,b) or the environment (W. R. Shek, personal communication). Research complications induced by RV are associated with its tropism for mitotically active cells of fetuses, neonates, cell cultures, and tumors. Rat virus has been shown to modulate immune function through its tropism for T-cell lymphocytes (McKisic et al., 1995). Rat virus infection in the diabetesresistant BioBreeding rat increases the expression of macrophage cytokines, leading to to an autoimmune diabetes (Chung et al., 1997). The effect of RV on the immune system has been shown to be rat strain dependent for natural killer cell activity. Natural killer cell-mediated cytotoxicity is increased in Brown Norway rats, whereas it is decreased in Wistar-Furth rats (Darrigrand et al., 1984). The effects, if any, that RPV may have on research are essentially unknown.

d.

Pneumonia Virus o f Mice Infection

Pneumonia virus of mice (PVM) is a pneumovirus in the family Paramyxoviridae. Contrary to the virus's name, serological evidence indicates infectivity in mice, rats, hamsters, gerbils, guinea pigs, and rabbits. The prevalence of seropositive rat colonies was reported in 1982 to exceed 50%; however, today serological evidence of PVM infection is infrequently observed in rats. Diagnosis is typically accomplished by ELISA or HAI testing (National Research Council, 1991a). The virus does not cause clinical disease, but multifocal, nonsuppurative vasculitis and interstitial pneumonitis with necrosis are prominent lesions seen in the acute phase of the disease. These lesions persist for several weeks. The virus may be a significant copathogen with other respiratory agents such as M y c o p l a s m a pulmonis, and cross species transmission is a potential concern (Percy and Barthold, 1993b).

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e.

Rotavirus-Like Agent Infection

Diarrhea in suckling rats has been associated with a virus morphologically identical but antigenically and genomically distinct from group A rotaviruses (Eiden et al., 1985). The authors who first reported this disease named it infectious diarrhea of infant rats (Vonderfecht et al., 1984). Affected infant rats excreted feces that varied from liquid to being poorly formed, and the animals displayed erythema and bleeding of the perianal skin. Pathology associated with infection included small intestinal villous atrophy, villous epithelial necrosis, and syncytial cell formation. This same agent was found to be associated with diarrhea in humans and has been shown by enzyme immunoassay inhibition assay to be prevalent in children and adults. Human isolates were shown to induce diarrhea in infant rats (Eiden et al., 1985; Vonderfecht et al., 1985). This suggests that under nonexperimental conditions there may be cross-infectivity between humans and rats. f

Hantavirus Infection

Hantaviruses are enveloped RNA viruses of the genus Hantavirus, family Bunyviridae. Rodents serve as the natural reservoirs for hantaviruses, with each virus in the genus being associated with a specific rodent species. Hantavirus infections in rodents are characterized by being chronic and subclinical, with virus being shed persistently in the feces and urine. Hantaviruses pose as significant zoonotic agents. Rattus norvegicus is the natural host for the Seoul Hantavirus, causing hemorrhagic fever with renal syndrome (HFRS) in humans. Hantavirus has been isolated from wild rats in Baltimore and several other cities in the United States. One report cites evidence of human infection with a rat-associated Hantavirus (Childs et al., 1987). Cotton rats, Sigmodon hispidus, are reservoirs for a Hantavirus that has induced hantavirus pulmonary syndrome in individuals living in Florida (Hutchinson et al., 1998). Transmission of Hantavirus from laboratory rats to laboratory personnel has been reported in Japan, Belgium, and the United Kingdom (Desmyter et al., 1983; Lloyd et al., 1984). In both reports, multiple cases occurred that resulted in hemorrhagic fever with renal syndrome. Unidentified Viral Agent Associated with Lymphohistiocytic Lung Lesions Lymphohistiocytic lung lesions probably attributable to a virus etiology have been reported in barrier-maintained commercial breeding colonies. The lesions appear as multiple small gray to tan foci on the pleural surface of the lung. Histologically, mild to moderate multifocal histiocytic alveolitis and perivascular cuffing are observed. The lesions occur in 8- to 18-weekold animals with the most severe lesions seen at 8-12 weeks. Bacteriological culturing and PCR assays for Mycoplasma, CAR bacillus, and eubacteria genera suggest that the lesions are

not associated with bacteria. A viral etiology is suggested, because inoculation of lung tissue homogenates that pass through bacteriologic filters induces cytopathic effects in tissue culture (Riley and Franklin, 1997; Slaoui et al., 1998; C. Clifford personal communication, 1999). h.

Other Viral Infections

There are several rodent viruses for which there is serological evidence of infection in the rat, but for which there are negligible data demonstrating any clinical or pathological importance. These viruses include mouse adenovirus, mouse encephalomyelitis virus, reovirus 3, parainfluenza virus 3, and endogenous retroviruses (Kohn and Barthold, 1984; National Research Council, 1991; Percy and Barthold, 1993b). 3.

Parasitic Infections

a.

Protozoa

Protozoa are of little consequence in laboratory rats in recent decades (National Research Council, 1991a; Kohn and Barthold, 1984). Reasons for this are several. First, no spontaneous disease due to any naturally occurring enteric protozoa of laboratory rats has been reported. Second, parenteral infections are rare in laboratory rats because of absence of vectors. Third, there is almost universal use of high-quality diets, which are generally subjected to heat disinfection prior to use. The days of giving rats fresh produce are happily slipping into the past. Protozoa of potential significance in rodent facilities include Encephalitozoon cuniculi and two enteric flagellates, Spironucleus muris and Giardia muris, which may produce disease and alter immune responses in mice. Encephalitozoon cuniculi, formerly Nosema cuniculi, is a microsporidian parasite of a wide variety of mammalian hosts, including rodents, lagomorphs, carnivores, and primates, including humans. It has also been reported in birds (Poonacha et al., 1985; Reetz, 1993). Encephalitozoonosis is common in conventional rabbit colonies and most guinea pig colonies but is rare in rats. It is transmitted by ingestion, and possibly inhalation, of spores shed in urine (Wilson, 1979). Vertical transmission has been proposed in primates, foxes, mice, rabbits, and guinea pigs but not in rats (Boot et al., 1988; Liu et al., 1988). Resistance to infection and the outcome of infection are dependent on T-cell function, which is strain dependent (Liu et al., 1989; Niederkorn et al., 1981). Athymic nude mice, and presumably athymic nude rats, are more susceptible to lethal infection than are euthymic animals. Encephalitozoon cuniculi has also been recovered from transplantable ascites tumors in rats (Petri, 1969). Clinical signs and gross lesions of E. cuniculi infection are not reported in rats. On histopathologic examination (Majeed

148

and Zubaidy, 1982), rats with E. cuniculi infection may have nonsuppurative or granulomatous meningoencephalitis in any or all parts of the brain and occasionally the spinal cord. Interstitial nephritis may also be observed Less frequently, similar lesions may be observed in other tissues. Spores may be observed in, or more frequently adjacent to, any of the lesions. Spores stain poorly with hematoxylin and eosin but are strongly gram-positive. Diagnosis of E. cuniculi infection is usually based on serology (Pakes et al., 1984). Screening of colonies by ELISA is probably the most efficient method, because infected colonies normally have a high prevalence (Gannon, 1980). As with all serologic assays, positive serologic results should be confirmed by a second method or by repeating the assay on groups of animals to establish a pattern of positive results. Histopathologic observation of the organism is definitive. The primary histopathologic differential diagnosis for E. cuniculi infection in rats is toxoplasmosis. Encephalitozoon cuniculi measures 1 • 2 gm, stains well with Gram stain and poorly with hematoxylin and eosin. Toxoplasma gondii measures 2 • 4 gm, stains well with hematoxylin and eosin, and poorly with Gram stain (Wilson, 1979). Encephalitozoonosis is controlled by purchasing only animals that are free of Encephalitozoon and by maintaining them away from infected animals. There is currently no effective treatment. Research complications of E. cuniculi infections have not been reported in rats, although it is potentially a confounding factor if histopathologic evaluation of the central nervous system and kidney is part of the study (Majeed and Zubaidy, 1982). Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii. Toxoplasmosis in rats is usually subclinical. The definitive host is the domestic cat and other felids, which shed oocysts in the feces. Rats, like many other vertebrates, serve as intermediate hosts. Transmission to rats is via ingestion of cat feces. Ingestion of infected intermediate hosts might also horizontally transmit the infection, although it would not be expected as a mode of transmission in a well-managed rat colony. Infected rats can transmit T. gondii vertically, but only very poorly. Therefore, in order for a rat colony to remain infected with T. gondii, cat feces would need to be repeatedly introduced. As a result, T. gondii is an organism is of little current significance in research facilities, and routine monitoring for toxoplasmosis in rats continues only in some geographic areas (Rehbinder et al., 1996). Numerous enteric flagellates have been reported in laboratory rats over the years, but none are of significance. The life cycle of all flagellates, and Entamoeba muris, is direct (Flynn, 1973c; Levine, 1961), with fecal-oral transmission. Trophozoites, the feeding form, are present in the gastroinestinal tract. Reproduction is asexual and produces resistant cyst forms, which are shed in the feces (Kunstyr, 1977). Spironucleus muris colonizes mice, rats, and hamsters, where it inhabits glandular crypts and the lumen of the small intes-

DENNIS F. KOHN AND CHARLES B. CLIFFORD

tine (Gruber and Osborne, 1979; Wagner et al., 1974). A g e infection relationships have not been reported for rats but are probably similar to that of mice, in which animals under 6 weeks of age are more susceptible to infection. Transmission of cloned S. muris between rats and mice has been attempted (Schagemann et al., 1990). An isolate from rats was not infective to hamsters, immunocompetent mice, or athymic nude mice. Similarly, rats were not persistently colonized by isolates from mice or hamsters. Cysts of S. muris are resistant to drying (room temperature for 14 days), freezing ( - 2 0 ~ for 6 months), pH 2.2 for 1 day, or 0.1% glutaraldehyde for 1 hr (Kunstyr and Ammerpohl, 1978). Spironucleus muris infection is diagnosed by examination of wet mounts of duodenal scrapings of weanling rats. Phase-contrast microscopy is especially helpful in observing the trophozoites. Identification is usually based on the size, 3 - 4 • 10-15 gm, and characteristic rolling motion of the flagellated trophozoites. Cysts may be observed in wet mounts or in fecal smears. These measure 4 • 7 gm and are reported to have a characteristic banded pattern (Kunstyr, 1977). Giardia spp. are ancient, with one of the most highly conserved genomes of all eukaryotes (Yu et al., 1996, 1998). Giardia also has its own microflora, including mycoplasma-like particles and bacteria (Feely et al., 1988) and viruses (Tai et al., 1991, 1996). Giardia muris colonizes a wide variety of mammalian hosts, including rats, mice, hamsters, and humans (Levine, 1961). Recent evidence has suggested that G. muris may actually be a form of G. duodenalis (Sharma and Mayrhofer, 1988b). Although this conclusion seems to have implications for zoonotic potential, because G. duodenalis is a human pathogen, there does appear to be some host specificity, albeit incomplete. Giardia muris isolated from mice and hamsters, for example, did not produce infection when inoculated into rats (Kunstyr et al., 1992). Trophozoites were previously reported to attach to the surface of intestinal epithelial cells by means of a flat suction disk (Levine, 1985a). It is now thought that attachment is via a surface membrane mannose-binding lectin and can occur via any point on the parasite surface, without requiring the disk (Inge et al., 1988). Cysts stored in liquid feces have remained infective for at least 1 year (Craft, 1982). No naturally occurring clinical disease has been reported in rats infected with G. muris. Experimental infection with G. lamblia and G. duodenalis has resulted in secretion of specific immunoglobulin A into bile (Loftness et al., 1984; Sharma and Mayrhofer, 1988a). Giardiasis is diagnosed similarly to spironucleosis. Trophozoites, measuring 7-13 • 5 - 10 gm (Levine, 1961), have a characteristic piriform or teardrop shape, with a broad, rounded anterior tapering to a pointed posterior end (Levine, 1985a). The trophozoites have a slight curvature toward the ventral side, which causes the motion of their multiple flagella to impart a rolling motion to the organisms in wet mounts (National Research Council, 1991a). In stained preparations, the darkly

4. BIOLOGY AND DISEASES OF RATS

stained dual nuclei are prominent. Two small dark median bodies are also visible, immediately posterior to the nuclei. Cysts may also be identified on fecal smears or with fecal flotation methods. Entamoeba muris is a nonpathogenic commensal amoeba of rats, mice, and hamsters (National Research Council, 1991a; Levine, 1985b). Trophozoites, measuring 8 - 3 0 ~tm in length, are found in wet-mount preparations of contents from the cecum and colon, where they feed on bacteria (Levine, 1961). Cysts 9 - 2 0 ~tm in diameter have eight nuclei and can be observed in feces. Control measures in rats for all intestinal flagellates and Entamoeba muris are similar. Rederivation, either by cesarean section or by embryo transfer, is effective. Contaminated animal rooms should be thoroughly cleaned, then disinfected with chlorine dioxide solutions or other suitable disinfectants (Wickramanayake and Sproul, 1991), prior to repopulation introduction. All materials brought into the room, which may have had prior exposure to rodents or rodent feces should be autoclaved. All animals should be monitored for infection prior to introduction. This should include examination of rats of appropriate age, i.e., 3 - 6 weeks. Treatment of animals to eliminate infection with intestinal flagellates has met with limited success. Metronidazole (Flagyl) or dimetridazole (National Research Council, 1991a) can be added to the drinking water but is ineffective against cysts in the environment. Other authors have reported success in eliminating Giardia spp., using metronidazole in rats and mice (Sharma and Mayrhofer, 1988b). Significantly, however, metronidazole has been shown to be carcinogenic in rats and mice (Goldman, 1980). b.

Nematodes

i. Oxyuriasis Three species of oxyurid nematodes (pinworms)--Syphacia muris, S. obvelata, and Aspicularis tetrap t e r a I o c c u r in the laboratory rat. Their continued occurrence, despite the dramatic progress in eliminating viral and bacterial pathogens, is due both to the persistence of the eggs in the environment and to the low degree of attention paid to these parasites. Syphacia muris is the most common oxyurid of the rat (National Research Council, 1991 a; Owen, 1992a). Syphacia obvelata is more frequently found in mice, hamsters, and gerbils but is also occasionally found in the rat, especially when housed in the same room with infested mice. Syphacia spp. have a direct life cycle, requiring 11-15 days for completion (Flynn, 1973a). Transmission is horizontal via ingestion of eggs. Eggs, which remain viable at room conditions for weeks to months, are deposited around the anus and in the colon and become infective in approximately 6 hr. They are ingested during self-cleaning and hatch in the small intestine. The larvae then mature in the cecum in 10-11 days. The mor-

149

phology of adults of both species is similar, although S. muris is slightly smaller and the male has a longer tail, measured as a proportion of body width (Flynn, 1973a). Eggs vary more markedly between the species, with eggs of S. muris being 7 2 - 8 2 x 25-36 ~tm and those of S. obvelata being 118-153 X 3 3 55 ~tm. In addition, the eggs of S. obvelata are almost completely flat along one side, whereas those of S. muris are only slightly flattened on one side. Aspicularis tetraptera is also transmitted horizontally by ingestion of eggs, which are extremely persistent in the environment (Flynn, 1973a). The direct life cycle is longer than that of Syphacia, requiring 23-25 days. Also unlike in Syphacia, Aspicularis eggs are passed in the feces and are not deposited around the anus. Adult A. tetraptera are readily recognized by the four alae present at the anterior end of the body. Eggs of A. tetraptera are approximately the same size as S. muris eggs, measuring 89-93 X 3 6 - 4 2 Bm, and are bilaterally symmetrical. Gross lesions of oxyuriasis are very rare (Flynn, 1973a), and histologic lesions of oxyuriasis have not been reported. Diagnosis of oxyuriasis is most practically accomplished by direct examination of macerated cecum and colon under low magnification with a stereomicroscope. This is almost as sensitive as complete direct examination of the large bowel and is significantly less time-consuming. Examination for eggs must be tailored to the infesting species suspected. The perianal tape test is effective only for Syphacia spp., and fecal flotation is effective only for A. tetraptera. Screening for oxyurid eggs is significantly less sensitive than direct examination of the bowel for the adult helminths (West et al., 1992; Klement et al., 1996). Oxyuriasis can be eliminated in individual rats with ivermectin (Huerkamp, 1993; Klement et al., 1996; Zenner, 1988; Hasslinger and Wiethe, 1987). However, the source of the original infestation should be identified, and the premises should be thoroughly disinfected so as to prevent reinfestation. Ivermectin is not effective against eggs, which can persist for long periods in the environment. Oxyuriasis can also be eliminated by rederivation. It is readily excluded by proper adherence to modern practices of barrier room technology (Hasslinger and Wiethe, 1987). Numerous research effects of oxyuriasis have been described. In rats, oxyuriasis has been reported to interfere with adjuvant arthritis (Pearson and Taylor, 1975), growth rate (Wagner, 1988), and intestinal electrolyte transport (Lubcke et al., 1992). ii. Trichosomoides crassicauda This trichurid nematode is found only in the rat (Flynn, 1973a). Although geographically widespread, Trichosomoides crassicauda is very rare in barriermaintained rodents that have been rederived by cesarean section or embryo transfer. Adult females, approximately 10 mm long, live in the urinary bladder, either free in the lumen or embedded in the mucosa (Flynn, 1973a; Antonakopoulos et al., 1991; Cornish et al.,

150

DENNIS F. KOHN AND CHARLES B. CLIFFORD

1988). The males are anatomically degenerate and exist symbiotically in the vagina or uterus of the females. Embryonated eggs are laid and pass in the urine. Transmission of T. crassicauda is via ingestion of these eggs and probably occurs from dam to pups prior to weaning. The eggs hatch in the stomach, where the larvae penetrate the wall and pass through the peritoneal cavity or bloodstream to reach the lungs and other tissues. Most larvae lodge in tissues other than the kidneys and may cause hemorrhages or granulomas. Only those that reach the kidney or bladder survive and develop to maturity. The entire life cycle is 8 - 9 weeks, so eggs are not present in the urine until the rats are 8-12 weeks of age. Infestation with T. crassicauda, although persistent, is usually clinically inapparent (Flynn, 1973a). Usually very few worms, perhaps averaging 3 in number (Barthold, 1996a), are present in the bladder, where they cause mild uroepithelial hyperplasia (Zubaidy and Majeed, 1981; Antonakopoulos et al., 1991). When found in the renal pelvis, they are associated with mild pyelitis and pyelonephritis. Trichosomoides crassicauda infestation is diagnosed in live rats by filtration of urine and then examination of the filter medium for the eggs. Diagnosis in recently killed rats is by direct examination of the bladder wall, histopathology, scanning electron microscopy, or microscopic examination of cryostat sections stained with acridine orange (Barthold, 1996a; Cornish et al., 1988). The last two methods are purported to be more reliable but are probably not practical for routine, large-scale screening. Treatment for T. crassicauda infestation has been reported, using a single dose of ivermectin (Summa et al., 1992). Followup found that the infestation was not eliminated in 1 of 30 rats, perhaps because of reinfection. Once a colony is free of this parasite, however, there should be little chance of reintroduction if no infected rats enter the colony. No confirmed research effects of T. crassicauda infestation have been reported in the scientific literature, although proliferative changes in the urothelium would render these animals unsuitable for research involving the urinary system (Cohen et al., 1998). Early speculation concerning the possible etiologic role of T. crassicauda infestation in causing bladder tumors in a famous study in rats that were administered high doses of saccharin in the diet (Homburger, 1977) has not been supported by later investigators (Barthold, 1996a). However, proliferative changes in uroepithelium caused by T. crassicauda infestation are identical to those produced early in carcinogenesis by chemical compounds such as N-methylnitrosourea (MNU) (Pauli et al., 1996). c.

Cestodes

There are only two adult cestodes that are likely to be encountered in laboratory rats: Hymenolepis nana and H. diminuta. The primary differences of consequence between the two

species are that H. nana is zoonotic and can have a direct life cycle, whereas H. diminuta always has an indirect life cycle, utilizing an intermediate host, and is not zoonotic. Fortunately, both are rare in laboratory rats. Hymenolepis nana lives in the small intestine of rats, mice, hamsters, and primates, including humans (Hsu, 1979). It is primarily the ability to parasitize humans that gives H. nana significance, for it causes little damage in rats or mice (National Research Council, 1991a). Hymenolepis nana averages 2 0 40 mm long but can vary greatly. It is slender and less than 1 mm wide. The scolex has four suckers, and a rostellum armed with 20-27 hooks. Mature proglottids are trapezoidal and contain as many as 200 eggs, which are thin-shelled, oval, and colorless and have six visible polar filaments. Within the eggs, the embryo, or oncosphere, has three pairs of hooklets within an inner envelope. The eggs are approximately 30-56 ~m • 4 4 - 6 2 ~tm and do not persist for long periods outside the host. In the direct life cycle (Hsu, 1979), which requires 14-16 days, embryonated eggs are ingested and hatch in the small intestine. The oncospheres penetrate villi and develop into cysticercoid larvae in 4 - 5 days. These larvae reenter the lumen, the scolex evaginates, and they attach to the mucosa. An additional 10-12 days are required before mature proglottids are formed. Adults live only a few weeks. Infection normally results in some level of immunity, which prevents autoinfection. When autoinfection occurs, eggs hatch in the small intestine and develop, without being passed in the feces, and can result in very high worm burdens. In the indirect life cycle, grain beetles (Tenebrio molitor and T. obscurus) and fleas (Pulex irritans, Ctenocephalus canis, Xenopsylla cheopis) serve as intermediate hosts. Rats and other definitive hosts are infected by ingesting the intermediate host. Hymenolepis diminuta has a similar host range: mice, rats, hamsters, and primi~tes, including humans (Hsu, 1979). Hymenolepis diminuta is larger than H. nana, 2 0 - 6 0 mm long and 3 - 4 mm wide. The scolex of H. diminuta also has four suckers but the rostellum is unarmedmit has no hooks. Eggs of H. diminuta are 6 0 - 8 8 X 52-81 ~tm, and the oncosphere has three pairs of hooks, but no polar filaments. The life cycle of H. diminuta is always indirect and is similar to the indirect life cycle of H. nana. Both Hymenolepis nana and H. diminuta are pathogenic in rats only in severe infections, where retarded growth, weight loss, impaction, and death have been reported in the older literature (Hsu, 1979), although no recent reports excluding the contributions of other potential pathogens have been published. In humans (Jueco, 1982), infection is common in some geographic areas but is usually subclinical. As in rats, the adult worms live 2 5 - 6 0 days before dying, but human cases may persist as long as 22 months because of autoinfection. Infection is diagnosed by detection of the adult cestodes on direct examination of the small intestine, by observation of the eggs in feces (smear or fecal flotation), or by histopatho-

151

4. BIOLOGY AND DISEASES OF RATS

logic detection of the cysticercoid in the small intestine (Hsu, 1979). Infection is most common in recently weaned rats and young adults, probably because of acquired immunity in older animals. Hymenolepis nana and H. diminuta infection is prevented by purchase of clean stocks of rodents, by adequate disinfection of barrier room supplies, and thorough insect control and exclusion of wild rodents (National Research Council, 1991 a). Treatment of infected animals is not generally recommended, because of the zoonotic implications of this disease. No indirect interference with research has been reported for hymenolepiasis in rats. In addition to the adult cestodes, one may occasionally encounter larvae of Taenia taeniaformis, also called cysticercus fasciolaris. The cysts are found in the livers of rats, mice, and hamsters and are up to several centimeters in diameter. They are readily identified by the presence in the cyst of a scolex, strobila, and bladder (Hsu, 1979; Wescott, 1982). Although considered nonpathogenic (Hsu, 1979), the cyst may be associated with the development of hepatic sarcomas, probably in a mechanism similar to the induction of sarcomas in the rat by a variety of foreign bodies (Altman and Goodman, 1979). Because the definitive host is the cat, detection of the cysticercus is evidence that materials in the animals' immediate environment, usually feed, were contaminated with unsterilized feces from an infected cat. Control, therefore, is simple.

d.

Trematodes

Numerous trematodes have been reported in wild rats, including Plagiorchis muris, P. philippinensis, and P. javensis. Some are zoonotic (Hong et al., 1996), but none are significant in laboratory rats (Wescott, 1982).

e.

Mites

Radfordia ensifera is the only ectoparasite of rats likely to be encountered in a laboratory animal environment, although other acarids, such as Radfordia affinis or Myobia musculi, could possibly be harbored on the pelage. Acariasis, or mite infestation, is transmitted by eggs, which can persist in the environment for long periods. The eggs hatch in 7-8 days, and females can begin to lay eggs after another 16 days. Infestation can result in pruritus, self-excoriation, and secondary bacterial infection. In mice (Weisbroth, 1979), acariasis has also been associated with increased mitotic activity in the skin, immunologic alterations, and amyloidosis. Acariasis is most practically diagnosed by direct examination of the animals with a dissecting microscope (Flynn, 1973b). As an alternative, dead rats or their pelts can also be placed in a sealed clear glass or plastic container and refrigerated overnight, then examined against a black background. Control of acariasis is similar to that for other parasitic meta-

zoa, and acariasis can be eliminated in individual rats with ivermectin (West et al., 1992). However, the source of the original infestation should be identified, and the premises thoroughly disinfected, so as to prevent reinfestation. Ivermectin is not effective against eggs, which can persist for long periods in the environment. Infestation can also be eliminated by rederivation and is readily excluded by proper adherence to modern practices of barrier room technology (Weisbroth, 1979).

f . Lice Pediculosis in the laboratory rat is currently rare and is attributed to only one species, Polyplax spinulosa (Flynn, 1973f; Owen, 1992b). Polyplax spinulosa females are approximately 0.6-1.5 mm long; females are larger than males. Like all insects, they have six legs. The female lays eggs, called nits, which are cemented to hairs. The eggs have a distinct operculum, with a row of pores near the operculated end. The eggs hatch by a pneumatic mechanism in 5 - 6 days; the larvae ingest air through the pores, pass it through the body, and then use that pressure to force open the operculum (Owen, 1992b). The young nymphs are paler than the yellow-brown adults but are morphologically similar. After three ecdyses, or molts, they become adults. Depending on environmental conditions, the ecdyses require 1-3 weeks. The entire life cycle is completed in 2 - 5 weeks. Adults live only 25-28 days. Transmission is by direct contact (Hsu, 1979). Pediculosis is usually inapparent, although heavily parasitized animals may appear unthrifty and pruritic. Polyplax spinulosa is also the vector of Haemobartonella muris (National Research Council, 1991b). Diagnosis is by direct examination of the pelt for adults, nymphs, and eggs (Hsu, 1979). Any time that infestation with P. spinulosa is detected, blood smears should be screened for Haemobartonella muris. Pediculosis is prevented by introducing only animals free of the condition. Pyrethrins or organophosphates may be used effectively to treat infestations (Hsu, 1979) but would probably be advisable only in especially valuable rats in the absence of significant intercurrent infections. 4.

Fungal Infections

Fungal infections in the rat have been infrequently reported and are associated with predisposing factors that reduce immunocompetence. In one report, about one-fifth of Wistar rats on a 2-year carcinogenesis study had chronic rhinitis associated with Aspergillus fumigatus (Rehm et aL, 1988). The predisposing factor in these animals was thought to be Sendai virus infection. Clinical signs included sniffing and nasal exudation.At necropsy, yellowish, friable material was present either unilaterally or bilaterally in the nasal cavities, and in the most severe cases, the nasal cavities were completely blocked. The A. fumigatus-induced rhinitis was, in most cases, limited to the

152

naso- and maxilloturbinates. A bronchial abscess containing hyphae and multiple fruiting heads occurred in one rat. Tracheobronchial aspergillosis was reported in an aged F-344 rat with concomitant large granular-cell leukemia. Immunodeficiency due to the leukemia was thought to be involved with the multifocal, transmural necrotic lesions of the trachea and bronchi (Hubbs et al., 1991). Pneumocystis carinii is classified as a fungus based upon DNA base sequences in genes encoding ribosomal RNAs (Feldman et al., 1996). This agent is latently present in the lungs of immunocompetent laboratory rats and humans, causing pneumonitis in hosts that are severely immunosuppressed. The immunocompromised rat is commonly used as a model of P. carinii pneumonitis that occurs in AIDS patients (Oz and Hughes, 1996). The agent is naturally acquired by rats through airborne transmission (Hughes, 1982) and is commonly present latently in rats from both conventional and barrier-maintained commercial sources. Diagnosis of infection in immunocompetent rats usually requires at least 6 weeks of treatment with a corticosteroid or cyclophosphamide to elicit a histologically detectable level of infection. Special stains such as methenamine silver demonstrate the fungal cysts within the alveoli. More recently, polymerase chain reaction (PCR) has been used to detect P. carinii infection in rat lungs consistently after only 1 week of treatment with corticosteroids or cyclophosphamide (Feldman et al., 1996). Pneumocystis carinii has a life cycle consisting of four morphologically distinct stages: trophozoite (1.5-2 gm), precyst ( 2 - 4 gm), cyst (5-7 gm), and sporozoites (1-1.7 gm) that develop within cysts (National Research Council, 1991 a). Histopathology may vary from multifocal alveolar aggregates of cysts with interstitial and perivascular infiltrates in less severe cases to pulmonary consolidation with foamy, eosinophilic, honeycombed alveolar exudation and severe interstitial fibrosis (GV-SOLAS, 1999). Control of the disease in immunocompromised rats can be achieved by treatment with trimethoprim-sulfamethoxazole. Royals et al. (1999) reported 2 cases of fungal-induced rhinitis in rats that had no known immunosuppression. Corncob and hardwood bedding from 2 sources were tested to determine if the source of the Aspergillus infection was bedding material. A range of 700 to 5400 fungal spores per gram of nonautoclaved corncob bedding was found. Six genera of fungi (Cladosporidium, Acremonium, Penicillium, Aspergillus, Fusarium, and Scolobasidium) were isolated from the samples of corncob bedding, whereas only negligible counts were isolated from hardwood bedding samples. The authors suggested that either the use of autoclaved or 7-irradiated corncob bedding should be considered as a means to eliminate fungal contamination of bedding. Dermatomycosis (ringworm) due to Trichophyton mentagrophytes has been reported in wild and laboratory rats. However, it has not been reported in laboratory rats for many years. In rats, dermatomycosis may be presented clinically by patchy hair

DENNIS F. KOHN AND CHARLES B. CLIFFORD

loss and scurfy or erythematous papular-pustular lesions (Weisbroth, 1979).

B. I.

Noninfectious Diseases

Metabolic and Nutritional Diseases

a.

Genetic Anomalies

One investigator's meat (desirable trait) is another investigator's poison. Every stock and strain of laboratory rat has been carefully selected for specific genetic traits for decades. Common among these are albinism, behavioral characteristics, such as docility and willingness to breed in captivity, and certain tumor profiles. Overt metabolic diseases such as obesity (Zucker rat), diabetes (BB rat), and hypertension (SHR and fawn-hooded and Dahl rats) make these strains valuable models in biomedical research, whereas spontaneous appearance of the same characteristics in outbred stocks may complicate other research studies. More subtle strain-related tendencies, such as immunologic responsiveness characteristics in Brown Norway and Lewis rats, are exploited by researchers in particular areas of research. In recent years, genetic manipulation has allowed further development of specifically tailored metabolic disease to model critical human defects. It is beyond the scope of this chapter to catalog the innumerable genetic traits or strain-related variations that occur in laboratory rats, and the reader is encouraged to consult large electronic databases, such as the National Library of Medicine, for specific and current information on particular genes, strains, and conditions. In addition to known and characterized, spontaneous or induced, genetic variation in rats, isolated colonies of breeding rats inevitably experience some degree of genetic drift. Although this may be monitored to some degree in inbred rats by molecular techniques such as restriction fragment length polymorphisms, it is more difficult to assess the degree to which it has occurred in outbred stocks, where expected interindividual variation may obscure intercolony differences. Nonetheless, any two colonies started from the same source will vary increasingly with time unless there is a sufficient and ongoing exchange of breeders between the colonies. Genetic drift can also be reduced by careful adherence to specific outbreeding programs, such as line breeding with systematic exchange of breeders between multiple lines. The inevitability of some degree of genetic drift should not, however, blind researchers to the large role played by environmental, husbandry, dietary, and experimental variables in apparent differences between succeeding groups of animals. These extraneous factors can also have a major impact on the expression of underlying genetic traits. An example of modification of lesion prevalence is the impact of ad libitum over-

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4. BIOLOGY AND DISEASES OF RATS

feeding on increasing the incidence of progressive renal disease (Keenan et al., 1996, 1998). b.

Nutritional deficiencies.

Frank dietary deficiencies are uncommon, probably for several reasons. First, high-quality commercial diets are in almost universal use. Second, rats store fat-soluble vitamins and vitamin B12, manufacture vitamin C, and can fulfill many of their requirements for other B vitamins by coprophagy. However, heat and moisture, such as are associated with autoclaving, can reduce vitamin levels, particular lysine, vitamin A, vitamin E, riboflavin, and thiamin. Prolonged storage can have similar effects. In addition, diets designed for maintenance of adult rodents may be too low in protein and fat for optimal growth of young animals or successful reproduction. Clinical evidence of dietary insufficiency may include decreased reproductive performance, litter loss, poor growth, and sparse hair coat. Signs of severe deficiencies of specific vitamins are rare. If they occur, they would include squamous metaplasia of salivary ducts with hypovitaminosis A, disseminated hemorrhage with hypovitaminosis K, and embryonic death and testicular degeneration with hypovitaminosis E. Nutritional deficiencies can also alter disease susceptibility and severity. In addition, feed qualities, aside from total levels of calories and specific nutrients, must be considered, including contaminating chemicals, microbes, and the size and hardness of pellets. For example, feeding a powdered diet will result in an increased incidence of malocclusion. 2.

Management-Related Diseases: Nonnutritional

There is essentially no limit to the number of health problems that may be caused by suboptimal care and management, including those relating to experimental manipulations. Only a few of the most common will be mentioned. Sanitation of the animal's cage, bedding, water, and feed, as well as of experimental equipment, is critical. High moisture content in bedding leads to rapid growth of bacteria, which can increase the incidence of urinary tract infections and, possibly, mastitis and skin lesions. Some softwood bedding materials emit aromatic compounds that may increase hepatic microsomal levels, although these compounds are usually completely sublimated during the drying phase in bedding manufacture. Some organisms are airborne and can enter or spread within a facility via air currents. Considerations of air quality might also include factors such as ammonia, other bedding gases, dusts, fungal spores, disinfectant vapors, and pollutants. Management consideration must be given to organisms carried by human caretakers and investigators. Humans are commonly colonized by, and can transmit, Streptococcus spp, Staphylococcus spp., Escherichia coli, Klebsiella spp., Pseudomonas spp., Campylobacter spp., and so on. In addition, humans can transmit viruses that may result in

serologic cross-reactions, if not outright infection, and can also serve as fomites for many other organisms. Rats are sensitive to temperature, humidity, noise, light, room activity, and generally to any changes in their environment. Low humidity, considered to be relative humidity of less than 40%, together with high temperature, has been linked to the poorly characterized condition known as ringtail. Unfortunately, experimental reproduction of ringtail has not been reported, nor has the pathogenesis of the condition been elucidated. Ringtail is primarily a condition of young rats, usually sucklings, characterized by the formation of prominent annular constrictions of the tail and occasionally of the digits. Portions of affected extremities distal to the constrictions often become necrotic and are sloughed. This condition should not be confused with bite wounds or the normal, more subtle annulations of rat tails which develop with age. The hearing range of rats has been given as 0.25-76 kHz (Sales and Milligan, 1992); correspondingly many vocalizations of rats also are in the ultrasonic range. Therefore, low audible noise levels for humans do not indicate that noise levels are acceptable to rats. Rats, especially albino rats are susceptible to retinal degeneration when exposed to ambient light levels above a threshold of between 130 and 270 lux (Semple-Rowland and Dawson, 1987). As a result, recommended room light levels are 325-400 lux (National Research Council, 1996b). Note that these recommended levels are as measured 1 m above the floor and do not necessarily reflect actual light levels to which rats are exposed in individual cages at various levels in racks at varying distances from light fixtures. Exposure to very high light levels of 1600 lux for 12 hours each day for 8 days resulted in necrosis in Harderian glands (Kurisu et al., 1996). In addition, exposure to constant light may cause anestrus and other breeding problems as described in Section II,D,3. Exposure (contamination) during the dark phase of the light cycle, with light levels as low as 0.21 lux, has been reported to interfere with growth and metabolism of tumors (Dauchy et al., 1997).

C.

Traumatic and Iatrogenic Diseases

Traumatic lesions are uncommon in the rat. Rats housed in wire-bottom cages may develop pododermatitis or lesions on their hocks if housed long-term in such caging. Occasionally, wire-grid floors will allow a rat's foot to become entrapped in the wire grid causing severe edema and injury to the foot and leg. One of the authors (DK) has observed this most often when rats are allowed to recover from anesthesia in a wire-bottom cage. Group housing of rats is much less likely to result in traumatic injuries due to fighting than is seen in mice. Ulcerative dermatitis, associated with Staphylococcus aureus and selfinduced trauma from scratching, has been reported (Fox et al., 1977; Wagner, 1977). In one report (Fox et al., 1977), the skin

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lesions were observed only in rats originating from two breeding colonies of one commercial vendor, leading to the hypothesis that the lesions may have been associated with specific Staphylococcus phage types or host susceptibility factors. Adynamic ileus, sometimes leading to death, may occur subsequent to intraperitoneal administration of chloral hydrate. Clinical signs occur several days after anesthesia and include lethargy, anorexia, and abdominal distension. The most prominent dilatation occurs in the jejunum, ileum, and cecum. The usual anesthetic dose of chloral hydrate is 400 mg/kg; however, the concentration of the drug, not the dosage, appears to be correlated with the induction of ileus (Fleischman et al., 1977).

D.

N e o p l a s t i c Diseases

The prevalence of neoplastic disease in the rat is well defined because this species has been routinely used for decades in large-scale carcinogenic, aging, and toxicological studies. Stock and strain-specific differences in the prevalence of some types of tumors are well documented (MacKenzie and Garner, 1973; Burek, 1978; Goodman et al., 1979, 1980). However, the overall prevalence of neoplasia and that of specific tumor types may vary considerably within stocks or strains because of genetic variation, environmental influences, and differences in laboratory methodologies and diagnostic criteria (MacKenzie and Garner, 1973; Altman and Goodman, 1979). The age at which rats are surveyed is also important, because most tumors, other than mammary gland fibroadenomas in many stocks and testicular tumors in F-344 rats, occur in animals greater than 18 months old (Kohn and Barthold, 1984). Table VI compares the incidence of the most frequently occurring tumors in SpragueDawley and F-344 rats. Among the environmental influences, diet has been found to be an extremely important factor in modulating tumor prevalence. A 20% reduction in food intake was found to significantly reduce the overall tumor incidence in male and female Wistar rats, primarily to differences in the incidence of pituitary and mammary gland tumors (Tucker, 1979). In another study (Morris and Bras, 1971), tumor incidence in Sprague-Dawley rats was compared among 3 groups: rats fed ad libitum, rats foodrestricted throughout life, and rats food-restricted between 21 and 70 days of age. Both food-restricted groups had a significantly reduced prevalence of tumors. The proportion of rats in the nonrestricted group with multiple tumor types was 16.7%, about twice that as for each of the other two groups. Interestingly, the group that was food-restricted up to day 70 had a persistent reduction in food consumed thereafter on an ad libiturn diet. Another environmental influence on the prevalence of tumors is the pathogen or disease status of the rats in a particular report. Data on tumor risk can be significantly influenced by the effect that some infectious diseases may have on longevity, preneo-

Table VI

Incidence of Most Prevalent Tumors in Two-Year-OldRatsa Incidence (%) Sprague-Dawley (Crl:CDBR) Organ/tissue Testes Interstitial cell tumor Uterus Endometrial stromalpolyp Ovary Granulosa cell/theca cell tumor Mammary gland Fibroadenoma Carcinoma Liver Hepatocellular adenoma/carcinoma Lymphoreticular Large granular lymphocyticleukemia Histiocytic sarcoma Pituitary Adenoma/carcinoma, pars distalis Adrenal gland Cortical adenoma Pheochromocytoma,benign Pheochromocytoma,malignant Pancreas Islet cell adenoma Islet cell carcinoma a

F-344 (CDF/CflBR)

Male Female Male Female 4.8

--

78.3

m

4.1

--

14.3

--

1.0

--

0.8

2.0 1.0

31.4 17.7

2.5 0

12.0 1.5

6.8

2.6

1.4

1.0

0.2 1.6

0.3 1.5

16.5 0.6

10.4 0

67.1

82.6

16.3

19.7

2.9 15.0 1.9

6.0 3.9 0.6

0.4 6.7 0.5

1.0 0.9 0.3

8.3 2.0

3.8 1.4

9.3 0.6

2.0 0

Adapted from Lang (1990, 1992).

plastic changes, and masking of small tumors (Kohn and Barthold, 1984).

1.

Mammary Gland Tumors

Mammary gland tumors are the most frequently occurring tumors in most stocks and strains of rats. Sprague-Dawley stocks often have an incidence of 50% in aged female animals, whereas F-344 have a relatively low incidence of about 15% (Goodman et al., 1979; Lang, 1990, 1992). Most mammary tumors are benign fibroadenomas, with carcinomas occurring less frequently. Both types can occur in aged males; however, the incidence is usually less than 1%. The tumors may arise in mammary tissue at any point from the neck to the inguinal area, and they tend to attain a large size and become ulcerated unless surgically excised. On gross examination, fibroadenomas are freely movable in subcutaneous tissues, circumscribed, firm, and lobulated. Histologically, they are characterized by well-differentiated acinar epithelial components surrounded by inter- and intralobular connective tissue components (Altman and Goodman, 1979; Percy and Barthold, 1993).

155

4. BIOLOGY AND DISEASES OF RATS 2.

Testicular Tumors

Interstitial cell tumors occur in about 80% of aged F-344 rats (Goodman et al., 1979; Lang, 1990). They are discrete, soft, and yellow to brown, with areas of hemorrhage, and may occur in multiple sites unilaterally or bilaterally. Histologically, their Leydig's cell origin is apparent. Tumors have two cell types that are arranged in solid sheets or in an organoid pattern. The cell types are (1) polyhedral to elongated cells with granular to vacuolated cytoplasm and (2) smaller cells with hyperchromatic nuclei and scanty cytoplasm (Altman and Goodman, 1979; Percy and Barthold, 1993). The incidence of interstitial cell tumors in most other stocks and strains is quite low.

3.

Pituitary Tumors

Pituitary tumors occur frequently in aged rats of some stocks and strains, most notably in Sprague-Dawley and Wistar rats (Percy and Barthold, 1993). Most pituitary tumors are classified as chromophobe adenomas, originating from the pars distalis. Carcinomas of the pars distalis are reported with much less frequency; however, their reported prevalence may vary considerably because of differences in classification protocols by pathologists. Surveys reflect an incidence in F-344 rats of about 20% (Goodman et al., 1979; Lang, 1990), and a 75% incidence in Sprague-Dawley rats (Lang, 1992). In some reports, the prevalence of pituitary tumors is greater in female F-344 and Sprague-Dawley rats. As was previously noted, diet restriction significantly reduces the incidence of pituitary tumors in rats. Chromophobe adenomas vary in size, often reaching 0.5 cm in diameter. Grossly, the tumors are soft and dark red due to prominent hemorrhagic areas (Fig. 10). They are well circumscribed and, because of their size, often compress adjacent brain tissue and induce hydrocephalus. Microscopically, they consist of large polygonal cells with prominent vesicular nuclei and eosinophilic cytoplasm. The architecture of the tumors consists of cells arranged in nests, cords, or sheets separated by vascular sinusoids (Altman and Goodman, 1979).

4.

5.2% in female Sprague-Dawley rats, and the incidence in F-344 was somewhat less (Lang, 1990, 1992). Grossly, islet cell tumors may be either single or multiple and are circumscribed and reddish brown. Islet cell carcinomas are distinguished from adenomas by capsular invasion and metastases. Tumors of the exocrine pancreas are rare (Altman and Goodman, 1979). 6.

Lymphoreticular System

Large granular lumphocytic leukemia is a major cause of death in F-344 rats (Ward and Reynolds,. 1983), with a reported incidence of 10-16% (Coleman et al., 1977; Goodman et al., 1979). The initial site of malignancy is thought to be the spleen. The neoplastic cells are transplantable to rats of the same strain. Unlike leukemia in mice, this leukemia in rats is not associated with a retrovirus. Diagnosis is based upon clinical signs of anemia, jaundice, weight loss, and laboratory findings of splenomegaly, elevated leukocyte counts of 70,000-180,000/ml, and

Adrenal System

Lang (1990, 1992) reported a cortical adenoma incidence of 2.9% in male and 6% in female Sprague-Dawley rats, and a benign pheochromocytoma incidence of 15% in males and 3.9% in females of this stock. The incidence in F-344 rats was less than half that seen in Sprague-Dawley rats (Table VI).

5.

Pancreas

Pancreatic islet cell tumors are relatively common in some stocks of rats. An incidence of 10.3% was reported in male, and

Fig. 10. Chromophobeadenoma of the brain of a rat. The large hemorrhagic mass is visible on the ventral aspect of the brain. (Courtesyof Dr. Robert Jacoby.)

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DENNIS F. KOHN AND CHARLES B. CLIFFORD

diffuse infiltration of malignant lymphocytes in various organs (Percy and Barthold, 1993). In the Sprague-Dawley rat and most other stocks, the incidence is quite low. Other lymphoreticular tumors include histiocytic sarcoma, which has an incidence in Sprague-Dawley rats of about 1.5% (Lang, 1992), and myelomonocytic leukemia in BN/Bi rats, with an incidence of 5% in females and 11% in males (Burek, 1978).

E. 1.

Miscellaneous Conditions

Congenital/Hereditary Lesions

It is beyond the scope of this chapter to catalog congenital defects in rats. The incidence of such defects is obviously influenced by administration of mutagenic and teratogenic substances, but it also varies with strain, age of mother, disease status, coincidences of statistics, and human terminology. As noted above, rats are susceptible to a wide variety of genetic diseases, some of which make them valuable models and others of which are confounding variables. Only a few spontaneous defects, involving the urinary tract, heart, and central nervous system, will be mentioned here. The growing range of genetically engineered diseases, and their unintentional side effects, will not be addressed. Researchers and supporting animal resource professionals are strongly urged to investigate, with due scientific scrutiny, background information concerning specific stocks and strains, prior to embarking on courses of research involving any laboratory animal. Large databases of defects observed in reproductive studies are available from the Middle Atlantic Reproduction and Teratology Association and Midwest Teratology Association (1996), for example, and should be consulted. Hydronephrosis is one of the more commonly reported congenital defects of rats, characterized by unilateral or bilateral dilation of the renal pelvis. Although it may be inherited as a single dominant gene in the Gunn rat, it appears to be polygenic in the Brown Norway and Sprague-Dawley rat (Van Winkle et al., 1988). The right kidney is affected more often than the left. Severity of hydronephrosis can vary from a slight dilation of the renal pelvis to such severe dilation that the kidney appears as a transparent cystic structure. The ureter may also be affected to varying degrees. The normal renal pelvis of young animals may appear to be dilated, however, so some caution is required in identifying hydronephrosis (Maronpot, 1996). Hydronephrosis may also be mistaken for pyelonephritis, in which the material in the dilated pelvis is typically cloudy; for polycystic kidneys; and for renal papillary necrosis. Culture and histopathology of the affected site will distinguish among these conditions. Congenital lesions of the cardiovascular system are less frequently reported but include ventricular and atrial septal defects, dextrocardia, and defects of the valves and endocardial

cushion, as well as various anomalies of the great vessels. Overall incidence of cardiac defects has been estimated in one colony of Sprague-Dawley rats at 2.3% (Johnson et al., 1993). The most common anomaly of the central nervous system of the rat is dilation of the cerebral ventricles (hydrocephalus), estimated at 2.6% in Sprague-Dawley rats (Middle Atlantic Reproduction and Teratology Association and Midwest Teratology Association, 1996). Seizures have also been reported in a variety of stocks and strains of rat but have been reported most frequently in various Wistar stocks (Nunn and MacPherson, 1995). Wistar rats are especially used in investigation of audiogenic seizures (Garcia-Cairasco et al., 1998). Congenital and genetically determined ocular defects are very common in some strains of rats. In albino rats, the lack of a pigmented tapetum predisposes for the development of retinal atrophy. Fischer rats (F-344) have an incidence of corneal mineralization that varies from 10 to 100%, depending on subline (Bruner et al., 1992; Yoshitomi and Boorman, 1990). This is characterized by deposition of calcium salts, often visible in routinely stained sections as basophilic granules, along the interface of the corneal epithelium and the stroma. Other ocular abnormalities reported in laboratory rats include retinal degeneration, cataracts, osseous and cartilaginous metaplasia of the sclera, and colobomas. Several abnormalities of the reproductive tract have been reported in laboratory rats, including transverse vaginal septum in female Wistar and Sprague-Dawley (Barbolt and Brown, 1989; De Schaepdrijver et al., 1995). Affected animals are functionally sterile if the septum is complete, and subfertile if the septum only partially prevents spermatozoa from entering the uterus. Pseudohermaphroditism is occasionally observed in rats, most often male pseudohermaphroditism, also known as testicular feminization; i.e., testes are present internally, but the external genitalia are approximately female. Affected rats are karyotypically XY but express the default feminine phenotype. Although mutant strains have been selected for this characteristic (Allison et al., 1965), it is also occasionally observed in other strains as well. In the testicular-feminized rat (~m), the defect is a lack of androgen receptors due to a point mutation (Yarbrough et al., 1990), although defects in other genes could potentially result in similar syndromes. Brown Norway rats have a high incidence of eosinophilic granulomatous pulmonary inflammation, nearing 100% incidence in both males and females at 3 - 4 months of age. Brown Norway rats from colonies worldwide are affected, including those maintained in isolators. Affected colonies are seronegative for all known agents, and rats of other strains maintained with the Brown Norway rats do not develop lung lesions. The lung lesions are scattered throughout the parenchyma and are characterized by generally well-organized granulomas of Langhans' giant cells, macrophages, and eosinophils. No foreign material, fungi, or bacteria are routinely visible or can be demonstrated by polarized light or special stains.

4. BIOLOGY AND DISEASES OF RATS

2.

Age-Related Diseases

Laboratory rats are subject to a wide range of neoplastic and nonneoplastic age-related diseases, as are most aging mammals. Because of the use of rats in 2-year carcinogenicity studies, and as models of gerontology for humans, diseases of the geriatric rat have particular significance to the laboratory animal professional. The type, incidence, and severity of these lesions vary greatly with stock or strain of rat, infectious disease status, experimental manipulation, and husbandry practices, including dietary restriction. Only a few of the most common nonneoplastic conditions will be discussed here, and readers are encouraged to consult the scientific literature, including excellent reviews for additional information concerning the particular stock or strain with which they are concerned (Mohr et al., 1992; Boorman et al., 1990). Neoplastic conditions are afforded a separate section in this chapter. Chronic progressive nephropathy (CPN) is the most important age-related disease of rat kidneys and is among the most common causes of death in rats in lifetime studies. Synonyms abound, including chronic progressive nephrosis and old rat nephropathy. The condition is more common in males than in females and is progressive, as correctly indicated by its appellation. Gross lesions of CPN are first observed in rats more than 6 months of age and are characterized by pitting of the cortical surface. Because of cortical interstitial fibrosis, removal of the renal capsule may tear the cortical parenchyma. As it becomes more severe in rats more than 1 year of age, the cortical surface becomes increasingly irregular and may develop areas of pallor. Microscopically, glomerular changes are characterized by thickened basement membranes, thickening of the capillary tufts, adhesions to the parietal layer of Bowman's membrane, and segmental glomerulosclerosis (Short and Goldstein, 1992). As the disease advances, numeroUs tubules in both the cortex and medulla are often dilated and filled with eosinophilic proteinaceous casts. Secondary hyperparathyroidism may occur subsequent to renal functional compromise in advanced cases, resulting in widespread dystrophic mineralization. The etiopathogenesis of CPN is poorly understood and is probably multifactorial. However, several of the major contributing factors have been described (Barthold, 1996b; Percy and Barthold, 1993b). First, the reported incidence varies with strain. This indicates probably at least some genetic predisposition for the development of CPN. Sprague-Dawley and F-344 rats have high incidences, whereas Wistar and Long-Evans stocks have a lower incidence. Reported incidences, however, are difficult Ito interpret, because of geographic variation in use of different stocks and strains (Wistar rats have been used more predominantly in Europe, and Sprague-Dawley rats in the United States), which could lead to other factors, such as housing and diet, actually causing what otherwise appears to be a strainrelated change. For example, when many of the reports of the incidence of CPN in European rats were published, rats were

157

housed 5 per cage, which is known to result in decreased feed consumption and decreased weight gain, relative to single housing. Second, gender is a determining factor in the development of CPN. Male rats have an earlier onset, higher incidence at any given age, and greater severity of lesions than do females. Third, diet is a critical factor and is also the factor that may be the most amenable to management solutions. It is now clear that moderate dietary restriction will greatly reduce the incidence and severity of CPN at any given age, relative to ad libitum overfeeding. The mechanism is hypothesized to be that overfeeding results in prolonged increases in renal blood flow and glomerular filtration rate (Gumprecht et al., 1993). These increases cause glomerular hypertrophy, leading to macromolecule filtration deficits, mesangial damage, glomerulosclerosis, and protein leakage. Whatever the mechanism, however, 25-30% reduction in caloric intake, relative to ad libitum, results in decreased incidence and severity of CPN in female rats, and decreased severity of CPN in male rats, as well as increased survival in both sexes (Keenan et al., 1995a). Nephrocalcinosis is defined as the deposition of calcium phosphate in renal tissue, although a variety of additional terms are sometimes employed to reflect the localization of the mineral in the cortex, medulla, and so on. In contrast to CPN, which is more common in males, nephrocalcinosis is more common in female rats. In addition to gender, the incidence varies with age and strain and may occur in F-344 rats as young as 7 weeks old. The incidence in F-344 rats may reach 50%, whereas the lower incidence of 0 - 7 % is reported in stocks of Sprague-Dawley and Wistar rats (Montgomery and Seely, 1990). An especially high incidence is observed in BDIX rats. The incidence and severity of nephrocalcinosis may be increased by several dietary manipulations, including high levels of calcium, high phosphorus, low calcium/phosphorus ratios, or low magnesium (Percy and Barthold, 1993b). However, it is not clear if dietary levels of these minerals are a key determining factor in the background incidence of nephrocalcinosis. Histologically (Short and Goldstein, 1992), mineral deposition is observed most frequently at the corticomedullary junction, in cells of the pars recta and thin loops of Henle, as well as in the lumen of these tubules. Chronic myocardial disease is a major cause of death in aged male rats of multiple strains, including Sprague-Dawley, when fed ad libitum (Keenan et al., 1995b). The condition is often known as cardiomyopathy, or chronic progressive cardiomyopathy, and may be observed as early as 3 months of age. Grossly, the heart is enlarged, occasionally with pale streaks visible. Increased weight of the heart correlates well with the degree of damage observed on histologic examination. Microscopically (Lewis, 1992), there is necrosis of myocardial fibers and an interstitial infiltration of mononuclear cells. Later in the course of the disease, fibrosis may be more prominent. Large reactive nuclei are also observed in myofibers. The most commonly affected myocardial sites are the papillary muscles and interventricular septum. As with chronic progressive nephropathy, the

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DENNIS F. KOHN AND CHARLES B. CLIFFORD

incidence of chronic progressive cardiomyopathy can be dramatically reduced at any age by moderate dietary restriction, i.e., reduction of 2 5 - 3 0 % of total caloric intake relative to a d lib i t u m overfed rats (Keenan et al., 1995b). Changes in skin and pelage are often observed but rarely reported in geriatric laboratory rats, which may cause concern to the inexperienced observer. The most c o m m o n change is thinning or loss of hair, especially over the back (Elwell et al., 1990). This may be observed in any stock or strain but is especially c o m m o n in the Brown Norway rat. Old albino rats also have a more yellow appearance at times, because of the accumulation of sebum in the skin. The rings of scales covering the tail increase in number with age to 190 at 1 year (English and Munger, 1992). They continue to become more prominent and more yellowed with time after that. The yellowish material which accumulates on the tail and adjacent to the ear also may become black with time, probably from oxidation and/or bacterial action. In addition, male rats accumulate brown-pigmented foci on the skin, termed scales (Tayama and Shisa, 1994). These scales can be detached and can overlay skin of "normal" color. They are found on the dorsum, with some on the tail and perineum. Scale formation is abrogated by gonadectomy. The nature of the pigment is unclear, but it may be oxidized lipid or amino acids. Alveolar histiocytosis is a very c o m m o n incidental finding in the lung of aging rats of m a n y stocks and strains (Dungworth et al., 1992). Grossly, alveolar histiocytosis is visible as white to pale tan foci, usually about 1 m m in diameter, visible on the pleural surface. The foci may extend slightly above the pleural surface in uninflated lung. Microscopically (Boorman and Eustis, 1990), clusters of alveoli, often in a subpleural locations or adjacent to a terminal bronchiole, contain increased numbers of large, pale, foamy-appearing macrophages. Occasionally, cholesterol clefts may be visible in the more dense aggregates of macrophages, and a slight infiltration of lymphocytes may be present around adjacent vessels, probably as a response to proinflammatory mediators released by the macrophages. Alveolar histiocytosis should not be mistaken for any of the viral pneumonias of rats, because affected animals are seronegative, and any lymphoid infiltrate is slight and localized to the areas of macrophage aggregation. The cause of alveolar histiocytosis is not known, but it does not appear to be infectious.

References

Allison, J. E., Stanley, A. J., and Gumbreck, L. G. (1965). Sex chromatin and idiograms from rats exhibiting anomalies of the reproductive organs. Anat. Rec. 153, 85-91. Altman, N. H., and Goodman, D. G. (1979). "The Laboratory Rat," Vol. 1. "Biology and Diseases" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), pp. 334-376. Academic Press, New York.

Amao, H., Komukai, Y., Akimoto, T., Sugiyama, M., Takahashi, K. W., Sawada, T., and Saito, M. (1995). Natural and subclinical Corynebacterium kutscheri infection in rats. Lab. Anim. Sci. 45, 11-14. Anderson, L. C., Leary, S. L., and Manning, P. J. (1983). Rat-bite fever in animal research laboratory personnel. Lab. Anim. Sci. 33, 292-294. Antonakopoulos, G. N., Turton, J., Whitefield, P., and Newman, J. (1991). Hostparasite interface of the urinary bladder-inhabiting nematode Trichosomoides crassicauda: Changes induced in the urothelium of infected rats. Int. J. ParasitoL 21, 187-193. Ayala, M. E., Monroy, J., Morales, L., Castro, M. E., and Dominguez, R. (1998). Effects of a lesion in the dorsal raphe nuclei performed during the juvenile period of the female rat, on puberty. Brain Res. Bull 47, 211-218. Baker, D. E. J. (1979). "The Laboratory Rat," Vol. 1. "Biology and Diseases" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), pp. 153-168. Academic Press, New York. Barbolt, T. A., and Brown, G. L. (1989). Vaginal septum in the rat. Lab. Anim. 18, 47-48. Barthold, S. W. (1996a). "Urinary System" (T. C. Jones, G. C. Hard, and U. Mohr, eds.), 2nd ed., pp. 463-465. Springer, Berlin. Barthold, S. W. (1996b). "Urinary System" (T. C. Jones, G. C. Hard, and U. Mohr, eds.), 2nd ed., pp. 228-233. Springer, Berlin. Barthold, S. W. (1997). "Digestive System" (T. C. Jones, J. A. Popp, and U. Mohr, eds.), 2nd ed., pp. 416-418. Springer-Verlag, Berlin. Barthold, S. W., and Brownstein, D. G. (1988). The effect of selected viruses on Corynebacterium kutscheri infection in rats. Lab. Anim. Sci. 38, 580-583. Bennett, J. P., and Vickery, B. H. (1970). "Reproduction and Breeding Techniques for Laboratory Animals" (E. S. E. Hafez, ed.), pp. 299-315. Lea and Febiger, Philadelphia. Besch-Willford, C. L., Wagner, J. E., and Templeman, C. C. (1987). Establishment of a commercial production colony of Sprague-Dawley rats free of Sendai virus. Lab. Anim. Sci. 37, 666-667. Besselsen, D. G., Besch-Williford, C. L., Pintel, D. J., Franklin, C. L., Hook, R. R., Jr., and Riley, L. K. (1995a). Detection of H-1 parvovirus and Kilham rat virus by PCR. J. Clin. Microbiol. 33, 1699-1703. Besselsen, D. G., Besch-Williford, C. L., Pintel, D. J., Franklin, C. L., Hook, R. R., and Riley, L. K. (1995b). Detection of newly recognized rodent parvoviruses by PCR. J. Clin. Microbiol. 33, 2859-2863. Beys, E., Hodge, T., and Nohynek, G. J. (1995). Ovarian changes in SpragueDawley rats produced by nocturnal exposure to low intensity light. Lab. Anim. 29, 335-338. Bihun, C. G., and Percy, D. H. (1994). Coronavirus infections in the laboratory rat: Degree of cross protection following immunization with a heterologous strain. Can. J. Vet. Res. 58, 224-229. Bivin, W. S., Crawford, M. P., and Brewer, N. R. (1979). Morphophysiology. In "The Laboratory Rat," Vol. 1, "Biology and Diseases" (J. R. Lindsey, H. J. Baker, and S. H. Weisbroth, eds.), pp. 74-103. Academic Press, New York. Blandau, R. J., Boling, J. L., and Young, W. C. (1941). The length of heat in the albino rat as determined by the copulatory response test. Anat. Rec. 79, 453-463. Blom, H. J. M., VanTintelen, G., Van Vorstenbosch, C. J. A. H. V., Baumans, V., and Beynen, A. C. (1995). Preferences of mice and rats for types of bedding material. Lab. Anim. 30, 234-244. Boivin, G. P., Wagner, J. E., and Besch-Williford, C. L. (1990). Use of cyclophosphamide in diagnostic provocation of Tyzzer's disease in hamsters. Lab. Anim. Sci. 40, 545-545. (Abstract) Boorman, G. A., and Eustis, S. L. (1990). "Pathology of the Fischer Rat: Reference and Atlas" (G. A. Boorman, S. L. Eustis, M. R. Elwell, C. A. Montgomery, and W. E MacKenzie, eds.), pp. 339-367. Academic Press, San Diego. Boorman, G. A., Eustis, S. L., Elwell, M. R., Montgomery, C. A., and MacKenzie, W. F., eds. (1990). "Pathology of the Fischer Rat: Reference and Atlas." Academic Press, San Diego.

4. BIOLOGY AND DISEASES OF RATS

Boot, R., van Knapen, E, Kruijt, B. C., and Walvoort, H. C. (1988). Serological evidence for Encephalitozoon cuniculi infection (nosemiasis) in gnotobiotic guineapigs. Lab. Anim. 22, 337-342. Brammer, D. W., Dysko, R. C., Spilman, S. C., and Oskar, P. A. (1993). Elimination of sialodacryoadenitis virus from a rat production colony by using seropositive breeding animals. Lab. Anim. Sci. 43, 633-634. Brown, H. R., and Leininger, J. R. (1992). "Pathobiology of the Aging Rat" (U. Mohr, D. L. Dungworth, and C. C. Capen, eds.), Vol. 2, pp. 309-322. ILSI Press, Washington, D. C. Brudzynski, S. M., and Chiu, E. M. (1995). Behavioural responses of laboratory rats to playback of 22 kHz ultrasonic calls. Physiol. Behav. 57, 1039-1044. Brudzynski, S. M., and Ociepa, D. (1992). Ultrasonic vocalization of laboratory rats in response to handling and touch. Physiol. Behav. 52, 655-660. Brudzynski, S. M., Bihari, E, Ociepa, D., and Fu, X.-W. (1993). Analysis of 22 kHz ultrasonic vocalization in laboratory rats: Long and short calls. Physiol. Behav. 54, 221. Bruner, R. H., Keller, W. E, Stitzel, K. A., Sauers, L. J., Reer, P. J., Long, P. H., Bruce, R. D., and Alden, C. L. (1992). Spontaneous corneal dystrophy and generalized basement membrane changes in Fischer-344 rats. Toxicol. Pathol. 20, 357-366. Burek, J. D. (1978). "Pathology of Aging Rats." CRC Press, Boca Raton, Florida. Burek, J. D., Jurcher, C., Van Nunen, M. C. J., and Hollander, C. E (1977). A naturally occurring epizootic caused by Sendai virus in breeding and aging rodent clonies. 2. Infection in the rat. Lab. Anim. Sci. 27, 963-971. Carter, G. R. (1984). "Bergey's Manual of Systematic Bacteriology" (N. R. Krieg, and J. G. Holt, eds.), Vol. 1, pp. 552-557. Williams and Wilkins, Baltimore. Carthew, P., and Aldred, P. (1988). Embryonic death in pregnant rats owing to intercurrent infection with Sendai virus and Pasteurella pneumotropica. Lab. Anim. 22, 92-97. Carthew, P., and Slinger, R. P. (1981). Diagnosis of sialodacryoadenitis virus infection of rats in a virulent enzootic outbreak. Lab. Anim. 15, 339-342. Cassell, G. H., Lindsey, J. R., Davis, J. K., Davidson, M. K., Brown, M. B., and Mayo, J. G. ( 1981). Detection of natural Mycoplasma pulmonis infection in rats and mice by an enzyme linked immunosorbent assay (ELISA). Lab. Anim. Sci. 31, 676-682. Cassell, G. H., Davis, J. K., Simecka, J. W., Lindsey, J. R., Cox, N. R., Ross, S., and Fallon, M. T. (1986). "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 87-130. Academic Press, Orlando, Florida. Childs, J. E., Glass, G. E., Korch, G. W., Arthur, R. R., Shah, K. V., Glasser, D., Rossi, C., and LeDuc, J. W. (1987). Evidence of human infection with a rat-associated hantavirus in Baltimore, Maryland. Am. J. Epidemiol. 127, 875-878. Chmiel, D. J., and Noonan, M. (1996). Preference of laboratory rats for potentially enriching stimulus objects. Lab. Anim. 30, 97-101. Chung, Y. H., Jun, H. S., et al. (1997). Role of macrophages and macrophagederived cytokines in the pathogenesis of Kilham rat virus-induced autoimmune diabetes in diaabetes-resistant BioBreeding rats. J. Immunol. 159, 466-471. Cohen, S. M., Anderson, T. A., de Oliveira, L. M., and Arnold, L. L. (1998). Tumorigenicity of sodium ascorbate in male rats. Cancer Res. 58, 25572561. Coleman, G. L., Barthold, S. W., Osbaldiston, G. W., Foster, S. J., and Jonas, A. M. (1977). Pathological changes during aging in barrier-reared Fischer 344 male rates. J. Gerontol. 32, 258-278. Coleman, G. L., Jacoby, R. O., et al. (1983). Naturally occurring lethal parvovirus infection of juvenile and young-adult rats. Vet. Pathol. 20, 49-56. Compton, S. R., Smith, A. L., and Gaertner, D. J. (1999). Comparison of the pathogenicity in rats of rat coronaviruses of different neutralization groups. Lab. Anim. Sci. 49, 514-519.

159 Cornish, J., Vanderwee, M. A., Findon, G., and Miller, T. E. (1988). Reliable diagnosis of Trichosomoides crassicauda in the urinary bladder of the rat. Lab. Anim. 22, 162-165. Craft, J. C. (1982). Experimental infection with Giardia lamblia in rats. J. Infect. Dis. 145, 495-498. Cundiff, D. D., Besch-Williford, C. L., Hook, R. R., Franklin, C. L., and Riley, L. K. (1994). Detection of cilia-associated respiratory bacillus by PCR. J. Clin. Microbio132, 1930-1934. Cundiff, D. D., Besch-Williford, C. L., Hook, R. R., Franklin, C. L., and Riley, L. K. (1995a). Characterization of cilia-associated respiratory bacillus in rabbits and analysis of the 16S rRNA gene sequence. Lab. Anim. Sci. 45, 22-26. Cundiff, D. D., Riley, L. K., Franklin, C. L., Hook, R. R., and Besch-Williford, C. L. (1995b). Failure of a soiled bedding sentinel system to detect ciliaassociated respiratory bacillus infection in rats. Lab. Anim. Sci. 45, 219-221. Dameron, G. W., Weingand, K. W., Duderstdt, J. M., Odioso, L. W., Dierckman, T. A., Schwecke, W., and Baran, K. (1992). Effect of bleeding site on clinical laboratory testing of rats: Orbital venous plexus versus posterior vena cava. Lab. Anim. Sci. 42, 299-301. Darrigrand, A. A., Singh, S. B., et al. (1984). Effects of Kilham rat virus on natural killer cell-mediated cytotoxicity in Brown Norway and Wistar-Furth rats. Am. J. Vet. Res. 45, 200-202. Dauchy, R. T., Sauer, L. A., Blask, D. E., and Vaughan, G. M. (1997). Light contamination during the dark phase in "photoperiodically controlled" animal rooms: Effect on tumor growth and metabolism in rats. Lab. Anim. Sci. 47, 511-518. Davidson, M. K., Davis, J. K., Lindsey, J. R., and Cassell, G. H. (1988). Clearance of different strains of Mycoplasma pulmonis from the respiratory tract of C3H/HeN mice. Infect. Immun. 56, 2163-2168. Davis, J. K., and Cassell, G. H. (1982). Murine respiratory mycoplasmosis in LEW and F-344 rats: Strain differences in lesion severity. Vet. Pathol. 19, 280-293. DeLong, D., and Manning, P. J. (1994). "The Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), 2nd ed., pp. 129-170. Academic Press, San Diego. Del Vecchio, F. R. (1992). "Pathobiology of the Aging Rat" (U. Mohr, D. L. Dungworth, and C. C. Capen, eds.), Vol. 1, pp. 331-336. ILSI Press, Washington, D.C. De Schaepdrijver, L. M., Fransen, J. L., Van der Eycken, E. S., and Coussement, W. C. (1995). Transverse vaginal septum in the specific pathogen-free Wistar rat. Lab. Anim. Sci. 45, 181-183. Desmyter, J., Johnson, K. M., Deckers, C., LeDuc, J. W., Brasseur, F., and van Ypersele de Strihon, C. (1983). Laboratory rat associated outbreak of haemorrhagic fever with renal syndrome due to Hantan-like virus in Belgium. Lancet 2, 1445-1448. Dewsbury, D. A. (1970). "Reproduction and Breeding Techniques for Laboratory Animals" (E. S. E. Hafez, ed.), pp. 123-136. Lea and Febiger, Philadelphia. Duncan, A. J., Carman, R. J., Olson, G. J., and Wilson, K. H. (1993). Assignment of the agent of Tyzzer's disease to Clostridium piliforme comb. nov. on the basis of 16S rRNA sequence analysis. Int. J. Syst. Bacteriol. 43, 314-318. Dungworth, D. L., Ernst, H., Nolte, T., and Mohr, U. (1992). "Pathobiology of the Aging Rat" (U. Mohr, D. L. Dungworth, and C. C. Capen, eds.), Vol. 1, pp. 143-160. ILSI Press, Washington, D.C. Eaton, K. A., Dewhirst, F. E., Paster, B. J., Tzellas, N., Coleman, B. E., Paola, J., and Sherding, R. (1996). Prevalence and varieties of Helicobacter species in dogs from random sources and pet dogs: Animal and public health implications. J. Clin. Microbiol. 34, 3165-3170. Eckstein, B., Golan, R., and Shani Mishkinsky, J. (1973). Onset of puberty in the immature female rat induced by 5a-androstane-313,1713-diol. Endocrinology 92, 941-945.

160 Eiden, J., Vonderfecht, S., et al. (1985). Evidence that a novel rotavirus-like agent of rats can cause gastroenteritis in man. Lancet 2, 8-11. Elwell, M. R., Stedham, M. A., and Kovatch, R. M. (1990). "Pathology of the Fischer Rat: Reference and Atlas" (G. A. Boorman, S. L. Eustis, M. R. Elwell, C. A. Montgomery, and W. F. MacKenzie, eds.), pp. 261-277. Academic Press, San Diego. Enders, A. C. (1970). "Reproduction and Breeding Techniques for Laboratory Animals" (E. S. E. Hafez, ed.) pp. 137-156. Lea and Febiger, Philadelphia. English, K. B., and Munger, B. L. (1992). "Pathobiology of the Aging Rat" (U. Mohr, D. L. Dungworth, and C. C. Capen, eds.), Vol. 2, pp. 363-389. ILSI Press, Washington, D.C. Fallon, M. T., Reinhard, M. K., Gray, B. M., Davis, T. W., and Lindsey, J. R. (1988). Inapparent Streptococcus pneumoniae type 35 infections in commercial rats and mice. Lab. Anim. Sci. 38, 129-132. Feely, D. E., Chase, D. G., Hardin, E. L., and Erlandsen, S. L. (1988). Ultrastructural evidence for the presence of bacteria, viral-like particles, and mycoplasma-like organisms associated with Giardia spp. J. Protozool. 35, 151-158. Feldman, M. L. (1992). "Pathobiology of the Aging Rat" (U. Mohr, D. L. Dungworth, and C. C. Capen, eds.), Vol. 2, pp. 121-147. ILSI Press, Washington, D.C. Feldman, S. H., Weisbroth, S. P., and Weisbroth, S. H. (1996). Detection of Pneumocystis carinii in rats by polymerase chain reaction: Comparison of lung tissue and bronchoalveolar lavage specimens. Lab. Anim. Sci. 46, 628-633. Fleischman, R. W., McCracken, D., and Forbes, W. (1977). Adynamic ileus in the rat induced by chloral hydrate. Lab. Anim. Sci. 27, 238-243. Flynn, R. J. (1973a). "Parasites of Laboratory Animals," 1st ed., pp. 203-320. Iowa State University Press, Ames. Flynn, R. J. (1973b). "Parasites of Laboratory Animals," 1st ed., pp. 435-492. Iowa State University Press, Ames. Flynn, R. J. (1973c). "Parasites of Laboratory Animals," 1st ed., pp. 3-35. Iowa State University Press, Ames. Flynn, R. J. (1973f). "Parasites of Laboratory Animals," 1st ed., pp. 376-397. Iowa State University Press, Ames. Fox, J. G., and Lee, A. (1997). The role of Helicobacter species in newly recognized gastrointestinal tract diseases of animals (special topic overview). Lab. Anim. Sci. 47, 222-255. Fox, J. G., Niemi, S. M., Murphy, J. C. and Quimby, F. W. (1977). Ulcerative dermatitis in the rat. Lab. Anim. Sci. 27, 671-678. Fox, J. G., Niemi, S. M., Ackerman, J., and Murphy, J. C. (1987). Comparison of methods to diagnose an epizootic of Corynebacterium kutscheri pneumonia in rats. Lab. Anim. Sci. 37, 72-75. Fox, J. G., Yan, L. L., Dewhirst, F. E., Paster, B. J., Shames, B., Murphy, J. C., Hayward, A., Belcher, J. C., and Mendes, E. N. (1995). Helicobacter bilis sp. nov., a novel Helicobacter species isolated from the bile, liver, and intestines of aged, inbred mice. J. Clin. Microbiol. 33, 445-454. Fox, J. G., Yan, L., Shames, B., Campbell, J., Murphy, J. C., and Li, X. (1996b). Persistent hepatitis and enterocolitis in germfree mice infected with Helicobacter hepaticus. Contemp. Top. 23, 62-62. (Abstract). Fox, R. R., and Laird, C. W. (1970). "Reproduction and Breeding Techniques for Laboratory Animals" (E. S. E. Hafez, ed.), pp. 107-122. Lea and Febiger, Philadelphia. Franklin, C. L., Kinden, D. A., Stogsdill, P. L., and Riley, L. K. (1993). In vitro model of adhesion and invasion by Bacillus piliformis. Infect. Immun. 61, 876-883. Franklin, C. L., Motzel, S. L., Besch-Williford, C. L., Hook, R. R., and Riley, L. K. (1994). Tyzzer's infection: Host specificity of Clostridium piliforme isolates. Lab. Anim. Sci. 44, 568-572. Franklin, C. L., Pletz, J. D., Riley, L. K., Livingston, B. A., Hook, Jr., R. R., and Besch-Williford, C. L. (1999). Detection of cilia-associated respiratory (CAR) bacillus in nasal-swab specimens from infected rats by use of polymerase chain reaction. Lab. Anim. Sci. 49, 114-117. Fujinaga, M., Baden, J. M., and Mazze, R. I. (1992). Reproductive and

DENNIS F. KOHN AND CHARLES B. CLIFFORD

teratogenic effects of a major earthquake in rats. Lab. Anim. Sci. 42, 209 - 210. Gaddum-Rosse, P., Blandau, R. J., Langley, L. B., and Battaglia, D. E. (1984). In vitro fertilization in the rat: Observations on living eggs. Fertil. Steril. 42, 285-292. Gaertner, D. J., Jacoby, R. O., et al. (1989). Persistence of rat parvovirus in athymic rats. Arch. Virol. 105, 259-268. Gaertner, D. J., Jacoby, R. O., Johnson, E. A., Paturzo, F. X., and Smith, A. L. (1995). Persistent rat virus infection in juvenile athymic rats and its modulation by immune serum. Lab. Anim. Sci. 45, 249-253. Gaertner, D. J., Smith, A. L., and Jacoby, R. O. (1996). Efficient induction of persistent and prenatal parvovirus infection in rats. Virus Res. 44, 67-78. Ganaway, J. R. (1980). Effect of heat and selected chemical disinfectants upon infectivity of spores of Bacillus piliformis (Tyzzer's disease). Lab. Anim. Sci. 30, 192-196. Gannon, J. (1980). A survey of Encephalitozoon cuniculi in laboratory animal colonies in the United Kingdom. Lab. Anim. 14, 91-94. Garcia-Cairasco, N., Oliveira, J. A., Wakamatsu, H., Bueno, S. T., and Guimaraes, F. S. (1998). Reduced exploratory activity of audiogenic seizures in susceptible Wistar rats. Physiol. Behav. 64, 671-674. Garlinghouse, L. E., and Van Hoosier, G. L. (1978). Studies on adjuvant-induced arthritis, tumor transplantability, and serologic response to bovine serum albumin in Sendai virus-infected rats. Am. J. Vet. Res. 39, 297-300. Garside, D. A., Charlton, A., and Heath, K. J. (1996). Establishing the timing of implantation in the Harlan Porcellus Dutch and New Zealand White rabbit and the Han Wistar rat. ReguL Toxicol. Pharmacol. 23, 69-73. Giusti, A. M., Crippa, L., Bellini, O., Luini, M., and Scanziani, E. (1998). Gastric spiral bacteria in wild rats from Italy. J. Wildl. Dis. 34, 168-172. Goelz, M. F., Thigpen, J. E., Mahler, J., Rogers, W. P., Locklear, J., Wiegler, B. J., and Forsythe, D. B. (1997). Efficacy of various therapeutic regimens in eliminating Pasteurella pneumotropica from the mouse. Lab. Anim. Sci. 46, 280-285. Goldman, P. (1980). Drug therapy: Metronidazole. N. Engl. J. Med. 303, 1212-1218. Gonzalez, D. E., and Deis, R. P. (1990). The capacity to develop maternal behavior is enhanced during aging in rats. Neurobiol. Aging 11, 237-241. Goodman, D. G., Ward, J. M., Squires, R. A., Chu, K. C., and Linhart, M. S. (1979). Neoplastic and non-neoplastic lesions in aging F-344 rats. Toxicol. Appl. Pharmacol. 48, 237-248. Goodman, D. G., et al. (1980). Neoplastic and nonneoplastic lesions in aging Osborne-Mendel rats. Toxicol. Appl. Pharmacol. 55, 433-447. Greene, E. C. (1949). "The Rat in Laboratory Investigation" (E. J. Ferris, and J. Q. Griffith, eds.), pp. 24-50. J. B. Lippincott, Hafner Press, New York. Greene, E. C. (1963). "Anatomy of the Rat." Hafner Publ. Co., New York. Griffith, J. W., White, W. J., Danneman, P. J., and Lang, C. M. (1988). Ciliaassociated respiratory (CAR) bacillus infection of obese mice. Vet. Pathol. 25, 72-76. Gruber, H. E., and Osborne, J. W. (1979). Ultrastructural features of spironucleosis (hexamitiasis) in X-irradiated rat small intestine. Lab. Anim. 13, 199-202. Gumprecht, L. A., Long, C. R., Soper, K. A., Smith, P. F., Haschek-Hock, W. M., and Keenan, K. P. (1993). The early effects of dietary restriction on the pathogenesis of chronic renal disease in Sprague-Dawley rats at 12 months. Toxicol. Pathol. 21, 528-537. GV-SOLAS Working Group on Hygiene. (1999). Pneumocystis carinii: Implications for animal experiments. Lab. Anim. 33(Suppl. 1), 77-78. Haines, D. C., Gorelick, P. L., Battles, J. K., Pike, K. M., Anderson, R. J., Fox, J. G., Taylor, N. S., Shen, Z., Dewhirst, F. E., Anver, M. R., and Ward, J. M. (1998). Inflammatory and large bowel disease in immunodeficient rats naturally and experimentally infected with Helicobacter bilis. Vet. Pathol. 35, 202-208. Hansen, A. K., Dagnaes-Hansen, F., and Mollegaard-Hansen, K.-E. (1992b). Correlation between megaloileitis and antibodies to Bacillus piliformis in laboratory rat colonies. Lab. Anim. Sci. 42, 449-453.

4. BIOLOGY AND DISEASES OF RATS Hansen, A. K., Skovgaard-Jensen, H.-J., Thomsen, P., Svendsen, O., DagnaesHansen, E, and Mollegaard-Hansen, K.-E. (1992a). Rederivation of rat colonies seropositive for Bacillus piliformis and the subsequent screening for antibodies. Lab. Anim. Sci. 42, 444-447. Hasslinger, M. A., and Wiethe, T. (1987). [Oxyurid infestation of small laboratory animals and its control with ivermectin]. Tierarztl. Prax. 15, 93-97. Hirsjarvi, P., and Valiaho, T. (1995). Effects of gentling on open-field behaviour of Wistar rats in fear-evoking test situation. Lab. Anim. 29, 380-384. Homburger, E (1977). Saccharin and cancer. N. Engl. J. Med. 297, 560-561. Hong, S. J., Woo, H. C., and Chai, J. Y. (1996). A human case of Plagiorchis muris (Tanabe, 1922: Digenea) infection in the Republic of Korea: Freshwater fish as a possible source of infection. J. Parasitol. 82, 647-649. Hook, R. R., Franklin, C. L., Riley, L. K., Livingston, B. A., and BeschWilliford, C. L. (1998). Antigenic analyses of cilia-associated respiratory (CAR) bacillus isolates by use of monoclonal antibodies. Lab. Anim. Sci. 48, 234-239. Hsu, C.-K. (1979). "The Laboratory Rat," Vol. 1. "Biology and Diseases" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), pp. 307-331. Academic Press, New York. Hubbs, A. F., Hahn, E E, and Lundgren, D. L. (1991). Invasive tracheobronchial aspirgillosis in an F-344/N rat. Lab Anim. Sci. 41, 521-523. Huerkamp, M. J. (1993). Ivermectin eradication of pinworms from rats kept in ventilated cages. Lab. Anim. Sci. 43, 86-90. Hughes, W. T. (1982). Natural mode of acquisition for de novo infection with Pneumocystis carinii. J. Infect. Dis. 145, 842-847. Hutchinson, K. L., Rollin, P. E., et al. (1998). Pathogenesis of a North American hantavirus, Black Creek Canal virus, in experimentally infected Sigmondon hispidus. Am. J. Trop. Med. Hyg. 59, 58-65. Inge, P. M., Edson, C. M., and Farthing, M. J. (1988). Attachment of Giardia lamblia to rat intestinal epithelial cells. Gut 29, 795-801. Itoh, T., Kohyama, K., Takakura, A., Takenouchi, T., and Kagiyama, N. (1987). Naturally occurring CAR bacillus infection in a laboratory rat colony and epizootiological observations. Exp. Anim. 36, 387-393. Jacoby, R. O. (1986). Rat coronavirus. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C., Morse, III, and A. E. New, eds.), pp. 625-638. Academic Press, Orlando, Florida. Jacoby, R. O., Bhatt, P. N., and Jonas, A. M. (1979). Viral diseases. In "The Laboratory Rat," Vol. 1, "Biology and Diseases" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), pp. 272-306, Academic Press, New York. Jacoby, R. O., Gaertner, D. J., Bhatt, P. N., Paturzo, E X. and Smith, A. L. (1988). Transmission of experimentally induced rat virus infection. Lab. Anim. Sci. 38, 11-14. Jacoby, R. O., Johnson, E. A., et al. (1991). Persistent rat parvovirus infection in individually housed rats. Arch. Virol. 117, 193-205. Jacoby, R. O., Ball-Goodrich, L. J., Besselsen, D. G., McKisic, M. D., Riley, E K., andSmith, A. L. (1996). Rodent parvovirus infections (special topic overview). Lab. Anita. Sci. 46, 370-380. Johnson, P. D., Dawson, B. V., and Goldberg, S. J. (1993). Spontaneous congenital heart malformations in Sprague-Dawley rats. Lab. Anim. Sci. 43, 183-188. Jueco, N. L. (1982). "Handbook Series in Zoonoses" (L. Jacobs, and P. Arambulo, eds.), pp. 283-287. CRC Press, Boca Raton, Florida. Keenan, K. P., Laroque, P., Ballam, G. C., Soper, K. A., Dixit, R., Mattson, B. A., Adams, S. P., and Coleman, J. B. (1996). The effects of diet, ad libitum overfeeding, and moderate dietary restriction on the rodent bioassay: The uncontrolled variable in safety assessment. Toxicol. Pathol. 24, 757-768. Keenan, K. P., Soper, K. A., Hertzog, P. R., Gumprecht, L. A., Smith, P. F., Mattson, B. A., Ballam, G. C., and Clark, R. L. (1995a). Diet, overfeeding, and moderate dietary restriction in control Sprague-Dawley rats: 2. Effects on age-related proliferative and degenerative lesions. Toxicol. Pathol. 23, 287-302. Keenan, K. P., Soper, K. A., Smith, P. E, Ballam, G. C., and Clark, R. L. (1995b).

161 Diet, overfeeding, and moderate dietary restriction in control SpragueDawley rats: 1. Effects on spontaneous neoplasms. Toxicol. Pathol. 23, 269-286. Keenan, K. P., Laroque, P., and Dixit, R. (1998). Need for dietary control by caloric restriction in rodent toxicology and carcinogenicity studies. J. Toxicol. Environ. Health, Part B 1, 135-148. Kelly, J. B., and Masterton, B. (1977). Auditory sensitivity of the albino rat. J. Comp. Physiol. Psychol. 91, 930-936. Kiska, D. L., and Gilligan, P. H. (1999). "Manual of Clinical Microbiology" (P. R. Murray, E. J. Baron, M. A. Pfaller, E C. Tenover, and R. H. Yolken, eds.), 7th ed. pp. 517-525. ASM Press, Washington, D.C. Klement, P., Augustine, J. M., Delaney, K. H., Lement, G., and Itz, J. I. (1996). An oral ivermectin regimen that eradicates pinworms (Syphacia spp.) in laboratory rats and mice. Lab. Anim. Sci. 46, 286-290. Kohn, D. E, and Kirk, B. E. (1969). Pathogenicity of Mycoplasma pulmonis in laboratory rats. Lab. Anim. Care 19, 321-330. Kohn, D. E (1971). Bronchiectasis in rats infected with Mycoplasma pulmonis: An electron microscopic study. Lab. Anim. Sci. 21, 856-861. Kohn, D. E, and Barthold, S. W. (1984). "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 91-122. Academic Press, San Diego. Kojima, A., and Okaniwa, A. (1991). Antigenic heterogeneity of sialodacryoadenitis virus isolates. J. Vet. Med. Res. 53, 1059-1063. Koolhass, J. M. (1999). The laboratory rat. In "The UFAW Handbook on the Care and Management of Laboratory Animals," pp. 313-330. Blackwell Science Ltd., Malden, Massachusetts. Koto, M., Miwa, M., Togashi, M., Tsuji, K., Okamoto, M., and Adachi, J. (1987a). [A method for detecting the optimum for mating during the 4-day estrous cycle in the rat: measuring the value of electrical impedance of the vagina]. Jikken Dobutsu 36, 195-198. Koto, M., Miwa, M., Tsuji, K., Okamoto, M., and Adachi, J. (1987b). [Change in the electrical impedance caused by cornification of the epithelial cell layer of the vaginal mucosa in the rat]. Jikken Dobutsu 36, 151-156. Kubisch, H. M., and Gomez-Sanchez, E. P. (1999). Embryo transfer in the rat as a tool to determine genetic components of the gestational environment. Lab. Anim. Sci. 49, 90-94. Kunstyr, I. (1977). Infectious form of Spironucleus (Hexamita) muris: Banded cysts. Lab. Anim. 11, 185-188. Kunstyr, I., and Ammerpohl, E. (1978). Resistance of faecal cysts of Spironucleus muris to some physical factors and chemical substances. Lab. Anim. 12, 95-97. Kunstyr, I., Schoeneberg, U., and Friedhoff, K. T. (1992). Host specificity of Giardia muris isolates from mouse and golden hamster. Parasitol. Res. 78, 621-622. Kurisu, K., Sawamoto, O., Watanabe, H., and Ito, A. (1996). Sequential changes in the Harderian gland of rats exposed to high intensity light. Lab. Anim. Sci. 46, 71-76. Laliberte, E, Mucchielli, A., and Laliberte, M. E (1984). Dynamics of antibody transfer from mother to fetus through the yolk-sac cells in the rat. Biol. Cell 50, 255-261. Lang, P. L (1990). "Spontaneous Neoplastic Lesions in the CDF(F-344)/CrlBR Rat." Charles River Laboratories, Wilmington, Massachusetts. Lang, P. L. (1992). "Spontaneous Neoplastic Lesions and Selected Nonneoplastic Lesions in the CrhCDBR Rat." Charles River Laboratories, Wilmington, Massachusetts. La Regina, M., Woods, L., Klender, P., Gaertner, D. J., and Paturzo, E X. (1992). Transmission of sialodacryoadenitis virus from infected rats to rats and mice through handling, close contact, and soiled bedding. Lab. Anim. Sci. 42, 344-346. Leader, R. W., Leader, I., and Witschi, E. (1970). Genital mycoplasmosis in rats treated with testosterone propionate to produce constant estrus. J. Am. Vet. Med. Assoc. 157, 1923-1925. LeChevallier, M. W., Cawthon, C. D., and Lee, R. G. (1988). Inactivation of biofilm bacteria. Appl. Environ. Microbiol. 54, 2492-2499.

162 Lee, A., Phillips, M. W., O'Rourke, J. L., Paster, B. J., Dewhirst, E E., Fraser, G. J., Fox, J. G., Sly, L. I., Romaniuk, E J., Trust, T. J., and Kouprach, S. (1992). Helicobacter muridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents. Int. J. Syst. Bacteriol. 42, 27-36. Le Minor, L. (1984). Genus III. Salmonella. In I. N. R. Krieg and J. G. Holt (eds.), Bergey's Manual of Systematic Bacteriology p. 427-448. Baltimore: Williams and Wilkins. Levine, N. D. (1961). "Protozoan Parasites of Domestic Animals and of Man," pp. 107-128. Burgess Publ. Co., Minneapolis. Levine, N. D. (1985b). "Veterinary Protozoology," 1st ed., pp. 109-129. Iowa State Univ. Press, Ames. Levine, N. D. (1985a). "Veterinary Protozoology," 1st ed., pp. 89-108. Iowa State Univ. Press, Ames. Lewis, D. J. (1992). "Pathobiology of the Aging Rat" (U. Mohr, D. L. Dungworth, and C. C. Capen, eds.), pp. 301-309. ILSI Press, Washington, D.C. Liang, S. C., Schoeb, T. R., Davis, J. K., Simecka, J. W., Cassell, G. H., and Lindsey, J. R. (1995). Comparative severity of respiratory lesions of sialodacryoadenitis virus and Sendai virus infections in LEW and F-344 rats. Vet. Pathol. 32, 661-667. Liang, S. C., Simecka, J. W., Lindsey, J. R., Cassell, G. H., and Davis, J. K. (1999). Antibody responses after Sendai virus infection and their role in upper and lower respiratory tract disease in rats. Lab. Anim. Sci. 49, 385-394. Lindsey, J. R. (1979). "The Laboratory Rat," Vol. 1, "Biology and Diseases" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), pp. 1-36. Academic Press, New York. Lindsey, J. R., Baker, H. J., Overcash, R. G., Cassell, G. H., and Hunt, C. E. (1971). Murine chronic respiratory disease: Significance as a research complication and experimental production with Mycoplasma pulmonis. Am. J. Pathol. 64, 675-716. Lindsey, J. R., Cassell, G. H., and Davidson, M. K. (1982). "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), pp. 2141. Academic Press, New York. Lindsey, J. R., Davidson, M. K., Schoeb, T. R., and Cassell, G. H. (1985). Mycoplasma pulmonis-host relationships in a breeding colony of SpragueDawley rats with enzootic murine respiratory mycoplasmosis. Lab. Anim. Sci. 35, 597-608. Liu, J. J., Greeley, E. H., and Shadduck, J. A. (1988). Murine encephalitozoonosis: The effect of age and mode of transmission on occurrence of infection. Lab. Anim. Sci. 38, 675-679. Liu, J. J., Greeley, E. H., and Shadduck, J. A. (1989). Mechanisms of resistance/susceptibility to murine encephalitozoonosis. Parasite Immunol. 11, 241-256. Livingston, R. S., Riley, L. K., Besch-Williford, C. L., Hook, R. R., and Franklin, C. L. (1998). Transmission of Helicobacter hepaticus infection to sentinel mice by contaminated bedding. Lab. Anim. Sci. 48, 291-293. Lloyd, G., Bowen, E. T. W., Jones, N., and Pendry, A. (1984). HFRS outbreak associated with laboratory rats in U.K. Lancet 1, 1175-1176. Loeb, W., and Quimby, E, eds. (1999.) "The Clinical Chemistry of Laboratory animals," 2nd ed. Taylor and Francis, Philadelphia. Loftness, T. J., Erlandsen, S. L., Wilson, I. D., and Meyer, E. A. (1984). Occurrence of specific secretory immunoglobulin A in bile after inoculation of Giardia lamblia trophozoites into rat duodenum. Gastroenterology 87, 1022-1029. Lubcke, R., Hutcheson, E A. R., and Barbezat, G. O. (1992). Impaired intestinal electroyte transport in rats infested with the common parasite Syphacia muris. Digest. Dis. Sci. 37, 60-64. Lukas, V., Ruehl, W. W., and Hamm, T. E. (1987). An enzyme-linked, immunosorbent assay to intact serum IgG in rabbits naturally exposed to ciliaassociated respiratory bacillus. Lab. Anim. Sci. 37, 553. (Abstract). Lussier, G. (1991). Detection methods for the identification of rodent viral and mycoplasmal infections. Lab. Anim. Sci. 41, 199-225. MacKenzie, W. E, and Garner, E M. (1973). Comparison of neoplasms from six sources of rats. J. Natl. Cancer Inst. 50, 1243-1257.

DENNIS F. KOHN AND CHARLES B. CLIFFORD MacKenzie, W. E, Magill, L. S., and Hulse, M. (1981). A filamentous bacterium associated with respiratory disease in wild rats. Vet. Pathol. 18, 836839. Majeed, S. K., and Zubaidy, A. J. (1982). Histopathological lesions associated with Encephalitozoon cuniculi (nosematosis) infection in a colony of Wistar rats. Lab. Anim. 16, 244-247. Makino, S., Seko, S., Nakao, H., and Mikazuki, K. (1972). An epizootic of Sendai virus infection in a rat colony. Exp. Anim. 22, 275-280. Manser, C. E., Elliot, H., Morris, T. H., and Broom, D. M. (1995a). The use of a novel operant system to determine the strength of preference for flooring in laboratory rats: Lab. Anim. 30, 1-6. Manser, C. E., Morris, T. H., and Broom, D. M. (1995b). An investigation into the effects of solid or grid cage flooring on the welfare of laboratory rats. Lab. Anita. 29, 353-363. Manser, C. E., Broom, D. M., Overend, E, and Morris, T. H. (1998a). Operant studies to determine the strength of preference in laboratory rats for nestboxes and nesting materials. Lab. Anim. 32, 36-41. Manser, C. E., Broom, D. M., Overend, E, and Morris, T. H. (1998b). Investigations into the preferences of laboratory rats for nest-boxes and nesting materials. Lab. Anim. 32, 23-35. Maronpot, R. R. (1996). "Urinary System" (T. C. Jones, G. C. Hard, and U. Mohr, eds.), 2nd ed. pp. 306-309. Springer, Berlin. Martin, M. G., Wu, S. V., and Walsh, J. H. (1997). Ontogenetic development and distribution of antibody transport and Fc receptor mRNA expression in rat intestine. Digest. Dis. Sci. 42, 1062-1069. Martinez-Gomez, M., Cruz, Y., Salas, M., Hudson, R., and Pacheco, E (1994). Assessing pain threshold in the rat: Changes with estrus and time of day. Physiol. Behav. 55, 651-657. Matsushita, S., and Joshima, H. (1989). Pathology of rats intranasally inoculated with the cilia-associated respiratory bacillus. Lab. Anim. 23, 89-95. Matsushita, S., Kashima, M., and Joshima, H. (1987). Serodiagnosis of cilia-associated respiratory bacillus infection by the indirect immunofluorescence assay technique. Lab. Anita. 21, 356-359. Matsushita, S., Joshima, H., Matsumoto, T., and Fukutsu, K. (1989). Transmission experiments of cilia-associated respiratory bacillus in mice, rabbits, and guinea pigs. Lab. Anim. 23, 96-102. Mattheij, J. A. M., and Swarts, J. J. M. (1991). Quantification and classification of pregnancy wastage in 5-day cyclic young through middle-aged rats. Lab. Anim. 25, 30-34. Matuszczyk, J. V., Appa, R. S., and Larsson, K. (1994). Age-dependent variations in the sexual preference of male rats. Physiol. Behav. 55, 827-830. McKisic, M. D., Paturzo, E X., and Gaertner, D. J. (1995). A nonlethal rat parvovirus infection suppresses rat T lymphocyte effector functions. J. Immunol. 155, 3979-3986. Medina, L. V., Chladny, J., Ortman, J. D., Artwohl, J. E., Bunte, R. M., and Bennett, B. T. (1998). Rapid way to identify the cilia-associated respiratory bacillus: Tracheal mucosal scraping with a modified microwave Steiner silver impregnation. Lab. Anim. Sci. 46, 113 - 115. Mendes, E. N., Queiroz, D. M., Dewhirst, E E., Paster, B. J., Moura, S. B., and Fox, J. G. (1996). Helicobacter trogontum sp. nov., isolated from the rat intestine. Int. J. Syst. Bacteriol., 46, 916-921. Mercier, O., Perraud, J., and Stadler, J. (1987). A method for routine observation of sexual behaviour in rats. Lab. Anita. 21, 125-130. Meyer, T. K., Yurchak, M. R., Pliego, J. E, Wincek, T. J., Dukelow, W. R., and Kuehl, T. J. (1997). Cryopreservation of murine zygotes for use in testing culture environments. Lab. Anim. Sci. 47, 496-499. Middle Atlantic Reproduction and Teratology Association and Midwest Teratology Association. (1996). Historical control data ( 1992-1994) for developmental and reproductive toxicity studies using the Crl:CD(SD)BR rat. Charles River Laboratories, Wilmington, Massachusetts. Mohr, U., Dungworth, D. L., and Capen, C. C., eds. (1992). "Pathobiology of the Aging Rat," Vols. 1 and 2. ILSI Press, Washington, D.C. Montes, G. S., and Luque, E. H. (1988). Effects of ovarian steroids on vaginal smears in the rat. Acta Anat. (Basel) 133, 192-199.

4. BIOLOGY AND DISEASES OF RATS Montgomery, C. A., and Seely, J. C. (1990). "Pathology of the Fischer Rat: Reference and Atlas" (G. A. Boorman, S. L. Eustis, M. R. Elwell, C. A. Montgomery, and W. E MacKenzie, eds.), pp. 127-153. Academic Press, San Diego. Moore, D. M. (1995). Chapter 11. In "The Experimental Animal in Biomedical Research" (B. E. Rollin, and M. J. Kesel, eds.), Vol. 2. CRC Press, Boca Raton, Florida. Moore, H. D., and Akhondi, M. A. (1996). Fertilizing capacity of rat spermatozoa is correlated with decline in straight-line velocity measured by continuous computer-aided sperm analysis: Epididymal rat spermatozoa from the proximal cauda have a greater fertilizing capacity in vitro than those from the distal cauda or vas deferens. J. AndroL 17, 50-60. Morris, R. H., and Bras, G. J. (1971). Lasting influence of early caloric restriction on prevalence of neoplasms in the rat. J. Natl. Cancer Inst. 47, 1095-1113. Mottola, M. E, Plust, J. H., Christopher, P. D., and Schachter, C. L. (1992). Effects of exercise on maternal glycogen storage patterns and fetal outcome in mature rats. Can. J. Physiol. Pharmacol. 70, 1634-1638. Motzel, S. L., and Riley, L. K. (1991). Bacillus piliformis flagellar antigens for serodiagnosis of Tyzzer's disease. J. Clin. Microbiol. 29, 2566-2570. Motzel, S. L., and Riley, L. K. (1992). Subclinical infections and transmission of Tyzzer's disease in rats. Lab. Anim. Sci. 42, 439-443. Naot, Y., Merchav, S., Ben-David, E., and Ginsburg, H. (1979a). Mitogenic activity of Mycoplasma pulmonis. 1. Stimulation of rat B and T lymphocytes. Immunology 36, 399-406. Naot, Y., Simon-Tou, R., and Ginsburg, H. (1979b). Mitogenic activity of Mycoplasma pulmonis. 2. Studies on the biochemical naure of the mitogenic factor. Eur. J. Immunol. 9, 149-151. National Research Council. (1976). Long-term holding of laboratory rodents. ILAR News 19, L1-L25. National Research Council. (1991a). Respiratory system. In "Infectious Diseases of Mice and Rats," pp. 33-84. National Academy Press, Washington, D.C. National Research Council. (1991 b). Multiple systems. In "Infectious Diseases of Mice and Rats," pp. 236-256. National Academy Press, Washington, D.C. National Research Council. (1991c). "Infectious Diseases of Mice and Rats" (J. R. Lindsey, G. A. Boorman, M. J. Collins, C.-K. Hsu, G. L. Van Hoosier, Jr., and J. E. Wagner, eds.), pp. 85-163. National Academy Press, Washington, D.C. National Research Council (1991d). "Infectious Diseases of Mice and Rats" (J. R. Lindsey, G. A. Boorman, M. J. Collins, C.-K. Hsu, G. L. Van Hoosier, Jr., and J. E. Wagner, eds.), pp. 187-190. National Academy Press, Washington, D.C. National Research Council. (1991e). "Infectious Diseases of Rats and Mice" (J. R. Lindsey, G. A. Boorman, M. J. Collins, C.-K. Hsu, G. L. Van Hoosier, Jr., and J. E. Wagner, eds.), pp. 198-221. National Academy Press, Washington, D.C. National Research Council. (1995). "Nutritional Requirements of Laboratory Animals," 4th ed. National Academy Press, Washington, D.C. National Research Council. (1996a). "Guide for the Care and Use of Laboratory Animals." National Academy Press, Washington, D.C. National Research Council. (1996b). "Laboratory Animal ManagementmRodents." National Academy Press, Washington, D.C. Niederkorn, J. Y., Shadduck, J. A., and Schmidt, E. C. (1981). Susceptibility of selected inbred strains of mice to Encephalitozoon cuniculi. J. Infect. Dis. 144, 249-253. Niggeschulze, A., and Kast, A. (1994). Maternal age, reproduction, and chromosomal aberrations in Wistar derived rats. Lab. Anim. 28, 55-62. Nunn, G., and MacPherson, A. (1995). Spontaneous convulsions in Charles River Wistar rats. Lab Animal 29, 50-53. Orcutt, R. P. (1980). Bacterial diseases: Agent, pathology, diagnosis, and effects on research. Lab Animal 9, 28-43. Orihuela, P. A., Ortiz, M. E., and Croxatto, H. B. (1999). Sperm migration into

163 and through the oviduct following artificial insemination at different stages of the estrous cycle in the rat. Biol. Reprod. 60, 908-913. Owen, D. G. (1992b). "Parasites of Laboratory Animals," pp. 13-38. Royal Society of Medicine Services, Ltd., London. Owen, D. G. (1992a). "Parasites of Laboratory Animals," pp. 89-90. Royal Society of Medicine Services, Ltd., London. Oz, H. S., and Hughes, W. T. (1996). Effect of sex and dexamiethasone dose on the experimental host for Pneumocystis carinii. Lab. Anim. Sci. 46, 109-110. Pakes, S. P., Shadduck, J. A., Feldman, D. B., and Moore, J. A. (1984). Comparison of tests for the diagnosis of spontaneous encephalitozoonosis in rabbits. Lab. Anim. Sci. 34, 356-359. Paturzo, E Y., Jacoby, R. O., Bhatt, P. N., Smith, A. L., Gaertner, D. J., and Ardito, R. B. (1987). Persistence of rat virus in seropositive rats as detected by explant culture. Arch. Virol. 95, 137-142. Pauli, B. U., Gruber, A. D., and Weinstein, R. S. (1996). "Urinary System" (T. C. Jones, G. C. Hard, and U. Mohr, eds.), 2nd ed., pp. 381-392. Springer, Bedim Pearson, D. J., and Taylor, G. (1975). The influence of the nematode Syphacia obvelata on adjuvant arthritis in rats. Immunology. 29, 391-396. Peluso, J. J. (1992). "Pathobiology of the Aging Rat." (U. Mohr, D. L. Dungworth, and C. C. Capen, eds.), pp. 337-349. ILSI Press, Washington, D.C. Percy, D. H., and Barthold, S. W. (1993a). "Pathology of Laboratory Rodents and Rabbits," 1st ed., pp. 3-69. Iowa State Univ. Press, Ames. Percy, D. H., and Barthold, S. W. (1993b). "Pathology of Laboratory Rodents and Rabbits," 1st ed., pp. 70-114. Iowa State Univ. Press, Ames. Percy, D. H., Bond, S. J., Paturzo, F. X. and Bhatt, P. N. (1990). Duration of protection from reinfection following exposure to sialodacryoadenitis virus in Wistar rats. Lab. Anim. Sci. 40, 144-149. Perez, C., Canal, J. R., Dominguez, E., Campillo, J. E., Guillen, M., and Torres, M. D. (1997). Individual housing influences certain biochemical parameters in the rat. Lab. Anim. 31, 357-361. Petri, M. (1969). Studies on Nosema cuniculi found in transplantable ascites tumours with a survey of microsporidiosis in mammals. Acta Pathol. Microbiol. Scand. Suppl. 204, 1-91. Pleasants, J. R. (1974). "Handbook of Laboratory Animal Science" (E. C. Melby, and N. H. Altman, eds.), pp. 119-174. CRC Press, Cleveland. Poonacha, K. B., William, P. D., and Stamper, R. D. (1985). Encephalitozoonosis in a parrot. J. Am. Vet. Med. Assoc. 186, 700-702. Pucak, G. J., Lee, C. S., and Zaino, A. S. (1977). Effects of prolonged high temperature on testicular development and fertility in the male rat. Lab. Anim. Sci. 27, 76-77. Reetz, J. (1993). Naturliche microsporidien-(Encephalitozoon-cuniculi-)Infektionen bei Huhnern. Tierarztl. Prax. 21, 429-435. Rehbinder, C., Baneux, P., Forbes, D., Van Herck, H., Nicklas, W., Rugaya, Z., and Winkler, G. (1996). FELASA recommendations for the health monitoring of mouse, rat, hamster, gerbil, guinea pig, and rabbit experimental units. Lab. Anim. 30, 208. Rehm, S., Waalkes, M. P., and Ward, J. M. (1988). Aspergillus rhinitis in Wistar (Cfl:(WI)BR) rats. Lab. Anim. Sci. 38, 162-166. Richter, C. P. (1954). The effects of domestication and selection on the behavior of the Norway rat. J. Natl. Cancer Inst. 15, 727-738. Riley, L. K., and Franklin, C. L. (1997). "Digestive System" (T. C. Jones, J. A. Popp, and U. Mohr, eds.), 2nd ed., pp. 201-209. Springer-Verlag, Berlin. Riley, L. K., Franklin, C. L., Hook, R. R., and Besch-Williford, C. (1996). Identification of murine helicobacters by PCR and restriction enzyme analysis. J. Clin. Microbiol. 34, 942-946. Riley, L. K., Carty, A. J., and Besch-Williford, C. L. (1999). PS 45 PCR-based testing as an alternative to MAP testing. Lab. Anim. Sci. 49, 443-444. Ristic, M., and Kreier, J. P. (1984). "Bergey's Manual of Systematic Bacteriology" (I. N. R. Krieg, and J. G. Holt, eds.), pp. 719-739. Robinson, 1965. Reprint, Williams and Wilkins, Baltimore. Robinson, R. (1965). "Genetics of the Norway Rat." Pergamon Press, New York.

164 Roe, E J. C., Lee, P. N., Conybeare, G., Kelly, D., Matter, B., Prentice, D., and Tobin, G. (1995). The biosure study: Influence of composition of diet and food consumption on longevity, degenerative diseases, and neoplasia in Wistar rats studied for up to 30 months post weaning. Food Chem. Toxicol. 33 (Suppl. 1), 1S-100S. Romero, A., Villamayor, E, Grau, M. T., Sacristan, A., and Ortiz, J. A. (1992). Relationship between fetal weight and litter size in rats: application to reproductive toxicology studies. Reprod. Toxicol. 6, 453-456. Rosen, M., Kahan, E., and Derazne, E. (1987). The influence of the first-mating age of rats on the number of pups born, their weights, and their mortality. Lab. Anim. 21, 348-352. Rottinghaus, A. A., Gibson, S. V., and Wagner, J. E. (1986). Comparison of serological tests for detection of antibodies to Sendai virus in rats. Lab. Anim. Sci. 36, 496-948. Rouleau, A. M., Kovacs, P. R., Kunz, H. W., and Armstrong, D. T. (1993). Decontamination of rat embryos and transfer to specific pathogen-free recipients for the production of a breeding colony. Lab. Anim. Sci. 43, 611-615. Royals, M. A., Getzy, D. M., and Vandewoude, S. (1999). High fungal spore load in corncob bedding associated with fungal-induced rhinitis in two rats. Contemp. Top. 38, 64-66. Russell, L. D. (1992). "Pathobiology of the Aging Rat" (U. Mohr, D. L. Dungworth, and C. C. Hapen, eds.), Vol. 1, pp. 395-405. ILSI Press, Washington, D.C. Russell, L. D., Alger, L. E., and Nequin, L. G. (1987). Hormonal control of pubertal spermatogenesis. Endocrinology 120, 1615-1632. Saibaba, P., Sales, G. D., Stodulski, G., and Hau, J. (1996). Behavious of rats in their home cages: daytime variations and effects of routine husbandry procedures analysed by time sampling techniques. Lab. Anim. 30, 13-21. Sales, G. D., and Milligan, S. R. (1992). Ultrasound and laboratory animals. Anim. Technol. 43, 89-98. Savage, D. C. (1971). "Defining the Laboratory Animal. IV Symposium, International Committee on Laboratory Animals" (National Research Council, ed.), pp. 60-73. National Academy of Sciences, Washington, D.C. Savage, N. (1984). "Bergey's Manual of Systematic Bacteriology," (I. N. R. Krieg, and J. G. Holt, eds.), pp. 598-600. Williams and Wilkins, Baltimore. Schagemann, G., Bohnet, W., Kunstyr, I., and Friedhoff, K. T. (1990). Host specificity of cloned Spironucleus muris in laboratory rodents. Lab. Anim. 24, 234-239. Schoeb, T. R., Davidson, M. K., and Lindsey, J. R. (1982). Intracage ammonia promotes growth of Mycoplasma pulmonis in the respiratory tract of rats. Infect. Immun. 38, 212-217. Schoeb, T. R., Kervin, K. C., and Lindsey, J. R. (1985). Exacerbation of murine respiratory mycoplasmosis in gnotobiotic F344/N rats by Sendai virus infection. Vet. Pathol. 22, 272-282. Semple-Rowland, S. L., and Dawson, W. W. (1987). Retinal cyclic light damage threshold for albino rats. Lab. Anim. Sci. 37, 289-298. Shalev, U., Feldon, J., and Weiner, L. (1998). Gender- and age-dependent differences in latent inhibition following pre-weaning non-handling: Implications for a neurodevelopmental animal model of schizophrenia. Int. J. Dev. Neurosci. 16, 279-288. Sharma, A. W., and Mayrhofer, G. (1988a). Biliary antibody response in rats infected with rodent Giardia duodenalis isolates. Parasite Immunol. 10, 181-191. Sharma, A. W., and Mayrhofer, G. (1988b). A comparative study of infections with rodent isolates of Giardia duodenalis in inbred strains of rats and mice and in hypothymic nude rats. Parasite Immunol. 10, 169-179. Shoji, Y., Itoh, T., and Kagiyama, N. (1988). Enzyme-linked immunosorbent assay for detection of serum antibody to CAR bacillus. Exp. Anim. 37, 67-72. Short, B. G., and Goldstein, R. S. (1992). "Pathobiology of the Aging Rat" (U. Mohr, D. L. Dungworth, and C. C. Capen, eds.), Vol. 1, pp. 211-225. ILSI Press, Washington, D.C. Skelton, H., Smith, K., Hilyard, E., Hadfield, T., Tuur, S., Wagner, K., Decker, C., and Angritt, P. (1995). Bacillus piliformis (Tyzzer's disease) in an

DENNIS F. KOHN AND CHARLES B. CLIFFORD

HIV-1+ patient: Confirmation with 16S rRNA sequence analysis. J. Invest. Dermatol. 104, 687-687. (Abstract). Slaoui, M., Dreef, H. C., and van Esch, E. (1998). Inflammatory lesions in the lungs of Wistar rats. Toxicologic Pathology 26, 712-713. Smith, E. R., Stefanick, M. L., Clark, J. T., and Davidson, J. M. (1992). Hormones and sexual behavior in relationship to aging in male rats. Horm. Behav. 26, 110-135. Songsasen, N., and Leibo, S. P. (1998). Live mice derived from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa. Lab. Anim. Sci. 48, 275-281. Sorden, S. D., and Castleman, W. L. (1991). Brown Norway rats are high responders to bronchiolitis, pneumonia, and bronchiolar mastocytosis induced by parainfluenza virus. Exp. Lung Res. 17, 1025-1045. Suber, R. L., and Kodell, R. L. (1985). The effect of three phlebotomy techniques on hematologic and clinical chelmcal evaluation in Sprague-Dawley rats. Vet. Clin. Pathol. 14, 23- 30. SultanDosa, A. B., Bryner, J. H., and Foley, J. W. (1983). Pathogenicity of Campylobacterjejuni and Campylobacter coli strains in the pregnant guinea pig model. Am. J. Vet. Res. 44, 2175-2178. Summa, M. E. L., Ebisui, L., Osaka, J. T., and de Tolosa, E. M. C. (1992). Efficacy of oral ivermectin against Trichosomoides crassicauda in naturally infected laboratory rats. Lab. Anim. Sci. 42, 620-622. Suzuki, E., Mochida, K., and Nakagawa, M. (1988). Naturally occurring subclinical Corynebacterium kutscheri infection in laboratory rats: Strain and age related antibody response. Lab. Anim. Sci. 38, 42-45. Swing, S. P., Davis, J. K., and Egan, M. L. (1995). In vitro effects of Mycoplasma pulmonis on murine natural killer cell activity. Lab. Anim. Sci. 45, 352-356. Tada, N., Sata, M., Mizorogi, T., Kasai, K., and Gawa, S. (1995). Efficient cryopreservation of hairless mutant (bald) and normal Wistar rat embryos by vitrification. Lab. Anim. Sci. 45, 323-325. Tai, J. H., Wang, A. L., Ong, S. J., Lai, K. S., Lo, C., and Wang, C. C. (1991). The course of giardiavirus infection in the Giardia lamblia trophozoites. Exp. Parasitol. 73, 413-423. Tai, J. H., Chang, S. C., Chou, C. E, and Ong, S. J. (1996). Separation and characterization of two related giardiaviruses in the parasitic protozoan Giardia lamblia. Virology 216, 124-132. Takeichi, N., Hamada, J., et al. (1988). Depression of T cell-mediated immunity and enhancement of autoantibody production by natural infection with microorganisms in spontaneously hypertensive rats (SHR). Microbiol. lmmunol. 32, 1235-1244. Tanemura, K., Wakayama, T., Kuramoto, K., Hayashi, Y., Sato, E., and Ogura, A. (1997). Birth of normal young by microinsemination with frozen-thawed round spermatids collected from aged azoospermic mice. Lab. Anim. Sci. 47, 203-204. Tayama, K., and Shisa, H. (1994). Development of pigmented scales on rat skin: Relation to age, sex, strain, and hormonal effect. Lab. Anim. Sci. 44, 240-244. Thomas, D. S., and Pass, D. A. (1997). Chronic ulcerative typhlocolitis in CBHrnu/rnu (athymic nude) rats. Lab. Anim. Sci. 47, 423-427. Tidehag, P., Hallmans, G., Sjostrom, R., Sunzel, B., Wetter, L., and Wing, K. (1988). The extent of coprophagy in rats with differing iron status and its effect on iron absorption. Lab. Anim. 22, 313-319. Tucker, M. J. (1979). The effect of long-term food restriction on tumors in rodents. Int. J. Cancer 23, 803-807. Tvedten, H. W., Whitehair, C. K., and Langham, R. E (1973). Influence of vitamins A and E on gnotobiotic and conventionally maintained rats exposed to Mycoplasma pulmonis. J. Am. Vet. Med. Assoc. 163, 605-612. Tyzzer, E. E. (1917). A fatal disease of Japanese Waltzing mice caused by a spore-forming bacillus (Bacilluspiliformis n. sp.). J. Med. Res. 37, 307-338. Urano, T., Maejima, K., Okada, O., Takashina, S., Syumiya, S., and Tamura, H. (1977). Control of Pseudomonas aeruginosa infection in laboratory mice with gentamicin. Jikken Dobutsu 26, 259-262.

4. BIOLOGY AND DISEASES OF RATS Utsumi, K., Satoh, E., and Iritani, A. (1991). Sexing of rat embryos with antisera specific for male rats. J. Exp. Zool. 260, 99-105. Van Andel, R. A., Franklin, C. L., Besch-Williford, C. L., Hook, R. R., and Riley, L. K. (1996). Cytokine expression in subclinical murine Tyzzer's disease. Contemp. Top. 35(4), 67. (Abstract). Vanderhyden, B. C., Rouleau, A., Walton, E. A., and Armstrong, D. T. (1986). Increased mortality during early embryonic development after in-vitro fertilization of rat oocytes. J. Reprod. Fertil. 77, 401-409. Van Ruiven, R., Meijer, G. W., Wiersma, A., Baumans, V., van Zutphen, L. E M., and Ritskes-Hoitinga, J. (1988). The influence of transportation stress on selected nutritional parameters to establish the necessary minimum period for adaptation in rat feeding studies. Lab. Anim. 32, 446-456. Van Winkle, T. J., Womack, J. E., Barbo, W. D., and Davis, T. W. (1988). Incidence of hydronephrosis among several production colonies of outbred Sprague-Dawley rats. Lab. Anim. Sci. 38, 402-406. Van Zwieten, M. J., Solleveld, H. A., Lindsey, J. R., de Groot, E G., Zurcher, C., and Hollander, C. E (1980). Respiratory disease in rats associated with a filamentous bacterium: A preliminary report. Lab. Anim. Sci. 30, 215-221. Vonderfecht, S. L., Huber, A. C., et al. (1984). Infectious diarrhea of infant rats produced by a rotavirus-like agent. J. Virol. 52, 94-98. Vonderfecht, S. L., Miskuff, R. L., et al. (1985). Enzyme immunoassay inhibition assay for the detection of rat rotavirus-like agent in intestinal and fecal specimens obtained from diarrheic rats and humans. J. Clin. Microbiol. 22, 726-730. Waggie, K. S., Spencer, T. H., and Allen, A. M. (1987). Cilia associated respiratory (CAR) bacillus infection in New Zealand White rabbits. Lab. Anim. Sci. 37, 533. (Abstract). Wagner, J. E. (1977). Self trauma and Staphylococcus aureus in ulcerative dermatitis of rats. J. Am. Vet. Med. Assoc. 171, 839-841. Wagner, M. (1988). The effect of infection with the pinworm (Syphacia muris) on rat growth. Lab. Anim. Sci. 38, 476-478. Ward, J. M., and Reynolds, C. W. (1983). Large granular lymphocyte leukemia. A heterogeneous lymphocytic leukemia in F-344 rats. Am. J. PathoL 111, 1-10. Watson, D. S. B. (1993). Evaluation of inanimate objects on commonly monitored variables in preclinical safety studies for mice and rats. Lab. Anim. Sci. 43, 378-380. Weir, E. C., Jacoby, R. O., Paturzo, E X., and Johnson, E. A. (1990). Infection of SDAV-immune rats with SDAV and rat coronavirus. Lab. Anim. Sci. 40, 363-366. Weisbroth, S. H. (1979). "The Mouse in Biomedical Research" Vol. 2. "Diseases" (H. L. Foster, D. Small, and J. G. Fox, eds.), pp. 385-402. Academic Press, New York. Weisbroth, S. H. (1982). "The Laboratory Rat," Vol. 1. "Biology and Diseases" (H. L. Foster, D. Small, andJ. G. Fox, eds.), pp. 192-241. Academic Press, New York. Weisbroth, S. H., Peters, R., Riley, L. K., and Shek, W. (1998). Microbiological assessment of laboratory rats and mice. ILAR J. 39(4), pp. 272-290. National Research Council.

165 Weisse, I. (1992). "Pathobiology of the Aging Rat" (U. Mohr, D. L. Dungworth, and C. C., Capen, eds.), Vol. 2, pp. 65-119. ILSI Press, Washington, D.C. Wescott, R. B. (1982). "The Mouse in Biomedical Research," Vol. 2. "Diseases" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), pp. 373-384. Academic Press, New York. West, W. L., Schofield, J. C., and Bennett, B. T. (1992). Efficacy of the "microdot" technique for adn~.nistering topical 1% ivermectin for the control of pinworms and fur mites in mice. Contemp. Top. 31, 7-10. Wickramanayake, G. B., and Sproul, O. J. (1991). "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.), 4th ed., pp. 72-84. Lea and Febiger, Philadelphia. Will, L. A. (1994). "Handbook of Zoonoses, Section A: Bacterial, Rickettsial, Chlamydial, and Mycotic" (G. W. Beran, ed.), 2nd ed., pp. 231-240. CRC Press, Boca Raton, Florida. Wilson, J. M. (1979). The biology of Encephalitozoon cuniculi. Med. Biol. 57, 84-101. Wray, C. (1994). "Handbook of Zoonoses, Section A: Bacterial, Rickettsial, Chlamydial, and Mycotic" (G. W. Beran, ed.), 2nd ed., pp. 289-302. CRC Press, Boca Raton, Florida. Wullenweber, M. (1995). Streptobacillus moniliformisma zoonotic pathogen. Taxonomic considerations, host species, diagnosis, therapy, geographical distribution. Lab. Anim. 29, 1-15. Wyand, D. S., and Jonas, A. M. (1967). Pseudomonas aeruginosa infection in rats following implantation of an indwelling jugular catheter. Lab. Anim. Care 17, 261-267. Yang, E C., Paturzo, E X., and Jacoby, R. O. (1995). Environmental stability and transmission of rat virus. Lab. Anim. Sci. 45, 140-144. Yarbrough, W. G., Quarmby, V. E., Simental, J. A., Joseph, D. R., Sar, M., Lubahn, D. B., Olsen, K. L., French, E S., and Wilson, E. M. (1990). A single base mutation in the androgen receptor gene causes androgen insensitivity in the testicular feminized rat. J. Biol. Chem. 265, 8893-8900. Yoshitomi, K., and Boorman, G. A. (1990). "Pathology of the Fischer Rat. Reference and Atlas" (G. A. Boorman, S. L. Eustis, M. R. Elwell, C. A. Montgomery, and W. E MacKenzie, eds.), pp. 239-259. Academic Press, San Diego. Yu, D. C., Wang, A. L., and Wang, C. C. (1996). Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia. J. Virol. 70, 8752-8757. Yu, D. C., Wang, A. L., Botka, C. W., and Wang, C. C. (1998). Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA. Mol. Biochem. Parasitol. 96, 151-165. Zenner, L. (1988). Effective eradication of pinworms (Syphacia muris, Syphacia obvelata, and Aspicularis tetraptera) from rodent breeding colony by oral anthelmentic therapy. Lab. Anim. 32, 337-342. Zubaidy, A. J., and Majeed, S. K. (1981). Pathology of the nematode Trichosomoides crassicauda in the urinary bladder of laboratory rats. Lab. Anim. 15, 381'384.

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Chapter 5 Biology and Diseases of Hamsters F Claire Hankenson and Gerald L. Van Hoosier Jr.

Syrian Hamster I.

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Fig. 1. The use of hamsters in the United States has declined by more than 50% since 1973.

Approximately 217,079 hamsters were used in research in 1997 in the United States, which represents a 53% decrease in use since 1973 (Fig. 1) (Report of the Secretary of Agriculture to the President of the Senate and the Speaker of the House of Representatives, 1998). The species used in research include the Syrian (golden), Mesocricetus auratus; the Chinese (stripedback), Cricetulus griseus; the Armenian (gray), C. migratorius; the European, Cricetus cricetus; and the Djungarian, Phodopus campbelli (Russian dwarf) and P. sungorus (Siberian dwarf) (Fig. 2). The family Cricetidae is a member of the order Rodentia. Animals in this family are characterized by large cheek pouches, thick bodies, short tails, and an excess of loose skin. They have incisors that grow continuously and cuspidate molars

that do not continue to grow (I 1/1, C 0/0, PM 0/0, M 3/3 X 2 = 16).

SYRIAN HAMSTER

I.

INTRODUCTION

The publication by Hoffman et al. (1968) is a good, comprehensive source of information and reference to the literature

Fig. 2. The appearance and comparative size of adult species of hamsters; from left to right, Chinese (39 gm), Armenian (65 gm), and Syrian (120 gm).

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prior to 1963 on the biology and experimental uses of the hamster. This text also contains a master bibliography and includes a stereotaxic atlas of the brain of the Syrian hamster. Instructional audiotapes and 2 X 2 colored slides from the Laboratory Animal Medicine and Science Series, containing information about the use of hamsters in biomedical research, are recommended as an introduction to the hamster for a variety of learning and instructional settings (Autotutorial Committee, 1994).

A.

Description

The Syrian or golden hamster (Mesocricetus auratus) is native to the arid, temperate regions of southeast Europe and Asia Minor. In their natural environment, hamsters live in deep tunnels that ensure a cooler temperature and higher humidity than the general desert environment. They are nocturnal animals. The adult Syrian hamster usually grows to a length of 6 to 8 inches (14 to 19 cm) and weighs between 110 and 140 gm. The adult female of this breed tends to be larger than the male. The hamster has a small blunt tail and smooth, short hair. Normal coloration is reddish gold, with a grayish white ventrum. Haircoat colors also include cream, albino, piebald, and cinnamon; the length of hair can also vary (Harkness and Wagner, 1995). The ears are pointed, with dark pigmentation, and the eyes are small, dark, and bright. Male hamsters can be identified by prominent flank glands and by large testicles that protrude behind the body on each side of the tail. The posterior of the male hamster's body is pointed and protuberant. The normal gross anatomy has been described (Hoffman et al., 1968).

B.

Use in Research

Practically all Syrian hamsters now in use as laboratory animals originated from one litter captured in Syria in 1930. The use of the golden hamster as a laboratory animal was initiated by Saul Adler, who sought a laboratory animal susceptible to infection with Leishmania (1948). Only 3 littermates, 1 male and 2 females, were retained in captivity, and it is the progeny of these 3 animals that were first imported to the United States in 1938. By 1973, the hamster had become the third most commonly used laboratory animal in the United States, behind mice and rats. Since that time, the use of the hamster in research has also been surpassed by that of the guinea pig and rabbit (Report of the Secretary of Agriculture to the President of the Senate and the Speaker of the House of Representatives, 1998). The four major reasons given for selection of the Syrian hamster for research are (1) availability and ease of reproduction, (2) relative freedom from spontaneous diseases coupled with susceptibility to many introduced pathogenic agents,

(3) anatomical and physiological features with unique potential for study, and (4) rapid development with short life cycle. 1.

Availability

The Syrian hamster reproduces readily, with females of some strains able to produce more than 6 litters of 4 - 1 2 pups during their breeding life of approximately 1 year. The short breeding cycle has encouraged development of inbred strains that are used as animal models in studies of genetics, carcinogenesis, and infectious disease processes. Muscular dystrophy was first reported in an inbred line of BIO 1.50 Syrian hamsters in 1962 (Homburger, 1972b). A new dystrophic line from these animals was established, BIO 14.6, which develops congestive heart failure and is used extensively in cardiomyopathy research. An additional inbred line, identified as BIO 4.24, is characterized by obesity in females, with multiple endocrine anomalies and a high incidence of benign adenomas of the adrenal cortex. In the BIO 2.4 and BIO 87.20 inbred lines, cystic prostatic hypertrophy occurred that resembled that seen in canines. In the BIO 12.14 line, male animals develop progressive pelvic limb paralysis by the age of 10 months. Homburger (1972b) also reported that certain inbred lines have an exceptionally high rate of susceptibility to carcinogens. These strains represent the original inbred lines, but the current biomedical community still uses these and further derivatives in comparative biomedical research studies. Additional cardiomyopathic hamster lines include BIO 82.62, BIO-TO-2, BIO 53.58 (a model of idiopathic dilatative cardiomyopathy), as well as UMX( )7-1 (a model of severe childhood autosomal recessive muscular dystrophy) (Nonaka, 1998). The inbred line BIO 15.16 is used in smoke-inhalation studies. Many strains also exist as normal control animals, including BIO RB and CLAC lines. 2.

Susceptibility to Pathogens and Induced Agents

Hamsters have relatively few spontaneous diseases when compared to other laboratory rodents; however, they are susceptible to many experimentally induced diseases and infections (Frenkel, 1979). The continued use of the Syrian hamster in biomedical research is, in large part, attributable to their susceptibility to tumor induction by viruses of other species, e.g., polyomavirus of mice, simian virus 40 (SV40) of monkeys, and adenoviruses of humans. Hamsters are susceptible to most deep fungal infections. Histoplasmosis has been studied extensively in hamsters as they are sensitive to small inocula and are useful for diagnostic purposes. Most of the fungi grow in spleen, lymph nodes, and liver. Conchoid Schaumann bodies are produced during mycobacterial and leishmanial infections. Hamsters infected with Mycoplasma pneumoniae are used as models of local infection in the respiratory tract (Brunner, 1997).

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F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

Other infections to which hamsters are susceptible include tuberculosis, leprosy, atypical mycobacterial infections, and leptospirosis, as well as viral, protozoal, and helminthic infections. Hamster strains continue to be used in the study of prion diseases because of their susceptibility to scrapie, transmissible mink encephalopathy (TME), Creutzfeldt-Jakob disease, and Gerstmann-Staussler syndrome (GSS) (Lowenstein et al., 1990). These prion agents cause slow, progressive, degenerative diseases in the central nervous system (CNS). Hamsters develop amyloid-like deposits in their brains, which may be similar to extracellular deposits of amyloid found in human Alzheimer's disease (Czub et al., 1986). Scrapie prions replicate to high titers in the brains of several species of hamsters, making it possible to compare the human and hamster forms of the disease in a single host (Lowenstein et al., 1990; Marsh and Hanson, 1978). Further information about the prion diseases can be obtained in the review by Prusiner (1991). 3.

Special Anatomical and Physiological Features

a.

Cheek Pouch

The cheek pouches, bilateral invaginations of the oral mucosa, are found in the lateral buccal walls. Often these highly distensible pouches are used by the hamster for temporary storage of food and bedding materials. These pouches do not contain glands but are rich in mast cells (deArruda and Montenegro, 1995). Blood supply to the pouches is carried by branches from the external carotid artery (Davis et al., 1986). More specifically, the pouches are supplied directly or indirectly by six small arteries in the neck and face that are potentially important in controlling cheek pouch blood flow (Davis et al., 1986). The pouches can easily be everted (Fig. 3), with their blood flow intact, and have been used extensively for microvascular studies of inflammation, tumor growth, and vascular smooth muscle function (Svensjo, 1990; Hedqvist et al., 1990). These pouches lack an intact lymphatic drainage pathway and are therefore described as "immunologically privileged." Studies have shown that the surface density of Langerhans cells in the cheek pouches is markedly decreased, which may contribute to the specialized immune status of the tissue (Bergstresser et al., 1980). The pouch tissue will support the longterm survival of transplanted foreign tissue without immunological rejection. The Syrian hamster model of carcinogenesis in the cheek pouch is likely the best animal system for the evaluation of human oral cancer development (Gimenez-Conti and Slaga, 1993). b.

Immunological System

The immune systems of Syrian hamsters have unique features that contribute to the continued use of these hamsters in biomedical research. Some examples include the immunologically privileged cheek pouch, the decrease in rejection of skin allografts as compared to rejection in other laboratory animals, and

the enhanced susceptibility to certain viral infections (Streilein, 1978). Streilein et al. (1980) have determined, based upon skingrafting experiments, that the 3 littermates isolated in 1930 had very little alloantigenic variation. In addition, few mutational changes in this defined gene pool have occurred since the introduction of the hamster into biomedical use (Streilein et al., 1980). Many immunological studies have focused on the organization of major histocompatibility complex (MHC) class I in hamsters. While diversity exists at the MHC class II locus, the region is likely similar among the strains of Syrian hamsters currently available (Hixon et al., 1996). Related to the short gestation period, the ontogeny of the thymic system and associated cellular immunity in Syrian hamsters is delayed compared to other rodents. In addition, only four of the five immunoglobulin (Ig) classes have been described in the hamster, i.e., IgM, IgG, IgA, and IgE, while IgD remains to be defined, and at least two strains of inbred hamsters are deficient in the sixth component of complement. Recently, another IgG isotype, classified as IgG3, has been isolated from some strains of inbred Syrian hamsters. This immunoglobulin is differentiated from IgG1 and IgG2 by its affinity for protein A (Coe et al., 1995). Immunodeficiency has not been linked to deficiencies in IgG3. c.

Hibernation

Hibernation varies among strains and between individual animals; however, exposure to cold stimulates the hamster to gather food, and it will often hibernate at a temperature of approximately 5~ (__+2~ This behavior, not exhibited in mice, rats, or guinea pigs, enables hamsters to be used for a variety of unique experimental objectives in behavioral and physiological research (Lyman, 1979). Because cold exposure and hibernation in the hamster are associated with desaturation of white adipose tissue, this animal is useful for studies of factors controlling the saturation of fat. Hibernation in the hamster has also been linked to modification of disease entities, such as Mycobacterium tuberculosis and Treponema pallidum (Lyman and Fawcett, 1954). A hamster does not fatten prior to hibernation, and it will starve unless it awakens periodically to eat. There is evidence that inability to gather a store of food delays hibernation. Hibernating animals remain sensitive to external stimuli and usually are aroused if handled. d.

Radioresistance

The Syrian and Chinese hamster strains are among the most radioresistant mammals ever studied, with respect to lethality and survival time after irradiation (Eddy and Casarett, 1972). e.

Dentition

Syrian hamsters develop dental caries under defined conditions of diet and oral flora, making them useful for the study of

1 71

5. BIOLOGY AND DISEASES OF HAMSTERS

Fig. 3. The cheek pouch has been manually everted for illustrativepurposes. Note the vasculature supplyingthe pouch.

etiological factors. Studies show that the caries rate in hamsters is influenced not only by the amount of carbohydrate in the diet but also by the form of carbohydrate. The presence or absence of vitamins in the diet is also suggested to be a contributing factor (Shklar, 1972). Reports have suggested that caries may be infectious and transmissible among rodents (Jordan and van Houte, 1972). Hamsters may be useful for testing the anticaries or caries-inhibiting effects of agents such as fluorine and iodoacetic acid. f.

sponse to androgen production. When the male is excited, hair over these glands becomes wet, and the animal scratches and rubs itself as if the area were irritated. There is evidence that the glandular secretions are used for territorial marking. The female also has dorsal sebaceous glands, but they are not as easily identified, and the secretions are associated with the estrous cycle. Flank glands are resistant to locally applied carcinogens but are susceptible to malignant transformation associated with the simultaneous systemic administration of estrogens and androgens (Homburger, 1972a).

Flank Glands

Coarse hair over darkly pigmented skin can be readily observed in the costovertebral area in males (Fig. 4). The flank glands of the Syrian hamster are dermal structures composed of microscopic sebaceous glands that produce secretions in re-

g.

Respiratory Tract

The conductive airways of the Syrian hamster contain a limited number of glandular structures, primarily in the proximal trachea. This makes Mesocricetus auratus a potential model for

172

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

Fig. 4. The flank glands in the male hamster are used as sex glands and for olfactory marking. Females also have these glands, although they are less prominent.

studies of chronic bronchitis (Hayes et al., 1977). The pulmonary vascular bed is similar to that of human beings in many ways, and hamsters develop pulmonary lesions that resemble human centrilobular emphysema (Kleinerman, 1972). Spontaneous bronchiogenic and pulmonary cancers are rare; hence, M. auratus is a good animal in which to study chemical carcinogenesis in the respiratory tract (Homburger, 1968). Similar to that of other rodents, the respiratory tract appears less sensitive than skin to topical carcinogen applications. Because the hamster is resistant to pulmonary infection and able to decompose nicotine, it is a good subject for study on the effects of longterm smoke inhalation.

h.

Gastrointestinal System

The digestive process of a Syrian hamster is different from that of other rodents, such as the rat. The esophagus enters between a forestomach and a glandular stomach compartment. The nonglandular forestomach is similar to that of ruminants and contains an elevated pH level and microorganisms that contribute to digestion through a fermentation process. Although wild-type strains of mice, rats, and guinea pigs have not been especially useful for studies of spontaneous neoplasms of the gastrointestinal tract, such tumors are more common in the hamster (Fortner, 1957). Despite reports of gastrointestinal neoplasia occurring in current transgenic laboratory animals,

these tumors are not considered to be the result of a spontaneous disease process. The experimental production of papillomas and adenocarcinoma in the forestomach and intestines, as well as adenomatous polyps in the colon, suggests that the hamster may be useful for studies of gastrointestinal carcinogenesis (Homburger, 1968). A novel urease-positive helicobacter, Helicobacter aurati, along with two other microaerobes, has been associated with gastritis and intestinal metaplasia in Syrian hamsters (Patterson et al., 2000a,b); the primary niche of these three species is believed to be the lower bowel of infected hamsters. Syrian hamsters respond predictably to intragastric administration of purified cholera enterotoxin, presenting with intraluminal accumulation of fluid in the small bowel, cecum, and proximal colon (Lepot and Banwell, 1976). Therefore, this model is appropriate for the study of pharmacological agents, such as indomethacin, polymyxin B sulfate, glucose electrolyte solutions, and colchicine, that might inhibit intestinal fluid secretions.

i.

Pancreas/Gallbladder/Biliary Tract

In the hamster, the major pancreatic ducts join the common bile duct shortly before it enters the duodenum. This anatomical configuration is similar to that of mice and rats, but is distinct from that of other mammals, including humans. The pan-

5. BIOLOGY AND DISEASES OF HAMSTERS

creas of the Syrian hamster is similar in function to that of the mouse and rat. The Syrian hamster serves as a model for pancreatic carcinogenesis because it is the only animal model in which pancreatic tumors can be induced that are comparable to those of humans, both morphologically and clinically (Pour and Birt, 1979; Mohr, 1979). Induction of exocrine pancreatic tumors has been possible in Syrian hamsters since 1974, and tumor latency can be as short as 12 to 15 weeks. Bile tract carcinomas have been noted in hamsters following injection of bile from patients with cancer of the bile duct, and a high incidence of adenocarcinoma is produced by implantation of methylcholanthrine pellets into the gallbladder. The pancreatic carcinogens used effectively in the hamster have not affected other laboratory animals to the same extent. j.

Kidneys and Urinary Bladder

In the Syrian hamster, the reproductive and urogenital tracts develop from the same embryonic germinal ridge, rendering the kidneys highly responsive to estrogen. The administration of estrogen to male hamsters causes renal tumors, which represents one of the best animal models for human renal cancer (Li et al., 1993). Next to the dog, the Syrian hamster may be the most reliable model for studying the effect of chemical carcinogens on the urinary bladder. k.

Endocrine System

Hamsters are reported to be the first model in which the equivalent of Addisonian adrenal necrosis could be studied. The adrenal glands show a distinct difference in size by 4 weeks of age, depending on the sex of the animal. Male adrenal glands reportedly have a greater number of reticular cells within the adrenal cortex, accounting for the size double that of the female adrenal gland (Militzer et al., 1990). The pituitary gland is of interest because of estrogen-induced adenomas in the intermediate lobe. l.

Harderian Glands

The Harderian glands are pigmented lacrimal glands located posteriorly to the eyes. These secretory glands release lipid- and porphyrin-rich material that lubricates the eyes and lids. Marked sexual dimorphism of the glands in Syrian hamsters was first reported in the 1950s and has not been shown to exist in the Chinese, Armenian, or Djungarian species. Female and male Syrian hamsters differ most significantly in the type of lipid droplets secreted and in the relative concentrations of secreted porphyrin (females secrete 100-1000 times more porphyrin than males) (Buzzell, 1996). This glandular dimorphism is androgen-dependent and exhibits seasonal variation. Acom-

173

plete histologic description of the glands has been published (Buzzell, 1996). m.

Oocytes

In 1976, the hamster oocyte was discovered to be penetrable by human spermatozoa (Yanagimachi et al., 1976). Since that time, one of the main uses of Syrian hamsters in the biomedical setting has been to aid in the assessment of human fertility using the zona-free hamster oocyte assay (Barros et al., 1978). The oocytes can also be used to investigate treatments for human male infertility. 4.

Short Life Cycle and Rapid Development

The short life cycle of the Syrian hamster, ranging between 18 and 24 months, makes it an excellent animal for the study of development and the effect of teratogenic agents. The eighth day of pregnancy is the optimal time for teratogenic studies, when hourly development of the fetal pups can be observed (Ferm, 1967). Using the diabetogenic agent, streptozotocin, diabetes can be induced in the Syrian hamster in advance of mating, and insulin therapy can be instituted without interrupting the teratogenic effects of the drug. While conception is not reported to be affected, the decreased birth size and weight of the litter, as well as increased fetal death and resorption, demonstrate effects of streptozotocin on the development of the young (Connor et al., 1981). The hamster thus makes an excellent model for studying embryonic development in diabetic pregnant females.

II.

A.

BIOLOGY

Development and Physiology

A newborn M. auratus pup weighs 2 - 3 gm. It is hairless, with eyes and ears closed. It has incisor teeth at birth. On approximately the fifth day, the ears open; at the ninth day, hair growth is first observed; and at the fifteenth day, the eyes open. By age of weaning at 21 days, the pup weighs 3 5 - 4 0 gm. By maturity at 6 - 8 weeks, males weigh 85-110 gm and females, 95-120 gm. As the animals age further, there is some increase in weight. Male and female hamsters can be identified by comparing the anogenital distances, and by observing either mammae on the ventrum of the female or the posterior scrotum of the male

(Fig. 5).

The reproductive life span is from 6 - 8 weeks to 15 months of age, and the total life span averages 2 years, with a 3-year maximum reported. It is of interest to note that the average life span

174

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

Fig. 5. Sexingof female (left) and male (right) hamsters is based on the shorter anogenital distance and mammaeof the female versus the longer anogenital distance and large posterior scrotumof the male.

of the female golden hamster may be markedly shorter than that of males, depending on strain and source of the animals (Bernfeld et al., 1986). Physiological data, such as heart rate and respiration, can be found in Table I. Serum blood chemistry values have been provided in Table II. It should be mentioned that serum chemistry parameters may differ between sexes and strains of hamsters (Maxwell et al., 1985).

B.

Genetics

Golden hamsters have a diploid chromosome number of 44. Numerous mutations have been introduced since the establishment of the Syrian hamster as a laboratory animal in the 1930s (Yoon and Peterson, 1979). Eighteen of the mutations involve coat and eye color; the earliest mutations produced brown, cream, piebald, and white hamsters. Six mutations involve the neuromuscular system, and 6 are identifiable by quantity or texture of hair. Breeders have also developed inbred strains of hamsters, some of which are of value to researchers because of genetically transmitted diseases or conditions, and unique susceptibility to teratogenic and carcinogenic agents (Homburger, 1972b).

C.

Nutrition

Commercial rodent feed is generally used as the basic diet for omnivorous hamsters, sometimes in combination with alfalfa cubes, to provide a balance of 16-24% protein, 6 0 - 6 5 % carbohydrate, and 5 - 7 % fat. Male and female hamsters consume approximately the same amount of food, between 5.5 and 7 gm per day, during growth and development. Although hamsters seem to grow and reproduce normally with this type of diet, research has demonstrated that Syrian hamsters have nutritional requirements that differ from those of mice and rats. The hamster differs from other rodents in that it has a forestomach in which the first stage of digestion is a fermentation process that affects the utilization of nutrients. For hamsters, unlike other rodents, soybean meal was shown to offer better nutritional efficiency than fish meal. Carbohydrates in the diet can induce changes in both the glucose and lipid metabolism in hamsters (Kasim-Karakas et al., 1996). Cornstarch provides a good source of energy, and 3 0 - 4 0 % provided in the diet is associated with good growth, reproduction, maintenance, and longevity. The mineral requirements for zinc, copper, and potassium are increased in the Syrian hamster, although the levels of other minerals are similar to those of the rat (Newberne

175

5. BIOLOGY AND DISEASES OF HAMSTERS Table I

Table II

Normative DatamSyrian (Golden) Hamster a

Serum Blood Chemistry Values for Adult Syrian Hamsters a

Adult weight Male Female Life span Average Maximum expected Chromosome number (diploid) Water consumption Food consumption Body temperature Puberty Male Female Gestation Litter size Birth weight Eyes open Weaning Heart rate Respiratory frequency Leukocyte counts Total Neutrophils Segmented Nonsegmented Lymphocytes Monocytes Eosinophils Basophils Erythrocyte sedimentation rate Platelets Red blood cells Hemoglobin a

85 - 140 gm 95-120 gm 2 years 3 years 44 30 ml/day 10-15 gm/day (adult) 36.2 ~176 6 - 8 weeks (90 gm) 8-12 weeks (90-100 gm) 15 - 18 days 4 - 1 2 pups 2 - 3 gm 15 days 21 days (35-40 gm) 280-412 74 (33 - 127) 7.62 • 103/mm 21.9% 8.0% 73.5% 2.5% 1.1% 1.1% 1.64 mm/hr 670.0 • 103/mm (indirect) 7.50 • 106/mm 16.8%

Serum analyte

Units

Glucose Urea nitrogen Creatinine Sodium Potassium Chloride Bicarbonate Calcium Phosphorus Magnesium Alanine aminotransferase Aspartate aminotransferase Alkaline phosphatase Lactate dehydrogenase Creatinine kinase Protein, total Albumin Cholesterol Triglycerides Bilirubin, total Bile acids Uric acid Luteinizing hormone

mg/dl mg/dl mg/dl mEq/1 mEq/1 mEq/1 mEq/1 mg/dl mg/dl mg/dl IU/liter

Follicle stimulating hormone

Male 84.0 23.2 0.40 148.0 6.50 104.0 29.9 12.6 5.40 2.50 44.7

___ 18.5 _ 4.1 _ 0.89 ___3.70 _+ 0.75 +__3.10 + 2.9 b ___0.59 ___ 1.00 ___0.20 ___25.9

IU/liter

61.2 _+ 39.1

IU/liter IU/liter IU/liter g/liter g/liter mg/dl mg/dl mg/dl p~mol/1

257 469 63 43 143 209 0.3

+ 63.6 _+ 174 __. 3.2 ___2.2 ___23.5 ___53.3 ___0.09

ng/ml

4.6 ___0.5 10-30

ng/ml

200-300

Prolactin

ng/ml

5-10

Thyroid stimulating hormone Thyroxine (T4) Triiodothyronine ( T 3 ) Cortisol

ng/ml

300 b

t~g/dl ng/dl txg/dl

3 -7 b 30-80 b 2.75 ___0.44

Progesterone

ng/ml

Estradiol

pg/ml

Testosterone

ng/ml

From Aeromedical Review (1975).

and McConnell, 1979). Syrian hamsters require sources of many of the B vitamins and also need a source of nonnutritive bulk (Warner and Ehle, 1976). Vitamin E has been reported as essential for preventing myocytolysis in cardiomyopathic hamsters; deficiencies in this vitamin, combined with oxidative stress, may play a role in the pathogenesis of heart disease in hamsters (Sakanashi et al., 1991). In addition, vitamin E can reduce fatty streak accumulation in hypercholesterolemic hamsters (Xu et al., 1998). For animals used in research, it is imperative that the diet be adequate to ensure that the biological responses obtained are, in fact, related to the experimental procedure (Newberne and Fox, 1980). Studies of hamster nutrition have shown that increased rates of survival for male and female hamsters are linked to long-term diets of 20 gm lactalbumin /100 gm of food (Birt et al., 1982). In addition, variations in dietary components can influence the outcome of spontaneous disease (Birt and Pour, 1985; Birt et al., 1985). Studies have shown that hamsters

Female 100.0 27.5 0.50 148.0 6.40 104.0

___ 16.6 _ 4.6 +_ 0.15 ___3.70 ___0.73 ___3.60

13.2 5.50 2.20 50.3

___ 1.38 ___ 1.09 +__0.10 __+ 18.3

53.3 __+22.7 126 ___6 208 ___54.7 520 _ 184 59 + 3.4 41 ___2.8 158 _ 35.3 212 ___52.7 0.3 ___0.13 0.9 _+ 0.2 4.4 +__0.5 2 0 - 4 0 (basal) 1500-2000 (late proestrus) 100-200 (basal) 400 - 600 (preovulatory, estrus) 10-15 (basal) 30 (late proestrus)

0.33 _ 0.04 (start of light) 1.0 (basal) 10 - 12 (proestrus) 6 - 8 (estrus, diestrus) 5 -10 (basal) 3 0 0 - 400 (proestrus)

1.5-2.0

Summarized from Loeb and Quimby (1999). b Gender not specified. a

changed from a diet of rodent chow to semipurified feed are susceptible to colocolic intussusception within 7 to 10 days of the change to the nutritionally refined diet (Cunnane and Bloom, 1990). Nutritional information is presented in Tables III-V. Although it is generally recommended that laboratory animals be fed in a manner that minimizes food contamination

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

176

Table III Nutritional Valuesa Dietary components

Arrington et al. (1966) Rogerset al. (1974)

Casein Sucrose Cornstarch Cellulose fiber Vegetable oil Mineral mix Sodium chloride Protein content

18.0 28.0 35.5 5.0 6.0 5.0 b 2.0 C 16.1

24.0 21.9 40.0 5.0 3.0 5.0 d 11.0e 21.0

aFrom Newberne and McConnell (1979). bUSP XIV mixture. cVitamin diet fortification, Nutritional Biochemicals, Cleveland, Ohio. dSalt mixture gm/kg diet: sodium chloride, 5.254; potassium citrate, 11.4; potassium phosphate, 3.867; calcium phosphate, 17.777; magnesium carbonate, 2.044; ferric citrate, 0.800; cupric sulfate, 0.027; manganesesulfate, 0.027; aluminum potassium sulfate, 0.0044; potassium iodide, 0.0022; cobalt chloride, 0.0044; zinc carbonate, 0.0176; sodium fluoride, 0.000044. eVitamin mixture gm/kg diet: cornstarch, 7.94; choline chloride, 2.0; thiamin hydrochloride, 0.025; riboflavin, 0.015; niacin, 0.100; calcium pantothenate, 0.040; pyridoxine hydrochloride, 0.006; biotin, 0.0006; folic acid, 0.004; menadione, 0.004; vitamin B12 (0.1% trituration with mannitol), 0.050; inositol, 0.20; p-aminobenzoic acid, 0.006; DL-tropherol (1.1001U/g), 0.600; vitamin D2, 2,44,00IU.

with excreta, Syrian hamsters are an exception. If food hoppers are used for hamsters, the feed pellets must be able to fall through the slots to the floor of the cage (Harkness e t al., 1977). In a hamster study that began with observations of failing health, decreased conception, and increased cannibalism, the problems were traced to a change in feeders. The feeders that

contributed to these problems had ~16-inch-wide slots that prevented the food from dropping to the cage floor. Because hamsters have a broad muzzle, the animals were forced to bite the food simultaneously from both sides of the individual metal strips of the feeder. The situation resulted in broken teeth and severe weight loss due to starvation. Placement of the food directly on the floor of the cage, in addition to or in lieu of the use of a feeder, is preferred for adults and young hamsters, who will begin to eat solid dry food at about 7 - 1 0 days of age if they can reach it. Like many other rodents, hamsters are naturally coprophagic. Fluid requirement is approximately 30 ml per day per animal, and these animals are well adapted for water conservation due to their desert origins (Committee on Rodents, 1996). Glass dispenser tubes are contraindicated for hamsters since they are able to bite through the glass. Therefore, use of a stainless steel sipper tube is advised. The location of the sipper tube must be sufficiently low for the smallest animal that is caged, as even nursing pups need fluids in addition to milk from the dam, to prevent gastrointestinal disturbances.

D.

Pharmacology

Hamsters are apparently more sensitive to the metabolic effects of corticosteroids than some other laboratory animals, and are less responsive to histamine. Hamsters are very resistant to morphine; it generally has no sedative or hypnotic effects.

E.

Mating and Reproduction

A male hamster is sexually mature when it reaches a weight of approximately 90 gm. In the female, estrus begins within 6 to 8 weeks, yet it is recommended that breeding be withheld until

Table IV Mineral Content of Satisfactory Hamster Diets in Comparison to Rat NRC Requirementsa Mineral

NRC rat requirement

Banta et al. (1975)b

Arrington et al. (1966)c

Rogers et al. (1974)r

Calcium (%) Phosphorus (%) Magnesium (%) Potassium (%) Sodium (%) Iron (mg/kg) Manganese (mg/kg) Copper (mg/kg) Zinc (mg/kg) Iodine (mg/kg) Cobalt (mg/kg) Fluoride (mg/kg)

0.50 0.40 0.04 0.18 0.05 35.00 50.00 5.00 12.00 0.15 -1.00

0.54 0.58 0.13 0.79 0.19 180.00 15.90 12.60 9.40 0.02 0.02 --

0.59 0.30 0.09 0.82 0.15 140.00 3.65 1.60 u 1.60 1.60 m

0.41 0.39 0.06 0.61 0.21 154.00 9.00 7.00 9.20 1.70 1.70 0.02

aNational Research Council (NRC) bNatural product diet. cSemipurified diet.

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5. BIOLOGY AND DISEASES OF HAMSTERS Table V

Vitamin Content of Diets Satisfactory for Golden Hamsters

Vitamin

Natural product (mg/kg diet) Banta et al. (1975)

Semipurified diets (mg/kg diet) Arrington et al. (1966)

A C D E K Choline PABA Inositol B12(txg/kg) Niacin Pantothenate Riboflavin Thiamin Pyridoxine Folic acid Biotin

15.3 m w 110.0 5.2 2000.0 100.0 100.0 32.0 92.0 54.0 12.0 14.0 10.1 3.1 0.9

90.0 9O0.0 5.0 100.0 45.0 150.0 100.0 100.0 28.0 90.0 60.0 20.0 20.0 20.0 1.8 0.4

Rogers et al. (1974) 2.0 62.1 600.0 4.0 2000.0 6.0 200.0 50.0 100.0 40.0 15.0 25.0 6.0 4.0 0.6

the hamster reaches a weight of 90-100 gm. Copulation activity may begin as early as 4 weeks of age, but it is unusual for pregnancy to occur before 8 weeks of age. The ability to reproduce decreases at approximately 14 months of age in both sexes. However, senescent females can often be successfully bred with younger males, even though there is a notable increase in defective ova and a decreasein number of young produced. Reproductive activity varies seasonally according to the strain. The female has a 4-day estrous cycle that can be assessed by evaluation of the vaginal discharge. The end of ovulation (usually day 2 of the cycle) is marked by the appearance of a copious postovulatory discharge that fills the vagina and may extrude through the vaginal orifice. The discharge is creamy white, opaque, and very viscous, with a distinct odor. The female can be successfully mated in the evening of the third day after this postovulatory discharge. Hamsters are usually test-mated by trial placement to determine if the female is receptive to the male. All animals are caged individually for at least 1 week, allowing males to establish cage dominance and the females to cycle normally. Approximately 2 hr after the beginning of the dark cycle, a female is introduced into a cage with a male. It has been reported that the females are receptive to mating for approximately 16 hr from early evening until mid-afternoon on the following day (Ciaccio and Lisk, 1971). If the female is ready for mating, she will quickly assume a position of lordosis with hindlegs spread and tail erect, and will hold it quietly as long as the male is interested. If mating does not occur within 5 min, or if the female is aggressive, she is removed and another female can be tried. If

copulation occurs, the pair can be left together until the following light cycle. With a normal dark cycle, ovulation and fertilization generally occur during the early morning hours, and this (the day of separation) is considered day 1 of gestation. Gestation in the Syrian hamster is from 15 to 18 days in length. Usually the female is placed in a solid-bottom cage and left undisturbed when pregnant. Colony-raised females can be returned to the colony until the fourteenth day if they do not fight. The female should be moved to a separate nesting cage for at least 1 week prior to and 10 days after parturition; otherwise, she may neglect or cannibalize the litter. Another breeding mechanism is to cage 1 male and 1 or 2 females jointly for 7 to 14 days, followed by the removal of the female(s) to a separate cage for parturition. Since Syrian female hamsters tend to be aggressive, measures should be taken to reduce chances for injury or death as a result of fighting. It is reco.rnrnended that breeding pairs have a male hamster that is older than the female. In addition, breeding hamsters should be checked daily for fight wounds. Female hamsters may show pseudopregnancy, usually as a result of an infertile mating. The animal can be examined for postovulatory discharge on days 5 and 9 after mating. If the discharge is present, she is having normal estrous cycles and is not pregnant. A hamster that is pregnant will have a distinct gain in weight, with abdominal distension, 10 days after mating. Studies have shown that the time of mating and the light-dark cycle under which the animals are housed have effects on the time of parturition (Viswanathan and Davis, 1992). Just prior to parturition, the female becomes restless and alternates between eating, grooming, and nest building. An increase in respiratory rate is also a sign that the young can be expected within the next several hours. The most common time for parturition is on the sixteenth day of gestation, and parturition itself usually lasts for more than 3 hr. A change toward maternal behavior occurs abruptly in late gestation for female Syrian hamsters; this differs from the gradual onset of maternal behavior observed throughout gestation in rats and mice (Buntin e t al., 1984). Litters range in size from 4 to 12 pups, with 6 to 8 being the most common. It is possible to sex the pups at birth by comparing the distance from the external urethral orifice to the anus (greater in males), but it is preferable to leave the litter undisturbed for the first 7 to 10 days. During this time, fresh food and water are provided for the mother, but no cage changes are performed. If it is necessary to disturb the litter, the dam should be provided with fresh food with which she can stuff her cheek pouches. This will decrease the likelihood of cannibalism of newborn pups by the mother. Pups are left with the mother until they are at least 19 days of age. Normal weaning time is 21-28 days, and the estrous cycle does not usually resume for the mother until 1 to 8 days following parturition (Battles, 1985). Young from different litters can usually be housed together until 40 to 50 days of age, when it becomes necessary to separate the females. Males from the

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F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

same litter can usually be kept together for a longer period of time.

F. 1.

M a n a g e m e n t and H u s b a n d r y

Caging and Environment

Hamsters can be maintained in colonies; however, mature animals are usually caged separately because of their tendency to fight. Females to be mated must be given some degree of isolation from adult males and pregnant or lactating females. A hamster weighing 60 gm or less requires about 10 in 2 of space. An animal over 60 gm should have 13-19 in 2 depending on body weight. A female with a litter should have approximately 150 in 2. The height of the cage for hamsters must be 6 inches from the cage floor to the cage top (Institute of Laboratory Animal Resources, 1996). Caging used for other laboratory rodents is acceptable for hamsters provided it is escape-proof. Hamsters are capable of chewing through thick wood and aluminum. Doors and corners

Fig. 6.

must be close-fitting, and latches must be secure. Plastic shoebox cages with locking lids are recommended (Fig. 6). It is essential to have a solid bottom for nesting females and for their young. Preference testing of hamsters found that solid-floored cages with litter material were more readily inhabited than wire cages; however, age and/or prior experience may have affected the choice by the animals (Arnold and Estep, 1994). Recommended bedding materials include processed hardwood chips, sawdust, shavings, corncobs, and beet pulp. Cedar and pine shavings may contain sharp edges and excessive dust, and should therefore be avoided. Aromatic hydrocarbons in these materials may induce nonspecific hepatic enzymes in the hamster (Harkness, 1994). Pregnant animals will use soft paper for nest building. Normal urine output is slight, and hamsters tend to consistently use one corner of the cage for elimination. Replacement of bedding materials can be routinely done once or twice weekly, and can be left for as long as 10 to 14 days when it is desirable to leave a litter undisturbed. Cages used for housing adult hamsters are maintained in an environment of approximately 640-79 ~F with 4 0 - 6 0 % humidity. Breeding rooms are kept slightly warmer, with a recom-

Shoe-boxcaging for hamsters. Note the placementof food on the cage bottom.

179

5. BIOLOGY AND DISEASES OF HAMSTERS

mended range of 71 ~176 F. Hamsters are fairly adaptable to cooler temperatures, and adult animals may actually prefer temperatures a few degrees above their hibernating environment. These animals are much less adaptable to excessively warm housing temperatures. A daily light period of 12 to 14 hours is recommended. The longer 14-hr period is required for breeding colonies. If natural daylight can be eliminated from the room, it is possible for the animals to adapt to an artificial light-dark cycle, which may be more convenient for laboratory management. A light intensity of 323 lux (30 ft-candles) measured approximately 1 meter above the floor has been recommended for rodents (Committee on Rodents, 1996). 2.

Handling and Restraint

Hamsters are nocturnal animals, so they tend to be quite inactive during the light cycle in the animal facility. Males are more docile and easier to handle than females. Frequent handling seems to reduce aggressiveness, but a startled or awakened hamster is likely to bite.

To move hamsters, place a small can or cup in the cage. The animal will usually enter the container, and the container with the hamster can be quickly moved to another cage. The easiest method of hand restraint is to grasp the hamster around the head and shoulders, approaching the animal carefully from the rear. Another method is to approach the animal in much the same way, but grasp only the skin. With the loose skin bunched securely in the hand, the skin is taut over the thorax and abdomen. As the animal is lifted, the hand holding the hamster is rotated so that the hamster's body is supported (Fig. 7). An alternative to this method is to approach from the animal's head, so that the thumb and forefinger are gripping the base of the tail; as before, the loose skin is secured between the fingers and the palmar surface before lifting. Still another method is to approach from the head and enclose the entire body with one hand. The thumb is placed at the base of the rear leg, with the first and second fingers on the opposite side at the base of the tail. The third and fourth fingers restrain the head and forelegs.

Fig. 7. One-handrestraint of hamsters is demonstrated. The excessive loose skin is gathered tautly around the neck as the animal is lifted.

180

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR. IIl.

A. 1.

DISEASES

Infectious Diseases

Bacterial Infections

a.

Enteritis

Infections of the alimentary tract include proliferative enteritis/ileitis, regional enteritis, enzootic intestinal adenocarcinoma, transmissible ileal hyperplasia, and "wet tail." i. Etiology. Proliferative enteritis is a fairly common spontaneous disease of hamsters (Renshaw et al., 1975). The importance of this disease in laboratory animal facilities appears to have diminished since it was initially reported in the mid-1960s (Cooper and Gebhart, 1998). Many different organisms have been implicated as the causative agents of the disease, including Campylobacter fetus subsp, jejuni, Escherichia coli, and Chlamydia trachomatis, strain SFPD; however, none of these have been consistently demonstrated in affected animals (Fox et al., 1981; Frisk et al., 1981; Fox et al., 1993, Zhang, et al., 1993; Dillehay et aL, 1994). Initial isolates of the putative causative organism were found to be curved or comma-shaped and morphologically similar to gram-negative rods like Campylobacterfetus (Jacoby, 1978). Using 16S rRNA sequencing and comparative analysis data, it was shown that proliferative enteritis in hamsters is not caused by a Campylobacter species (Fox et al., 1994; Peace et al., 1994). The causative organism isolated from hamsters with proliferative enteritis has now been identified as being nearly identical to Lawsonia intracellularis by 16S rDNA sequence analysis (Stills, 1991; Cooper, et al., 1997). Lawsonia intracellularis, related to Desulfovibrio desulfuricans, causes proliferative enteritis in swine and is an obligate intracellular bacterium that is very difficult to culture and manipulate (Cooper and Gebhart, 1998). ii. Clinical signs. Clinical signs of experimentally induced disease are observed within 2 weeks of inoculation. Watery diarrhea results in characteristic moist, matted fur on the tail, perineum, and ventral abdomen. Death occurs in 50 to 90% of cases, usually within 7 days after onset of clinical signs (Frisk et al., 1977). Chronic courses of proliferative enteritis have also been observed in hamsters with mild diarrhea and weight loss (Frisk et al., 1977). It is important to note that the disease may be self-limiting without clinical signs. iii. Transmission and epizootiology. Natural transmission most likely occurs by ingestion of contaminated fecal material. Animals from weaning to 2 months of age are most frequently affected. The disease can be transmitted experimentally by oral inoculation of tissue homogenate (Jacoby, 1978). Cross-species transmission has been shown to occur between infected swine

and hamsters (McOrist and Lawson, 1987). Vertical transmission has not been evaluated; however, it is not considered likely that L. intracellularis can cross the placenta to infect fetuses (Cooper and Gebhart, 1998). In addition, it is unknown how long L. intracellularis can survive in the environment and if this is important in natural infections (Cooper and Gebhart, 1998). iv. Necropsy findings. The gross lesions can include a segmental thickening and congestion of the ileum, enlargement of the mesenteric lymph nodes, peritonitis, and adhesions, although lesions are not always observed (Fig. 8). Histopathologic changes are characterized by hyperplasia of columnar mucosal epithelial cells in the terminal ileum, proliferation of glandular epithelium, and lymphadenitis with lymphoid hyperplasia, edema, and leukocytic infiltration of sinusoids (Frisk et al., 1977). Intestinal crypts may be lengthened, with increased mitosis, decreased numbers of goblet cells, and villar atrophy (Fig. 9). Finally, L. intracellularis organisms can often be identified, using Warthin-Starry silver stain, in the apical cytoplasm of crypt enterocytes (Cooper and Gebhart, 1998). v. Pathogenesis and diagnosis. Because of the susceptibility of young weanling hamsters to this organism, immunosuppression is probably a factor in the clinical manifestation of disease. The lesions observed in the ileum develop in two phases following the experimental transmission of the disease (Jacoby, 1978). The initial phase is characterized by hyperplasia, which begins as a focal lengthening of villi. Approximately 3 weeks following transmission, an inflammatory phase begins, associated with focal or segmental necrosis of crypt epithelium. The evolution of the lesions is closely associated with a particulate bacterial antigen that can be detected by immunoperoxidase staining or in situ hybridization in the cytoplasm of mucosal epithelial cells. It is not clear what mechanism is utilized by L. intracellularis to localize to the gastrointestinal tract; however, cellular receptors or factors in the microenvironment may be important (Cooper and Gebhart, 1998). The proposed model for entry into the crypt epithelial cells involves attachment of the bacteria to the microvillus brush border, ingestion by endocytosis, and release from vacuoles into the cytoplasm of the cell. Released bacteria may then multiply within the epithelial cells prior to cell rupture. Additional bacteria may then attach to neighboring epithelial cells and spread the infection more rapidly (Jasni et al., 1994). Serum antibodies have been detected that are specific for the intracytoplasmic antigen, which may be of diagnostic value (Stills, 1991). A sensitive and specific polymerase chain reaction (PCR) assay can also detect L. intracellularis in fecal samples (Jones etal., 1993). vi. Differential diagnosis. Other infectious diseases that should be considered for hamsters with diarrhea are Tyzzer's disease (Clostridium piliforme), Clostridium difficile enterotoxemia, antibiotic-associated diarrhea, and salmonellosis. Entero-

5. BIOLOGY AND DISEASES OF HAMSTERS

181

Fig. 8. The abdominal viscera of a hamster with proliferative enteritis. The arrows denote the thickening of the terminaljejunum and ileum. (Reprinted with permission from Harold E Stills Jr.)

cecocolitis has also been linked to [3-hemolytic E. coli, which may contribute to the pathologic lesions seen in proliferative enteritis (Dillehay et al., 1994). Microbiologic and pathologic findings should distinguish between the various possibilities. vii. Prevention, control and treatment. The history of potential suppliers with regard to enteritis should be reviewed before obtaining hamsters for the biomedical facility. Animals should be purchased from a colony with minimal disease his-

tory, and they should not be mixed with animals from other sources. Hamsters with diarrhea should be separated and isolated from other animals. Treatment should be supportive and aggressive to correct nutritional and electrolyte imbalances. Appropriate antibiotic therapy indicated for L. intracellularis should be administered. Tetracycline (10 mg/kg PO q12 hr for 5 - 7 days), enrofloxacin (10 mg/kg PO or IM q12 hr for 5 7 days), and trimethoprim-sulfa combinations (30 mg/kg PO q12 hr for 5 - 7 days) have been recommended; these can be

Fig. 9. The crypt epitheliumin an animalwith proliferativeenteritis. There are an increased number of mitotic figures (arrows)coupled with cellular immaturity in the epithelium. (Reprintedwith permission from Harold E Stills Jr.)

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F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

added to drinking water to control infections (Donnelly, 1997). Significantly, proliferative enteritis in hamsters is a multifactorial disease, and the clinical response to antibiotics may actually be the result of effects on other gastrointestinal flora (Cooper and Gebhart, 1998). viii. Research complications. Enteritis in hamsters can be a major problem in their experimental use because of its prevalence, variable morbidity (20-60%), and high mortality (approximately 90%). b.

Pneumonia

i. Etiology and prevalence. In an early survey of diseases of the hamster, pneumonia was the second most common clinical disease reported after diarrhea (Renshaw et al., 1975). The respective roles of bacteria, mycoplasmas, and viruses, or combinations thereof, in the etiology of hamster pneumonia are unclear. Possible bacterial etiologies include Pasteurella pneumotropica, Streptococcus pneumoniae, and other Streptococcus spp. Infection with Corynebacterium paulometabulum has been reported to cause acute pneumonia in hamsters; however, nasal infections with another strain, C. kutscheri, are subclinical in hamsters (Tansey et al., 1995). The role of Mycoplasma spp. in respiratory disease in hamsters has not been clearly defined. ii. Clinical signs. Overt manifestations of disease may include depression, anorexia, and nasal and ocular discharges, with "chattering" and respiratory distress. iii. Pathogenesis. Various causes of stress, including significant variations from recommended environmental temperatures, are contributing and predisposing factors to respiratory disease in the hamster. iv. Differential diagnosis. A judicious assessment of clinical signs, lesions, and the results of microbiology laboratory reports is essential to definitively diagnose the etiologic agent of pneumonia in hamsters. v. Prevention, control and treatment. Stressful situations should be avoided, and affected animals should be isolated. If treatment is necessary, the use of antibiotics to which the etiologic organism is sensitive may be appropriate. A number of antibiotics are associated with fatal enterocolitis in this species; therefore, careful selection of antimicrobials is imperative. c.

Tyzzer's Disease

This condition was first reported in Japanese Waltzing mice but has since been diagnosed in several other species, such as rats, guinea pigs, and hamsters (Ganaway et al., 1971; Waggie et al., 1987). The disease is caused by Clostridium piliforme, formerly Bacillus piliformis, an intracellular bacterium. Transmission is believed to occur through the oral ingestion of C. pil-

iforme spores from the feces of infected animals (Waggie et al., 1987). Clinical signs include roughened hair coats, diarrhea, and high mortality in animals that tend to be of weaning age or immunosuppressed (Donnelly, 1997). Reported necropsy lesions include enterocolitis, lymphadenitis, and multifocal necrotizing hepatitis (Fig. 10) (Nakayama et al., 1975). The diagnosis depends on the demonstration of the characteristic organism in the affected tissue, particularly in the epithelial and smooth muscle cells of the ileum, cecum, and colon, following special staining with Giemsa or silver techniques (Waggie et al., 1987). In experimental infections, inflammatory lesions may be present within 2 days of inoculation, while foci of liver necrosis occur within 4 days (Waggie et al., 1987). Infection with C. piliforme may not always manifest into clinical disease in the hamster. Outbreaks may have lesions localized only to the intestines, only to the intestines and liver, or primarily to cardiac muscle, with or without intestinal involvement (Nakayama et al., 1976; Magaribuchi et al., 1977; Zook et al., 1977). d.

Clostridium difficile

Enteritis associated with this bacterium has been linked to inappropriate antibiotic administration, stress, experimental manipulation, and heavy environmental contamination with C. difficile (Ryden et al., 1991; Rehg and Lu, 1982; BlankenshipParis et al., 1995b). Clostridium difficile does not readily colonize the normal, healthy intestinal tract; however, different toxigenic strains vary in their pathogenicity (Borriello et aL, 1987). One outbreak with toxigenic, cytotoxin B-positive C. difficile resulted in diarrhea that ranged from profuse and watery to hemorrhagic, and was highly associated with mortality (Chang and Rohwer, 1991). Histologic findings included typhlitis and colitis in these adult hamsters. In experimental infections, treatment with vancomycin and bismuth subsalicylate (BSS, PeptoBismol at 15 mg PO BID for 5 days) was found to delay death (Chang et al., 1990). Hamster models of atherosclerosis placed on high-fat and cholesterol diets may be prone to development of enteric disease associated with toxigenic C. difficile (Blankenship-Paris et al., 1995b). Necrohemorrhagic typhlitis and cecal mucosal hyperplasia were commonly seen in these hamsters. Alterations in diet may be risk factors in disease development due to changes in intestinal microflora, pH, and ability to mount immune responses (Blankenship-Paris et al., 1995a). The development of antibodies against the virulence factors, toxins A and B, has proved useful in preventing disease relapse and subsequent reinfections in hamsters (Kink and Williams, 1998). e.

Salmonellosis

The naturally occurring disease is rare in hamsters now, although outbreaks have been reported in the literature (Innes et al., 1956). The current rarity of salmonellosis is likely attrib-

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5. BIOLOGY AND DISEASES OF HAMSTERS

Fig. 10. Grosslesions in Tyzzer's disease includehepatomegalyand multifocalhepatic necrosis (arrows)as seen on the left. Intestinallesions, seen on the right, involve the ileum throughthe colon and include loss of tone and serosal edema. In some cases, hyperemiaand hemorrhagemay occur. (Reprintedwith permission from Sherri L. Motzel.) utable to well-managed facilities, improved quality of animals, regulated diets, and better animal care (Percy, 1987). At necropsy, there is multifocal necrosis of the liver, but no enteritis. Histologically, the disease is characterized by septic thrombi involving the veins and venules, an unusual feature of bacterial infection in hamsters. Preventive procedures should include the isolation of hamsters from other rodents and quality control procedures to preclude the introduction of contaminated food or bedding.

ence of PVM may be a complicating factor in the experimental use of the hamster, particularly in studies involving the respiratory tract.

2.

ii. Clinical signs. The type of disease observed, if any, varies due to a number of factors, including the age of the hamster at the time of infection, the strain of animal and virus, the route and dose of infection, and the immune status of the host. Approximately half of the hamsters infected congenitally or as newborns develop a chronic, progressive fatal disease characterized by inactivity and weight loss (wasting disease). Other animals infected in vitro or as newborns and young adults develop subclinical infections. An impairment of reproductive performance has been reported for chronically infected female hamsters (Parker et al., 1976).

Viral Infections

a.

Prevalence

Current recommendations indicate that several viral infections should be monitored serologically in hamster breeding units. These include, but are not limited to, lymphocytic choriomeningitis virus (LCMV), pneumonia virus of mice (PVM), reovirus type 3 (Reo 3), Sendai virus, and simian virus 5 (SV5) (Federation of European Laboratory Animal Science Associations Working Group on Animal Health, 1994; Committee on Rodents, 1996). Three different groups have reported on the presence in Syrian hamsters of antibodies to 10 or 11 murine viruses. As there is only one anecdotal report of PVM causing natural disease in the hamster, it appears that the infection in hamsters is usually subclinical. Nonetheless, the potential pres-

b.

Lymphocytic Choriomeningitis Virus

i. Etiology. The infection is caused by an RNA virus of the arenavirus group. Mice are most commonly associated with the disease, but the virus has been isolated from a variety of other species.

iii. Transmission. The implantation of virus-containing tumors has been the principal method of transmission in experimental hamsters. Other methods of spread are by direct contact,

184

and by fomites and aerosols, which are attributed to the excretion of high viral titers in urine for varying lengths of time (Fox et al., 1984). Congenital infections have also been reported.

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

transplanted tumors, while the third episode was related to pet hamsters.

c. iv. Necropsy findings. The histopathology of animals developing disease, subsequent to congenital or neonatal infections, consisted of chronic glomerulonephropathy and widespread vasculitis. v. Pathogenesis. The experimental infection of young adult hamsters results in a viremia that decreases in titer over a period of 3 months. The levels of virus detectable in the urine exceed viral amounts detected in the blood. Virus excreted in the urine persists longer than that in the blood. Complement-fixing antibodies appear by the tenth day postinfection, reach peak levels at the sixtieth day, and decline slowly. Some hamsters infected neonatally remain healthy and follow a pattern of infection similar to that of young adults. However, other neonates develop disease with persistent viremia and lower levels of both complement-fixing and neutralizing antibodies. The presence of viral antigen and y-globulin in the glomeruli of affected hamsters suggests an immune complex mechanism for the glomerulonephropathy, analogous to that reported for LCMV disease in mice (Parker et al., 1976). vi. Differential diagnosis. Other potential causes of wasting disease include graft versus host disease and any procedures resulting in suppression of normal immune responses. The renal lesions should be differentiated from glomerular amyloidosis. vii. Prevention, control and treatment. A quality-assurance program that includes the regular testing of hamster colonies for antibodies and transplantable tumors for virus, with the elimination of infected animals or tumors, is the principal means of prevention. Since wild house mice can be reservoirs of infection, their direct or indirect contact with experimental animal colonies should be avoided. Lymphocytic choriomeningitis virus is zoonotic and can be transmitted to humans through contact with rodents. The spectrum of disease manifested in humans varies from asymptomatic infection to rare cases of severe infection localized to the central nervous system. Humans that work with large numbers of rodents, particularly in laboratory animal facilities, should be aware of the risk of transmission and follow appropriate personal protective measures. viii. Research complications. Superinfection of the spontaneous hamster tumor designated Fortner fibrosarcoma No. 2 resuited in complement-fixing antibodies reactive with tumor extracts that mimic specific tumor antigens observed with virusinduced tumors. Especially noteworthy are three human epidemics involving 236 cases of LCMV that occurred in the United States during 1973-1974 (Gregg, 1975). Two of the episodes were associated with experimental hamsters bearing

Sendai Virus

i. Etiology. Parainfluenza 1 (Sendai) is a single-stranded pleomorphic RNA virus of the Paramyxoviridae family and is closely related to the human virus hemadsorption type 2 (HA-2). Although mice are believed to be the natural host and most common laboratory animal affected, rats, hamsters, and guinea pigs are also susceptible to natural infections. Initial reports of the condition in hamsters were from Japan (Matsumoto et al., 1954). ii. Clinical signs. Sendai infection may lead to mortality in newborn pups; however, most infections are subclinical in hamsters. iii. Epizootiology and transmission. An enzootic form of the infection has been reported at a research facility in association with the periodic, but continuous, introduction of susceptible hamsters from a commercial vendor (Profeta et al., 1969). iv. Necropsy findings. Consolidation of the lungs has been reported (Profeta et al., 1969). Experimental infections in hamsters have resulted in hyperplasia of the nasal mucosal epithelium, hyperplasia of bronchial epithelium, peribronchial edema, and peribronchial lymphocytic infiltration, which resolves within 2 weeks postinoculation (Percy and Palmer, 1997). These findings concurred with those seen in a Sendai viral vaccine study (Tagaya et al., 1995). Lesions and sites of viral replication within the respiratory tract are similar to those reported in strains of laboratory mice (Percy and Palmer, 1997). v. Pathogenesis. In the enzootic form of the disease in mice, virus can be recovered from the lungs for approximately 2 weeks, with 50% of the animals between 3 and 6 weeks of age yielding virus (Parker and Richter, 1982). Sendai virus infection in laboratory mice has effects on both the immune response and the antimicrobial activity of macrophages. These parameters have not yet been evaluated in hamsters infected with the virus. Complement-fixing and hemagglutining-inhibition antibodies develop in essentially 100% of the hamsters by 9 weeks of age and persist for 1 year or longer. vi. Differential diagnosis. Additional causes of pneumonia that should be excluded for differential diagnostic purposes include Corynebacterium spp. (Tansey et al., 1995), Streptococcus pneumoniae, Pasteurella pneumotropica, other Streptococcus spp., and PVM (Renshaw et al., 1975). vii. Prevention, control, and treatment. Based on the likelihood that other laboratory animal species are the source of Sendai virus infections observed in hamsters, experimental

185

5. BIOLOGY AND DISEASES OF HAMSTERS

hamsters should be housed in rooms separate from mice, rats, and guinea pigs. Hamsters from different sources should not be housed in the same room unless all sources are known to be free of the virus. In addition, analogous procedures described for mice should be applicable to hamsters (Parker and Richter, 1982). viii. Research complications. Sendai virus can be fatal in outbreaks in research colonies (Profeta et al., 1969). In addition, reports of immunosuppressive effects of the virus in other species may be extrapolated to infection in hamsters (Garlinghouse and Van Hoosier, 1978). Due to effects on the nasal mucosal epithelium, and given the importance of olfactory cues to the hamster, Sendai viral infection may complicate studies of behavior and olfactory function in hamsters (Murphy and Schneider, 1970; Percy and Palmer, 1997). d.

Type C Virus

i. Etiology. The type C oncovirus group includes the Moloney murine leukemia and sarcoma viruses, the feline sarcoma and leukemia viruses, gibbon ape leukemia virus, and the guinea pig and bovine type C oncoviruses. Syrian hamster type C virus antigens (SHCVA) were initially observed in human adenovirus-induced hamster tumors (Hatch et al., 1975). Syrian hamster reproductive tissues have been found to express unique retroviral sequences related to type C viruses (DeHaven et al., 1998). ii. Clinical signs. The development of tumors may occur in animals that have been inoculated with viral particles, but there are conflicting reports in the literature. Overt clinical signs have not been reported. iii. Epizootiology, transmission, and pathogenesis. Although specific information is not available for the hamster type C virus, other viruses of this group have copies of their genomes integrated into normal cell chromosomes. This viral genetic material may not ordinarily be expressed but may be activated by physical or chemical agents or by superinfection with other oncogenic viruses. iv. Research complications. The presence of hamster agents that resemble known leukemic agents complicates research that involves testing the oncogenic potential of other animal or human agents. The presence of type C virus particles in hamster pancreatic carcinoma lines could potentially complicate research conclusions in immunological or biochemical experiments. In these cell lines, viral protein production could be mistaken for tumor-associated antigens (Sindelar et al., 1983). e.

Adenovirus

Infections with adenovirus have been reported in mice, rats, and other rodents. Mice tend to be naturally infected with two

strains, mouse adenovirus strain FL (MAd FL) and K87 (MAd K87). Mouse adenovirus FL systemic infection can be chronic, with viral shedding in the urine occurring for several months; in contrast, MAd K87 infection is localized to the intestine, with fecal shedding of virus lasting for 3 - 4 weeks (Richter, 1986). Hamsters have been serologically positive for antibodies to MAd FL, although reports of adenoviral infections are sporadic (Suzuki et al., 1982). Naturally occurring enteric adenovirus infection in hamsters, closely resembling infection with MAd K87 in mice, is not associated with clinical disease and affects animals less than 24 days of age (Gibson et al., 1990). Adenoviral intranuclear inclusion bodies may be found in the intestinal epithelium in young hamsters. f

Hamsterpolyomavirus

Hamster polyomavirus (HaPV) was first described in Germany in association with spontaneous skin tumors (Graffi et al., 1967). When the virus was inoculated into newborn hamsters, lymphomas and leukemias developed in the colony (Graffi et al., 1969). Within the United States, similar reports have identified lymphomas of B- and T-cell origin (Coggin et al., 1983). However, later studies linked HaPV to epitheliomas and not to the etiology of the lymphomas (Coggin et al., 1985). Cloning experiments have found great similarity in the HaPV genome to that of mouse polyomavirus; however, there are major differences between the two viruses in their transformation properties in vivo (Delmas et al., 1985; Courtneidge et al., 1991). The incidence of tumors in hamsters is high (30-80%), with a 4- to 8-week latency period (Scherneck and Feunteun, 1990). Viral infection has been divided into three phases: (1) acute infection with high replication of HaPV DNA in hematopoietic organs and liver, (2) viral clearance at 10 days postinoculation, and (3) accumulation of HaPV DNA at sites of tumors (Prokoph et al., 1996). There appears to be a strong link between tissue specificity of viral replication and the type of tumor induced (Prokoph et al., 1996).

3.

Parasitic Diseases

a.

Protozoas

Fecal smears of Syrian hamsters are literally a "gold mine" for a protozoologist, as one can observe a large number and variety of organisms. Yet, their etiologic role in enteric disease remains a matter for speculation, as they have been found in comparable numbers in diverse species in both healthy and diseased animals. The presence of Hexamita sp. has been reported as an incidental finding (Wagner et al., 1974). Tritrichomonas muris has been successfully eradicated from the intestinal tract using a regimen of 80 mg of metronidazole administered intragastrically for 6 days (Taylor et al., 1993). This protocol is insufficient for the eradication of Giardia muris.

186 b.

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

Nematodes

The mouse pinworm, Syphacia obvelata, has been observed in hamsters (Taffs, 1976). Even though the reported prevalence is less than 1%, infection rates can be high in individual colonies (Wantland, 1955). Uninfected hamsters can become infected with S. muris, the rat oxyurid, as a consequence of direct contact with infected rats (Ross et al., 1980). Eradication has been reported by two treatment courses with piperazine citrate (10 mg/ml of drinking water) for 7 days separated by 5 days without treatment (Unay and Davis, 1980). Cross-infection of nematodes from other cricetid rodents has been reported, including infection by Dentostomella translucida (Greve, 1985). The life cycle of this parasite is direct, and transmission occurs through infected cages and bedding material (Wightman et al., 1978).

c.

Cestodes

i. Etiology (prevalence, host range). Hymenolepsis nana, the dwarf tapeworm, is the most important internal parasite found in hamsters; infection may be common in animals from commercial colonies. Hymenolepsis nana ranges in size from 25 to 40 mm in length and is usually found in the small intestine. The host range includes mice, rats, nonhuman primates, and humans. ii. Clinical signs. The consequences of infection are usually benign, but the effects depend on the number of parasites and degree of intestinal occlusion, as impactions have been reported. iii. Epizootiology and transmission. Hymenolepsis nana is the only known tapeworm with either a direct or an indirect cycle; flour beetles or fleas serve as the intermediate host. The direct cycle is 14-16 days, while the indirect life cycle is variable. Autoinfection can also occur. iv. Diagnosis. A diagnosis can be made by the demonstration of eggs in the feces or by isolation of the mature worm in the intestines at necropsy. Hymenolepsis nana can be distinguished from H. diminuta by the presence of hooks on the scolex in the former. v. Prevention, control and treatment. Preventive and control measures include isolation and quarantine of newly acquired animals, effective insect and wild-rodent control, and regular sanitation of cages and ancillary equipment. Yomesan (niclosamide) has been reported as safe and effective for treatment (Ronald and Wagner, 1975). vi. Research complications. Although any infection associated with morbidity and mortality may interfere with research, the primary significance of H. nana is its transmissibility to hu-

mans. Accordingly, personnel working with infected hamsters should be informed of potential transmission and receive instruction in appropriate hygienic procedures.

d.

Mites

i. Etiology and prevalence. Ascariasis in hamsters is predominantly associated with two species of the genus Demodex (D. criceti and D. aurati). In addition, infections with ear mites (Notoedres sp.), the tropical rat mite (Ornithonyssus bacoti), and a nasal mite (Spleorodens clethrionomys) have also been reported. Despite high infection rates with Demodex spp., clinical signs of skin disease are uncommon. ii. Clinical signs. Alopecia, predominantly of the rump and back, with dry, scaly skin has been noted in association with D. aurati and D. criceti (Estes et aL, 1971). Notoedric mange in the female hamster usually affects only the ears; however, in males, lesions may also be observed on the nose, genitalia, tail, and feet. iii. Pathology. The cases of demodectic mange reported by Estes et al. (1971) were characterized by dilated hair follicles that contained debris and mites, loss of hair shaft, an increase in thickness of the corneum, and little evidence of inflammation. iv. Pathogenesis. Demodectic mange has been observed in hamsters involved in a lymphosarcoma transmission study and a chemical carcinogenesis study, although lesions were apparently more related to increasing age than experimental procedures (Estes et al., 1971). Males may be more susceptible to infection and disease than females. Van Hoosier (1965) has personally observed clinical signs in hamsters thymectomized as newborns. v. Diagnosis and treatment. A diagnosis can be established by the demonstration of mites in skin scrapings, even though the presence of Demodex spp. in association with lesions does not necessarily establish a cause-and-effect relationship. Initial treatment involves combined therapy with 1% selenium sulfide shampoo and topical application of 0.013% amitraz (Hasegawa, 1995). vi. Research complications. Demodicosis, as a clinical condition, is a potential cause of complication in experimental protocols involving hamsters. B.

Neoplastic Diseases

1. Introduction The degree of background tumor incidence in Syrian hamsters has been a controversial topic in the literature. Although some reports state that hamsters have a low incidence of natu-

187

5. BIOLOGY AND DISEASES OF HAMSTERS

rally occurring tumors, e.g., m a m m a r y tumors and laryngeal carcinomas, other groups have found the occurrence of spontaneous tumors to be quite high (Homburger, 1983; Pour et al., 1979). The overall incidence of spontaneous malignant neoplasms in Syrian hamsters has been estimated to be 3.7%, based on 435 tumors in 11,972 animals (Van Hoosier and Trentin, 1979). Factors affecting the difference in numbers of tumors reported include the strain, age, and sex distribution of the animals observed; the environmental conditions and source of the colony; and the extent to which the animals are examined grossly and microscopically. Early tumor classification schemes have been provided in Table VI. 2.

Benign Neoplasms

Reported benign neoplasms of the Syrian hamster include intestinal polyps, adrenal adenomas, splenic hemangiomas, islet cell pancreatic tumors, hepatic adenomas, squamous papillomas of the forestomach, and fibroadenomas of the m a m m a r y gland. One report found the incidence of benign tumors to be 10% in males and 13% in females when 200 control animals from the BIO 15.16 line were examined (Homburger, 1983). Table VII lists the types of tumors for which 10 or more cases have been reported. 3.

Malignant Neoplasms

L y m p h o s a r c o m a is the most frequently reported malignant tumor of the Syrian hamster. Noteworthy is the observation of a 10:1 male-to-female ratio in a total of 30 reported melanomas. Other malignancies include adrenal cortical carcinoma, renal

Table VII

Benign Tumorsa Group (as in Table VI) I.

I. Tumors of epithelial tissue A. Glandularorigin B. Postulated, but unconfirmed, glandular origin C. Nonglandular II. Lymphomas A. Lymphosarcoma and/or lymphocytic leukemia, or both (with neoplastic involvement of organs and/or leukemic blood) B. Myelocyticleukemia (including chloroleukemia) C. Reticulumcell sarcoma D. Plasma cell tumor III. Tumors of connective tissues A. Known specific cell type, e.g., fibrosarcoma B. Nonspecific cell type, e.g., "mixed-cell, round cell, or sarcoma" IV. Tumors of melanin-forming tissue V. Tumorsof neural tissue VI. Tumors of mixed tissues VII. Tumors not classified elsewhere aFrom Stewart et al. (1959).

Polyps Adenoma

III.

Papilloma Hemangioma

IV.

Cholangioma Thecoma Cellular blue nevi

Intestine Adrenal Thyroid Parathyroid Stomach Spleen Liver Bile duct Ovary Skin

Relative frequencyb ++++ ++++ +++ ++ +++ +++ ++ ++ ++ ++

a From Van Hoosier and Trentin (1979). b'lt- -3t- -~- -1t-, most common; + + +, common; + +, occasional. cell carcinoma, and subcutaneous sarcoma (Homburger, 1983). Table VIII lists the types of malignant tumors for which 10 or more cases have been reported. Of special interest in this regard are the reported outbreaks of horizontally transmitted malignant lymphomas in a hamster colony, with an incidence of 50 to 90% in young inbred and random-bred animals (Ambrose and Coggin, 1975). The agent associated with the disease is the hamster polyomavirus (HaPV) (Coggin et al., 1983; Prokoph et al., 1996). These reports are important because the disease poses an epizootic threat to experimental colonies and because the disease condition in hamsters is a valuable model for understanding h o s t - v i r u s relationships in other species. Table VIII

Table VI

Classification of Tumors of Animals According to Site of Origin and Histologya

Site

Tumor type

Malignant Neoplasms of the Hamstera Group (as in Table VI) I.A.

II.A.

II.C. II.D.

Tumor type Adenocarcinoma Carcinoma 26 Spindle cell carcinoma 6 Adenocarcinoma 1 Carcinoma 2 Carcinoma 1 Lymphosarcoma

Reticulumcell sarcoma Plasmacell tumor

Site

Relative frequencyb

Intestine Adrenal Thyroid

+ + + + + + + +

Liver/intrahepatic bile duct Uterus Kidney Lymph node Small intestine Liver Kidney Spleen Lymph node

+ +

Extramedullary

+ +

+ + + + + + + +

+ + + + + + + + + + + + +

aFrom Van Hoosier and Trentin (1979). b+ + + +, most common; + + +, common; + +, occasional.

F. CLAIRE HANKENSONAND GERALD L. VAN HOOSIER JR.

188

C.

Miscellaneous Diseases

1. Amyloidosis and Associated Nephrotic Syndrome Amyloidosis, a disease in which normally soluble proteins polymerize as insoluble fibrils, is a principal cause of death in hamsters on long-term experiments. The two components of amyloid are amyloid A (AA), which is derived from amyloid fibrils, and amyloid P (AP), also known as female protein (FP), a member of the pantraxin family of plasma proteins (Tennent et al., 1993). Studies have shown that sex hormones regulate the expression of AP and that levels in females are normally 100- to 200-fold greater than levels in males (Coe et aL, 1981). When compared to male Syrian hamsters, female Syrian hamsters have a distinct predisposition to acquire amyloidosis, which is directly related to serum levels of AP, either normally with aging or experimentally (Coe and Ross, 1990). Testosterone has been linked to the inhibition of hepatic synthesis of AP, which is the homolog of primary importance in the deposition of amyloid (Coe and Ross, 1990). The incidence of nephrotic syndrome due to amyloidosis was reported to be 6% in a colony of Syrian hamsters, with ascites and anasarca observed (Murphy et al., 1984). The primary gross lesions are pale tan, enlarged, and misshapen kidneys (Fig. 11). Serum albumin is decreased and the total globulin component is increased in hamsters over 1 year of age. Proteinuria and hypercholesterolemia have been

reported. These clinical signs and laboratory findings are consistent with the nephrotic syndrome described in humans. Histologically, characteristic amyloid deposits are present initially in the glomeruli of the kidney and subsequently in a variety of tissues, especially the spleen, liver, and adrenals. Because amyloidosis in mice is strain-associated, genetic factors should be considered in the etiology and pathogenesis of the disease in hamsters. Amyloidosis in the Syrian hamster can be used as a model to understand the pathogenesis of the same disease in humans, including the form linked to Alzheimer's disease (Coe et al., 1997). 2. Polycystic Disease Cysts have been observed in 76% of hamsters over 1 year of age (Gleiser et al., 1970). The liver is a common site, but several organs may be affected (Table IX). The liver lesions are related to developmental defects of normal ductal structures (i.e., bile ducts), whereas cysts in other organs likely develop from dilations of the lymphatic system. The condition may be associated with distension of the abdomen, but other clinical signs have not been recorded. Typical findings include cysts of variable size that can be unilocular to multilocular (Kaup et al., 1990). At necropsy, the cysts are thin-walled with clear watery fluid varying in color from amber to green. Findings in one study mentioned that the proteinaceous nature of the fluid re-

Fig. 11. Classicappearanceof amyloidosisin the kidneys on the right. Normalhamsterkidneys are shownon the left for comparison. (Reprintedwith permission from J. Derrell Clark.)

189

5. BIOLOGY AND DISEASES OF HAMSTERS Table IX Frequency of Cysts at Various Sites Number (%)

Organ systems Gastrointestinal system Esophagus Liver Cecum Colon Reproductive system Seminal vesicle Epididymis Uterus Ovary Endocrine Adrenal Pancreas Other Kidney Spleen Gleiser e t al. (1970). b Kaup et al. (1990).

a

Syrian hamster a

European hamster b

1/40 (2.5 %) 17/40 (42.5%) m m

54/150 (36.0%) 18/150 (12.0%) 1/150 (0.7%)

4/17 8/17 1/23 1/23

(23.5%) (47.0%) (4.35%) (4.35%)

3.

m

13/150 (8.7%)

1/40 (2.5%) 5/40 (12.5 %) 2/40 (5.0%) ~

sulted in white solidified collections within cysts in the cecal walls (Kaup et al., 1990). Reports have found that a higher incidence of intraperitoneal cysts occurs in European hamsters than in Syrian hamsters.

7/150 (4.7%) 2/150 (1.3%)

Antibiotic-Associated Enterocolitis

Morbidity and mortality have been observed in hamsters following the administration of antibiotics generally considered to be selective for gram-positive bacterial organisms. Administration of penicillins, vancomycin, erythromycin, cephalosporins, and gentamicin can induce a fatal enterotoxemia. Clostridium difficile has consistently been found in high concentrations in the intestinal tracts of these animals, and cell-free supernatants can experimentally reproduce the disease (Bartlett et al., 1978). The administration of tetracycline and metronidazole are not associated with the disease (Bartlett et al., 1978). Anorexia and diarrhea can be detected within 4 to 19 days after dosing with oral antibiotics; ileal and cecal distension with hyperemia can be seen at necropsy (Fig. 12) (Small, 1:968).

Fig. 12. Lesions of Clostridium difficile enterocolitis. Note the distended cecum and markedly hemorrhagic distal small intestine (arrows). (Reprinted with permission from Susan V. Gibson.)

190 4.

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR. Chronic Hepatitis

Chronic hepatitis and cirrhosis were first described as an incidental finding during various carcinogen studies in 1965 (Chesterman and Pomerance, 1965). Disease has been linked to dietary contamination, infection with bacterial pathogens, and immune system abnormalities, yet the actual etiology is not known (Brunnert and Altman, 1991). The primary means of diagnosis has been from necropsy, since there are usually no clinical signs of disease, even in cirrhotic animals (Hamilton and Reynolds, 1983). In most strains, females are more commonly affected than males (Homburger, 1972b). Significant elevations of both alanine aminotransferase (ALT) and bile acids may be seen on serum clinical chemistries (Brunnert and Altman, 1991). Chronic hepatitis in laboratory animals other than hamsters has been linked to the bacterial pathogen, Helicobacter hepaticus. The role of Helicobacter spp. in liver disease of hamsters has not been studied; however, H. cholecystus, found in hamster gallbladders, is strongly associated with cholangiofibrosis (Franklin et al., 1996).

CHINESE HAMSTER

I.

INTRODUCTION

The Chinese hamster (Cricetulus griseus), also known as the striped-back hamster, was first used as a laboratory animal in 1919 (Yerganian, 1985). Benefits such as small size, polyestrous cycle, short gestation period, and low chromosome number encouraged the use of the Chinese hamster in biomedical research. Today, use of this animal in research is greatly overshadowed by the extensive use of cell lines derived from its ovarian cells. These Chinese hamster ovary (CHO) cells are used for cell culturing experiments to obtain heterologous protein products (Oka and Rupp, 1990). The Chinese hamster has been shown to be susceptible to a number of infectious disease agents, such as Streptococcus spp., mycobacterias, diphtheria, rabies, influenza, and equine encephalitis (Yerganian, 1958). Originally, the number of chromosomes of the Chinese hamster was thought to be 14; later studies defined 22 chromosomes. Because of the low incidence of spontaneous and endogenous viral infections, Chinese hamster tissue culture cells have become popular experimental tools for mutagenic and carcinogenic studies. These animals are also used as models for radiobiological research and have been shown to be more radioresistant than the Syrian hamster and many other common laboratory rodents (Corbascio et al., 1962). Spontaneous hereditary diabetes mellitus with similarities to the human disease has been described (Meier and Yerganian, 1959), thus providing

an animal model that has subsequently been studied extensively. The Chinese hamster has also been shown to be susceptible to experimental induction of stomach and esophageal cancer (Baker et al., 1974). Chinese hamsters can be reared under laboratory conditions and can be purchased commercially. Because their size is comparable to that of the mouse, a similar type of caging is adequate. These animals do very well under standard laboratory animal housing conditions. However, animals that are diabetic are very susceptible to stress; special precautions must therefore be taken in their routine care and feeding.

II.

BIOLOGY

The Chinese hamster, like the Syrian hamster, has a cheek pouch that can be utilized as an immunologically privileged site (Yerganian, 1958). Another unique biological feature of this animal is a chromosome number of 22, which is beneficial for cytogenetic studies. The 10 large pairs of autosomes and 2 sex chromosomes can be readily differentiated. The constant diploidy maintained in cell culture provides a stable cell system for assessment of agents with known or suspected mutagenic and carcinogenic properties. Adult animals weigh between 39.3 and 45.7 gm, and are approximately 9 cm long (Fig. 13). Newborns weigh 1.5 to 2.5 gm. The average normal life span under laboratory conditions is 2.5 to 3.0 years. Adult males have exceptionally large testicles; also, the spleen and brain in both sexes are relatively larger, with respect to overall body size, than those of the Syrian hamster (Festing, 1972). The normal hemogram is shown in Table X (Moore, 1966). There appear to be no unique dietary needs for Chinese hamsters. They do very well on standard rodent chow, but wheat germ may be used as a supplement for breeders. The average daily water intake was shown to be 11.4 ml per 100 gm body weight for males and 12.9 ml per 100 gm body for females (Thompson, 1971). Early attempts to breed Chinese hamsters under laboratory conditions were unsuccessful. However, in 1958, a reversed illumination schedule was used to successfully establish a production colony (Yerganian, 1958). Normal reproductive data are shown in Table XI (Moore, 1965). Sexual maturity is indicated by vaginal opening, with a mucus-like, creamy material frequently secreted at the beginning of estrus. The estrous cycle consists of four phases, with distinct behavioral characteristics associated with vaginal orifice changes (Yerganian, 1958). The use of tester males, as well as routine examinations of the vulva, has been a means of determining estrus and optimal breeding time. Several programs for managing breeding colonies have been

191

5. BIOLOGY AND DISEASES OF HAMSTERS

Table XI Reproductive Data for the Chinese Hamster 21-25 days a 8-12 weeks a Polyestrus b 4 days a 6-8hr a Immediately before estrus b 2 - 4 hr after start of dark period C 5 - 6 days b 20.5 days a 4.5-5.2 b 8b

Weaned Sexually mature Type of estrous cycle Duration of estrous cycle Length of estrus Ovulation time Copulation Implantation Gestation Average litter size Number of mammae Postpartum estrus

4 days a

Yerganian (1958). bFesting (1972). c Moore (1965).

a

Fig. 13. Appearance of the adult male Chinese hamster, Cricetulus griseus. Note the dark stripe of fur along the dorsal midline.

described. Because females can become very aggressive immediately following mating and even kill the male, hand mating was originally used. However, selective monogamous breeding with docile females having high fecundity was found to be very successful for establishing breeding colonies (Calland et aL, 1986). It has also been shown that Chinese hamsters can be mated in groups (Cisar et al., 1972). Temperatures above 82~ may increase the number of runts in litters (Yerganian, 1958). Infertility in young females in open-bottom cages may be due to an excess growth of hair Table X Normal Hemogram of the Chinese Hamster a Parameter

Value _ SD

Leukocytes Mature neutrophils Bands Lymphocytes Monocytes Eosinophils Basophils RBC Hemoglobin MCV MCHC PCV Bleeding time b

5500 105,600 _+ 12,100 935 ___385 413,600 11,500 -+- 1760 9350 _-Z-3850 825 _+ 220 7,120,000 ___ 1029 12.4 g/dl 59.4 ___5.8 29.49% 55 sec

Data from Moore (1966). bFrom the cheek pouch (Yerganian et al., 1955).

a

WBC (%)

19.2 _+ 2.2 0.17 _+ 0.07 75.2 2.1 ___0.32 1.7 _ 0.04 0.15 __+0.04 (4.4-9.1)

around the vulva, preventing penile penetration during copulation attempts. Progesterone levels are significantly different during the estrus cycle and pregnancy. During the 4-day cycle, maximal synthesis of progesterone occurs on day 3, which differs from the low levels found on day 3 of the Syrian hamster cycle (Sato et al., 1984). Progesterone levels in peripheral blood increase through day 12 of gestation, stabilize until day 18, peak on day 19, and then dramatically drop prior to parturition (Sato et al., 1984). Pregnancy is indicated by a closed vagina with dry, pale, and scaly perineal tissues at day 4 following mating. Dystocia may occur as a result of fetal wedging in the proximal portion of the vagina during parturition attempts. The fetuses can be saved by surgical removal. Newborn animals have front incisors. Body hair appears at 3 to 4 days of age, with complete coverage in 7 days. Eyes and ears open within 10 to 14 days, and testicles descend in males at about 30 days of age. As animals approach sexual maturity, aggressive females tend to fight. Dominance is established, and separation of the litter may be necessary to prevent trauma and possible deaths.

lIl.

A.

DISEASES

Infectious Diseases

The Chinese hamster appears to be experimentally susceptible to a number of infectious disease agents. However, very little has been reported concerning spontaneous infections. Tyzzer's disease is similar to that seen in Syrian hamsters. The animals may be more susceptible to infection due to environmental stresses of management.

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F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

The presence of antibodies against murine viruses has also been reported in Chinese hamsters. Parasitic infections may include persistent intestinal colonization with Trichomonas spp; however, few reports of other infecting endo- and ectoparasites exist in the literature. Despite the recent identification of Demodex sinocricetuli (Desch and Hurley, 1997) in Cricetulus barabensis (a species considered to be synonymus with C. griseus), there appears to be a very low susceptibility to demodectic mange in Chinese hamsters (Benjamin and Brooks, 1977). B. 1.

Metabolic/Genetic

Diseases

Diabetes Mellitus

Spontaneous diabetes mellitus was first recognized in during the course of inbreeding and was described in (Meier and Yerganian, 1959). The disease is similar in a ber of aspects to insulin-dependent diabetes of humans ganian, 1965).

1957 1959 num(Yer-

a. Etiology. The disease is associated with a degranulation of the [3 cells of the pancreatic islets of Langerhans, resulting in a primary defect in the biosynthesis of insulin. b. Clinical signs. Animals can show signs as early as 18 days of age, but the disease may occur at any age. Polydipsia and polyuria develop, with up to 50 to 70 ml of urine passed in 24 hours. The abdomen is usually soiled with urine, and an odoriferous smell may be apparent. At the onset of disease, there is an initial weight gain, but animals soon become sluggish and may die due to dehydration. Occasionally, hamsters develop blindness. Nonspecific conjunctivitis and alopecia may be seen. Animals are very susceptible to mild stress of any kind, and sudden death may be triggered by such procedures as cage transfer and changes in environmental temperature. Diabetic females may be infertile, but hamsters that do become pregnant tend to have increased numbers of abortions and fetal deaths at delivery. It is not unusual for an entire litter to die following parturition. c. Epizootiology. The disease appears to be transmitted as a recessive factor. When glucosuria was used to characterize diabetes, it was shown that 4 recessive genes were involved (Buffer and Gerritsen, 1970). If any 2 of the 4 genes were homozygous, glucosuria could result. Apparently the duration, severity, and constancy of glucosuria is controlled by modifier genes. It has also been shown that 100% of the offspring become diabetic if the parents are ketotic (Gerritsen et al., 1970). When parents are not ketotic, it is difficult to predict which offspring might become diabetic. Inbred lines of diabetic Chinese hamsters are now used in biomedical research.

d. Necropsy findings. Macroscopic lesions are confined mainly to the kidneys, which are slightly enlarged, spongy, and friable in diabetic animals. The renal pelvis may or may not be dilated. When hydronephrosis is seen, the retained urine is clear and odoriferous, and the urinary bladder is usually distended with urine. In some animals, the liver may be moderately enlarged with a yellow to gray color. Microscopically, the pancreatic islets of Langerhans are decreased in number (Meier and Yerganian, 1959). There is a decrease in the number of [3 cells; remaining cells stain lightly basophilic with cytoplasmic granulation and vacuolization. There is periodic acid-Schiff (PAS)-positive material within the cytoplasm that accumulates around pyknotic nuclei. Ultrastructural findings in the pancreas have been characterized (Boquist, 1969). Renal convoluted tubules contain much protein precipitate, and glomeruli are hypocellular with marked sclerosis. Intercapillary homogeneous material can be observed that is PAS positive. Bowman's capsule is slightly to moderately thickened, and adhesions between the glomerulus and capsule may be seen. PAS-positive material is also found in the basement membrane; the latter may be wrinkled and slightly thickened. The liver shows an intact lobular arrangement with extensive vacuolization of cells. Perinuclear haloes are seen. Intracytoplasmic material is PAS positive, but negative when stained for fat. PAS-positive material is occasionally found in pericardial adipose tissue. e. Pathogenesis. The basic defect is a degranulation of [3 cells, which results in a decreased amount of insulin production with a reciprocal increase in glucagon. In highly inbred glucosuric strains with diabetes, there is a greatly reduced level of pancreatic insulin and a significantly elevated level of glucagon in both pancreas and stomach. A decrease in lactate dehydrogenase (LDH) isozymes appears to be associated with severity of 9the diabetic condition (Chang et al., 1977). A contributing factor to the observed renal pathology may be subnormal levels of specific glycosidases in the kidneys, with a resulting change in turnover of tissue glycoproteins (Chang, 1981). f Differential diagnosis. Because diabetes mellitus is a spontaneous disease, it should be ruled out anytime a colony illness occurs. If the animals are being used as a model to study diabetes, the experimental protocol will dictate the diagnostic monitoring procedures. Certainly Tyzzer's disease must be considered both in the initial differential phases and also as a secondary complication, since diabetic animals are very susceptible to stress. g. Treatment and control. Treatment with hypoglycemic drugs may be indicated in breeding females in an attempt to maintain inbred lines (Meier and Yerganian, 1961).

5. BIOLOGY AND DISEASES OF HAMSTERS

h. Research complications. The disease could potentially occur in animals being used in research protocols unrelated to diabetes. Because cellular metabolism is affected, cytogenetic studies could produce unreliable data.

193

mation and necrosis of the anterior cerebral artery. A homogeneous PAS-positive material could be seen within the media of the diseased artery. The vessel wall was greatly thickened in chronic cases. 2.

C.

Female littermates can become very aggressive as they reach maturity. Severe bite wounds, especially about the tail and head area, can be inflicted, and death is not an uncommon occurrence. Litters should be separated before fighting becomes a problem. Following attempts at breeding, the female can become quite aggressive to the male, so some means of removing the male must be anticipated.

D.

Neoplastic Diseases

In general, Chinese hamsters have a low incidence of spontaneous tumors, with mainly the liver and reproductive organs involved. The rarity of spontaneous and induced leukemias may reflect the absence of innate tumor viruses. Uterine adenocarcinomas were detected in 30 of 120 females (Ward and Moore, 1969). The growths were firm and white with implantation frequently seen on the visceral and parietal peritoneum. Approximately 10% of affected hamsters had lung metastases. Another report showed 11 of 77 affected with similar characteristics except that no pulmonary metastasis was seen (Benjamin and Brooks, 1977). Vaginal bleeding was the sign most often seen initially. The incidence of ovarian tumors was significantly increased with radiation exposure, but was rarely reported in control animals (Kohn and Gultman, 1964). Hepatomas were found in 66 of 253 animals (Ward and Moore, 1969). These were benign and most often occurred as multiple nodules. Nodular hyperplasia, a nonneoplastic lesion, was seen in 111 of 157 animals in another survey (Benjamin and Brooks, 1977). Pancreatic adenocarcinomas were reported in three 3-year-old females that were partially inbred for the development of spontaneous diabetes mellitus (Poel and Yerganian, 1961). Later reports in nondiabetic animals show these tumors to be very rare. E. 1.

Periodontitis

Traumatic Diseases

Miscellaneous Diseases

This condition was found in a strain of Chinese hamster with hereditary diabetes mellitus (Cohen et al., 1961). The lesion is characterized by absorption of alveolar bone, inflammation, and pocket formation due to splitting of the epithelial attachment. The disease corresponds to that in humans with diabetes mellitus in whom periodontitis is seen. 3.

Nephrosclerosis

In a study of 157 animals, 46 had evidence of nephrosclerosis (Benjamin and Brooks, 1977). The pathology was different from the intercapillary glomerulosclerosis associated with diabetes mellitus. Grossly, pitting and a decrease in size were seen when kidneys were severely affected. Microscopically, tubular degeneration, mild interstitial fibrosis, and focal atrophy of the cortex were seen early in the disease. In more advanced conditions there was hyaline sclerosis of glomeruli, more severe interstitial fibrosis, and tubular degeneration. 4.

Spondylosis

The incidence and extent of spondylosis were increased in hamsters with spontaneous diabetes mellitus compared to nondiabetic control animals (Silberberg and Gerritsen, 1976). 5.

Pulmonary Granulomas

Pulmonary granulomas were observed in 54 of 157 animals (Benjamin and Brooks, 1977). Grossly, the lesions appeared as subpleural, yellowish gray foci, measuring 1 to 3 mm in diameter with variable involvement of the lung parenchyma. Microscopically, lesions consisted of alveolar collections of lipidfilled macrophages, mixed inflammatory cells, septal fibrosis, and occasional cholesterol clefts. The cause is not known. Affected animals were housed in both suspended wire cages and plastic shoe-box cages with different types of bedding.

ARMENIAN HAMSTER

Cerebral Hemorrhage

This lesion occurred in 20% of 253 hamsters given 1311(Ward and Moore, 1969). Deaths occurred at 1 to 2 years of age. Grossly, the hemorrhage was most evident between the cerebral hemispheres, with blood often in the lateral ventricles. Microscopically, the hemorrhage was shown to be caused by inflam-

I.

INTRODUCTION

The Armenian hamster (Cricetulus migratorius), also known as the gray hamster, was first introduced as a laboratory animal

194

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

in 1963. It was chosen as a laboratory research animal because of its susceptibility to mutagenic and carcinogenic agents. These hamsters are also important for studying meiosis due to their unique semisynchronous meiotic progression, which begins at 15 days of age (Yerganian and Lavappa, 1971). Like the Syrian hamster, the Armenian hamster is highly susceptible to oncogenic viruses and has high tolerance to both homologous and heterologous transplantable tumors. Cytological features are comparable to those of the Chinese hamster, so this species is of value for cytogenetic studies. Armenian hamsters are also used to study infection with prion diseases. Armenian hamster spleen cells can form stable hybridomas with mouse myeloma cell lines (Hurley and Desch, 1994).

imals from a West German industrial area were found to have bronchogenic squamous cell carcinoma. It has since been found to be susceptible to N-diethylnitrosamine (DEN), with the subsequent development of respiratory tumors (Mohr et al., 1973). The European hamster is believed to be a more suitable model than the Syrian hamster for highly concentrated and prolonged smoke-inhalation studies (Reznik et al., 1975). To the authors' knowledge, there are no breeding colonies of these animals presently housed in the United States. Most of their use in the biomedical field involves studies of hibernation.

II. lI.

BIOLOGY

Care and management procedures are similar to those of the Chinese hamster. Body size and weight (40 to 80 gm) are also similar. The diploid chromosome number is 22, with the X and Y chromosome of equal size. Captured animals are aggressive, but if reared in the laboratory, they can be bred successfully. The gestation period is 18 to 19 days, with an average litter size of 3 to 4 pups.

IIl.

DISEASES

Little has been reported concerning spontaneous infectious diseases in the Armenian hamster. The expression of spontaneous amyloidosis differs in gender-specific AP expression and susceptibility to AA amyloidosis from that seen in the Syrian hamster (de Beer et al., 1993). Hepatocellular carcinomas have been reported in animals exposed to estrogen (Coe et al., 1990). Skin lesions have been attributed to mite infestations, which have recently been identified as D e m o d e x cricetuli (Hurley and Desch, 1994). This mite is similar to D. aurati of the Syrian hamster and occupies hair follicles, particularly along the face and back.

EUROPEAN HAMSTER

I.

INTRODUCTION

The European hamster (Cricetus cricetus) developed some importance as a laboratory model when several wild-caught an-

BIOLOGY

These animals are nocturnal, and they hibernate during the winter months in the wild. They are the largest hamster species, being minimally 3 times the size of a Syrian hamster. Their outward appearance is like that of the Syrian hamster, except they have white faces and feet, bodies with reddish brown dorsums, cranioventral black patches, and caudolateral white patches. These hamsters tend to be very aggressive toward members of their own species and humans. They are easily frightened and will attack and give a painful bite. Those in captivity are less aggressive toward humans and with increased laboratory breeding, the animals have become much easier to handle. Each litter develops a defined social order, with the heaviest male being dominant (Reznik-Schuller et al., 1974). The average life span is 34 months for females and 31 months for males, which may be related to the higher reported incidence of neoplasia in males (Ernst et al., 1989). Other reports indicate that the life span ranges from 6 to 10 years (Reznik-Schuller et al., 1974). Like the Chinese hamster, the European hamster has a chromosome diploid number of 22. A normal hemogram is presented in Table XII. Water consumption is 5 ml/100 gm body weight, and average food consumption is 2.9 gm/100 gm body weight in summer (August) and 1.8 gm/100 gm body weight in winter (November) (Silverman and Chavannes, 1977). These animals are mainly seed eaters. Reproductive data are shown in Table XIII. Estrus is determined by vaginal smears and by test mating, using a steel mesh divider to keep the pair separated. When no aggressiveness is observed, hamsters may be mated (Mohr et al., 1973). Pseudopregnancy is seen after mating if conception does not occur. Females will gain 20 to 30 gm in 10 to 12 days, make nest preparations, and become very aggressive; but, if not pregnant, will begin to lose weight over the next 4 days. Estrus occurs 6 days later. Females tend to bear 1 to 2 litters per year, each with 6 to 9 pups (Reznik-Schuller et al., 1974). Newly weaned animals (25 days postpartum) have an average body weight of 75 gm, with 6-month-old females and males approaching 300 and

195

5. BIOLOGY AND DISEASES OF HAMSTERS Table X l l

Normal Hemogramof the European Hamster Emminger et al. (1975)

Silverman and Chavannes (1977) Hemogram

Value

Percentage

Leukocytes (103/ml) Neutrophils Lymphocytes Monocytes Eosinophils Basophils Thrombocytes (103/ml) RBC (106/ml) PCV (%) Hemoglobin (g/dl)

7.4 +__2.6 1.71 _ 0.06 5.47 ___0.06 0.192 • 0.015 0.005 • 0.002 0.001 • 0.002

23.2 _ 2.5 74.0 ___2.3 2.6 ___0.6 0.07 ___0.10 0.02 +_0.07

7.64 • 0.42 49.2 • 1.6 18.0 • 0.7

400 gm, respectively (Mohr et al., 1973). Sexual activity is not observed in winter months, during which time females and males are very aggressive toward each other. In the nonbreeding season, the female's vagina is closed and the male's scrotum is decreased in size with the testes situated in the abdominal cavity. Anatomy of the European hamster has been studied extensively. The exocrine pancreas was described in an attempt to determine the suitability of this animal as a model for pancreatic cancer (Spikermann and Althoff, 1980). The nasal cavity has been fully described, as have comparative analyses of organ weights (Reznik and Jensen, 1979; Reznik et al., 1973). Photoperiodic regulation of annual cycles has been described in European hamsters (Pevet, 1988). The critical photoperiod, at which time gonadal regression is induced, is between 15 and 15.5 hrs. Studies have also implicated a circannual rhythm in physiological variations in these animals, including changes in body weight and food intake even under conditions of constant photoperiods (Masson-Pevet et al., 1994; Wollnik and Schmidt, 1995). There is a slight reduction in activity in the winter months. European hamsters are true hibernators. Hibernation affects thrombocyte and leukocyte values, but there is no sig-

Table X l l I

Reproductive Data for the European Hamstera Sexual maturity Estrus cycle Gestation Litter size Weaning aData from Mohr et al. (1973).

Females, 80-90 days Males, 60 days 4-6 days 18-21 days (captured) 15-17 days (laboratory-born) 7-9 25 days 28 days

Value 8.3 • 2.2 2.87 • 3.74 5.02 _.+0.39 0.083 ___0.022 0.093 • 0.018 0 210 • 32 7.45 +__0.49

Percentage 34.6 _ 17 60.0 ___17.6 1.00 • 1.00 1.13 • 0.83 0

nificant difference in these values in nonhibernating animals during winter or summer (Reznik et al., 1979).

III.

DISEASES

Spontaneous neoplasia in the European hamster has been found to be slightly more prevalent in males than in females. The most frequent tumors in descending order are leukemias and lymphomas, adrenal pheochromocytomas, and granulosa cell tumors in females (Ernst et al., 1989). Thymomas discovered in a small number of examined European hamsters resembled benign human thymomas (Ghadially and Illman, 1965). Thymic tumors were associated with large numbers of mast cells, which are not normally seen in the human form (Ghadially and Illman, 1965). Similar to the Syrian and Chinese hamsters, the European hamster has a very low incidence of spontaneous pulmonary neoplasia (Ernst et al., 1989). Silverman and Chavannes (1977) tested 8 males and found them to be free of endoparasites, ectoparasites, and blood parasites. European hamsters are prone to developing cysts within the peritoneal cavity, particularly in the liver. Cysts tend to occur more often in females than in males (Table IX) (Kaup et al., 1990). Other locations for cysts include the cecum, ovaries, spleen, kidney, and colon. Spontaneous pathological processes in the head, and particularly in the jaws, of the European hamster have been reported. The development of such disorders as malocclusion, osteomyelitis, and dysplasia appears to increase with age and suggest that the European hamster could be of use in dental research (Kunstyr et al., 1987). These disease processes in the mouth are often complicated by secondary bacterial infections, which may be fatal (Ernst et al., 1989).

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F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR.

DJUNGARIAN HAMSTER

I.

INTRODUCTION

The Djungarian hamsters are Phodopus campbelli (Russian dwarf or striped hairy-footed hamster) and P. sungorus (Siberian dwarf). Initially these animals were assumed to be subspecies; however, P. sungorus and P. campbelli are now believed to be separate species. The two may be confused because the common name "Djungarian" is often used to denote either species. The Russian dwarf hamster has been traced to Siberia, China, and Mongolia and is a distant relative of the Syrian hamster (Cooper et al., 1991). The Siberian dwarf hamster is native to the steppes of Kazakhstan, Manchuria, and northern China (Wynne-Edwards and Lisk, 1984). These hamsters range from 50 to 100 mm in body length, with an additional 10 mm of tail. Body weights range from 18 to 25 gm. The dorsal fur is gray, and a dark stripe runs dorsally along the length of the body. The fur of the ventrum, limbs, and tail tends to be white. Unlike in some of the other hamster species, the feet and tail are covered with fur. Under conditions of natural lighting, some of these animals may turn more white, and are thus called "Winter White" hamsters. Djungarian hamsters generally live from 9 to 15 months, although survival up to 2 years has been reported (Lawrie and Megahy, 1991). The normal karyotype of the Djungarian species is 28 chromosomes. These hamsters have a high incidence of neoplasia, are susceptible to carcinogens, and can be infected with oncogenic viruses, particularly Rous sarcoma virus (RSV), human adenovirus-12 (Adeno-12), and simian virus 40 (SV40) (Pogosianz, 1975). The dwarf hamsters are extensively used in behavior and reproductive physiology studies.

II.

BIOLOGY

The dwarf hamsters have the most compressed reproductive cycle of any eutherian mammal. They can mate on the day of parturition and deliver the second litter, while weaning the first, within a 36-day time period (Newkirk et al., 1997). Similar to the Syrian and Chinese hamsters, the two Djungarian hamster species have a 4-day estrus cycle with spontaneous ovulation (Erb et al., 1993). The gestation period is 18 days. Pregnancy in P. campbelli is dependent on continued secretion of progesterone by the corpus luteum through late gestation (Edwards et al., 1995). Prolactin levels in this species are absent during midgestation and resume in late gestation. Possible roles for this reappearance of activity include influences on lactogenesis, mammary gland development, and the regulation of maternal

behavior toward newborns (Edwards et al., 1995). Successful reproduction in P. campbelli is dependent on monogamous parental care by both the male and female (Wynne-Edwards and Lisk, 1984, 1987). The nonaggressive behavior of females facilitates the maintenance of breeding pairs throughout life, with weaning of offspring occurring at 3 weeks of age. Females can bear between 1 and 18 litters, with each consisting of 1 to 9 pups, in their reproductive years (Pogosianz, 1975). Reproductive development in females is accelerated when exposed to males of the same species (Reasner and Johnston, 1988). Increased food hoarding has been observed in Siberian dwarf hamsters as a behavioral adaptation to provide accessible energy during pregnancy (Bartness, 1997). Endocrinology in P. sungorus is similar to that of other rodent species, yet differs from that of P. campbelli. This implies that the two species of Djungarian hamsters have undergone evolutionary selection pressures with respect to reproductive endocrinology (Mcmillan and Wynne-Edwards, 1998). Phodopus species show decreases in fertility and fecundity at the age of 8 to 10 months, the youngest age of all comparable mammalian models (Edwards et al., 1998). As adults, these hamsters are biologically dependent on the photoperiod under which they are housed. The critical photoperiod is approximately 13 hr (versus 12.5 hr in the Syrian and 15.5 hr in the European hamsters) (Pevet, 1988). Changes in photoperiod influence seasonal changes in breeding activities, thermoregulation, hair-coat growth, and fat metabolism (Pogosianz, 1975; Ebling, 1994). Djungarian hamsters have been widely used in studies of the pineal gland and melatonin secretion in mediating the effects of photoperiod. These hamsters are unusual in that they do not hibernate even when exposed to temperatures below - 4 0 ~ (Schlenker, 1985). Seasonal acclimation of blood-gas transport during periods of colder temperatures is facilitated by an increased relative heart weight, increased surface area of erythrocytes, and slightly altered hemoglobin content, all of which aid in oxygen transport (Puchalski and Heldmaier, 1986). These animals also decrease their metabolic resting rates and increase their capacity for nonshivering thermogenesis to adapt to inclement temperatures (Schlenker, 1985). No differences in dietary needs have been reported for Djungarian hamsters. They can be maintained on the same diet as Syrian hamsters and are less costly to maintain because of their smaller size (Pogosianz, 1975). These animals are omnivorous (Sawrey et al., 1984).

III.

DISEASES

High incidences of neoplasia, particularly of the oral cavity, skin, and mammary glands have been reported in Russian dwarf

197

5. BIOLOGY AND DISEASES OF HAMSTERS Table X I V

Spontaneous Neoplasms of Djungarian Hamsters a

Location Mammary glands Skin (muzzle) Lungs Uterus and ovaries Kidney Hematopoeitic system Other locations Benign neoplasms

Tumor type Adenocarcinoma Squamous cell carcinoma Adenocarcinoma Various types Carcinoma Not reported Not reported Hepatic adenoma; skin papilloma

Total neoplasms found

Female Male (n = 98) b (n = 32) b 72 16

0 17

22 9 0 4 0 6

1 0 4 2 3 9

129

36

a Total number of hamsters evaluated: female (634), male (643). bData from Pogosianz (1975); Pogosianz and Sokova (1982).

hamsters (Lawrie and Megahy, 1991). In a study of 30 animals ranging from 6 to 12 months in age, 30% were found to have metastatic tumors that consisted of fibromas (4), fibrosarcomas (2), mammary adenocarcinomas (2), and fibroma with liver cell carcinoma (1) (Cooper et al., 1991). Additional reports support the high occurrence of spontaneous neoplasms, particularly in the female hamster (Table XIV). The incidence of the tumors has changed over time, with mammary tumors increasing and skin tumors decreasing in frequency; this may reflect genetic alterations linked to further inbreeding (Pogosianz and Sokova, 1982). These affected dwarf hamsters are reported to exhibit rapid weight loss, similar to that seen with disease conditions of bronchopneumonia and incisor malocclusion. Dermatologic conditions of trichophytosis have been reported (Pogosianz, 1975). Other skin problems include alopecia and ventral dermatitis caused by demodectic mites. Female Russian dwarf hamsters prevented from breeding may develop cystic ovaries, with clinical presentations of swollen abdomens and bloody vaginal discharge (Lawrie and Megahy, 1991). Hypersensitivity to bedding materials, particularly cedar chips, has been empirically reported. Affected dwarf hamsters may develop alopecia with dry skin and have secondary bacterial infections (McGuire, 1993). Enteritis with rectal prolapse, although common in other varieties of hamsters, has not been noted in the Russian hamster (Lawrie and Megahy, 1991).

REFERENCES

Adler, S. (1948). Origin of the golden hamster, Cricetus auratus, as a laboratory animal. Nature. 162: 256-257. Aeromedical Review. (1975). The hamster. In "Selected Topics in Laboratory

Animal Medicine," pp. 27-35. USAF School of Aerospace Medicine, Brooks Air Force Base, San Antonio. Ambrose, K. R., and Coggin, J. H. J. (1975). An epizootic in hamsters of lymphomas of undetermined origin and mode of transmission. J. Natl. Cancer Inst. 54, 877-879. Arnold, C. E., and Estep, D. Q. (1994). Laboratory caging preferences in golden hamsters (Mesocricetus auratus). Lab. Anim. 28, 232-238. Arrington, L. R., Platt, J. K., and Shirley, R. L. (1966). Protein requirements of growing hamsters. Lab. Anim. Care. 18(2), 151-159. Autotutorial Committee. (1994). Laboratory Animal Medicine and Science Series II. Am. Coll. Lab. Anim. Med. 2, 27-31. Baker, J. R., Mason, M. M., Yerganian, G., Weisburger, E. K., and Weisburger, J. H. (1974). Induction of tumors of the stomach and esophagus in inbred Chinese hamsters by oral diethylnitrosamine. Proc. Soc. Exp. Biol. Med. 146, 291-293. Banta, C. A., Warner, R. G., and Robertson, J. B. (1975). Protein nutrition of the golden hamster. J. Nutr. 105(1), 38-45. Barros, C., Gonzalez, J., Herrera, E., and Bustos-Obregon, E. (1978). Fertilizing capacity of human spermatozoa evaluated by actual penetration of foreign eggs. Contraception 17, 87-92. Bartlett, J. G., Chang, T., Moon, N., and Onderdonk, A. B. (1978). Antibioticinduced lethal enterocolitis in hamsters. Studies with eleven agents and evidence to support the pathogenic role of toxin-producing clostridia. Am. J. Vet. Res. 39, 1525-1530. Bartness, T. J. (1997). Food hoarding is increased by pregnancy, lactation, and food deprivation in Siberian hamsters. Am. J. Physiol. 272, R118-R125. Battles, A. H. (1985). The biology, care, and diseases of the Syrian hamster. Compend. Contin. Educ. Pract. Vet. 7, 815-825. Benjamin, S. A., and Brooks, A. L. (1977). Spontaneous lesions in Chinese hamsters. Vet. Pathol. 14, 449-462. Bergstresser, P. R., Toews, G. B., Gilliam, J. N., and Streilein, J. W. (1980). Unusual numbers and distribution of Langerhans cells in skin with unique immunologic properties. J. Invest. DermatoL 74, 312-314. Bernfeld, P., Homburger, E, Adams, R. A., Soto, E., and Van Dongen, C. (1986). Base-line data in a carcinogen-susceptible first generation hybrid strain of Syrian golden hamsters: FID Alexander. J. Natl. Cancer Inst. 77, 165-171. Birt, D. E, and Pour, P. M. (1985). Interaction of dietary fat and protein in spontaneous diseases of Syrian golden hamsters. J. Natl. Cancer Inst. 75, 127133. Birt, D. E, Schuldt, G. H., and Salmasi, S. (1982). Survival of hamsters fed graded levels of two protein sources. Lab. Anim. Sci. 32, 363-366. Birt, D. E, Patil, K., and Pour, P. M. (1985). Comparative studies on the effects of semipurified and commercial diet on longevity and spontaneous and induced lesions in the Syrian golden hamster. Nutr. Cancer 7, 167-177. Blankenship-Paris, T. L., Chang, J., Dalldorf, E G., and Gilligan, P. H. (1995a). In vivo and in vitro studies of Clostridium difficile-induced disease in hamsters fed an atherogenic, high fat diet. Lab. Anim. Sci. 45, 47-53. Blankenship-Paris, T. L., Walton, B. J., Hayes, Y. O., and Chang, J. (1995b). Clostridium difficile infection in hamsters fed an atherogenic diet. Vet. Pathol. 32, 269-273. Boquist, L. (1969). Pancreatic islet morphology in diabetic Chinese hamsters. Acta Pathol. Microbiol. Scand. 75, 399-414. Borriello, S. P., Ketley, J. M., Mitchell, T. J., Barclay, F. E., Welch, A. R., Price, A. B., and Stephen, J. (1987). Clostridium difficilema spectrum of virulence and analysis of putative virulence determinants in the hamster model of antibiotic-associated colitis. J. Med. Microbiol. 24, 53-64. Brunner, H. (1997). Models of mycoplasma respiratory and genital tract infections. Wien. Klin. Wochenschr. 109, 569-573. Brunnert, S. R., and Altman, N. H. (1991). Laboratory assessment of chronic hepatitis in Syrian hamsters. Lab. Anim. Sci. 41, 559-562. Buntin, J. D., Jaffe, S., and Lisk, R. D. (1984). Changes in responsiveness to newborn pups in pregnant, nulliparous golden hamsters. Physiol. Behav. 32, 437-439.

198 Butler, L., and Gerritsen, G. C. (1970). A comparison of the modes of inheritance of diabetes in the Chinese hamster and the KK mouse. Diabetologia 6, 163-167. Buzzell, G. R. (1996). Sexual dimorphism in the Harderian gland of the Syrian hamster is controlled and maintained by hormones, despite seasonal fluctuations in hormone levels: Functional implications. Microsc. Res. Tech. 34, 133-138. Calland, C. J., Wightman, S. R., and Neal, S. B. (1986). Establishment of a Chinese hamster breeding colony. Lab. Anim. Sci. 36, 183-185. Chang, A. Y. (1981). Biochemical abnormalities in the Chinese hamster (Cricetulus griseus) with spontaneous diabetes. Int. J. Biochem. 13, 41-43. Chang, J., and Rohwer, R. G. (1991). Clostridium difficile infection in adult hamsters. Lab. Anim. Sci. 41, 548-552. Chang, A. Y., Noble, R. E., and Wyse, B. M. (1977). Comparison of highly inbred diabetic and nondiabetic lines in the Upjohn colony of Chinese hamsters. Diabetes 26, 1963-1971. Chang, T. W., Dong, M. Y., and Gorbach, S. L. (1990). Effect of bismuth subsalicylate on Clostridium difficile colitis in hamsters. Rev. Infect. Dis. 12(Suppl. 1), $57-$58. Chesterman, E C., and Pomerance, A. (1965). Cirrhosis and liver tumours in a closed colony of golden hamsters. Br. J. Cancer 19, 802-811. Ciaccio, L. A., and Lisk, R. D. (1971). Hormonal control of cyclic estrus in the female hamster. Am. J. Physiol., 221(3), 936-942. Cisar, C. E, Gumperz, E. P., Nicholson, E S., and Moore, W. Jr. (1972). A practical method for production of breeding of Chinese hamsters (Cricetulus griseus). Lab. Anim. Sci. 22, 725-727. Coe, J. E., and Ross, M. J. (1990). Amyloidosis and female protein in the Syrian hamster. Concurrent regulation by sex hormones. J. Exp. Med. 171, 1257-1267. Coe, J. E., Margossian, S. S., Slayter, H. S., and Sogn, J. A. (1981). Hamster female protein. A new pentraxin structurally and functionally similar to C-reactive protein and amyloid P component. J. Exp. Med. 153, 977-991. Coe, J. E., Ishak, K. G., and Ross, M. J. (1990). Estrogen induction of hepatocellular carcinomas in Armenian hamsters. Hepatology 11, 570-577. Coe, J. E., Schell, R. E, and Ross, M. J. (1995). Immune response in the hamster: Definition of a novel IgG not expressed in all hamster strains. Immunology 86, 141-148. Coe, J. E., Cieplak, W., Hadlow, W. J., and Ross, M. J. (1997). Female protein, amyloidosis, and hormonal carcinogenesis in Turkish hamster: Differences from Syrian hamster. Am. J. Physiol. 273, R934-R941. Coggin, J. H. J., Bellomy, B. B., Thomas, K. V., and Pollock, W. J. (1983). Bcell and T-cell lymphomas and other associated diseases induced by an infectious DNA viroid-like agent in hamsters (Mesocricetus auratus). Am. J. Pathol. 110, 254-266. Coggin, J. H. J., Hyde, B. M., Heath, L. S., Leinbach, S. S., Fowler, E., and Stadtmore, L. S. (1985). Papovavirus in epitheliomas appearing on lymphoma-bearing hamsters: Lack of association with horizontally transmitted lymphomas of Syrian hamsters. J. Natl. Cancer Inst. 75, 91-97. Cohen, M. M., Shklar, G., and Yerganian, G. (1961). Periodontal pathology in a strain of Chinese hamster, Cricetulus griseus, with hereditary diabetes mellitus. Am. J. Med. 31, 864-867. Committee on Rodents, Institute of Laboratory Animal Resources. (1996). "Laboratory Animal Management: Rodents." National Academy Press, Washington, D.C. Connor, J. S., Lavine, R. L., and Rose, L. I. (1981). Mesocricetus auratus: A new animal model for studying diabetes mellitus in pregnancy. Lab. Anim. Sci. 31, 35-38. Cooper, D. M., and Gebhart, C. J. (1998). Comparative aspects of proliferative enteritis. J. Am. Vet. Med. Assoc. 212, 1446-1451. Cooper, J. E., Knowler, C., and Pearson, A. J. (1991). Tumours in Russian hamsters (Phodopus sungorus). Vet. Rec. 128, 335-336. Cooper, D. M., Swanson, D. L., Barns, S. M., and Gebhart, C. J. (1997). Comparison of the 16S ribosomal DNA sequences from the intracellular agents of proliferative enteritis in a hamster, deer, and ostrich with the sequence of

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR. a porcine isolate of Lawsonia intracellularis. Int. J. Syst. Bacteriol. 47, 635-639. Corbascio, A. N., Kohn, H. I., and Yerganian, G. (1962). Acute X-ray lethality in the Chinese hamster after whole-body and partial-body irradiation. Radiat. Res. 17, 521-530. Courtneidge, S. A., Goutebroze, L., Cartwright, A., Heber, A., Scherneck, S., and Feunteun, J. (1991). Identification of characterization of the hamster polyomavirus middle T antigen. J. Virol. 65, 3301-3308. Cunnane, S. C., and Bloom, S. R.(1990). Intussusception in the Syrian golden hamster. Br. J. Nutr. 63, 231-237. Czub, M., Braig, H. R., and Diringer, H. (1986). Pathogenesis of scrapie: Study of the temporal development of clinical symptoms, of infectivity titers and scrapie-associated fibrils in brains of hamsters infected intraperitoneally. J. Gen. Virol. 67, 2005-2009. Davis, M. J., Ferrer, P. N., and Gore, R. W. (1986). Vascular anatomy and hydrostatic pressure profile in the hamster cheek pouch. Am. J. Physiol. 25tl, H291-H303. deArruda, M. S. P., and Montenegro, M. R. (1995). The hamster cheek pouch: An immunologically privileged site suitable to the study of granulomatous infections. Rev. Inst. Med. Trop. Sao Paulo 37, 303-309. de Beer, M. C., de Beer, F. C., Beach, C. M., Gonnerman, W. A., Carreras I., and Sipe, J. D. (1993). Syrian and Armenian hamsters differ in serum amyloid A gene expression. Identification of novel Syrian hamster serum amyloid A subtypes. J. Immunol. 150, 5361-5370. DeHaven, J. E., Schwartz, D. A., Dahm, M. W., Hazard, E. S., Trifiletti, R., Lacy, E. R., and Norris, J. S. (1998). Novel retroviral sequences are expressed in the epididymis and uterus of Syrian hamsters. J. Gen. Virol. 79, 2687-2694. Delmas, V., Bastien, C., Scherneck, S., and Feunteun, J. (1985). A new member of the polyomavirus family: The hamster papovavirus. Complete nucleotide sequence and transformation properties. EMBO J. 4, 12791286. Desch, C. E., Jr., and Hurley, R. J. (1997). Demodex sinocricetuli: New species of hair follicle mite (Acari: Demodicidae) from the Chinese form of the striped hamster, Cricetulus barabensis (Rodentia: Muridae). J. Med. Entomol. 34, 317-320. Dillehay, D. L., Paul, K. S., Boosinger T. R., and Fox, J. G. (1994). Enterocecocolitis associated with Escherichia coli and Campylobacter-like organisms in a hamster (Mesocricetus auratus) colony. Lab. Anim. Sci. 44, 12-16. Donnelly, T. M. (1997). Disease problems of small rodents. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 307-327. Saunders, Philadelphia. Ebling, E J. P. (1994). Photoperiodic differences during development in the dwarf hamsters Phodopus sungorus and Phodopus campbelli. Gen. Comp. Endocrinol. 95, 475-482. Eddy, H. A., and Casarett, G. W. (1972). Pathology of radiation syndromes in the hamster. Prog. Exp. Tumor Res. 16, 98-119. Edwards, H. E., Reburn, C. J., and Wynne-Edwards, K. E. (1995). Daily patterns of pituitary prolactin secretion and their role in regulating maternal serum progesterone concentrations across pregnancy in the Djungarian hamster (Phodopus campbelli). BioL Reprod. 52, 814-823. Edwards, H. E., Tweedie, C. J., Terranova, P. E, Lisk, R. D., and WynneEdwards, K. E. (1998). Reproductive aging in the Djungarian hamster, Phodopus campbelli. Biol. Reprod. 58, 842-848. Emminger, A., Reznik, G., Reznik-Schuller, H., and Mohr, U. (1975). Differences in blood values depending on age in laboratory-bred European hamsters (Cricetus cricetus L.). Lab. Anim. 9, 33-42. Erb, G. E., Edwards, H. E., Jenkins, K. L., Mucklow, L. C., and WynneEdwards, K. E. (1993). Induced components in the spontaneous ovulatory cycle of the Djungarian hamster (Phodopus campbelli). Physiol. Behav. 54, 955-959. Ernst, H., Kunstyr, I., Rittinghausen, S., and Mohr, U. (1989). Spontaneous tumours of the European hamster (Cricetus cricetus L.). Z. Versuchstierkd. 32, 87-96.

5. BIOLOGY AND DISEASES OF HAMSTERS

Estes, E C., Richter, C. B. and Franklin, J. A. (1971). Demodectic mange in the golden hamster. Lab. Anim. Sci. 21(6), 825-828. Federation of European Laboratory Animal Science Associations (FELASA) Working Group on Animal Health (1994). Recommendations for the health monitoring of mouse, rat, hamster, guinea pig, and rabbit breeding colonies. Report of the Federation of European Laboratory Animal Science Associations (FELASA) Working Group on Animal Health Accepted by the FELASA Board of Management November 1992. Lab. Anim. 28, 1-12. Ferm, V. H. (1967). The use of the golden hamster in experimental teratology. Lab. Anim. Care 17, 452-462. Festing, M. (1972). Hamsters. In "The UFAW Handbook on the Care and Management of Laboratory Animals" (University Federation for Animal Welfare, ed.), pp. 242-256. Churchill-Livingstone, Edinburgh. Fortner, J. G. (1957). Spontaneous tumors, including gastrointestinal neoplasms and malignant melanomas, in the Syrian hamster. Cancer 10, 1152-1156. Fox, J. G., Zanotti, S., and Jordan, H. V. (1981). The hamster as a reservoir of Campylobacter fetus subspecies jejuni. J. Infect. Dis. 143, 856. Fox, J. G., Newcomer, C. E., and Rozmiarek, H. (1984). Selected zoonoses and other health hazards. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Leon, eds.), pp. 613-648. Academic Press, New York. Fox, J. G., Stills, H. E J., Paster, B. J., Dewhirst, E E., Yan, L., Palley, L., and Prostak, K. (1993). Antigenic specificity and morphologic characteristics of Chlamydia trachomatis, strain SFPD, isolated from hamsters with proliferative ileitis. Lab. Anim. Sci. 43, 405-410. Fox, J. G., Dewhirst, E E., Paster, B. J., Shames, B., Yan, L. L., and Murphy, J. C. (1994). Intracellular Campylobacter-like organism from ferrets and hamsters with proliferative bowel disease is a Desulfovibrio sp. J. Clin. MicrobioL 32, 1229-1237. Franklin, C. L., Beckwith, C. S., Livingston, R. S., Riley, L. K., Gibson, S. V., Besch-Williford, C. L., and Hook, R. R., Jr. (1996). Isolation of a novel Helicobacter species, Helicobacter cholecystus sp. nov., from the gallbladders of Syrian hamsters with cholangiofibrosis and centrilobular pancreatitis. J. Clin. Microbiol. 34, 2952-2958. Frenkel, J. K. (1979). Immunity and infection: Hamster. In "Inbred and Genetically Defined Strains of Laboratory Animals," Vol. II, pp. 473-480. Fed. Am. Soc. Exp. Biol., Bethesda, Maryland. Frisk, C. S., Wagner, J. E., and Owens, D. R. (1977). Hamster enteritis: A review. Lab. Anim. 11, 79-85. Frisk, C. S., Wagner, J. E., and Owens, D. R. (1981). Hamster (Mesocricetus auratus) enteritis caused by epithelial cell-invasive Escherichia coli. Infect. Immun. 31, 1232-1238. Ganaway, J. R., Allen, A. M., and Moore, T. D. (1971). Tyzzer's disease.Am. J. PathoL 64, 717-732. Garlinghouse, L. E., Jr., and Van Hoosier, G. L., Jr. (1978). Studies on adjuvantinduced arthritis, tumor transplantability, and serologic response to bovine serum albumin in Sendai virus infected rats. Am. J. Vet. Res. 39, 297-300. Gerritsen, G. C., Needham, L. B., Schmidt, E L., and Dulin, W. E. (1970). Studies on the prediction and development of diabetes in offspring of diabetic Chinese hamsters. Diabetologia 6, 158-162. Ghadially, F. N., and Illman, O. (1965). Naturally occurring thymomas in the European hamster. J. Pathol. Bacteriol. 90, 465-469. Gibson, S. V., Rottinghaus, M. S., Wagner, J. E., Stills, H. E J., Stogsdill, P. S., and Kinden, D. A. (1990). Naturally acquired enteric adenovirus infection in Syrian hamsters (Mesocricetus auratus). Am. J. Vet. Res. 51, 143-147. Gimenez-Conti, I. B., and Slaga, T. J. (1993). The hamster cheek pouch carcinogenesis model. J. Cell Biochem. Suppl. F 17, 83-90. Gleiser, C. A., Van Hoosier, G. L., Jr., and Sheldon, W. G. (1970). A polycystic disease of hamsters in a closed colony. Lab. Anim. Care 20, 923-929. Graffi, A., Schramm, T., Graffi, I., Bierwolf, D., and Bender, E. (1967). Virusassociated skin tumors of the Syrian hamster. J. Natl. Cancer Inst. 40, 867873. Graffi, A., Bender, E., Schramm, T., Kuhn, W., and Schneiders, E (1969). Induction of transmissible lymphomas in Syrian hamsters by the application

199 of DNA from viral hamster papovavirus-induced tumors and by cell-free filtrates from human tumors. Med. Sci. 64, 1172-1175. Gregg, M. B. (1975). Recent outbreaks of lymphocytic choriomeningitis in the United States of America. Bull. World Health Organ. 52, 549-553. Greve, J. H. (1985). Dentostomella translucida, a nematode from the golden hamster. Lab. Anim. Sci. 35, 497-498. Hamilton, J. M., and Reynolds, T. (1983). Cholangiofibrosis in the Syrian golden hamster. Vet. Rec. 112, 359-360. Harkness, J. E. (1994). Small rodents. Vet. Clin. North Am. Small Anim. Pract. 21, 89-102. Harkness, J. E., and Wagner, J. E. (1995). "The Biology and Medicine of Rabbits and Rodents." Williams and Wilkins, Media, Pennsylvania. Harkness, J. E., Wagner, J. E., Kusewitt, D. E, and Frisk, C. S. (1977). Weight loss and impaired reproduction in the hamster attributable to an unsuitable feeding apparatus. Lab. Anim. Sci. 27, 117-118. Hasegawa, T. (1995). A case report of the management of demodicosis in the golden hamster. J. Vet. Med. Sci. 57, 337-338. Hatch, G. G., Casto, B. C., McCormick, and K. J., Trentin, J. J. (1975). RNA type C virus antigens in hamster cells transformed by carcinogenic DNA viruses and chemicals. Cancer Res. 35, 3792-3797. Hayes, J. A., Christensen, T. G., and Snider, G. L. (1977). The hamster as a model of chronic bronchitis and emphysema in man. Lab. Anim. Sci. 27, 762-770. Hedqvist, P., Raud, J., and Dahlen, S. E. (1990). Microvascular actions of eicosanoids in the hamster cheek pouch. Adv. Prostaglandin Thromboxane Leukot. Res. 20, 153-160. Hixon, M. L., Lewis, A. M. J., Levine, A. S., and Chattopadhyay, S. K. (1996). Limited diversity in the major histocompatibility complex class II loci of Syrian hamster DNA. Lab. Anim. Sci. 46, 679-681. Hoffman, R. A., Robinson, P. F., and Magalhaes, H. (1968). "The Golden Hamster: Its Biology and Use in Medical Research." Iowa State Univ. Press, Ames. Homburger, F. (1968). The Syrian golden hamster in chemical carcinogenesis research. Prog. Exp. Tumor Res. 10, 164-237. Homburger, F. (1972a). Chemical carcinogenesis in Syrian hamsters. Prog. Exp. Tumor Res. 16, 152-175. Homburger, F. (1972b). Disease models in Syrian hamsters. Prog. Exp. Tumor Res. 16, 69-86. Homburger, E (1983). Background data on tumor incidence in control animals (Syrian hamsters). Prog. Exp. Tumor Res. 26, 259-265. Hurley, R. J., and Desch, C. E., Jr. (1994). Demodex cricetuli: New species of hair follicle mite (Acari: Demodecidae) from the Armenian hamster, Cricetulus migratorius (Rodentia: Cricetidae). J. Med. Entomol. 31, 529533. Innes, J. R. M., Wilson, C., and Ross, M. A. (1956). Epizootic Salmonella enteritidis infection causing septic pulmonary phlebothrombosis in hamsters. J. Infect. Dis. 98, 133-141. Institute of Laboratory Animal Resources. (1996). "Guide for the Care and Use of Laboratory Animals." National Academy Press, Washington, D.C. Jacoby, R. O. (1978). Transmissible ileal hyperplasia of hamsters. I. Histogenesis and immunocytochemistry. Am. J. PathoL 91,433-450. Jasni, S., McOrist, S., and Lawson, G. H. K. (1994). Experimentally induced proliferative enteritis in hamsters: An ultrastructural study. Res. Vet. Sci. 56, 186-192. Jones, G. E, Ward, G. E., Murtaugh, M. P., Lin, G., and Gebhart, C. J. (1993). Enhanced detection of intracellular organism of swine proliferative enteritis, ileal symbiont intracellularis, in feces by polymerase chain reaction. J. Clin. Microbiol. 31, 2611-2615. Jordan, H. V., and van Houte, J. (1972). The hamster as an experimental model for odontopathic infections. Prog. Exp. Tumor Res. 16, 539. Kasim-Karakas, S. E., Vriend, H., Almario, R., Chow, L. C., and Goodman, M. N. (1996). Effects of dietary carbohydrates on glucose and lipid metabolism in golden Syrian hamsters. J. Lab. Clin. Med. 128, 208-213. Kaup, E J., Konstyr, I., and Drommer, W. (1990). Characteristics of sponta-

200 neous intraperitoneal cysts in golden hamsters and European hamsters. Exp. Pathol. 40, 205-212. Kink, J. A., and Williams, J. A. (1998). Antibodies to recombinant Clostridium difficile toxins A and B are an effective treatment and prevent relapse of C. difficile-associated disease in a hamster model of infection. Infect. Immun. 66, 2018-2025. Kleinerman, J. (1972). Some aspects of pulmonary pathology in the Syrian hamster. Prog. Exp. Tumor Res. 16, 287-299. Kohn, H. I., and Gultman, P. H. (1964). Life span, tumor incidence, and intercapillary glomerulosclerosis in the Chinese hamster (Cricetulus griseus) after whole-body and partial-body exposure to X-rays. Radiat. Res. 21, 622-643. Kunstyr, I., Ernst, H., Merkt, M., and Reichart, P. (1987). Spontaneous pathology of the European hamster (Cricetus cricetus). Malocclusion, dysplastic, and inflammatory processes on the jaws. Z. Versuchstierkd. 29, 171180. Lawrie, A. M., and Megahy, I. W. (1991). Tumours in Russian hamsters. Vet. Rec. 128, 411-412. Lepot, A., and Banwell, J. G. (1976). The Syrian hamster: A reproducible model for studying changes in intestinal fluid secretion in response to enterotoxin challenge. Infect. Immun. 14, 1167-1171. Li, J. J., Gonzalez, A., Banerjee, S., Banerjee, S. K., and Li, S. A. (1993). Estrogen carcinogenesis in the hamster kidney: Role of cytotoxicity and cell proliferation. Environ. Health Perspect. 101(Suppl. 5), 259-264. Loeb, W., and Quimby, E, eds. (1999). "The Clinical Chemistry of Laboratory Animals," 2nd ed. Taylor and Francis, Philadelphia. Lowenstein, D. H., Buffer, D. A., Westaway, D., McKinley, M. P., DeArmond, S. J., and Prusiner, S. B. (1990). Three hamster species with different scrapie incubation times and neuropathological features encode distinct prion proteins. Mol. Cell Biol. 10, 1153-1163. Lyman, C. P. (1979). Usefulness of the hamster in the study of hibernation. In "Inbred and Genetically Defined Strains of Laboratory Animals" (Fed. Am. Soc. Exp. Biol., ed.), p. 431. Bethesda, Maryland. Lyman, C. P., and Fawcett, D. W. (1954). Effect of hibernation on growth of sarcoma in hamsters. Cancer Res. 14, 25-28. Magaribuchi, T., Koshimizu, K., and Fujiwara, K. (1977). An outbreak of "wet tail" in hamsters due to the Tyzzer's organism. Exp. Anim. 26, 123-129. Marsh, R. E, and Hanson, R. P. (1978). The Syrian hamster as a model for the study of slow virus diseases caused by unconventional agents. Fed. Proc., Fed. Am. Soc. Exp. Biol. 37, 2076-2078. Masson-Pevet, M., Naimi, E, Canguilhem, B., Saboureau, M., Bonn, D., and Pevet, P. (1994). Are the annual reproductive and body weight rhythms in the male European hamster (Cricetus cricetus) dependent upon a photoperiodically entrained circannual clock? J. Pineal Res. 17, 151-163. Matsumoto, T., Nagata, I., Kariya, Y., and Ohaski, K. (1954). Studies on a strain of pneumotropic virus of hamster. Nagoya J. Med. Sci. 17, 93-97. Maxwell, K. O., Wish, C., Murphy, J. C., and Fox, J. G. (1985). Serum chemistry reference values in two strains of Syrian hamsters. Lab. Anim. Sci. 35, 67-70. McGuire, J. (1993). Phodopus sungorus (Russian dwarf hamsters). . Mcmillan, H. J., and Wynne-Edwards, K. E. (1998). Evolutionary change in the endocrinology of behavioral receptivity: Divergent roles for progesterone and prolactin within the genus Phodopus. Biol. Reprod. 59, 30-38. McOrist, S., and Lawson, G. H. K. (1987). Possible relationship of proliferative enteritis in pigs and hamsters. Vet. Microbiol. 15, 293-302. Meier, H., and Yerganian, G. (1959). Spontaneous hereditary diabetes mellitus in Chinese hamster (Cricetulus griseus). I. Pathological findings. Proc. Soc. Exp. Biol. Med. 100, 810-815. Meier, H., and Yerganian, G. (1961). Spontaneous diabetes mellitus in the Chinese hamster (Cricetulus griseus). III. Maintenance of a diabetic hamster colony with the aid of hypoglycemic therapy. Diabetes 10, 19-21. Militzer, K., Herberg, L., and Buttner, D. (1990). The ontogenesis of skin and organ characteristics in the Syrian golden hamster. II. Body and organ

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR. weights as well as blood glucose and plasma levels. Exp. Pathol. 40, 139153. Mohr, U. (1979). The Syrian golden hamster as a model in cancer research. Prog. Exp. Tumor Res. 24, 245-252. Mohr, U., Schuller, H., Reznik, G., Althoff, J., and Page, N. (1973). Breeding of European hamsters. Lab. Anim. Sci. 23, 799-802. Moore, W., Jr. (1965). Observations on the breeding and care of the Chinese hamster, Cricetulus griseus. Lab. Anim. Care 15, 95-101. Moore, W., Jr. (1966). Hemogram of the Chinese hamster. Am. J. Vet. Res. 27, 608-610. Murphy, M. R., and Schneider, G. E. (1970). Olfactory bulb removal eliminates mating behavior in the male golden hamster. Science 167, 302-304. Murphy, J. C., Fox, J. G., and Niemi, S. M. (1984). Nephrotic syndrome associated with renal amyloidosis in a colony of Syrian hamsters. J. Am. Vet. Med. Assoc. 185, 1359-1362. Nakayama, M., Saegusa, J., Itoh, K., Kiuchi, Y., Tamura, T., Ueda, K., and Fujiwara, K. (1975). Transmissible enterocolitis in hamsters caused by Tyzzer's organism. Jpn. J. Exp. Med. 45, 33-41. Nakayama, M., Machii, K., Goto, Y., and Fujiwara, K. (1976). Typhlohepatitis in hamsters infected perorally with the Tyzzer's organism. Jpn. J. Exp. Med. 46, 309-324. Newberne, P. M., and Fox, J. G. (1980). Nutritional adequacy and quality control of rodent diets. Lab. Anim. Sci. 30, 352-365. Newberne, P. M., and McConnell, R. G. (1979). Nutrition of the Syrian golden hamster. Prog. Exp. Tumor Res. 24, 127-138. Newkirk, K. D., Mcmillan, H. J., and Wynne-Edwards, K. E. (1997). Length of delay to birth of a second litter in dwarf hamsters (Phodopus): Evidence for post-implantation embryonic diapause. J. Exp. Zool. 278, 106-114. Nonaka, I. (1998). Animal models of muscular dystrophies. Lab. Anim. Sci. 48, 8-17. Oka, M. S., and Rupp, R. G. (1990). Large-scale animal cell culture: A biological perspective. Bioprocess Tech. 10, 71-92. Parker, J. C., and Richter, C. B. (1982). Viral diseases of the respiratory system. In "The Biology of the Laboratory Mouse" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), pp. 107-155. Academic Press, New York. Parker, J. C., Igel, H. J., Reynolds, R. K., Lewis, A. M. J., and Rowe, W. P. (1976). Lymphocytic choriomeningitis virus infection in fetal, newborn, and young adult Syrian hamsters. Infect. Immun. 13, 967-981. Patterson, M. M., Schrenzel, M. D., Feng, Y., and Fox, J. G. (2000a). Gastritis and intestinal metaplasia in Syrian hamsters infected with Helicobacter aurati and two other microaerobes. Vet. Pathol. 37, 589-596. Patterson, M. M., Schrenzel, M. D., Feng, Y., Xu, S., Dewhirst, F. E., Paster, B. J., Thibodeau, S. A., Versalovic, J., and Fox, J. G. (2000b). Helicobacter aurati sp. nov., a urease-positive Helicobacter species cultured from gastrointestinal tissues of Syrian hamsters. J. Clin. Microbiol. 38, 37223728. Peace, T. A., Brock, K. V., and Stills, H. F., Jr. (1994). Comparative analysis of the 16S rRNA gene sequence of the putative agent of proliferative ileitis of hamsters. Int. J. Syst. Bacteriol. 44, 832-835. Percy, D. H. (1987). Zoonoses in laboratory animals. Can. Vet. J. 28, 268-269. Percy, D. H., and Palmer, D. J. (1997). Pathogenesis of Sendai virus infection in the Syrian hamster. Lab. Anim. Sci. 47, 132-137. Pevet, P. (1988). The role of the pineal gland in the photoperiodic control of reproduction in different hamster species. Reprod. Nutr. Dev. 28, 443-458. Poel, W. E., and Yerganian, G. (1961). Adenocarcinoma of the pancreas in diabetes-prone Chinese hamsters. Am. J. Med. 31, 861-863. Pogosianz, H. E. (1975). Djungarian hamster--a suitable tool for cancer research and cytogenetic studies. J. Natl. Cancer Inst. 54, 659-664. Pogosianz, H. E., and Sokova, O. I. (1982). Tumours of the Dungarian hamster. IARC Sci. Publ. 34, 451-455. Pour, P., and Birt, D. (1979). Spontaneous diseases of Syrian hamstersmtheir implications in toxicological research: Facts, thoughts, and suggestions. Prog. Exp. Tumor Res. 24, 145-156. Pour, P., Althoff, J., Salmasi, S. Z., and Stepan, K. (1979). Spontaneous tumors

5. BIOLOGY AND DISEASES OF HAMSTERS

and common diseases in three types of hamsters. J. Natl. Cancer Inst. 63, 797-811. Profeta, M. L., Lief, E S., and Plotkin, S. A. (1969). Enzootic Sendai infection in laboratory hamsters. Am. J. Epidemiol. 89, 316-324. Prokoph, H., Arnold, W., Schwartz, A., and Scherneck, S. (1996). In vivo replication of hamster polyomavirus DNA displays lymphotropism in hamsters susceptible to lymphoma induction. J. Gen. Virol. 77, 2165-2172. Prusiner, S. B. (1991). Molecular biology and transgenetics of prion diseases. Crit. Rev. Biochem. Mol. Biol. 26, 397-438. Puchalski, W., and Heldmaier, G. (1986). Seasonal changes of heart weight and erythrocytes in the Djungarian hamster, Phodopus sungorus. Comp. Biochem. Physiol. A 84, 259-263. Reasner, D. S., and Johnston, R. E. (1988). Acceleration of reproductive development in female Djungarian hamsters by adult males. Physiol. Behav. 43, 57-64. Rehg, J. E., and Lu, Y. S. (1982). Clostridium difficile typhlitis in hamsters not associated with antibiotic therapy. J. Am. Vet. Med. Assoc. 181, 14221423. Renshaw, H. W., Van Hoosier, G. L., Jr., and Amend, N. K. (1975). A survey of naturally occurring diseases of the Syrian hamster. Lab. Anita. 9, 179-191. Report of the Secretary of Agriculture to the President of the Senate and the Speaker of the House of Representatives (1998). "Animal Welfare Report: Fiscal Year 1997," pp. 31-36. U. S. Department of Agriculture, Washington, D.C. Reznik, G., and Jensen, K. A. (1979). Anatomy of the nasal cavity of the European hamster (Cricetus cricetus). Z. Versuchstierkd. 21, 321-340. Reznik, G., Reznik-Schuller, H., and Mohr, U. (1973). Comparative studies of organs in the European hamster (Cricetus cricetus L.), the Syrian golden hamster (Mesocricetus auratus W.), and the Chinese hamster (Cricetulus griseus M.). Z. Versuchstierkd. Bd. 15, $272-$282. Reznik-Schuller, H., Reznik, G., and Mohr, U. (1974). The European hamster (Cricetus cricetus L.) as an experimental animal: Breeding methods and observations of their behaviour in the laboratory. Z. Versuchstierkunde 16, $48-$58. Reznik, G., Reznik-Schuller, H., Schostek, H., Deppe, K., and Mohr, U. (1975). Comparative studies concerning the suitability of European hamsters and Syrian golden hamsters for investigations on smoke exposure. Arzneimittelforschung 25, 923-926. Reznik, G., Reznik-Schuller, H., Emminger, A., and Mohr, U. (1979). Comparative studies of blood from hibernating and nonhibernating European hamsters (Cricetus cricetus L.). Lab. Anita. Sci. 25, 210-215. Richter, C. B. (1986). Mouse adenovirus, K virus, pneumonia virus of mice. In "Viral and Mycoplasmal Infections of Laboratory Rodents" (E N. Bhatt, R. O. Jacoby, and I. H. C. Morse, eds.), pp. 137-192. Academic Press, New York. Rogers, A. E., Anderson, G. H., Lenhart, G. M., Wolf, G., and Newberne, E M. (1974). Semisynthetic diet for long-term maintenance of hamsters to study effects of dietary vitamin A. Lab. Anita. Sci. 24, 495. Ronald, N. C., and Wagner, J. E. (1975). Treatment of Hymenolepsis nana in hamsters with yomesan (niclosamide). Lab. Anim. Sci. 25, 219-220. Ross, C. R., Wagner, J. E., Wrightman, S. R., and Dill, S. E. (1980). Experimental transmission of Syphacia muris among rats, mice, hamsters, and gerbils. Lab. Anim. Sci. 30, 35-37. Ryden, E. B., Lipman, N. S., Taylor, N. S., Rose, R., and Fox, J. G. (1991). Clostridium difficile typhlitis associated with cecal mucosal hyperplasia in Syrian hamsters. Lab. Anim. Sci. 41, 553-558. Sakanashi, T., Sako, S., Nozuhara, A., Adachi, K., Okamoto, T., Koga, Y., and Toshima, H. (1991). Vitamin E deficiency has a pathological role in myocytolysis in cardiomyopathic Syrian hamster (BIO 14.6). Biochem. Biophys. Res. Commun. 181, 145-150. Sato, T., Komeda, K., and Shirama, K. (1984). Plasma progesterone concentrations during the estrous cycle, pregnancy, and lactation in the Chinese hamster, Cricetulus griseus. Jikken Dobutsu 33, 501-508. Sawrey, D. K., Baumgardner, D. J., Campa, M. J., Ferguson, B., Hodges, A. W.,

201 and Dewsbury, D. A. (1984). Behavioral patterns of Djungarian hamsters: An adaptive profile. Anim. Learn. Behav. 12, 297-306. Scherneck, S., and Feunteun, J. (1990). The hamster polyomavirus--a brief review of recent knowledge. Arch. GeschwulsO~orsch. 60, 271-278. Schlenker, E. H. (1985). Ventilation and metabolism of the Djungarian hamster (Phodopus sungorus) and the albino mouse. Comp. Biochem. Physiol. A 82, 293-295. Shklar, G. (1972). Experimental oral pathology in the Syrian hamster. Prog. Exp. Tumor Res. 16, 518. Silberberg, R., and Gerritsen, G. C. (1976). Aging changes in intervertebral discs and spondylosis in Chinese hamsters. Diabetes 25, 477-483. Silverman, J., and Chavannes, J. M. (1977). Biological values of the European hamster (Cricetus cricetus). Lab. Anim. Sci. 27, 641-645. Sindelar, W. E, Tralka, T. S., Kurman, C. C., Hyatt, C. L., and Henson, E. R. (1983). Demonstration of type-R and type-C virus particles in hamster pancreatic adenocarcinomas. Cancer Lett. 18, 119-129. Small, J. D. (1968). Fatal enterocolitis in hamsters given lincomycin hydrochloride. Lab. Anim. Care 18, 411-420. Spikermann, V. A. R., and Althoff, J. (1980). The fine structure of the exocrine pancreas of the European hamster (Cricetus cricetus L.): Electron microscopic investigations. Z. Versuchstierkd. 22, 36-42. Stewart, H. L., Snell, K. C., Dunham, L. J., and Schylen, S. M. (1959). "Transplantable and Transmissible Tumors of Animals," p. 378. Armed Forces Institute of Pathology, Washington, D. C. Stills, H. E, Jr. (1991). Isolation of an intracellular bacterium from hamsters (Mesocricetus auratus) with proliferative ileitis and reproduction of the disease with a pure culture. Infect. lmmun. 59, 3227-3236. Streilein, J. W. (1978). Hamster immune responses: Experimental models linking immunogenetics, oncogensis, and viral immunity. Fed. Proc., Fed. Am. Soc. Exp. Biol. 37, 2023-2108. Streilein, J. W., Duncan, W. R., and Homburger, E (1980). Immunogenetic relationships among genetically defined, inbred domestic Syrian hamster strains. Transplantation 30, 358-361. Suzuki, E., Matsubara, J., Saito, M., Muto, T., Nakagawa, M., and Imaizumi, K. (1982). Serological survey of laboratory rodents for infection with Sendai virus, mouse hepatitis virus, reovirus type 3, and mouse adenovirus. Jpn. J. Med. Sci. Biol. 35, 249-254. Svensjo, E. (1990). The hamster cheek pouch as a model in microcirculation research. Eur. Respir. J. Suppl. 12, 595s-600s. Taffs, L. E (1976). Pinworm infections in laboratory rodents: A review. Lab. Anim. 10, 1-13. Tagaya, M., Morie, M. I., Miyadai, T., et al. (1995). Efficacy of temperaturesensitive Sendai virus vaccine in hamsters. Lab. Anim. Sci. 45, 233238. Tansey, G., Roy, A. E, and Bivin, W. S. (1995). Acute pneumonia in a Syrian hamster: Isolation of a Corynebacterium species. Lab. Anim. Sci. 45, 366367. Taylor, D. M., Farquhar, C. E, and Neal, D. L. (1993). Studies on the eradication of intestinal protozoa of Syrian hamsters in quarantine and their transfaunation to mice. Lab. Anim. Sci. 43, 359-360. Tennent, G. A., Baltz, M. L., Osborn, G. D., Butler, P. J., Noble, G. E., Hawkins, P. N., and Pepys, M. B. (1993). Studies of the structure and binding properties of hamster female protein. Immunology 80, 645-651. Thompson, R. (1971). The water consumption and drinking habits of a few species and strains of laboratory animals. J. Inst. Anim. Technicians 22, 2936. Unay, E. S., and Davis, B. J. (1980). Treatment of Syphacia obvelata in the Syrian hamster (Mesocricetus auratus) with piperazine citrate. Am. J. Vet. Res. 41, 1899-1900. Van Hoosier, G. L., Jr., and Trentin, J. J. (1979). Naturally occurring tumors of the Syrian hamster. Prog. Exp. Tumor Res. 23, 1-12. Viswanathan, N., and Davis, E C. (1992). Timing of birth in Syrian hamsters. Biol. Reprod. 47, 6-10. Waggie, K. S., Thornburg, L. P., Grove, K. J., and Wagner, J. E. (1987). Lesions

202 of experimentally induced Tyzzer's disease in Syrian hamsters, guinea pigs, mice, and rats. Lab. Anim. 21, 155-160. Wagner, J. E., Doyle, R. E., Ronald, N. C., Garrison, R. G., and Schmitz, J. A. (1974). Hexamitiasis in laboratory mice, hamsters, and rats. Lab. Anim. Sci. 24, 349-354. Wantland, W. W. (1955). Parasitic fauna of the golden hamster. J. Dent. Res. 34, 631-649. Ward, B. C., and Moore, W., Jr. (1969). Spontaneous lesions in a colony of Chinese hamsters. Lab. Anim. Care 19, 516-521. Warner, R. G., and Ehle, E R. (1976). Nutritional idiosyncrasies of the golden hamster (Mesocricetus auratus). Lab. Anim. Sci. 26, 670-673. Wightman, S. R., Pilitt, P. A., and Wagner, J. E. (1978). Dentostomella translucida in the Mongolian gerbil (Meriones unguiculatus). Lab. Anim. Sci. 28, 290-296. Wollnik, E, and Schmidt, B. (1995). Seasonal and daily rhythms of body temperature in the European hamster (Cricetus cricetus) under semi-natural conditions. J. Comp. Physiol. B 165, 171-182. Wynne-Edwards, K. E., and Lisk, R. D. (1984). Djungarian hamsters fail to conceive in the presence of multiple males. Anim. Behav. 32, 626-628. Wynne-Edwards, K. E., and Lisk, R. D. (1987). Male-female interactions across the female estrous cycle: A comparison of two species of dwarf hamster (Phodopus campbelli and Phodopus sungorus). J. Comp. Psychol. 101, 335-344. Xu, R., Yokoyama, W. H., Irving, D., Rein, D., Walzem, R. L., and German, J. B. (1998). Effect of dietary catechin and vitamin E on aortic fatty streak

F. CLAIRE HANKENSON AND GERALD L. VAN HOOSIER JR. accumulation in hypercholesterolemic hamsters. Atherosclerosis 137, 2936. Yanagimachi, R., Yanagimachi, H., and Rogers, B. J. (1976). The use of the zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biol. Reprod. 15, 471-476. Yerganian, G. (1958). The striped-back or Chinese hamster. J. Natl. Cancer Inst. 20, 705-727. Yerganian, G. (1965). Spontaneous diabetes mellitus in the Chinese hamster, Cricetulus griseus. Int. Cong. Ser. Excerpta Med. 84, 612-627. Yerganian, G. (1985). The biology and genetics of the Chinese hamster. In "Molecular Cell Genetics" (M. Gottesman, ed.), pp. 3-36. Wiley, New York. Yerganian, G., Klein, E., and Roy, A. (1955). Physiological studies on Chinese hamster, Cricetulus griseus. II. A method for study of bleeding time in hamsters. Fed. Proc., Fed. Am. Soc. Exp. Biol. 14, 424. Yerganian, G., and Lavappa, K. S. (1971). Procedures for culturing diploid cells and preparation of meiotic chromosomes from dwarf species of hamsters. Chem. Mutagens 2, 387-410. Yoon, C. H., and Peterson, J. S. (1979). Recent advances in hamster genetics. Prog. Exp. Tumor Res. 24, 157-161. Zhang, Y. X., Fox, J. G., Ho, Y., Zhang, L., Stills, H. E, Jr, and Smith, T. E (1993). Comparison of the major outer-membrane protein (MOMP) gene of mouse pneumonitis (MoPn) and hamster SFPD strains of Chlamydia trachomatis with other Chlamydia strains. Mol. Biol. Evol. 10, 1327-1342. Zook, B. C., Huang, K., and Rhorer, R. G. (1977). Tyzzer's disease in Syrian hamsters. J. Am. Vet. Med. Assoc. 171, 833-836.

Chapter 6 Biology and Diseases of Guinea Pigs John E. Harkness, Kathleen A. Murray, and Joseph E. Wagner

I.

II.

III.

Introduction .................................................

203

A.

T a x o n o m y and G e n e r a l C o m m e n t s . . . . . . . . . . . . . . . . . . . . . . . . . . .

203

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U s e s in R e s e a r c h

C.

Availability and S o u r c e s

D.

L a b o r a t o r y M a n a g e m e n t and H u s b a n d r y

Biology

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204 206

A.

U n i q u e P h y s i o l o g i c and A n a t o m i c Characteristics . . . . . . . . . . . . . . .

206

B.

Life C y c l e and P h y s i o l o g i c Values . . . . . . . . . . . . . . . . . . . . . . . . . . .

207

C.

Diets, Nutrition, and F e e d i n g B e h a v i o r . . . . . . . . . . . . . . . . . . . . . . . .

208

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Behavior ................................................

209

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Reproduction ............................................

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Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

212

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212

Infectious D i s e a s e s

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M e t a b o l i c and Nutritional Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . .

230

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Traumatic Lesions ........................................

236

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Iatrogenic and M a n a g e m e n t - R e l a t e d Disorders

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Neoplastic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237

F.

Miscellaneous Conditions ..................................

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INTRODUCTION

The guinea pig (Caviaporcellus), the only New World rodent used commonly in research, has contributed to studies of anaphylaxis, asthma, gnotobiotics, immunology, infectious and nutritional disease, and otology, among others. Several outbred and inbred strains are available. Husbandry considerations include noninjurious housing, appropriate food, prevention of intraspecies aggression, environmental stability, and reproductive aspects, including a long gestation. Although guinea pigs are susceptible to a wide range of diseases, current breeding and LABORATORY ANIMAL MEDICINE, 2nd edition

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References ..................................................

I.

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housing conditions have reduced greatly many spontaneous infectious diseases in these animals. Diseases of concern that do occur in research colonies may include respiratory diseases (Bordetella, Streptococcus, adenovirus), chlamydiosis, pediculosis, dermatophytosis, hypovitaminosis C, pregnancy toxemias, urolithiasis, traumatic lesions, dental malocclusion, ovarian cysts, and antibiotic-induced intestinal dysbiosis. A.

Taxonomy and General Comments

The order Rodentia is subdivided into three suborders: Sciuromorpha (squirrel-like rodents), Myomorpha (rat-like rodents), Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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and Hystricomorpha (porcupine-like rodents). The domestic guinea pig (Cavia porcellus) is classified as a New World hystricomorph rodent belonging to the family Caviidae. Although recent investigations involving DNA sequencing question the traditional phylogenetic position of the guinea pig, evidence suggesting that the Hystricomorpha be reclassified outside Rodentia is controversial and inconclusive. Further work in this area needs to be done (Wolf et al., 1993; Cao et al., 1997). The family of Caviidae consists of 5 genera and approximately 23 species of South American rodents. All Caviidae have four digits on the forefeet and three on the hindfeet. The soles of the feet are hairless, and the nails are short and sharp. Members of the genus Cavia have stocky bodies with a large head, short limbs and ears, a single pair of mammae, and a vestigial tail. Guinea pigs were domesticated first by the Andean Indians of Peru as a food source and as a sacrificial offering to the Incan gods (Morales, 1995). The Dutch introduced guinea pigs to Europe in the sixteenth century, where they were bred by fanciers. There are several color (white, black, brown, red, brindle, and roan) and hair-coat varieties of guinea pigs. They may be mono-, bi-, or tricolored and have short regular hair (shorthair or English); longer hair arranged in whorls (Abyssinian); long straight hair (Peruvian); or medium-length fine hair (silky). These varieties can interbreed (Figs. 1, 2, and 3) (Harkness, 1997).

B.

which is down from a high of 599,000 animals in 1985. Their gentle temperament, commercial availability, low maintenance expense, and extensive historical use as a research model underlie their popularity as research subjects. The guinea pig was the first laboratory animal species derived and maintained in an axenic state (Wagner and Foster, 1976). Guinea pigs have been used in a variety of studies, including anaphylaxis, asthma, delayed hypersensitivity, genetics, gnotobiotics, immunology, infectious disease, nutrition, otology, and pharmacology. They are used also as a source of serum complement in laboratories using the complement-fixation test to diagnose infectious disease.

C.

Availability and Sources

The shorthair, albino English or Hartley guinea pig is used commonly in biomedical research, testing, and teaching. Outbred animals are available commercially from many breeders of laboratory animals. Additional types of guinea pigs used in research include outbred stocks, albinos, a hairless (euthymic) Hartley guinea pig, and two inbred lines (strains 2 and 13). A periodically revised listing of sources of several stocks and strains of guinea pigs was published by the National Research Council (1979), and a database called "Animal Models and Genetic Stocks" is available on the website of the Institute for Laboratory Animal Research. No new publication, however, is planned (Dell, Personal Communication, 1999).

Uses in Research

Guinea pigs have been used in research for over 200 years. Approximately 505,000 were used in 2000 in biomedical research and teaching in the United States (U.S. Department of Agriculture, Animal and Plant Health Inspection Service),

D.

Laboratory Management and Husbandry

Commonly used caging systems for guinea pigs housed in research facilities include predominantly solid-sided, wire-mesh

Fig. 1. A guinea pig typical of the shorthair, English and American varieties.

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Fig. 2. An Abyssinianguineapig with rosette patterns in the hair coat.

or solid-floored cages stacked vertically on racks; individual microisolator cages; solid-bottom plastic caging; and solid-bottom plastic caging in a ventilated rack. Solid-bottom cages may be bedded with commercially available materials such as ground corncobs, hardwood chips, or shavings and paper products. Some bedding materials may interfere in animal test systems involving ascorbic acid depletion because of the presence of low levels of vitamin C in some bedding materials (Dunham et al., 1994). Commercial breeders often use large solid-bottom, plastic tubs with wire-bar or-mesh tops to house breeding groups. These tubs can be stacked vertically on racks. Wire-mesh flooring may result in injuries to feet and legs of smaller, younger animals and reduced production in breeding animals. Cage space requirements for guinea pigs are 390 cm 2 (60 in. z) of floor space for animals weighing 350 gm or less and 650 cm 2 (101 in. z) for animals weighing more than 350 gm. For all animals, the height of the primary enclosure should be at least 18 cm (7 in.). Generally recommended environmental parameters for housing guinea pigs include an ambient temperature of 17~176 (630-79 ~F), relative humidity of 30 to 70%, ventilation of 10 to 15 air changes per hr with no draft, and a 12 hr light-12 hr dark light cycle (National Research Council, 1996). Feed is usually provided in a ,J-type feeder, which hangs inside the cage or is built into the cage.door. It is important that the feeder provide easy access to feed. Guinea pigs do not adapt readily to changes in the presentation of their feed or water.

When changes are necessary, it is important to observe the animals often and closely to ensure that they are eating and drinking. Guineapig feed is generally supplemented with vitamin C to meet the guinea pig's nutritional requirement. In some situations, additional feedstuff high in vitamin C (e.g., orange wedges, kale, cabbage) is fed. Supplemental feed, such as hay, may be placed in a crock or similar feeder and be removed on a regular basis if it is not eaten. Water can be provided in water bottles or by an automatic watering system. Guinea pigs often manipulate their water bottles and spill water into their cages. With solid-bottom, bedded cages it is important to remove soiled, wet bedding and replace it as needed with fresh, dry bedding. Automatic watering valves used in solid-bottom caging systems should be located outside the cage to minimize wet or flooded cages. Guinea pigs are gentle, docile animals that rarely scratch or bite when handled. When guinea pigs are approached, their first response may be to become immobilized, followed by rapid running. Large guinea pigs should be picked up with two hands. One hand is placed beneath the chest and upper abdomen, and the other hand supports the hindquarters. The two-handed support is especially important to prevent injury of pregnant females and large adults. Rodent restraint devices used for rats and mice are not easily adaptable to guinea pigs because of their compact body shape. Hypnotic sedation has been suggested as an alternative

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Fig. 3. A Peruvianstrain guineapig with the long hair characteristicof this strain.

to chemical sedation during minor diagnostic procedures in guinea pigs, including needle puncture (Clifford, 1984).

II.

A.

BIOLOGY

Unique Physiologic and Anatomic Characteristics

Several aspects of the anatomy, physiology, and metabolism of the guinea pig are unique among domesticated rodents and are reviewed in detail by Cooper and Schiller (1975), Wagner and Foster (1976), Festing (1976a), Navia and Hunt (1976), and McCormick and Nuttall (1976). 1.

Circulatory and Lymphoreticular Systems

The erythrocytic indices of the guinea pig are relatively low compared with those of other laboratory rodents. Lymphocytes, small and large, are the predominant leukocyte in the peripheral blood. Neutrophils (heterophils) have distinct eosino'philic granules in the cytoplasm (Schalm et al., 1975; Sanderson and

Phillips, 1981). The Foa-Kufloff or Kurloff cell is an estradioldependent mononuclear leukocyte unique to the guinea pig (Fig. 4). These cells are found primarily in the thymus and in the sinusoids of the spleen, liver, and lung, with increased numbers in the peripheral circulation during pregnancy. Large numbers are seen also in the placenta, where they may have a role in preventing the maternal rejection of the fetal placenta during pregnancy (Marshall et al., 1971). The Kufloff cell has a large mucopolysaccharide, intracytoplasmic inclusion body, which is metachromatic and periodic acid-Schiff positive, containing proteoglycans (Landemore et al., 1994) and hydrolytic enzymes (Taouji et al., 1994), similar to the smaller intracytoplasmic granules found in natural killer (NK) cells. The Kufloff cell has NK cytotoxic activity in vitro and may be part of cancer resistance in the guinea pig (Debout et al., 1995). Guinea pigs, like ferrets and primates, are relatively resistant to the effects of steroids, and the numbers of thymic and peripheral lymphocytes are not reduced markedly by corticosteroid injections (Hodgson and Funder, 1978). The guinea pig is an established model for the study of genetic control of the histocompatibility-linked immune response (Chiba et al., 1978). Although the thymus of the guinea pig is located in the ventral

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Also, compared with the rat, the guinea pig myocardiocytes are not as "stiff" (Kapel'ko and Navikova, 1993). Brewer and Cruise (1994) provide more details on the comparative aspects of the guinea pig heart. 4. Respiratory System

The guinea pig has been used as a model of lung-function impairment and bronchial reactions, including airway hyperresponsiveness and reactions that resemble asthma in humans (Nagase et al., 1994; Martin, 1994; Cook et al., 1998). A thorough review of the guinea pig respiratory system with an emphasis on species differences is presented by Brewer and Cruise (1997). Blood-gas parameters, acid-base balance, and hemodynamic and respiratory functions are described in Barzago et al. (1994). 5. The Ear Fig. 4. A Foa-Kurloffcell in a peripheral blood smear of a guineapig. The

intracytoplasmicinclusionbody is large and conspicuous(arrow).

cervical region and is easy to remove surgically, accessory thymic islets exist in contiguous fascia. The thymus apparently has no afferent lymphatic vessels (Ernstrom and Larsson, 1967). 2.

Gastrointestinal System

The anatomy of the guinea pig has been reviewed by Cooper and Schiller (1975) and Breazile and Brown (1976). The guinea pig dental formula is 2(1 1/1 C 0/0 PM 1/1 M 3/3) = 20, with a diastema or gap between the incisors and premolars. All teeth are open-rooted and grow continuously (hypsodontic). The incisors are normally white, unlike those of other rodents. The upper incisors are shorter than the lower pair. The oral cavity is small and narrow, making endotracheal intubation difficult. Guinea pigs are monogastric. Unlike that of other rodents, the stomach is undivided and is lined entirely with glandular endothelium. The large cecum can hold up to 65% of the total gastrointestinal contents. The gastric emptying time is approximately 2 hr. Cecal emptying time is very slow, and total gastrointestinal transit time is approximately 20 hr (Manning et al., 1984). With coprophagy, the total transit time can be approximately 60 to 70 hr (Jilge, 1980). 3.

Cardiovascular System

Compared with the rat, the guinea pig has both a lower basal coronary blood flow and a lower peak coronary blood flow. The intercoronary collateral network is well developed; therefore, it is difficult to produce a cardiac infarct in the guinea pig by acute coronary artery occlusion (Brewer and Cruise, 1994).

The large, accessible guinea pig ear is used for several types of auditory studies (McCormick and Nuttal, 1976). The Preyer or pinna reflex, which involves a cocking of the pinnae in response to a sharp sound, may be used in otologic studies as a measurement of hearing function. Advantages of using the guinea pig ear include the large bullae, ease of surgical entry to the middle and inner ears, and protrusion of the cochlea and blood vessels into the cavity of the middle ear, which allows examination of the microcirculation of the inner ear (Manning et al., 1984). 6. Pituitary Gland

Pituitary growth hormone is responsible for postnatal growth in vertebrates. Surgical removal of the pituitary gland in most species results in alteration of the growth pattern. However, hypophysectomy does not alter the growth rate of guinea pigs. In addition, supplementation with guinea pig pituitary extract fails to alter the growth rate of both hypophysectomized and normal guinea pigs. Somatomedins insulin-like growth factor I (IGF-I) and IGF-II are responsible for growth in the guinea pig. Unlike in other species, the somatomedins in the guinea pig are not growth-hormone dependent. Hypophysectomy does not decrease the level of somatomedins. It is not known what regulates somatomedin expression in the guinea pig (Baumann, 1997).

B.

Life Cycle and Physiologic Values

Tables I and III list general normative, physiologic, and life cycle data for the guinea pig. Values may vary with age, strain, sex, environment, and method of data collection. For more detailed information regarding the source of the data and method of collection, the references should be consulted.

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C. Diets, Nutrition, and Feeding Behavior Guinea pigs are strict herbivores and cecal fermenters, as are horses and rabbits. Unlike rabbits, however, guinea pigs possess lactobacilli and produce propionic acid as the primary fatty acid (Smith, 1965). Guinea pigs must have a dietary source of vitamin C due to their inability to synthesize the vitamin. Guinea pigs are coprophagic (King, 1956; Navia and Hunt, 1976) and ingest fecal pellets directly from the anus. Obese or pregnant animals may ingest pellets from the floor (Hintz, 1969; Harper,

1976). Maternal feces are eaten by young animals, thereby inoculating their intestines with autochthonous (normal) flora. In the wild, guinea pigs are crepuscular, feeding at dawn and dusk. In laboratory conditions, with 12 hr light-12 hr dark light cycle, guinea pigs feed during the day and night with rest periods between meals (White et al., 1989). Feeding behavior consists of alternating between feed and water, which forms a slurry that may block the lumen of the sipper tube. Guinea pigs should receive a feed prepared specifically for the species and containing vitamin C. Commercially available

Table I Approximate Physiologic Values for Guinea Pigs a-f General data Body weight: adult male Body weight: adult female Birth weight Body surface area a'g'h

900 - 1000 gm 700 - 900 gm 60-115 gm 700-830 gm: 9.2 (wt in gm) 2/3 cm 2 200-680gm: 10.1 (wt in gm) 2/3 cm 2 37.2-39.5~ 64 3 - 4 years 6 - 7 years 60 months 6 gm/100 gm body weight/day 10 ml/100 gm body weight/day 13-30hr 30~ 2-31~

Rectal temperaturei Diploid number iJ Life span: usual Life span: extreme 50% survival Food consumption Water consumption Gastrointestinal transit time k Critical temperaturei Thermal neutrality range i Cardiovascular and respiratory systems l--o Respiratory rate 42-104/min Tidal volume 2.3-5.3 ml/kg body weight 0.76-0.83 ml/gm body weight/hr Oxygen use 18-26 mM/liter Plasma CO2 21-59 mm Hg CO2 pressure Plasma pH 7.17-7.53 Heart rate 230-380/min Blood volume 69-75 ml/kg body weight Cardiac output p 240-300 ml/min/kg body weight Blood pressure 80-94/55-58 mmHg Blood cells p'q 5.4 • 1 0 6 / n l l T l 3 -4- 12%r Erythrocytes Hematocrit 43 ___ 12% Hemoglobin 13.4 gm/dl _ 12% 81 ixm3 MCV MCH 25 pg MCHC 30% a Festing (1976b). bCharles River Breeding Laboratories (1982). cAltman and Dittmer (1974). dWhite and Lang (1989). e Clifford and White (1999). I Harkness and Wagner (1995). gHong et aL (1977). hKlaassen and Doull (1980). i Short and Woodnott (1969).

J Robinson (1971). kJilge (1980). /Schalm et al. (1975). mSisk (1976). nPayne et al. (1976). oSchermer (1967). PQuillec et al. (1977). qLaird (1974). Coefficient of variation. r

Leukocytes Neutrophils Lymphocytes Kurloff cells Eosinophils Monocytes Basophils Platelets Clinical chemistry (serum)c-e Total protein Albumin Globulin Glucose Blood urea nitrogen Creatinine Total bilirubin Lipids Phospholipids Total triglyceride Cholesterol Calcium Phosphorus Magnesium Sodium Potassium Chloride Alanine aminotransferase Alanine transaminase Alkaline phosphatase Aspartate aminotransferase Aspartate serum transaminase Creatine phosphokinase Lactate dehydrogenase

9.9 • 103/ram3___30% 28-44% 39-72% 3-4% 1-5% 3-12% 0-3% 250-850 • 103/mm3 4.5-5.9 gm/dl 2.3-3.0 gm/dl 1.7-2.6 gm/dl 80 -110 mg/dl 15.7-31.5 mg/dl 1.0-1.8 mg/dl 0.2-0.4 mg/dl 95-240m~dl 25-75 mg/dl 28-76mg/dl 20-43mg/dl 9.0-11.3 mEq/dl 4.2-6.5 mEq/dl 2.1-2.7 mg/dl 121-126 mEq/liter 4 - 6 mEq/liter 96-98 mEq/liter 31-51 IU/liter 32-51 IU/liter 68-71 IU/liter 38-57 IU/liter 38-58 IU/liter 80-130 IU/liter 37-63 IU/liter

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guinea pig chow is pelleted and contains approximately 18 to 20% crude protein and 9 to 18% fiber. Feed should be stored in a cool, dry, dark area and not used after 90 days postmilling without additional vitamin C supplementation. Diets and nutrition are discussed in Section III, B. There are comprehensive reviews of guinea pig nutrition by Mannering (1949), Reid and Bieri (1972), and Navia and Hunt (1976). A tabular summary of estimated nutritional requirements of guinea pigs and signs associated with several deficiency states are given in Tables II and IV, respectively.

D.

Behavior

Reviews of guinea pig behavior include those of Harper (1976) and Sachser (1998). In group-housed animals, a dominant male hierarchy develops with a less well-defined female hierarchy. Scent marking by anal and supracaudal gland secretions and urine delineates territory. Vocalization, agonistic displays, and occasional fighting can also be used to define territory. Social interactions consist primarily of following and grouping. Guinea pigs move, rest, and often eat in groups (Manning et al., 1984). In a cage, they will align themselves along the outside perimeter, end to end, with young pups near the end of the line. They prefer walking along the periphery of the cage and avoid crossing the middle of the cage, whenever possible (White et al., 1989). Guinea pig learning occurs progressively over several trials rather than within a single interval. This may be related to some yet poorly known memory-consolidation mechanism that requires distributed rather than massed practice (Sansone and Bovet, 1970; Harper, 1976). Guinea pig vocalizations can be divided into 11 call types based on physical structure of sonograms. When classified according to situations evoking the sounds, it has been suggested that guinea pig vocalizations can be divided into five functional categories: calls used to increase proximity, greeting and proximity-maintaining calls, proximity-regaining calls, distress calls, and alarm calls (Berryman, 1976).

E.

Reproduction

Comprehensive descriptions of the reproductive anatomy and physiology of the guinea pig are found in Phoenix (1970), Barnes (1971), Cooper and Schiller (1975), Breazile and Brown (1976), and Sisk (1976). Reproductive data are summarized in Table III. 1. Reproductive Anatomy and Physiology

Accessory sex glands in the male guinea pig include large, transparent, smooth seminal vesicles (up to 10 cm in length),

Table II Estimated Nutrient Requirements for Guinea Pigs a

Nutrient Dry matter Water Fiber Nitrogen-free extract Protein L-Arginine b L-Tryptophan L-Sulfur amino acids r Fat d Methyl linoleate Calcium Phosphate Magnesium e Sodium Potassium f Manganese Copper Iron Zinc c Vitamin A acetate Vitamin D g Vitamin E Vitamin Kh Vitamin C Biotin Choline Folic acid Niacin Pantothenic acid Riboflavin Thiamin Pyridoxine Vitamin liE

Nutrient/kg diet 900 gm 100 gm 10-18% 45-48% 20-30% 1.6% 0.2% 0.7% 0.4% 1.0% 0.6% 0.3 % 0.4% 0.5% 40 mg 6 mg 2.5 mg 20 mg 8.0 mg (0.04 mg) 100 mg 2 Ixg 200 mg Not required 1500 mg 6 mg 15 mg 15 mg 16 mg 2 mg 3 mg Or cobalt required

Nutrient/kg body weight/day 40 gm 100 ml total 4 - 7 . 2 gm 18-19.2 gm 8 - 1 2 gm

400 mg 240 mg 120 mg 160 mg 200 mg 1.6 mg 0.25 mg

0.4 mg 2.3 mg 0.09 Ixg 10 mg 60 mg 0.25 mg 0.6 mg 0.6 mg 0.64 mg 0.08 mg 0.12 mg

a Approximations based on information in Navia and Hunt (1976) and Reid and Bieri (1972). bWith 30% casein diet. c With 30% soybean diet. d Corn oil. e Higher with elevated phosphate. fHigher if cation deficient. g Needed if Ca:P inappropriate. h Intestinal synthesis occurs: includes other B vitamins.

prostate, coagulating, bulbourethral, and rudimentary preputial glands. Testes remain in inguinal pouches; inguinal canals are open for life. There is an os penis. Starting as early as 4 weeks of age, males begin mounting and thrusting behavior. Intromissions occur around 45 days of age with ejaculations at approximately 56 days of age. Boars are first used for breeding at about 600 to 700 gm or 3 to 4 months of age. The uterus is bicornate in sows. The uterine body terminates

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Table Ill Reproductive Values for Guinea Pigs First ovulation First ejaculation Breeding onset: male Breeding onset: female Cycle length Implantation Gestation period Postpartum estrus Litter size Litter interval Weaning age Breeding life Young production Preweanling mortality Milk compositionb Milk yield (maximum) r

a

4-5 weeks 8-10 weeks 600-700 gm (3-4 months) 350-450 gm (2-3 months) 15-17 days 6-7 days postovulation 59-72 days 60-80% fertile 2-5 96 days 180gm (14-28 days) 18 months to 4 years (4-5 litters) 0.7-1.3/sow/month 5-15% 3.9% fat, 8.1% protein, 3.0% lactose 45-65 ml/kg body weight/day

aphoenix (1970), Ediger (1976), Sisk (1976), Peplow et al. (1974), Laird (1974), and Festing (1976b). bNelson et al. ( 1951). cDavis et al. (1979).

into a single os cervix. Sows are bred first when they weigh between 350 to 450 gm or are 2 to 3 months of age. Guinea pigs are spontaneous ovulators and, under laboratory conditions, polyestrous breeders. The estrous cycle of the guinea pig lasts approximately 16 days (range of 13-21 days). Proestrus (1-1.5 days) is characterized by vaginal swelling, rupture of the vaginal closure membrane, increased activity, and a vaginal smear of nucleated and cornified epithelial cells (Stockard and Papanicolaou, 1917; Young et al., 1935; Harkness, 1986). Estrus lasts 8 to 11 hr and is indicated by vaginal swelling and congestion, lordosis, a perforate vaginal membrane, and cornified epithelial cells. Metestrus (3 days) and diestrus (11-12 days) complete the estrous cycle. A fertile postpartum estrus occurs from 2 to 10 hr after parturition (Sisk, 1976). 2.

Detection of Estrus and Pregnancy

Estrus is indicated by swollen congested vulva, a perforate vaginal membrane, and lordosis posture, with rear quarters elevated (Harper, 1968; Phoenix, 1970). A vaginal smear contains mucus and a preponderance of cornified epithelial cells. A vaginal smear can be used to confirm estrus, if desired. Vaginal impedance measurements can also be used to assess the stage of estrous cycle in female guinea pigs (Lilley et al., 1997). Pregnancy can be detected by gentle palpation of the uterus. At day 15 of gestation, firm, oval swellings of approximately 5 mm in diameter can be felt in the uterine horns. These swellings progress to 7 to 15 mm in diameter at 25 days of gestation and 25 mm at 35 days of gestation. Individual body parts of fe-

tuses can be palpated after 35 days. During late pregnancy, abdominal distension becomes evident, and the pubic symphysis separates during the last week. 3.

Breeding Systems and Husbandry

General guinea pig husbandry requirements are discussed in Section I, D. Size of cage and amount of floor space required depend on the breeding system used. Both monogamous (one male to one female) and polygamous (one male to several females) systems can be used. With either system, continuous cohabitation allows mating to occur during the sow's fertile postpartum estrus. This type of breeding system will result in an average of 5 litters per sow per year. If the pregnant female is separated from the male before parturition, the average number of litters per sow per year is reduced to 3.5. Heavily bred sows may cease hair growth, resulting in partial alopecia (Fig. 5). Both solid-bottom and wire-bottom cages can be used for breeding, although wire floors are most often associated with weight and hair loss among young guinea pigs, decreased production, cooler ambient temperatures, and fractured limbs (Ediger, 1976). If wire mesh is used, the opening in the mesh should be of a size to prevent injuries. Young guinea pigs will begin to eat and drink water as early as 2 to 3 days of age. The feeder and sipper tube may be lowered to provide access to the smaller animals. 4.

Mating and Gestation

During the sow's estrus, the boar approaches, sniffs, circles, nibbles, licks, and mounts. The sow assumes the lordosis posture. The boar makes one or two intromissions and then ejaculates. Coital completion is indicated by grooming, scooting, and perianal marking by the boar (Manning et al., 1984). A copulatory or vaginal plug may be found in the female or the bedding. Approximately 60 to 85% of matings, including postpartum matings, are fertile. The gestation period is an average of 68 days (ranging from 59 to 72 days). Blastocysts implant on day 6 or 7 of gestation. Placentation is labyrinthine hemomonochorionic, similar to that of humans (Harkness, 1986). 5.

Parturition

Gestation length is generally inversely proportional to litter size. Relaxin is produced by the placenta, beginning around day 30 of gestation and continuing to about day 63. Relaxin is responsible for the loosening of the fibrocartilaginous pelvic symphysis. During the last week of gestation, the separation increases to 3 cm (Zarrow, 1947). Sows do not build nests. Young are delivered quickly, generally at night, with pups being born every 3 to 7 min and completion of parturition in 30 min. The sow cleans the pups and eats the placentas. Boars also will eat

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Fig. 5. Hairthinning in a frequentlybred, individuallyhoused sow.

placentas. Like female rabbits, sows do not retrieve their young. Pups approach the mother, sometimes crawling under her, and initiate nursing (Harper, 1976; Hennessy and Jenkins, 1994). Large litters (3 or more) are associated with a higher incidence of stillbirths. It is rare for a sow to eat stillborn pups. Dystocia can occur in obese sows, sows bred for the first time after 7 months of age, and in sows with large fetuses (Hisaw et al., 1944). The primary cause of dystocia is usually the inability of the fetus to pass through the confining birth canal. If a cesarean section is attempted, it must be done quickly because of anoxia. Young guinea pigs can survive anoxia for only a few minutes in the isolated uterus. Partially extruded young can be gently pushed or pulled through the tract. Fetal membranes should be removed from the face (Manning et al., 1984).

6.

Early Development of the Newborn

Pups are born with hair, teeth, and open eyes and ears, and are fully mobile. Average birth weight ranges from 45 to 115 gm. Those pups weighing less than 60 gm at birth generally do not survive. Young do not nurse for the first 24 hr. Unlike in rats and mice, a period of pup licking is not required for nipple attachment to occur in the guinea pig (Konig, 1985). The immobile, crouched nursing posture described in the altricial rat occurs also for the precocial guinea pig (Hennessy and Jenkins, 1994). Lactation peaks between days 5 and 8. The milk contains high

levels of saturated, long-chain fatty acids and is approximately 4% fat, 8% protein, and 3% lactose (Harkness, 1986). Even though young guinea pigs begin eating solid food and drinking water when only a few days old, pup mortality of up to 50% can be seen if pups are undersized or do not receive milk from a sow during the first 3 to 4 days of life. Voluntary micturition does not occur until pups are between 7 to 14 days of age. Young are weaned at 21 days of age. The sow has a fertile estrus shortly after pups are weaned. 7.

Sexing

Females have a Y-shaped depression in the perineal tissue. The anus is located at the base of the Y, the membrane-covered vulvar opening is at the intersection of the branches, and the top branches of the Y surround the urethral opening. In immature males, the penis can be palpated just anterior to the preputial opening or extruded with gentle pressure at its base. Adult boars have large testes in obvious scrotal pouches (Hillyer et al., 1997). 8.

Artificial Insemination

Artificial insemination has been used successfully in guinea pigs. Electroejaculation produces 0.4 to 0.8 ml of semen, which can be placed through a bulbed pipette into the vagina (Row-

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lands, 1957; Freund, 1969). Artificial insemination with conception has been successful up to 16 hr postestrus. In some electroejaculated boars, the ejaculum coagulates in the urethra. 9.

Synchronization

There is no conclusive evidence of cycle synchronization among group-housed sows (Donovan and Kopriva, 1965; Harned and Casida, 1972). A fertile postpartum estrus lasting 3.5 hr occurs within 12 to 15 hr of parturition in most sows (Rowlands, 1949). Administration of 1000 mg estradiol for 6 days results in an "induced estrus" for an extended period of 2 to 3 days (Lilley et al., 1997). The purpose of this experimental manipulation was to extend the time guinea pigs could be vaginally immunized with an antigen for eliciting a local immune response. Further research is needed to determine if this induced estrus differs from true estrus.

fact now quite rare in research guinea pigs include Streptococcus pneumoniae and Streptobacillus moniliformis infections, salmonellosis, yersiniosis, pediculosis, dermatophytosis, and diseases resulting from dietary deficiencies. On the other hand, inapparent conditions, including marginal hypovitaminosis C, may be more common than realized and continue to affect the reliability and validity of research results derived from studies using guinea pigs. Effects of disease on research are noted in some descriptions following, but in most cases the presence of an active disease process in guinea pigs increases the variability of responses within a colony; may cause removal of animals from a study, thus decreasing the number of animals needed for convincing statistical analyses; lengthens study time; and may increase the pain and distress experienced by the animals themselves. 1.

Bacterial, Mycoplasmal, and Rickettsial Diseases

a. IIl.

A.

DISEASES

Infectious Diseases

Publications and review presentations have, at least over the past quarter century, described a disease prevalence profile for guinea pigs that is more historical or characteristic of the retail pet trade than for guinea pigs as they are presently housed in well-managed research colonies. Improvements in gnotobiotic derivation, barrier housing, diets, caging, environmental control, routine health surveillance, and information sharing have led to the virtual elimination of most of the disease conditions described in the following pages, although, of course, any of these diseases could occur in susceptible hosts. Comprehensive reviews of diseases in guinea pigs in research settings are found in Wagner and Manning (1976) and Percy and Barthold (1993), and the previous edition of this chapter (Manning et al., 1984), which remains a valuable resource. Conditions that remain concerns in research colonies of guinea pigs are food and water deprivation; asymptomatic infections; inappropriate antibiotic use; diseases of aging (e.g., neoplasia, nephrosis); marginal hypovitaminosis C; Bordetella bronchiseptica pneumonia; Streptococcus zooepidemicus infection; adenovirus infection; the presence of Pseudomonas, Staphylococcus, and various streptococci in the animals and their environments; and the detection of antibodies to cytomegalovirus (CMV), reovirus 3, and parainfluenza virus. Nonpathogenic Bordetella bronchiseptica are recovered occasionally from guinea pig respiratory tracts (Besch-Williford, personal communication, 1998; Weisbroth, personal communication, 1998). Clinical diseases identified previously as "common" but in

Bordetella bronchiseptica

i. Etiology. Bordetella bronchiseptica is a common commensal organism in many species, including guinea pigs, rats, rabbits, mice, dogs, swine, cats, turkeys, and primates. The organism is a short, gram-negative rod or coccobacillus, aerobic, motile, and non-spore-forming, as are all gram-negative bacilli. Growth in vitro is best at 30~ but is slow to poor at 37 ~C, with circular, pearlescent colonies minute at 24 hr and maximum at 72 hr. Colonies are embedded in the media and are surrounded variably by a zone of [3-hemolysis (Ganaway, 1976; Boot et al., 1994). Immunologic studies (Wullenweber and Boot, 1994) and macrorestriction digestion of DNA techniques, as well as evidence of phenotypic modulation of surface components, provide evidence for serotypic variation within the species. The organism variably dissociates in culture (isogenic mutation), and these isolates vary in hemolysin, dermonecrotoxin, proteases, adenylate cyclase, and hemagglutinin production, which may affect host specificity, virulence, and disease manifestation (Griffith et al., 1996). ii. Clinical signs. Although subclinical infections are encountered more commonly than are clinical outbreaks, the epizootic respiratory or septicemic disease can progress rapidly (often within 24-72 hr) and produce high mortality. All ages and both sexes are affected. There may also be sporadic deaths in enzootically affected colonies. Clinical disease signs include inappetence, depression, upper respiratory discharges, dyspnea, cyanosis, and death. A genital form causes infertility, stillbirths, and abortions (Ganaway, 1976). The incubation period is 5 to 7 days. iii. Epizootiology and transmission. The organism is found commonly in the respiratory tracts of many species and may, potentially, be transmitted among these species. The potential

6. BIOLOGY AND DISEASES OF GUINEA PIGS

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for transmission of Bordetella from rabbits to guinea pigs is the primary reason these two animal types should be housed in separate areas. Transmission is by fine particle aerosol onto the respiratory mucosa, by contaminated fomites, or by genital contact (Nakagawa et al., 1971; Trahan et al., 1987). Many guinea pigs carry Bordetella bronchiseptica as a commensal resident. Higher morbidity and mortality occur among the young and, perhaps, in strain 2 inbred animals.

fective, although it is not used widely in guinea pigs. Vaccination causes a localized upper respiratory infection (Stephenson et al., 1989).

iv. Necropsy findings. Bordetellosis is manifested by various degrees of pulmonary consolidation with respiratory exudation, purulent bronchitis, trachitis, and otitis media. Consolidated areas are dark red or red brown to gray. Peribronchiolar and perivascular regions contain inflammatory cells, all leading to a morphologic diagnosis of fibrinous or fibrinopurulent bronchopneumonia. In uterine infections there may be pyosalpinx and dead embryos or fetuses (Ganaway, 1976).

ix. Treatment. Bacterial infections in guinea pigs are treated with general supportive measures (e.g., fluid administration, forced feeding), adequate dietary vitamin C, and use of an antibiotic (e.g., fluoroquinolone, trimethoprim-sulfonamides) considered safe for use in guinea pigs.

v. Pathogenesis. The organism attaches firmly to ciliated respiratory epithelium, where it proliferates rapidly and causes ciliary paralysis, an inflammatory response, antiphagocytic activity, and dermonecrosis, presumably through the action of an intracellular, heat-labile toxin. Respiratory clearance of other organisms and particulate matter is reduced (Quinn et al., 1994). B. bronchiseptica may bind variably to antigen-presenting cells in the respiratory epithelium. Such binding may lead to chronicity through an altered immune response (Griffith et al., 1996). vi. Differential diagnosis. Although several bacterial and some viral agents may cause acute bronchopneumonia in guinea pigs, including Streptococcus pneumoniae, S. zooepidemicus, Klebsiella pneumoniae, and adenovirus, Bordetella infection is the most common clinical diagnosis. Definitive diagnosis is through swabbing of the lumen of the bronchi or lower trachea (presumably in dead animals) and aerobic culture on sheep blood and MacConkey's agar. Enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) serologic testing are more sensitive than is culture for detecting the organism, but various Bordetella antigenic variants should be used in serologic testing because of organism variations described above (Wullenweber and Boot, 1994). vii. Prevention. Because clinical disease arises often from a preexisting subclinical infection, the reduction or elimination of stressors, if possible given the circumstances of a study, is essential. Stressors include transport, crowding, chilling and drafts, pregnancy, hypovitaminosis C, protein or caloric deprivation, other diseases, or experimental manipulations. Purchasing Bordetella-free stock and screening existing colonies for carriers are essential diagnostic and preventive measures. Many bacterins have been tried for preventing Bordetella infection in guinea pigs, and a nonadjuvant (i.e., no aluminum hydroxide) bacterin used in dogs appears to be safe and ef-

viii. Control. Control is by isolation of animals infected with or susceptible to B. bronchiseptica, treatment of animals, health screening (if compatible with study requirements), and removal of the clinically ill.

x. Research complications. Any pathogenic organism that exists as a commensal in the respiratory tracts of several laboratory species poses a risk to a guinea pig colony, even if the animals are designated "specific pathogen free." Stressprecipitated clinical disease can eliminate a research colony before an effective treatment can be determined and initiated. b.

Streptococcus zooepidemicus

i. Etiology. Streptococcus equi subsp, zooepidemicus, a somewhat tentative designation that may be redesignated S. zooepidemicus subsp. S. equi as a biovar, is a Lancefield's group C streptococcus (Timoney et al., 1997). The [3-hemolytic, grampositive organism has an antiphagocytic capsule (M-like antigen) and produces several exotoxins, including hyaluronidase, a protease, and a streptokinase. The species or subspecies zooepidemicus survives longer off the host than does the obligate pathogen S. equi (Quinn et al., 1994). ii. Clinical signs. This pyogenic bacterium is associated with suppuration and abscess formation, usually in the cervical lymph nodes, which are evident on observation and careful palpation (Fig. 6). Other signs that may be present, depending on organs affected, are torticollis, nasal or ocular discharge, dyspnea and cyanosis, hematuria and hemoglobinuria, cyanotic and swollen mammary glands, abortions, stillbirths, and unexpected deaths, although the presence of enlarged cervical nodes ("lumps") in otherwise healthy guinea pigs is the usual and only sign. There may be inapparent upper respiratory infections (Kohn, 1974). iii. Epizootiology and transmission. Guinea pigs of all ages are affected, but the infection may be more common in certain strains (e.g., strain 2) than in others and in females. The commensal organism inhabits mucosal surfaces. Clinical signs of S. equi subsp, zooepidemicus infection are much more common in guinea pigs than are signs of S. pneumoniae infection. Transmission of the organism is via aerosol onto respiratory,

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Fig. 6. Swellingsin the ventralneckare enlargedlymphnodesinfectedwithStreptococcus zooepidemicus, the causal organismof mostcases of caseouslymphadenitis. (FromGuinea Pigs: Infectious Disease, LaboratoryAnimal Medicine and Science, Series II, American College of LaboratoryAnimal Medicine. Used with permission.)

oropharyngeal, conjunctival, or female genital epithelium. The disease is of low contagion (Murphy et al., 1991).

iv. Necropsy findings. The most common finding on necropsy is one or more abscessed and encapsulated cervical lymph node, although the node itself usually is destroyed. The abscesses may be up to several centimeters in diameter and contain a nonodorous, yellow-white to red-gray pus. Other conditions that may be caused by S. equi subsp, zooepidemicus include pneumonia, generalized lymphadenitis, focal hepatitis, otitis media, pleuritis, peri- and myocarditis, nephritis, mastitis, metritis, and arthritis with necrosis and hemorrhage (Ganaway, 1976; Kinkier et al., 1976; Harkness and Wagner, 1995). v. Pathogenesis. The organisms enter the animal through mucosal abrasions, although passage through intact epithelium may occur. The bacteria follow the lymphatic ducts to regional cervical nodes. A peracute septicemia may also occur, usually in the young (Murphy et al., 1991; Percy and Barthold, 1993). vi. Differential diagnosis. Another organism linked historically to cervical lymphadenitis in guinea pigs is Streptobacil-

lus moniliformis, which is carried by wild rats. This organism is seldom involved and is also of low contagion (Aldred et al., 1974). Diagnostic criteria include clinical and necropsy signs and isolation of [3-hemolytic streptococci from an abscess margin or heart blood. The stained organisms appear in chains. Other organisms that can cause upper respiratory lesions and death in guinea pigs include Streptococcus pneumoniae, Bordetella bronchiseptica, Klebsiella pneumoniae, adenovirus, and others. vii. Prevention and control. Methods of preventing streptococcal cervical lymphadenitis include obtaining disease-free stock, feeding nonabrasive feed (assuming crude fiber may abrade the pharyngeal mucosa), trimming overgrown or broken teeth, using feeders that do not abrade the skin of the neck, and palpating periodically for subcutaneous lumps in the cervical region. Control is effected by removing affected animals from the colony or replacing the entire colony. A killed bacterin was at one time effective but is not used (Mayora et al., 1978). Killed bacterins for one or more of the 15 serovars of Streptococcus zooepidemicus may not provide cross protection. The bacterin must be appropriate for the serovars involved.

6. BIOLOGY AND DISEASES OF GUINEA PIGS

viii. Treatment. Treatment of cervical lymphadenitis usually involves surgical removal of the abscess and its capsule. Antibiotics effective against the organism yet safe for use in guinea pigs (e.g., fluoroquinolones, trimethoprim-sulfonamides, gentamicin, or chloramphenicol) may be given. ix. Research Complications.

Guinea pigs with encapsulated abscesses remain usually in good flesh, but a systemic infection can result in several clinical manifestations and unacceptable research complications. S. equi subsp, zooepidemicus has been isolated from humans.

c.

Streptococcus pneumoniae

i. Etiology. Streptococcus pneumoniae, whose genus is known also as Diplococcus or Pneumococcus, is gram-positive, a-hemolytic, and oval to lancet shaped. It occurs in paired or chain formation. The two serotypes recovered most often from guinea pigs are types 4 and 19F, which are assumed to be identical with certain human serovars (Parker et al., 1977). ii. Clinical signs. Asymptomatic upper respiratory tract carrier states of S. pneumoniae in guinea pigs (and in humans) are high, often over 50% prevalence in some populations. This high carrier state accounts for sporadic epidemics occurring when animals are stressed or malnourished. Clinical signs, when they do occur, include high mortality or, in less acute cases, depression, anorexia, nasal and ocular discharge, sneezing and coughing, dyspnea, torticollis, or abortion and stillbirths. Epizootics may occur more in winter months, but with

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modern environmental control systems, such seasonal variations are unlikely (Percy and Barthold, 1993).

iii. Epidemiology and transmission. Streptococcus pneumoniae infections, clinical or inapparent, may be common in pet guinea pigs, but clinical cases or even carrier states are rarely reported or detected in research colonies. Transmission is by respiratory aerosol, by direct contact with infected animals (including humans, nonhuman primates, and rats), or by an infected reproductive tract during birth.

iv. Necropsy findings. Lesions seen at necropsy are primarily pyogenic processes occurring in one or more forms: fibrinopurulent pleuritis; pericarditis (Fig. 7); peritonitis; suppurative pneumonia; otitis media; endometritis; and arthritis, among others (Boot and Walvoort, 1986; Witt et al., 1988). The pulmonary lesion is an acute, fibrinopurulent bronchopneumonia with thrombosis of pulmonary vessels. v. Pathogenesis. The organism becomes established in the upper respiratory tract, where it is protected by a polysaccharide capsule and can activate an alternative complement pathway, which initiates some of the pathologic changes associated with the organism. vi. Differential diagnosis. Streptococcus pneumoniae can be seen easily on Gram-stained smears of affected tissue, or it can be cultured on blood agar incubated when necessary under 5 to 10% carbon dioxide. Matsubara et al. (1988) developed an ELISA test for streptococci. Definitive identification of

Fig. 7. Fibrinopurulentpericarditis in a guinea pig, caused by Streptococcuspneumoniae. The pericardial sac is thickened and opaque (arrow). (From Guinea Pigs: Infectious Disease, LaboratoryAnimal Medicine and Science, Series II, American College of Laboratory Animal Medicine. Used with permission.)

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S. pneumoniae requires serotyping among the 83 different capsular polysaccharides. The Quellung test may utilize a serum pool product or type-specific antisera. The capsule appears swollen in positive microprecipitin reactions occurring on the surface of the capsule. Organisms mixed with saline serve as controls (Koneman, 1997). Differential diagnoses include the various respiratory and systemic pathogenic microbes affecting guinea pigs, including Bordetella, other streptococci, salmonellae, Klebsiella, and adenovirus. vii. Prevention. Guinea pigs free from streptococcal exposure or infection should be purchased for research or teaching. Clinical disease may occur in carrier animals when they are stressed or malnourished. A stable environment and a fresh guinea pig diet with adequate vitamin C are essential, especially for young and pregnant guinea pigs. viii. Control and treatment. Treatment is more likely to cause reversion to a subclinical, carrier state than eliminate the infection. Clinically affected guinea pigs should be removed from the colony and efforts made to reduce predisposing factors. Because S. pneumoniae is found in humans and not in the environment or in most other animals, infected humans may act as a source for infection in guinea pigs. Antibiotics safe for use in guinea pigs may, in some cases, reverse the pathologic process, but inapparent infections may remain. ix. Research complications. Guinea pigs infected chronically with S. pneumoniae remain predisposed to clinical disease, which can compromise a research study or eliminate a colony. d.

Salmonella spp.

i. Etiology. Salmonellosis, seen rarely in research-housed guinea pigs, can be caused by several species or serovars of the gram-negative bacillus Salmonella; however, S. typhimurium and S. enteritidis are encountered most frequently (Ganaway, 1976). ii. Clinical signs. In peracute to acute infections the only signs of salmonellosis in an animal or colony may be high morbidity and mortality. Epizootic outbreaks occur more often in late pregnant, weanling, aged, and poorly fed animals (Wagner, 1976; Harkness and Wagner, 1995). In longer-term survivors or in sporadic clinical cases in colonies with endemic infection, guinea pigs may exhibit rough hair coats, weakness, conjunctivitis, abortion of small litters, and light-colored feces or intermittent diarrhea (Schaeffer and Donnelly, 1996). Mortality may be as high as 50 to 100% of the population. iii. Epizootiology and transmission. Pathogenic Salmonella spp. are found worldwide in a variety of vertebrates, and one species or serovar of Salmonella may affect a wide variety

of animal species. The pattern of infection may be epizootic, enzootic, or subclinical with shedding of infectious organisms. Inapparent carriers shed the organisms intermittently, which poses a continuing threat to other animals, including people. Transmission of salmonellae among animals may be fecaloral, blood- or tissue-oral, or via the conjunctiva. The organisms are shed in the feces of wild rodents or other animals and contaminate food (e.g., green vegetables, hay) intended for guinea pigs. Guinea pigs are highly susceptible to Salmonella, and the incubation period is 5 to 7 days.

iv. Necropsy findings. Gross lesions in guinea pigs dying from salmonellosis may not be present or may include hepatomegaly, splenomegaly, and small yellow necrotic foci throughout the viscera. There may also be a necrotic metritis, or at least a lymphocytic infiltration into the uterine wall (Percy and Barthold, 1993). v. Pathogenesis. Salmonellae enter the body through the gastrointestinal tract or via the conjunctiva and elicit histiocytosis, tissue necrosis, and abscess formation. vi. Differential diagnosis. Diagnosis requires recovery of the organism from feces, heart blood, spleen, or other affected organs through enrichment in a broth such as selenite F or tetrathionate, culture on MacConkey's or brilliant green agar, and identification of the organism. Serotyping identifies the species (Ganaway, 1976; Percy and Barthold, 1993). vii. Prevention. Salmonellosis in guinea pigs is now a rare disease in most research colonies because of the use of barrierraised stock, care in shipping, careful selection and storage of food, vermin elimination, health monitoring, and excellent animal room and equipment sanitation. Aging, other diseases, malnutrition, and enviromental stress are predisposing factors. viii. Control and treatment. Because of the ubiquity, persistence, zoonotic potential, and existence of endemic and carrier states, the best control and treatment recommendation for Salmonella-infected animals is to euthanatize the entire colony, sanitize caging and equipment thoroughly, and restock with animals known free of Salmonella. Antibiotic use may cause an infection to become subclinical and lead to antibiotic resistance. ix. Research complications. Salmonellosis is essentially incurable, treated animals may show no signs but can shed the zoonotic organisms into the environment, and clinical disease may be induced through stressing the host. e.

Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is a gram-negative, nonhemolytic, exotoxin- and enzyme-producing, pleomorphic rod. Opti-

6. BIOLOGY AND DISEASES OF GUINEA PIGS

mal incubation temperatures are 20 ~ to 30 ~C. Virulent strains may grow within macrophages (Quinn et al., 1994). The organism, which infects both sexes and all ages of guinea pigs and has otherwise a wide host spectrum, can cause (1) an acute, highly fatal septicemia; (2) chronic emaciation, diarrhea, and death within 3 to 4 weeks; (3) nonfatal lymphadenitis; or (4) a subclinical carrier state, usually following a clinical phase (Ganaway, 1976). The zoonotic disease, yersiniosis or pseudotuberculosis, is rare in research guinea pigs in the United States, although guinea pigs are very susceptible to this infection. Horses and sheep in the United States are commonly affected, however. Transmission is by ingestion of contaminated food, by inhalation, or through skin lacerations from fighting. Dams pass the organism to their young. In acute cases, gross lesions include an acute enteritis and mucosal ulceration with miliary, creamcolored nodules in the intestinal wall. In the subacute to chronic form, lymph nodes, spleen, liver, lung, and bone marrow contain gray-white, ovoid nodules ranging in size from a few millimeters to 2 to 3 cm. Palpation detects enlarged peripheral nodes. Microscopically, the lesions contain dead cells, inflammatory cells, and blood vessels with bacterial emboli. More chronic lesions are granulomas that do not calcify (Obwolo, 1977; Percy and Barthold, 1993). The causative organisms can be seen in and cultured from the lesions, and the enlarged mesenteric nodes can be palpated in guinea pigs. The disease can be prevented by obtaining disease-free stock kept apart from wild birds and rodents. A bacterin prepared from an avirulent strain of Yersinia has protected guinea pigs from subsequent lethal challenge. Yersinia pseudotuberculosis can infect human beings. Because of a persistent carrier state in guinea pigs, treatment for them is not advised.

f.

Clostridium difficile

i. Etiology. Enteropathies and deaths in guinea pigs occurring within 1 to 5 days of administration of certain antibiotics are assumed to result from (1) antibiotic-induced suppression of resident microflora, perhaps Bacteriodes, (2) loss of cecal colonization resistance, and (3) colonization, proliferation, and toxin production by transiting or resident commensals, usually one or more strains of the spore-former Clostridium difficile. Escherichia coli, not a normal intestinal inhabitant in guinea pigs, may proliferate in an antibiotic-caused dysbiosis and cause deaths (Farrar and Kent, 1965). Antibiotics most often implicated are the aminopenicillins, cephalosporins, clindamycin, streptomycin, and lincomycin. Penicillin at dosages as low as 2000 U or ampicillin at dosages over 6 mg/kg q8 hr for 8 days are known to cause deaths (Lowe et al., 1980; Young et al., 1987). A mechanism for loss of colonization resistance may involve effects on microbial populations, accumulation of

217 excessive carbohydrates and reduced short-chain fatty acids, followed by growth or toxin production of organisms that utilize those metabolites (Clausen, 1998). Clostridium difficileassociated typhlitis occurred also in gnotobiotic guinea pigs exposed to murine intestinal microflora (Boot et al., 1989).

ii. Clinical signs. Signs of C. difficile toxocosis include rapidly progressive lethargy, rough hair coat, possibly diarrhea, and death following exposure to certain antibiotics in the intestinal lumen. The disease, however, does not inevitably follow antibiotic administration. Consequences depend on drug dose, presence and strain of the pathogen, and host resistance. iii. Epizootiology and transmission. Clostridium difficile is a common, fecal-borne, anaerobic, gram-positive, commensal organism whose large, subterminal spores persist in the environment. Susceptible guinea pigs may carry small, resident populations of C. difficile, or, more likely, susceptible animals may ingest the spores, which encounter a receptive cecal environment. iv. Necropsy findings. The lesion in guinea pigs is a hemorrhagic cecitis, with the cecum distended and containing bloody, liquid feces. Histologically, there is a severe inflammatory reaction in the lamina propria and microulceration of the mucosa with inflammatory cell infiltration. v. Pathogenesis. Some strains of C. difficile produce protein exotoxins (cytotoxins) A and B, which bind to epithelial cell membrane receptors. Toxin B is more cytotoxic but requires toxin A (known also as an enterotoxin) to access mucosal cells. The toxins catalyze glucose binding to threonine in specific Rho proteins, which are essential for cytoskeletal architecture and cell movement. Toxin A causes fluid secretion, mucosal damage, and inflammation. Cell death occurs subsequently. Guinea pigs may also die suddenly from heat or cold stress, septicemia, pregnancy toxemia, pneumonia, or gastric or cecal volvulus or torsion (Dodson and Borriello, 1996; Kelly and LaMont, 1998). vi. Differential diagnosis. If death is known to follow antibiotic administration, then the cause is reasonably certain. A commercially available rapid test is an enzyme immunoassay for toxin A conducted on feces, although the toxin may not be present in feces of affected animals. A widely used test involves cytotoxicity with neutralization, which takes up to 48 hr to obtain a result. Culture for C. difficile is difficult, as the species name suggests (Rehg and Pakes, 1981; Surawicz, 1998). vii. Prevention and control. Proper selection of antibiotics (fluoroquinolones, gentamicin, trimethoprim-sulfonamide combinations, chloramphenicol) and meticulous husbandry

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and sanitation are preventive measures. Once an outbreak begins, antibiotic use must cease. Malnutrition, especially from deficient vitamin C, predisposes to clostridial enterotoxemia (Davis, 1993). viii. Treatment. Drug prophylaxis is not advised; the preferred drug in treating human cases, metronidazole, may exacerbate the toxicosis problem in guinea pigs. Treatment of antibiotic-induced cecitis in guinea pigs is symptomatic" fluids, a high caloric food supplement, and heat. Treatment for C. difficile disease in humans includes metronidazole or vancomycin in combination with the yeast Saccharomyces bonlardii (Anonymous, 1997; Cleary et al., 1998) ix. Research complications. Inappropriate administration of antibiotics that reach the cecum and elicit toxin generation can kill research animals. Also, C. difficile is a recognized cause of disease in humans. g.

Other Bacteria

i. Actinomyces pyogenes and Corynebacterium kutscheri. Corynebacterium pyogenes has been lihked to a fatal, septicemic disease, and C. kutscheri was isolated from the lungs of guinea pigs. Infections with Corynebacterium are rare (Ganaway, 1976). ii. Brucella spp. Guinea pigs are susceptible to experimental infections, but spontaneous disease from Brucella abortus, B. melintensis, or B. suis is reported rarely and not in the United States (Ganaway, 1976). Guinea pigs could contract the organism through contact with contaminated meat products. iii. Campylobacter-like organisms. Elwell et al. (1981) reported diarrhea, weight loss, and deaths in guinea pigs receiving steroids. Necropsy signs included segmented epithelial hyperplasia and adenomatous changes in the duodenum and ileum. Muto et al. (1983) reported a natural adenomatous, segmental, intestinal epithelial hyperplasia in guinea pigs. Clinical signs again included diarrhea, weight loss, and death. Lesions in the jejunum and ileum contained intracellular bacteria resembling Campylobacter spp. iv. Citrobacter freundii. Citrobacter freundii was associated with an enzootic septicemia with high mortality (Ocholi et al., 1988). Necropsy signs were pneumonia, pleuritis, enteritis, and gastric ulcers. v. Clostridium piliforme. Clostridium piliforme, the causative organism of Tyzzer's disease, is a gram-negative, curved rod and an obligate, intracellular anaerobe with subterminal spores that persist in the environment. The disease occurs in several species, including rodents, rabbits, cats, dogs, horses,

and some primates. This disease, reported rarely in guinea pigs, causes emaciation, dehydration, lethargy, diarrhea, and death. The organism causes a necrotizing ileitis, typhlitis, and hepatic necrosis in weanling guinea pigs. Necropsy shows multifocal necrosis and inflammation of the ileum, cecum, and colon. Prevention is to avoid stressors and to maintain good sanitation. Diagnosis is through identifying characteristic filamentous bacteria in a Giemsa- or Warthin-Starry-stained section of enterocytes. The organism has not been cultured in vitro. Reported spontaneous cases identify an unclassified spirochete occurring along with the Tyzzer's organism and lesions (Zwicker et al., 1978; Waggie et al., 1986; Harkness and Wagner, 1995). vi. Erysipelothrix rhusiopathiae. In one report (Okewole et al., 1989), Erysipelothrix rhusiopathiae, a gram-positive, non-spore-forming rod, caused abortion and death in guinea pigs. The organism entered through the alimentary tract and was cultured from blood in the left uterine horns and from miliary abscesses in the liver. vii. Escherichia coli. Escherichia coli infections in guinea pigs are facilitated by marginal nutrition, crowding, environmental stress, and oral antibiotic administration. The question remains, however, whether or not a facultative anaerobe E. coli in guinea pigs is a primary pathogen or opportunist. Clinical signs of enteric disease include anorexia, weight loss, rough hair coat, diarrhea, and death, especially in weanlings. Necropsy signs include yellow fluid in the gut, gas, peritoneal fluid, and focal hepatic necrosis (Ganaway, 1976). Mastitis (Kinkier et al., 1976) and cystitis may be caused by E. coli. viii. Haemophilus sp. Boot et al. (1999) reported V-factor dependent Pasteurellaceae (Haemophilus parainfluenzae and H. aphrophilus/paraphrophilus) infection in the respiratory tracts of healthy guinea pigs. Various Haemophilus antigens recovered from guinea pigs cross-reacted by ELISA with Pasteurella pneumotropica and rabbit and rat Haemophilus antisera. The authors noted also that Haemophilus sp.-caused subcutaneous abscesses were reported in guinea pigs between 1913 and 1929. ix. Helicobacter pylori. Sturegard et al. (1998) induced a severe gastritis in guinea pigs using fresh Helicobacter pylori isolates from human source biopsies or from strains passed in guinea pigs. Twenty-two of 29 inoculated guinea pigs had a specific immune response against H. pylori and had gastritis, with erosion of the gastric epithelium. The authors suggest that the guinea pig may be an appropriate model for studying H. pylori infections in humans. x. Klebsiella pneumoniae. Klebsiella pneumoniae is a gram-negative, nonmotile bacillus that causes rare epizootics in guinea pigs of all ages and both sexes. Predisposing factors are

6. BIOLOGY AND DISEASES OF GUINEA PIGS

uncertain, but malnutrition, magnitude of exposure, unsanitary environments, and genetic predisposition are several factors underlying epizootics in guinea pigs (Ganaway, 1976). Clinical signs of Klebsiella infection include anorexia, dyspnea, and death. Necropsy and histologic findings include seropurulent or serofibrinous lesions in the thoracic and abdominal cavities, mastitis, splenomegaly, thrombosis, coagulative necrosis of the liver, and granular degeneration of the renal tubule cells. Septicemias occur. The pulmonary lesion is an acute, necrotizing bronchopneumonia. Klebsiella can be isolated from blood, liver, spleen, peritoneal exudate, and cerebrospinal fluid of diseased animals. xi. Leptospira icterohaemorrhagiae. Leptospira icterohaemorrhagiae may affect guinea pigs that have been in contact with wild rats or their habitats. Jaundice and petechial to ecchymotic hemorrhages are seen in several organs, including skin and lungs. xii. Listeria monocytogenes. Listeriosis is rare in guinea pigs, with few literature reports describing infection and actual or possible clinical signs. The causative agent, the grampositive rod Listeria monocytogenes, is widespread in the environment, including in soil and bedding. Clinical signs linked to Listeria infection in hairless guinea pigs were unilateral or bilateral keratoconjunctivitis (Colgin et al., 1995) and reproductive disorders (Ganaway, 1976). Necropsy findings included keratoconjunctivitis with inflammation extending into the lacrimal gland, focal necrosis of internal organs, meningitis, and perhaps, reproductive tract disorders. Ocular lesions included ulcerated cornea, with edema and vascularization and a serous to purulent ocular discharge. Listeria is transmitted by the fecal-oral route from contaminated vegetation used as food. Animal to human transmission is not recognized; human to human transmission is considered rare. Diagnosis of listeriosis is by culture and recovery of L. monocytogenes on culture. A monocytic leukocytosis may occur. Prevention and control involve general precautions. Treatment is not recommended because of the zoonotic potential of the organism. xiii. Mycobacterium spp. Although guinea pigs are very susceptible to Mycobacterium infection, and guinea pigs were used widely as a diagnostic aide for tuberculosis, spontaneous cases in guinea pigs are rare. Guinea pigs are susceptible to both human and bovine strains of Mycobacterium, but transmission of the disease from humans and cattle is unlikely (Ganaway, 1976). xiv. Pasteurella multocida. Pasteurellosis is uncommon to rare in guinea pigs in well-managed colonies, and the prevalence of infection is unknown. An epizootic reported by Wright (1936) involved sporadic, unexpected deaths with pulmonary

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consolidation, fibrinopurulent serositis, and conjunctivitis. Diagnosis is by culture and identification of the characteristic gram-negative coccobacillary rods. xv. Pseudomonas aeruginosa. Pseudomonas infections are rare in guinea pigs but have been associated with pulmonary lesions involving lung consolidation and a severe, focal, necrotizing bronchopneumonia (Bostrum et al., 1969). Pseudomonas may also cause conjunctivitis and otitis media. Clusters of bacteria surrounded by necrotic debris (grossly, "sulfur granules") may be present in focal, suppurative lesions. Samii et al. (1996) reported a pet guinea pig with an abdomen painful on palpation and containing a 2 x 3 cm mass in the caudal abdomen. Necropsy revealed an enlarged, inflamed, fibrous prostate gland with local extension of the inflammation. Pseudomonas aeruginosa was isolated from the gland. Pseudomonas is ubiquitous and may be spread in the drinking water or in damp bedding or food. xvi. Serpulina-like organisms. Vanrobaeys et al. (1998) reported sudden deaths with guinea pigs exhibiting nervous signs or occasional yellow, slimy feces. Guinea pigs affected were in poor physical condition with parasitism, weight loss, and marginal to clinical hypovitaminosis C. Guinea pigs on necropsy had a catarrhal to hemorrhagic cecitis and colitis. Electron microscopy revealed large numbers of spirochetes (Serpulina-like organisms) adhering to the affected cecal mucosa of the animals. The organism was not isolated, so Koch's postulates could not be fulfilled, but the histologic evidence of a cause-effect relationship was convincing in these unhealthy animals. McLeod et al. (1977) and Zwicker et al. (1978) reported spirochetes in cases diagnosed as Tyzzer's disease, but those spirochetes were not identified further. xvii. Staphylococcus aureus. Staphylococcus aureus is probably present in the environment and as an inapparent respiratory or cutaneous infection in a large number of guinea pig colonies (Markham and Markham, 1966). Taylor et al. (1971) isolated Staphylococcus from chronic, ulcerative pododermatitis ("bumblefoot") lesions, which in chronic cases were associated with amyloid accumulation in liver, adrenal glands, spleen, and pancreatic islets. Volar surfaces of one or more feet may be enlarged, firm, ulcerated, and resistant to treatment (Gupta et al., 1972) (Fig. 8). Prevention and treatment of bumblefoot and hyperkeratosis involve provision of smooth wire or solidbottom cage floors with bedding, good sanitation, reduction of obesity, application of dimethyl sulfoxide (DMSO), and in severe cases, antibiotic treatment (usually abortive), softening with lotion, and surgical debulking. Use of DMSO in other species may in the rapid deposition phase inhibit amyloid deposition (Grauer and DiBartola, 1995). Staphylococcus aureus can also cause pneumonia, mastitis, conjunctivitis, cheilitis, and osteoarthritis. The bacterium has

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Fig. 8. Pododermatitis,usuallyinvolvinga chronicStaphylococcus aureus infection,of a forefoot(rightfoot). been associated with an exfoliative dermatitis characterized by alopecia, erythema, scabs, and epidermal cracks (Ishihara, 1980). Staphylococci apparently enter the skin through abrasions. The histologic lesion is parakeratosis with minimal inflammation (Percy and Barthold, 1993). Some animals die, whereas others recover and hair grows to cover the lesions.

xviii. Streptobacillus moniliformis. Streptobacillus, an organism of low contagion carried by wild rats and birds, rarely causes disease in research guinea pig colonies. Lesions include cervical adenitis with abscessation (see also Streptococcus equi subsp, zooepidemicus in Section III, A, 1, b) and a pyogranulomatous bronchopneumonia (Aldred et al., 1974; Kirchner et al., 1992).

h.

Mycoplasmas (Mycoplasma caviae, M. pulmonis, and others) and acholeplasmas may occur as latent infections in the reproductive tract, brain, and nasopharynx of guinea pigs (Stalheim and Matthews, 1975; Ganaway, 1976).

i.

Rickettsia

Clinical disease caused by rickettsia occurs only experimentally; however, latent infections in guinea pigs with undefined rickettsia may occur (Bozeman et al., 1968). 2.

Chlamydial and Viral Infections

a. xix. Streptococcus pyogenes. Okewole et al. (1991) reported an outbreak in Nigeria of a highly fatal, systemic infection of Streptococcus pyogenes. All ages were affected, and clinical signs included anorexia, lethargy, bleeding from body orifices, and death. Necropsy examination revealed a seropurulent pneumonia, hemopericardium and hemothorax, pyometra, and hepato- and splenomegaly. Mortality exceeded 40% of the colony.

Mycoplasmas

Chlamydia psittaci

i. Etiology. Chlamydia psittaci exists in at least eight biotypes and nine immunotypes, which have close correlation with host specificity and disease manifestation (Perez-Martinez and Storz, 1985). ii. Clinical signs. Subclinical infections are common, especially when the signs are mild reddening of the eyelids and

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intracytoplasmic inclusions in conjunctival epithelial cells. Most active infections occur in 4- to 8- week-old guinea pigs, and signs can include conjunctivitis with serous to purulent exudate, rhinitis, and genital tract infections (Deeb et al., 1989). Abortions and lower respiratory tract infections are reported. The clinical disease is self-limiting, with recovery and no residual damage. The disease may be exacerbated by streptococcal or Bordetella infections present in the host. iii. Epidemiology and transmission. Chlamydiosis in guinea pigs is a spontaneous, enzootic disease, often asymptomatic, and probably widespread in poorly managed colonies. Clean animals are infected by direct contact or by the young during cervical passage. iv. Necropsy findings. Histopathologic examination of the conjunctiva reveals intracytoplasmic inclusions, exudate, and mixed inflammatory cell infiltration. v. Pathogenesis. Chlamydia multiply within conjunctival epithelial cell cytoplasm, and clinical signs may be seen as early as 2 weeks of age, although inclusion bodies are uncommon under 4 weeks and after 8 weeks (Senyk et al., 1981; Deeb et al., 1989). VanHoosier and Robinette (1976) state that the signs occur at 1 to 3 weeks and are gone by 4 weeks, with inclusions seen from 15 hr to 17 days postinfection. Lymphocytes are present early in the infection, followed by neutrophils. Rank et al. (1979) and Senyk et al. (1981) describe ocular and genital humoral and cell-mediated immune responses following chlamydial infection. vi. Differential diagnosis. Diagnosis is by demonstration of intracytoplasmic inclusion bodies in Giemsa- or Macchiavellostained conjunctival epithelial cells (Deeb et al., 1989). Sera tested using microimmunofluorescence will detect antibodies, and the antigen is detected in conjunctival scrapings by immunofluorescence using specific monoclonal antibodies (Cherian and Magee, 1990). Differential diagnoses include causes of bacterial conjunctivitis in guinea pigs: streptococci, coliforms, Staphylococcus aureus, and Pasteurella multocida. vii. Prevention and control. Chlamydia are excluded best by establishing and maintaining colonies of pathogen-free guinea pigs. Control within a colony is through strict isolation of infected animals. Animals affected previously remain seropositive into adulthood. viii. Treatment. antimicrobials.

Chlamydia are sensitive to sulfonamide

ix. Research complications. The chlamydial organism from guinea pigs does not affect humans, but the conjunctival

and genital infections in guinea pigs have served as models for the human disease (Deeb et al., 1989). b.

Herpesviruses

i. Etiology. This enveloped herpesvirus (biovars of cavid Herpesvirus) (DNA), known also as the salivary gland virus because of its common occurrence in those glands, is a speciesspecific pathogen whose replication can result in large intranuclear (usually) inclusion bodies (Osborn, 1987). The agent is known also as the guinea pig cytomegalovirus (GPCMV). Two other herpeslike viruses have been identified in guinea pigs: a herpeslike virus (GPHLV), isolated from primary guinea pig kidney cell cultures; and guinea pig "X" virus (GPXV), isolated from leukocytes of strain 2 guinea pigs. Neither of these latter two viruses is a primary pathogen but may be a complicating factor in some research studies (Hsiung et al., 1987). ii. Clinical signs. The infection is usually subclinical, with no signs apparent; however, strain of host, pregnancy, and an immunocompromised state predispose to more serious illness. Hartley guinea pigs are said to be more susceptible than are strain 2 guinea pigs. Clinical signs may include weight loss and a lymphadenopathy. iii. Epizoology and transmission. Infection occurs naturally and is widespread (as detected on serologic tests) in guinea pig colonies. Infection with similar viruses occurs in primates, rats, hamsters, and mice. The acute infection is followed by a chronic, persistent infection (Isom and Gao, 1988). Transmission is by exposure to saliva carrying the virus, or transplacental transmission can occur throughout gestation. A preexisting maternal antibody does not prevent transmission to the fetuses. Cesarean section rederivation does not interrupt the transmission, presumably due to transplacental infection. iv. Necropsy findings. Experimental introduction of the virus causes more severe signs, but the natural disease in susceptible animals ranges from karyomegaly of salivary gland epithelium (submaxillary gland) to severe interstitial pneumonia, splenomegaly, lymphadenopathy, and fetal meningitis. Congenital abnormalities caused by GPCMV are known. v. Pathogenesis. A viremia within 2 days of exposure resuits in widespread, systemic dissemination of the virus, and although animals generally remain ostensibly healthy, the salivary gland, hepatic, and renal cells are the primary sites for replication. Many more organs show infection by 10 days. By 12 to 14 days the viremia ceases and the virus is more difficult to find in visceral organs. By 3 to 4 weeks postexposure, inclusion bodies are present in the salivary glands. A chronic, persistent phase continues in the salivary gland and thymus in adults and in the salivary glands and spleen of fetuses (Isom and Gao, 1988).

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vi. Differential diagnosis. Diagnosis of GPCMV is by microscopic identification of large, eosinophilic, usually intranuclear inclusion bodies in the ductal epithelial cells of the submaxillary salivary gland. The inclusions form at 5 days up to 3 weeks postexposure. Inclusion bodies may also be seen in the brain, lung, kidney, spleen, pancreas, thymus, and liver. Indirect fluorescent antibody techniques and histopathology are methods of diagnosis. vii. Prevention, control and treatment. Prevention and control are by selecting guinea pig stock known free of GPCMV and by screening new arrivals by selective necropsy or serologic testing. There is no treatment. viii. Research complications. The natural disease may be inapparent (unless detected by serology or necropsy) but could interfere with studies involving tissues harboring the virus. c.

Adenovirus

i. Etiology. Adenoviral respiratory tract infection in guinea pigs is attributed to an adenovirus (DNA) with the typical icosahedral symmetry and 252 capsomers. Polymerase chain reaction results indicate that the guinea pig adenovirus is genetically distinct from other species adenoviruses (Pring-Akerblom et al., 1997). ii. Clinical signs. The prevalence of the subclinical disease is unknown because of lack of specific serologic tests, but asymptomatic disease may be common. Clinical disease is rare. Affected animals usually die without prior signs, or they may show dyspnea, tachypnea, dry rales, crepitations, and lethargy (Eckhoff et al., 1998). iii. Epizootiology and transmission. Guinea pig adenovirus infection occurs worldwide and may have a higher prevalence than suspected. The clinical disease has no age predilection, is sporadic in endemically affected colonies, and is characterized by low morbidity and high mortality (Eckhoff et al., 1998). Transmission is via the respiratory route. iv. Necropsy findings. Lesions include well-demarcated areas of dark red pulmonary consolidation, compensatory emphysema, and in some cases a catarrhal exudate in air passages. Histologic effects include necrosis and sloughing of bronchiolar, bronchial, and tracheal epithelial cells, which contain large, oval, intranuclear inclusion bodies. The surviving epithelium and underlying lamina propria are underlain with a mixed population of inflammatory cells (Crippa et al., 1997; Eckhoff et al., 1998). v. Pathogenesis. Factors for predisposition to infection include stress, an immunologically compromised animal, strain

and site of replication of the virus, and perhaps anesthetic gas irritation of the respiratory tract. The virus enters the tracheal and bronchial epithelial cells, where replication and cell damage occur. Epithelial erosion, parenchymal inflammation, and exudation in airways follow (Pring-Akerblom et al., 1997). vi. Differential diagnosis. Diagnosis of adenovirus disease is by exclusion of other causes and by histologic and electron microscopic examination of air passageway epithelial tissue. There is no specific serologic test available, and the use of the mouse adenovirus strain FL antigen produces excessive falsepositive reactions (Pring-Akerblom et al., 1997). Other agents that may infect the respiratory system of guinea pigs are Bordetella bronchiseptica, Streptococcus sp., Klebsiella pneumoniae, cytomegalovirus, herpesvirus, and Sendai and parainfluenza viruses (Eckhoff et al., 1998). vii. Prevention, control and treatment. Obtaining guinea pig stocks without a history of clinical adenovirus infection, reduction of stress in a colony, and observation of immunocompromised animals are methods of prevention and control. There is no treatment for this highly fatal viral disease. viii. Research complications. Inapparent pulmonary infections that may become clinical problems when animals are stressed interfere with laboratory studies involving guinea pigs. d.

Poliovirus

The poliovirus affecting guinea pigs is an RNA-containing enterovirus with some antigenic cross reaction with the GDVII strain of mouse poliovirus. Genetic variants among host guinea pigs may affect predisposition to infection and clinical signs (Van Hoosier and Robinette, 1976). Clinical signs include depression, lameness in one or more limbs, flaccid paralysis, weight loss, and death over 2 weeks. A recent report (Hansen et al., 1997) indicates that this infection is reported in pet store populations and in the older literature. Nevertheless, poliovirus infection remains a possible diagnosis in guinea pigs with lameness. The infection is more common in the pet and non-barriermaintained population than in research colonies. Clinical signs are rare, and within colonies clinical disease is sporadic, if it exists at all. The transmission route of the virus is not proven, although fecal-oral transmission is common to all enteroviruses, but in mice and rats the endemic epizootic cycle of Theiler's murine encephalomyelitis virus (TMEV) is by fecal-oral transmission (Lipton and Rozhon, 1986). Necropsy signs of poliovirus infection are microscopic and include meningomyeloencephalitis, perineuronal inflammation, neuronal degeneration, and necrosis of the anterior horn cells of the lumbar spinal cord. In mice the virus replicates presumably

6. BIOLOGY AND DISEASES OF GUINEA PIGS in the gray matter of the cortex and progresses into the white matter and upper motor neuron pathways. Diagnosis is by a positive ELISA assay using the TMEV/ strain GDVII mouse virus antigen combined with histopathologic finding of central nervous system and lumbar spinal cord lesions (Hansen et al., 1997). Hansen et al. (1997) recommend continuing administrations of vitamin C for prevention, control, and treatment, given that vitamin C contributes to adrenocorticosteroid production and, presumably, protection of myelin. The infection may complicate research investigations of the central nervous system of the guinea pig. e.

Coronavirus-Like Particle (CVLP)

Two reports (Jaax et al., 1990; Marshall and Doultree, 1996) using negative-staining electron microscopy describe detection of membrane-fringed particles in guinea pig feces. Although morphologically similar to Coronaviridae, these particles are distinct from coronaviruses, toroviruses, and arteriviruses. Jaax et al. (1990) tentatively links these virions to a "wasting syndrome" in guinea pigs, with clinical signs of anorexia, severe weight loss, diarrhea, and high mortality. Coronaviruses (RNA) affect several laboratory animal species, and transmission is fecal-oral. The ingested virions affect specifically the enterocyte tips in the distal ileum and, sometimes, the proximal colon, intestinal segments that are reddened and thickened. The lesion is an acute to subacute, necrotizing regional enteritis. Microscopically, villi tips lose epithelium (Jaax et al., 1990). Diagnosis of CVLP infection is by detecting the particle in feces by transmission electron microscopy, by histologic signs, and by exclusion of protozoal and bacterial enteropathies in host animals. Prevention of CVLP infection is by monitoring feces for characteristic particles, which may be excreted chronically (Marshall and Doultree, 1996). As of this writing, a serologic test is not available. Treatment of diarrhea in guinea pigs is primarily by provision of fluids. The infection could confuse studies involving guinea pigs generally and their intestinal tracks specifically. f

Arenavirus

The RNA arenavirus causing the natural lymphocytic choriomeningitis in mice, dogs, and primates (including humans) is a rare pathogen in guinea pigs. The virus infection in guinea pigs is contracted probably through inhalation, ingestion, or possibly through the skin following exposure to biting insects or infected wild mice. Associated signs are central nervous system dysfunction and hindlimb paralysis. The virus may cause a lymphocytic infiltration in meninges, choroid plexi, ependyma, liver, and lungs. The liver is the best site for indirect fluorescent

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antibody (IFA) detection of the virus, and antibodies are detected by ELISA. The virus causes disease in humans and has many systemic effects in guinea pigs that would interfere with research projects (Van Hoosier and Robinette, 1976).

g.

Other Viruses

Overt viral diseases are rare in guinea pigs, but there are many reports of inapparent infections other than those described above. Reviews of these infections are found in Van Hoosier and Robinette (1976) and Bhat et al. (1986). The viruses include poxviruses, guinea pig retrovirus, parainfluenza viruses, pneumonia virus of mice, reovirus 3, simian virus 5, and Sendai virus. 3.

Parasitic Diseases

a.

Protozoa

i. Eimeria spp. Eimeria caviae, a protozoan of the phylum Apicomplexa, is a moderately pathogenic coccidium with ellipsoidal oocysts having a brown wall. Infection with E. caviae is seen often in connection with high populations of Balantidium coli, which is probably a secondary agent in producing clinical disease. Stress is a significant predisposing factor in the pathogenesis of coccidiosis. Clinical signs occur in weanlings and usually include lethargy, anorexia, and pasty stool diarrhea with a duration of 4 to 5 days. Oocysts often do not appear before 10 days postexposure, so diarrhea and even death may occur before oocysts are seen. Constipation may follow the diarrhea (Percy and Barthold, 1993). Oocysts are not infective immediately when passed in the feces but require approximately 48 hr outside the host to develop to the infective stage. Factors affecting this transition include oxygen, heat, and humidity. Necropsy findings include edema, congestion or hemorrhage, and white plaques (lymphatic or groups of oocysts) in the proximal colon and adjacent cecal wall. The colon is thickened. Intestinal contents are watery and often contain blood. Histologic examination shows colonic epithelial cell hyperplasia, enterocyte sloughing, and edema and congestion of the lamina propria. Ingested, sporulated oocysts invade the mucosa of the proximal colon and damage the epithelium during schizogony. The prepatent period lasts up to 10 or more days (Hurley et al., 1995). Diagnosis is by finding oocysts on fecal flotation [in a flotation medium of higher specific gravity (1.33) than conventional ova flotation media] or by examining mucosal scrapings or stained section for the organism. Other causes of similar signs include pantothenic acid or vitamin C deficiencies, crytosporidiosis, bacterial enteropathies, and coronavirus infection. Prevention is through good husbandry, reduction of stress, good sanitation, and provision of fresh, appropriate feed. Reduction

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of stress during shipping and reducing animal density in cages are essential also. Treatment involves use of sulfonamides with known antiEimeria activity and provision of adequate vitamin C. Any study involving young guinea pigs can be compromised when animals do not thrive, shed infectious organisms, and have intestinal lesions. ii. Encephalitozoon cuniculi. Encephalitozoon cuniculi is an intracellular microsporidian affecting canids, rabbits, rats, mice, nonhuman primates, guinea pigs, and other species. In guinea pigs there are no known clinical signs of infection and few if any gross necropsy signs. The inapparent infection and infrequent use of serologic screening for the organism in guinea pigs suggest that the prevalence is known poorly and could, in fact, be high (Vetteding, 1976; Percy and Barthold, 1993). Infective spores are disseminated in the urine and are then ingested or inhaled. Evidence of transplacental transmission has been suspected in several species but is unlikely nevertheless (Boot et al., 1988). Guinea pigs are resistant to large spore doses, and the source of spores may be exposure to rabbit urine. Microscopic lesions occur primarily in the brain and kidney. An affected brain has randomly distributed necrotic foci, microgranulomas, perivascular lymphoplasmacytic cuffs, and lymphocytic meningitis. Renal lesions, which may not occur, are multiple, 2 to 4 mm gray to white granulomatous foci seen as indentations or plaques just beneath the renal capsule, signs that could be confused with nephrosis in older guinea pigs. The histologic lesion is an interstitial mononuclear nephritis (Wan et al., 1996; Percy and Barthold, 1993). The ingested organism undergoes merogony and then sporogony in the cytoplasm of endothelial cells, peritoneal macrophages, renal tubular epithelium, and oligodendrocytes. Spores are found intracellularly and, after cell rupture, extracellularly. Diagnosis involves characteristic histologic signs, birefringence of organisms under polarized light, organism staining with Goodpasture-carbol fuchsin stain, and indirect ELISA, IFA, and the India-ink immunoreaction assay. Serologic screening is the preferred method (Wan et al., 1996). Lesions may be confused with those of toxoplasmosis. Prevention and control of encephalitozoonosis involves purchase or breeding of seronegative animals, housing away from seropositive rabbits, a regular program of serologic screening and removal of seropositive animals, and strict sanitation. There is no effective treatment (Wan et al., 1996). iii. Toxoplasma gondii. Toxoplasma infections in guinea pigs are rare and, when they occur, are primarily subclinical. Clinical signs include vulvar bleeding and abortion (Vetterling, 1976; Green and Morgan, 1991). Markham (1937) reported an encephalitis. The asexual stages of the organism are distributed in most tissues, with tachyzoites in virtually every organ and bradyzoites in brain, heart, and skeletal muscle, where they may

be detected histologically. Modest immune responses occur in the host, and antibodies to Toxoplasma can be measured. Infection of the uterus, placenta, and fetus can cause a blood-filled uterus, fetal deaths, and abortion. iv. Cryptosporidium wrairi. Cryptosporidium wrairi is a coccidium of guinea pigs that has a prolonged phase of endogenous replication. Subclinical infection may be common. Clinical signs are seen most often in young animals (under 300 gm or up to 16 weeks of age), and may be exacerbated by concomitant Escherichia coli enterotoxemia. Clinical signs may include weight loss (most common sign), potbellied appearance, watery diarrhea staining the rear quarters, rectal prolapse, and death. Mice, lambs, and calves may also be infected (Lindsey, 1990). Transmission is fecal-oral. Necropsy findings are those of a diffuse enteritis from duodenum through the cecum. Infections are patent for 2 weeks and clear by 3 to 4 weeks postingestion. Intestinal signs include hyperemia, edema, necrosis of villus tips, and hyperplasia of crypt epithelium. Cryptosporidial bodies are seen intracellularly in the brush border epithelium near villus tips and are most numerous in the anterior ileum. The bodies are basophilic and round to oval, 1-4 gm in diameter. Detection of the organism is by identification in mucosal scrapings examined on phase contrast microscopy or on stained section (Gibson and Wagner, 1986). Oocysts themselves may not have been found, but oocyst proteins have been identified (Vetterling, 1976). Prevention and control are by strict sanitation and periodic screening for the organism. Sulfonamides are not an effective treatment. Any research project involving use of known carriers could result in clinical disease and the resulting health variations among colony animals. v. Balantidium caviae. Balantidium caviae is a nonpathogenic, ciliated protozoan possessing a micro- and a macronucleus (Flynn, 1973). It inhabits the cecum, and its trophozoites may be an opportunistic pathogen in bacterial enteropathies. The organism is identified in intestinal lesions and in ingesta. vi. Klossiella cobayae. Klossiella cobayae is distributed widely and has a predilection for the kidney in the guinea pig, but other organs may be involved. Sporozoites excyst in the gut lumen and return to the circulation and pass to the kidney. The first generation of schizonts is located in the endothelial cells of glomerular capillaries. Schizonts contain 8 to 12 merozoites, which on host cell rupture or pass by the circulatory system to the proximal tubule epithelium, where second generation schizogony occurs. Large schizonts, containing up to 100 merozoites, cause significant enlargement of the infected epithelial cells. Gametogonous and sporogonous forms occur in the epithelium of the loop of Henle. Schizogonous stages may be seen in epithelial cells of the proximal convoluted tubules and in the glomerular epithelium. Merozoites in the loop of Henle produce

6. BIOLOGYAND DISEASES OF GUINEA PIGS zygotes, which undergo sporogony (Vetterling, 1976). Histologic signs include presence of protozoal forms and irregular accumulations of inflammatory cells. There are no clinical or gross necropsy signs except in heavy infections, when the renal surface is irregular with gray motfling. Prevention involves good sanitation and removal of susceptible animals from exposure to the sporocysts in the urine of infected animals. b.

Nematodes

i. Paraspidodera uncinata. Paraspidodera uncinata, the cecal worm (and only common helminth) of the guinea pig, inhabits but does not penetrate the cecal and colonic mucosa. The worms mature in 45 days, and the ellipsoidal egg to egg life cycle is around 51 to 66 days. The eggs, which can be seen in the feces of infected animals, become infectious 3 to 5 days after shedding (Fig. 9). Removing fresh feces and maintaining good sanitation are essential in infected colonies. Adult male worms are 11 to 22 mm long, and the females are 16 to 28 mm. Infections are encountered worldwide but are uncommon in the United States. Reported clinical signs seen with heavy infections are weight loss, debility, and diarrhea (Wescott, 1976).

225 ii. Cerebral larva migrants. Larvae of Baylisascarisprocyonis of raccoons can infect guinea pigs. With B. procyonis infection, migration of larvae in the central nervous system cause progressive neurologic disease manifested variably as torticollis, ataxia, anorexia, stupor, and hyperexcitability (Van Andel et al., 1995). Raccoon feces contain the embryonated ascarid eggs, which are ingested. In the small intestine, larvae are released from the egg and migrate aggressively through tissues (Craig et al., 1995). Infected raccoons are common in the American Midwest. Ova remain viable for years in soil and for weeks to months in straw. Eggs are resistant to most chemical disinfectants. Humans are susceptible to the disease if eggs from raccoons are ingested. c.

Cestodes, Acanthocephala, and Pentastomes

Flynn (1973) describes no cestodes or acanthocephala in guinea pigs, but he noted the occurrence of the pentastome Linguatula serrata nymphs ("tongue worm") in guinea pigs. d.

Trematodes

Fasciola hepatica and rarely F. gigantica may infect guinea pigs exposed to snail-dropped cercariae, which swim to a vegetation substrate, lose their tails, and encyst, becoming metacercariae on leafy vegetables. Adults live in the liver and shed eggs, which enter the gut and the host's feces. Eggs require a moist environment to develop and hatch. The consequent biliary and hepatic damage may cause anorexia, debilitation, and death (Voge, 1973; Wescott, 1976). e.

Mites

i. Etiology. Mites reported to infest guinea pigs include the listrophorid Chirodiscoides caviae, the demodex mite Demodex caviae, the myocoptid Myocoptes musculinus, and the sarcoptids Trixacarus caviae, Sarcoptes scabiei, and Notoedres muris. Among these mites, only Chirodiscoides and Trixacarus are reported commonly, and then usually in pet guinea pigs. The remaining mites are known only from very few publications, and in some cases the identification of the mite was in error (Ronald and Wagner, 1976).

Fig. 9. Paraspidoderauncinata ovumrecoveredfromguineapig feces.

ii. Clinical signs. Chirodiscoides caviae infestation is usually asymptomatic, although a dense mite population moving on hair shafts is apparent (Fig. 10). Heaviest infestations occur on the posterior trunk and may cause pruritis and alopecia. Adult males are usually coupled in a noncopulatory position with nymphal females (Wagner et al., 1972; Ronald and Wagner, 1976). Trixacarus caviae can produce an intensely pruritic, generalized dermatitis, but the presence and severity of lesions may be related more to the immune response and self-traumatization

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Fig. 10.

Chirodiscoidescaviae infestation of the hair of the guinea pig. Mites are about 0.4 to 0.5 mm in length.

than to the mite population (Fig. 11). Also, secondary infections contribute to the severity and distribution of signs. Trixacarus lesions occur most often on the trunk, inner thighs, neck, and shoulders, and may be patchy or generalized. The skin is dry to oily, crusty, with alopecia or patchy hair loss.

D e m o d e x occurred in the conjunctiva but produced no signs, and Myocoptes, Sarcoptes, and Notoedres may cause a pruritic dermatitis (Ronald and Wagner, 1976). With Trixacarus infection, severely affected animals selfmutilate, lose weight, are lethargic or run, bump into objects,

Fig. 11. Exfoliative dermatitis and hair loss in a guinea pig infested with Trixacarus caviae. Dark areas (right) are abrasions due to self-traumatization.

6. BIOLOGYAND DISEASES OF GUINEA PIGS

227

convulse, and die (Kummel et al., 1980; Zajac et al., 1980). Guinea pigs less susceptible to mite effects show fewer intense signs and may carry the mites while skin lesions heal. The stress of the disease may cause infertility and abortion. Histologic lesions caused by Trixacarus are confined to the stratum corneum and consist of epidermal hyperplasia (or thinning) and orthokeratotic and parakeratotic hyperkeratosis. Folds in the stratum corneum contain the mites and eggs. Mites are found in short tunnels rather than in more extensive burrows. Spongiosis and leukocytic infiltration occur in the dermis (Dorrestein and VanBronswijk, 1979; Percy and Barthold, 1993). The blood differential count may show heterophilia, eosinophilia, and basophilia (Rothwell et aL, 1991). iii. Epizootiology and transmission. Chirodiscoides caviae was reported first in 1917, and Trixacarus caviae was reported in the United Kingdom in 1972 and in the United States in 1979. Both genera are distributed widely in North America and Europe and probably occur elsewhere. Transmission of mites is by direct contact or via pelage, cage debris, or bedding. Sows pass the mites to weanlings, infected animals pass to naive adults, and cool carcasses pass mites to live, warmer cagemates (Ronald and Wagner, 1976). iv. Necropsy findings. There are no abnormal necropsy findings (except for mites and ova on hair shafts) in guinea pigs infested with C. caviae. Animals with severe cutaneous lesions of T. caviae may have, in addition to skin lesions, loss of body fat, pale liver, and subcutaneous signs associated with secondary bacterial infection, e.g., staphylococcal pyoderma (Kummel et al., 1980). v. Pathogenesis. Chirodiscoides caviae and its ova are attached to hair shafts. They do not burrow into the skin. Trixacarus "burrows" into the stratum corneum. The pruritis response is due apparently to an initial allergic response by some guinea pigs to mite antigen and the consequent inflammation. vi. Differential diagnosis. Diagnosis of specific mite infestations is by examining hair shafts or skin scrapings and identifying the specific mites. Chirodiscoides is ovoid and elongated with a triangular anterior (Ronald and Wagner, 1976). The paired adult male and female nymphs are also characteristic of this mite. Trixacarus caviae infestation is indicated by the clinical signs, especially pruritis, and by finding the mites themselves in skin scrapings or in biopsy section. These mites are shorter (135-200 mcm) than Sarcoptes scabiei (200-450 ~tm), and skin and hair may have to be dissolved in 10% potassium hydroxide and then filtered (No. 80 mesh) to retain the small mites (Fig. 12). The best body places to examine for mites are the lumbar region and the lateral aspects of the rear legs (Zajac et al., 1980). Other skin conditions in guinea pigs that may resemble Trix-

Fig. 12. The sarcoptid mite of guinea pigs, Trixacarus caviae. This specimen is an adult male.

acarus lesions include pediculosis, dermatophytosis, consequences of barbering, and sarcoptic and notoedric mange. vii. Prevention and control. Acariasis is more likely to occur in guinea pigs maintained in unsanitary conditions and not provided adequate veterinary care. Trixacarus lesions seem to occur in some strains more than in others and in stressed animals. Control of an outbreak is by repeated treatment of all animals and thorough cleaning and sanitization of the environment. viii. Treatment. Treatment of Chirodiscoides is by twice dusting with permethrin or carbamate compounds. Trixacarus is treated with ivermectin 200 to 500 ~tg/kg SC twice at a 7to 10-day interval. Treated guinea pigs may be bathed in a medicated shampoo to loosen and remove cutaneous debris (Henderson, 1973; McKellar et al., 1992). The infestation may persist despite ivermectin treatment (K. Parton, personal communication, 2000). ix. Research complications. Trixacarus caviae can cause transient, pruritic papulovesicular lesions in humans (Kummel

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JOHN E. HARKNESS, KATHLEEN A. MURRAY, AND JOSEPH E. WAGNER

et al., 1980). Guinea pigs with untreated, severe acariasis would not be useful in research, especially if the study involves cutaneous responses to drugs or, e.g., electromagnetic radiation. f . Lice The two lice that affect guinea pigs worldwide are members of the suborder Mallophaga, or the chewing or biting lice. Gliricola porcelli is a slender louse (Fig. 13), and Gyropus ovalis is ovoid. The lice abrade the skin and ingest fluids. Clinical signs, other than seeing the 1.0-1.5 mm lice attached to hair shafts, are seen occasionally, but heavy louse infestations may cause scratching, partial alopecia, and scabbing around the ears and nape of the neck (Ronald and Wagner, 1976). Adults and ova are cemented to hair shafts. Gliricola is seen more often than is Gyropus, and mixed infections occur. Pet guinea pigs are infested frequently. Transmission is by direct contact with infected host or via contaminated bedding. On death of the host, lice migrate along the hair shafts away from the cooling body. Diagnosis is through viewing with a hand lens the adult or immature mites. Gliricola has a narrow head and body (0.3 mm wide), whereas Gyropus is broader (0.5 mm) and ovoid. Lice infestation is prevented by obtaining clean stock and by maintaining good sanitation. Control involves isolation; treatment with dust, dip, or ivermectin; and cleaning of the environment.

g.

Fleas and Ticks

Ronald and Wagner (1976) report that Ctenocephalides felis, the cat flea, and Nosopsyllusfasciatus, the northern rat flea, can inhabit Cavia porcellus, but occurrence in laboratory guinea pigs would be rare, assuming separation from infested cats or wild rodents. The authors do not describe lesions.

Ctenocephalides felis is an intermediate host for the cestode Dipylidium caninum, and N. fasciatus for the hymenolepid tapeworms. Neither Flynn (1973) nor Ronald and Wagner (1976) specifically mention tick infestations on guinea pigs, but some tick genera, e.g., Dermacentor, could possibly affect guinea pigs. 4.

Mycoses

a.

Dermatophytes

i. Etiology. Dermatophytosis or epizootic ringworm in guinea pigs is caused in most cases by the zoophilic dermatophyte Trichophyton mentagrophytes, an aerobic (as are fungi), ubiquitous, saprophytic fungus. Microsporum canis and several species of both genera have been reported rarely as causes of disease in guinea pigs (Sprouse, 1976). The organism is not known to live in soil (Medlean and Ristic, 1992). ii. Clinical signs. Dermatophyte lesions are seen most often in young guinea pigs or in guinea pigs genetically predisposed, malnourished, or living in unsanitary or stressful circumstances. Subclinical infections exist, and early clinical manifestations occur around the orbits, on the nose, and on or beneath the pinnae (Fig. 14). The irregularly shaped areas of flaky skin, hyperkeratosis, reddening, and hair loss may extend to the back and sides but rarely to the limbs (Sprouse, 1976; McAleer, 1980; Valiant and Frost, 1984). Lost hair does regrow. Associated with the lesion may be vesicles, pustules, and abscesses usually caused by a secondary bacterial infection. Lesions are often self-limiting, usually nonpruritic, and can last up to 30 or more days. iii. Epizootiology and transmission. Dermatophytoses occur in many warm-blooded species, especially in younger animals in close contact. Primates, dogs, cats, horses, swine, ruminants, rodents, and birds are common hosts. Transmission occurs by contact with spores either on the animal itself or on fomites, such as bedding. The disease has an incubation period of around 9 to 12 days. This zoonotic disease is of public health concern. iv. Necropsy findings. Changes in the skin caused by dermatophytes are confined to the keratin layers and structures of the skin and hair follicles.

Fig. 13. The louse Gliricolaporcelli is a commoninfestation of pet guinea pigs.

v. Pathogenesis. The fungi solubilize keratin with proteases, which produces the scale accumulation on and around the lesion and the loosening and weakening of the hair shaft. The dermatophyte penetrates the stratum corneum or invades hair follicles. Growth continues down the hair shaft to the keratogenous zone until the growth inward equals hair-growth rate outward (Medlean and Ristic, 1992).

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6. BIOLOGY AND DISEASES OF GUINEA PIGS

Fig. 14. A facial lesion of dermatomycosisin a guineapig caused by Trichophytonmentagrophytes. Rearlimb (stifle). vi. Differential diagnoses. Several conditions cause or are related to hair loss in guinea pigs, including protein and caloric deficiency, chewing and barbering, bacterial dermatopathies (e.g., Staphylococcus and Streptococcus), cystic ovary effects, acariasis, and continuous breeding. The irregularly shaped, nonpruritic, flaky-skin lesions on the face, recovery of the organism from hair and epithelial debris on Sabouraud's dextrose agar or dermatophyte test media (DTM), or observation (in vitro) of species-characteristic morphologic features and macroconidia in Microsporum (or microconidia in Trichophyton) provide a genus diagnosis (Sprouse, 1976; McAleer, 1980; Harvey, 1995). Epithelial debris and hair are best obtained by vigorous brushing with a toothbrush. Culture of the fungus is the most reliable diagnostic method, and growth usually occurs within 10 days (Medlean and Ristic, 1992).

removed from the environment. The disease is usually selflimiting, but it may take months.

vii. Prevention. Prevention of dermatophytoses involves selection of nonsusceptible animals; good husbandry, including appropriate feed and clean environment; and alleviation of stress. Dark, moist environments support survival and replication of dermatophytes.

x. Research complications. Guinea pigs infected heavily with a zoonotic fungus, even if the lesions are superficial, cause concern for public health and research use.

viii. Control. Control involves improved husbandry and removal of heavily infected animals. Infected hairs must be

Cryptococcus neoformans in one report caused a meningitis and pneumonia and in another (Van Herck et al., 1988), caused

ix. Treatment. Given that the clinical disease is usually selflimiting, treatment is often not pursued, especially in guinea pigs used in research. Among treatments deemed effective are griseofulvin 7.5 mg/kg PO q24 hr for 5 - 6 weeks; topical 1.5% griseofulvin in dimethyl sulfoxide solution for 5 - 7 days; 1% tolnaftate topically; or butenafine topically for 10 days (Post and Saunders, 1979; Valiant and Frost, 1984; Schaeffer and Donnelly, 1996). Griseofulvin pediatric is also effective. Oral griseofulvin is poorly absorbed in the intestine unless given with a high-fat meal, and the drug is teratogenic. Other drugs currently used to treat ringworm include thiabendazole, ketoconazole (with hair clipped), and itraconazole.

b.

Other Mycoses

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JOHN E. HARKNESS, KATHLEEN A. MURRAY, AND JOSEPH E. WAGNER

a severe pruritic, miliary, crusty dermatitis with confluent ulcerations. The granulomatous lesions had focal ulceration and were localized in the cutis and subcutis. The epithelium had sloughed. The lesions resemble those caused by Trixacarus caviae. Spontaneous infections with Histoplasma capsulatum and Candida albicans are recorded. Histoplasma caused emaciation, lameness, gastroenteritis, and lymphadenopathy. Candida infection was associated with occlusive capillary embolism and tissue infarction. Guinea pigs are susceptible to experimental infections with Coccidioides immitis, Blastomyces dermatitidis, and Aspergillus. A normal stomach inhabitant, Torulopsis pintolopesii, may cause an enteritis (Kunst~[ et al., 1980). Aflatoxicosis in guinea pigs is also reported.

B.

Metabolic and Nutritional Diseases

Well-managed colonies of guinea pigs rarely if ever encounter primary nutritional deficiencies or excesses, except perhaps after accidental feeding of out-of-date feed with low levels of vitamin C, feeding rabbit pellets, failing to fill water bottles, or dispensing multivitamin supplement instead of vitamin C only. Malnutrition and its consequences are much more common in pet guinea pigs than in research animals. Marginal deficiencies, however, are more common in some research colonies, and the consequences are increased susceptibility to infectious disease, especially streptococcal infections and enteropathies. Signs of conjunctivitis or upper respiratory disease should always suggest a marginal vitamin C deficiency, and treatment should include vitamin C supplementation. Signs frequently associated with many specific dietary deficiencies are failure to gain weight, weight loss, rough hair coat, pale mucous membranes, lethargy, anemia, and various signs of opportunistic infectious disease. Induced nutritional disorders are listed in Table IV. 1.

Hypovitaminosis C

a. Etiology. Hypovitaminosis C, known also as scorbutus or scurvy, is a multisystemic disease occurring in the small number of species that lack the genetic code to produce the hepatic enzyme L-gulonolactone oxidase. This enzyme converts L-gulonolactone into the isomers L-ascorbate (AH) and L-dehydroascorbic acid (DHA) (Marcus and Coulston, 1990). Hepatocytes convert normally extracellular DHA into extracellular AH, which may contribute to vitamin C homeostasis (Upstonet al., 1999). Probable primary roles of vitamin C are acting as a cofactor in hydroxylation and amidation reactions by transferring electrons to enzymes that provide reducing equivalents (i.e., protons) and scavenging both intracellularly and extracellulary superoxide radicals and singlet oxygen, whose activity results in tissue damage (Chakrabarty et al., 1992). It

maintains vitamin E in vitro by reducing a-tocopherol radicals. Vitamin C is carried in leukocytes and (30%) in erythrocytes.

b. Clinical signs. Hypovitaminosis C in laboratory guinea pigs may be subclinical, accompanied by overt signs of an infectious disease (e.g., diarrhea, upper respiratory infection), or a primary vitamin C deficiency. Marginal deficiencies are particularly disturbing in research animals because of an increased susceptibility to infectious disease. Signs of secondary (usually bacterial) infection include unexpected death, diarrhea, weight loss, swollen and reddened orbital margins, dehydration, and dyspnea. Signs of the primary hypovitaminosis include weight loss, reluctance to move, screaming when restrained, and swollen joints (Clark et al., 1980). c. Epizootiology and transmission. The absence or deficiency of the enzyme L-gulonolactone oxidase, or some other enzyme in the glucose to vitamin C pathway, is reported in primates, guinea pigs, fruit-eating bats, a few birds and fish, and to some degree in cetaceans. d. Necropsy findings. The most common gross necropsy findings include hemorrhage in the subperiosteum, adrenal cortex, skeletal muscles, joints (especially stifles and costochondral junctions), and intestine (Figs. 15 and 16). The gut is atonic and hyperemic. Histologic changes are extensive and are related in many cases to the absence of hydroxyproline and hydroxylysine elements in connective tissues. Epiphyseal growth centers of long bones are deranged with osteoid formation greatly reduced, chondrocytes deranged and degenerating, bony trabeculae absent in the marrow cavity, reduced osteoclastic and increased osteoblastic activity, and multiple microfractures (Percy and Barthold, 1993). Myofilaments are fragmented and mitochondria swollen (Kim, 1977). Hemorrhage occurs in many tissues. e. Pathogenesis. With defects in amino acid (including tyrosine and phenylalanine) metabolism, fibroblasts and osteoprogenitor cells produce defective intracellular architecture and the products dentin, collagen, and osteoid. Junctional defects and cytoplasmic disruption occur between endothelial cells; within muscle, liver, and connective tissue cells; in pericapillary fibrous tissue; and in arterial intimae. Subendothelial cholesterol deposition increases, as does lipid peroxidation of cardiac muscle. Iron absorption in the gut and steroidgenesis in the adrenal gland, which may be related to increased macrophage cytotoxicity, decrease (Thurnham, 1997). Macrophage migration and heterophil phagocytosis are decreased (Percy and Barthold, 1993). Cholesterol catabolism is slowed, reducing bile acid production and consequently fat-soluble vitamin assimilation, and cholesterol accumulates in the liver. f. Differential diagnosis. Weakness, pain, and death in young guinea pigs can be due to infectious disease, osteoarthri-

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6. BIOLOGY AND DISEASES OF GUINEA PIGS Table IV

Experimentally Induced Hypovitaminoses of Guinea Pigs a Vitamin

Major clinical signs, gross and microscopic lesions

Thiamin (B1)

Anorexia followed by tremors, ataxia, opisthotonus

Riboflavin (B2)

Poor growth, rough hair coat, pallor of extremities, corneal vascularization

Comments and suggested replacement dosage b Unstable in diets containing oxidizing agents: e.g., K2HPO4; 0.6 mg p e r os or im daily as necessary Quantitative requirements not determined; 1 mg or im daily as needed

p e r os

Niacin (nicotinic acid)

Poor growth, pallor of extremities, drooling, anemia

No ocular, anal, or skin lesions noted; niacin is produced from tryptophan; 6 mg per os or im daily as needed

Pyridoxine (B6)

No notable clinical signs; poor growth

0.6-1.0 mg per os or im daily as as needed

Folic acid (pteroglutamic acid)

Lethargy, weight loss, anemia, and leukopenia; after 5 weeks profuse salivation with terminal convulsions

1 mg p e r os or im daily

Pantothenic acid

Anorexia, weight loss, rough hair coat, GI and/or adrenal hemorrhage

2.5 mg p e r os or im daily as needed

Choline

Poor growth, anemia, myasthenia; occasional fatty liver in adults but not in young

Turnover of choline is slow because of lack of or low levels of hepatic choline oxidase; choline chloride 150 mg daily

Vitamin C

Weakness, anorexia, anemia, defective collagen synthesis, maintenance, and repair, and impaired clotting result in disturbed growth centers in long bones and ribs, and widespread hemorrhages primarily within superficial fascia, gingiva, skeletal muscles, and around joints

25-50 mg daily per os or im

Vitamin A (deficiency)

Poor growth, weight loss, desiccation of edge of pinna, edema, xerophthalmic keratitis; extensive squamous metaplasia of epithelium of trachea, urinary bladder, and uterus

1.5 mg vitamin A acetate daily p e r os or im

Vitamin A (excess)

Degeneration of epiphyseal cartilage; 200,000 USP units/kg (120mg/kg) in female 14 to 20 days pregnant is teratogenic, producing mainly agnathia, synotia, and microstomia

Vitamin D

Broadened cartilage plates in epiphysis of long bones; enamel hypoplasia of incisors, weight loss; may be unessential if calcium/phosphorus ratio is normal

6 mg (240 IU) per os or im daily

Vitamin E

Myasthenia, paralysis; fetal malformation and resorption skeletal muscle degeneration; testicular atrophy and degeneration

15 mg p e r os or im daily

Vitamin K

Unknown

None

See Table II for requirements. bMost suggested dosages are based on providing vitamins in an amount approximately fivefold in excess of that contained in the diet formulated by NIH (Navia and Hunt, 1976) assuming a daily intake of 40 gm/kg for a 750 gm animal. a

tis, heat stress, and toxemias. A history of inappropriate feed, decreased prothrombin time, and a serum vitamin C level below 0.55 mg/dl (normal around 2.01 mg/dl) indicate hypovitaminosis C (Kim, 1977).

be stored at cool temperatures and used within 90 days of milling. "Microencapsulated" vitamin C food products can be stored longer (Eva e t al., 1976). Guinea pigs drinking alcohol have increased need for vitamin C (Zloch and Ginter, 1995).

g. C o n t r o l a n d p r e v e n t i o n . Foods providing at least 6 mg vitamin C per day are adequate; a vitamin C level of 2 5 0 500 mg/liter in the drinking water provides adequate levels if the water is replenished daily (Groves, 1992). Vitamin C "halflife" in solution in glass bottles is around 24 hr only, and in food stored at 72~ vitamin C has only 33% original activity at 30 days postmilling and 14% at 90 days. Therefore, food should

h. P r e v e n t i o n . Dietary considerations for vitamin C are discussed earlier in the chapter, but vitamin levels in food must be adequate; lesions develop in 7 to 10 days with no dietary vitamin C and in approximately 3 weeks on marginally deficient diets. Improper compounding and storage, autoclaving, and feeding food for other species are c o m m o n errors that lead to vitamin C deficiencies in laboratory guinea pigs. Pregnant

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JOHN E. HARKNESS, KATHLEEN A. MURRAY, AND JOSEPH E. WAGNER

Fig. 15. Limbhemorrhagesdue to vitaminC deficiencyin a youngguineapig.

guinea pigs may require up to 30 mg/kg daily, but most levels given commonly (e.g., 10 mg/kg daily) probably exceed requirements. i. Treatment. Treatment of guinea pigs with scorbutus involves provision of vitamin C daily at levels up to 30 mg/kg. Recovery occurs rapidly over 1 to 2 weeks. j. Research complications. Because hypovitaminosis C causes such profound and extensive changes, including decreased disease resistance, research using scorbutic guinea pigs is compromised in multiple ways. 2.

Toxemias of Pregnancy

a. Etiology. Recent literature reviews of pregnancy toxemia in guinea pigs describe two conditions similar in many clinical and pathologic aspects but different in primary causation (Percy and Barthold, 1993). Both conditions are referred to often as "pregnancy ketosis," but are described best separately as (1) preeclampsia, eclamptogenic toxemia, or the circulatory form; and (2) fasting ketosis or the metabolic-nutritional form

(Van Beek and Peters, 1998). The circulatory form begins, presumably, from abnormal vascular changes that lead to ischemia of the uteroplacental unit; and the nutritional form progresses from hypoglycemia and hyperlipidemia (Seidl et al., 1979). Given, however, the many similarities between the pathogenesis and consequences of the two forms, there may be a common pathogenesis, perhaps involving generalized endothelial dysfunction related to maternal responses to fetal antigens, vasoconstriction due to chemical factors, or triglyceride effects on endothelial cells. b. Clinical signs. Preeclampsia occurs in late pregnancy (last 2 weeks) and more often in multiparous, obese, stressed sows with a large fetal load, but normal sows may also succumb. Cases may also be seen in immediately postpartum sows. Affected animals may die without abnormal signs or may be dehydrated, depressed, anorexic, and underweight. Proteinuria, acidic urine (pH 5 - 6 , normal pH 8), ketonuria, elevated serum creatinine, and increased or decreased plasma triglyceride levels occur. Unlike preeclampsia in humans (who also have a hemochorial placentation), guinea pigs exhibit hypertension variably and edema rarely if ever (Ganaway and Allen, 1971; Golden et al., 1980). The guinea pig placenta is labyrinthine

6. BIOLOGY AND DISEASES OF GUINEA PIGS

233

Fig. 16. Hemorrhagesat costochondraljunctions associated with vitamin C deficiencyin a guineapig.

hemomonochorial with maternal blood circulating around a single trophoblastic layer over fetal capillaries. Fasting ketosis occurs in the last trimester (over 45 days) of pregnancy but is seen more often in the last 1 to 2 weeks. Deaths occur following a 1- to 3-day fast, and affected animals are weak, depressed, and dehydrated. Urine is acidic and contains ketone bodies, and clinical pathology changes include variable plasma glucose levels, hyperlipidemia, and elevated alkaline phosphatase and ornithine carbamyl transferase serum levels (Bergman and Sellars, 1960). c. Epizootiology and transmission. Many mammalian species, including humans, nonhuman primates, rabbits, dogs, ruminants, and guinea pigs, exhibit similar conditions in late pregnancy or early lactation, but characteristics vary. d. Necropsy findings. Necropsy findings are similar between the two forms, but preeclamptic animals usually exhibit more severe changes. In the preeclamptic or circulatory form, the uterus, placenta, and adrenal cortices show petechial and ecchymotic hemorrhage and focal necrosis. Placental attachment sites, which detach easily, are also affected. Fetuses are dead and decomposing. Livers are enlarged, yellow tan, and have ne-

crotic foci. Kidneys show subcapsular hemorrhage. Ketosis can cause gastric ulcers in guinea pigs (Wagner, 1976). Lesions in fasting ketosis or the metabolic-nutritional form include marked fatty infiltration of the liver, kidney, adrenal glands, and vessel walls (Assali et al., 1960). The uterus and placentae have petechial and ecchymotic hemorrhages, but these organs are not affected as severely as those in preeclamptic animals. Fasting-induced ketotic animal livers show fewer necrotic areas, if any. e. Pathogenesis. Preeclampsia in guinea pigs has been induced experimentally by constricting the abdominal aorta or severing or ligating arteries supplying the pregnant uterus. Pathogenesis beyond the occurrence of uteroplacental ischemia is poorly defined and contains in current descriptions only certain elements of what is known of preeclampsia in humans. The proposed course in guinea pigs involves thrombocytopenia, thromoplastin release, alterations in the renin-angiotensin system, vasoconstriction, deposition of fibrin, and disseminated intravascular coagulation. The initial causes of preeclampsia in humans are multifactorial but involve reduced placental perfusion due to absence of vascular dilation (uterine vessels in late pregnancy only

234

JOHN E. HARKNESS, KATHLEEN A. MURRAY, AND JOSEPH E. WAGNER

40% normal size) and defective trophoblastic replacement of vessel endothelium accompanied by fibrinoid accumulation in and around vessels. This process in turn proceeds to generalized endothelial dysfunction through a maternal response to trophoblast antigens or vasoconstriction and activation of a clotting cascade caused by oxygen-free radicals, lipid peroxides, and proteases. Triglyceride levels increase within endothelial cells. There is apparently a genetic predisposition to these events (Van Beek and Peters, 1998). f. Differential diagnosis. Clinical signs of depression and death in late pregnancy suggest a diagnosis of pregnancy ketosis. Acidic urine, absence of evidence of acute septicemic disease (e.g., salmonellosis, bordetellosis), and nonresponse to treatment confirm the diagnosis. g. Prevention. Pregnant guinea pigs should be fed a nutritious and balanced diet continuously without changes. Guinea pig breeding stock with no history of obesity or deaths during pregnancy should be selected and housed in a reduced-stress environment. h. Treatment. Many treatments for toxemias of pregnancy have been tried, with rare success. Administration of electrolyte fluids, glucose, calcium gluconate, corticosteroids, and various combinations of fruits and vegetables provide no certain cure. i. Research complications. Any research project involving breeding guinea pigs, using or having obese guinea pigs, or housing animals in a stressful environment can have many sudden deaths, which, if another end point is sought, can devastate a study. 3.

Urolithiasis and Cystitis

a. Etiology. The specific cause of mineral crystallization and urolith growth in the urinary system of guinea pigs is unknown, but probably involves genetic, dietary, or urinary tract environmental factors. Some urolith formation may be associated with cystitis or nephritis, decreased urine flow, and elevated urine pH, but the uroliths themselves may predispose to cystitis and the other problems. Proteinaceous urethral plugs found occasionally in older male guinea pigs probably originate from seminal vesicular content (Wagner, 1976). A review of calcium nephrolithiasis (Baggio et al., 1997) postulated that the condition in humans may derive from an increase in A6-desaturase activity, which leads to lower linoleic acid and higher arachidonic acid concentrations in plasma and membrane phospholipids. Kok (1997) suggests that the formation of renal uroliths is related to altered membrane ion transport and consequent mineral crystallization in the tubules of the nephron. One of the few animal models of the human condition is the sea squirt Mogula manhattensis, which harbors a microbe Nephromyces, which in turn contains another bacte-

rium. These microbes process the invertebrate's uroliths (Anonymous, 1994). Peng et al. (1990) identified several types and causes of cystitis in guinea pigs. Bacteria involved in most infections were Escherichia coli and Staphylococcus sp. b. Clinical signs. Urolithiasis is usually subclinical and occurs in older sows, but when urinary tract blockage or infection occurs, weakness, vocalization, straining, anuria or dysuria, anorexia, and hematuria may be seen, with some signs progressing over several weeks. Untreated animals may die (Ball et al., 1991). c. Epizootiology and transmission. Urolithiasis in guinea pigs occurs more often in aged (over 30 months) females. Urinary tract blockage by proteinaceous plugs occurs in aged males (Wagner, 1976). There may be an inherited predisposition to urolithiasis. d. Necropsy findings. In addition to finding unilateral or bilateral uroliths in the kidneys or ureters, fine "sand" particles on up to 2 cm diameter concretions may occur in the bladder or urethra. Proteinaceous plugs are found in the urethra or bladder. With concurrent cystitis, the bladder may be distended with urine and have thickened, hemorrhagic walls, with calculi adherent to the mucosa (Peng et al., 1990; Okewole et al., 1991). Continued occlusion of urinary output results in hydroureter or hydronephrosis with the fluid containing white to brown mineral sediment or solid masses. On analysis, the stones may be calcium carbonate, calcium phosphate, calcium oxalate, magnesium ammonium phosphate hexahydrate, or carbonate apatite (Peng et al., 1990; Ball et al., 1991). e. Pathogenesis. Kok (1997) described a progression of ionic saturation, supersaturation, nucleation, crystallization, and crystal growth in urolith formation. In the bladder, urinary protein may provide a nucleus for crystal formation (Ball et al., 1991). f. Differential diagnosis. Diagnosis of urolithiasis is based on clinical signs of cystitis and on detection by radiography or ultrasonography of urinary tract masses (Gaschen et al., 1998). Other conditions causing hematuria or urinary tract blockage in guinea pigs are infection, neoplasia, and trauma to the genitalia. g. Prevention and control. Means of prevention include selection of stock known free of a history of urolithiasis, provision of appropriate food and ample water, and immediate clinical care of guinea pigs with cystitis. h. Treatment. Treatment involves provision of fluids and appropriate systemic antibiotics (e.g., fluoroquinolones), and, if indicated, surgical removal (Stuppy et al., 1979).

6. BIOLOGYAND DISEASESOF GUINEAPIGS i. Research complications. Guinea pigs with clinical cystitis and urolithiasis are unsuitable research subjects. 4.

Malnutrition

a.

Protein and Caloric Deprivation

Protein, caloric, and fatty acid deficiencies occur occasionally when feeding is restricted or neglected. The usual consequences of these deficiencies are reproductive impairment, both infertility and death of low weight (under 50 gm) neonates, and hair loss. Pregnancy and lactation may cause a negative energy balance and consequent hair loss in frequently bred sows. The limiting amino acid for guinea pigs is arginine, then methionine and tryptophan. Guinea pigs can produce niacin from tryptophan, and tryptophan-deficient diets can produce cataract formation (Reid and Sallman, 1960). Deficiencies in essential fatty acids result in weight loss, ulcerative dermatitis, hair loss, and visceral abnormalities (Navia and Hunt, 1976). b.

Vitamin Deficiencies and Excesses

Hypovitaminosis C was discussed in Section III,B,1. Hypovitaminosis A, which is rare in herbivores, leads to poor growth, keratitis, squamous metaplasia, crusty eyelids and pinna, and loss of organization in tooth-forming elements. Hypervitaminosis A, which can be caused by giving a multivitamin supplement, leads to degeneration of cartilaginous epiphyseal plates in long bones, abnormal bone repair, and teratogenic effects during organogenesis at 14 to 20 days (Navia and Hunt, 1976). The effects of vitamins D and K in guinea pigs are not well defined; guinea pigs may synthesize sufficient vitamin K (and most B vitamins) to prevent overt abnormalities. Experimental vitamin D deficiency produces wider epiphyseal cartilage plates, enamel hypoplasia, and weight loss. Rickets is not a spontaneous disease in guinea pigs. Thiamin (B1) deficiency leads to central nervous system disorders, including tremors and imbalance. In scorbutic animals, increased muscle weakness occurs in thiamin-deficient guinea pigs. Riboflavin (B2) deficiency leads to corneal vascularization, skin lesions, and myocardial hemorrhage, including the general effects. Niacin (nicotinic acid) deficiency in young guinea pigs produces anemia and diarrhea. Pyridoxine (B6) deficiency in young animals causes depression in phagocytic activity of myeloid cells; and folic acid deficiency, again in young animals, causes the general signs as well as profuse salivation and terminal convulsions. Pantothenic acid deficiency leads to anorexia, weight loss, and intestinal hemorrhage, and if deficient during weeks 9 and 10 of gestation, leads to abortion and sometimes death of dams. Choline deficiency produces poor growth and fatty liver. Deficiencies of one or both of vitamin E and selenium cause

235

similar signs in many species. In guinea pigs, primary, distinctive signs are hindlimb weakness through myasthenia or muscular dystrophy, reduced reproductive performance, and death (Percy and Barthold, 1993). Underlying or associated signs include coagulative necrosis of muscle, testicular degeneration, degenerative changes in seminiferous tubules and reduction of spermatozoa and spermatids, and elevated serum creatine phosphokinase. Muscular dystrophy precedes testicular degeneration. Lethargy and conjunctivitis are seen in debilitated animals. c.

Mineral Deprivation and Excesses

Interactions among magnesium, potassium, phosphates, calcium, and hydrogen ions are complex and are well described in guinea pigs (Navia and Hunt, 1976). Calcium and phosphates in excess increase the requirement of magnesium, which contributes to the problem of metastatic mineralization, described below. Guinea pigs use cation exchange and phosphate anions for removing excess hydrogen ions rather than remove protons by excretion of ammonium ions. Calcium turnover is rapid in guinea pigs. Phosphate ions are a critical component of the causes of metastatic mineralization. Magnesium supplementation is essential to offset hyperphosphatemia. High phosphate levels lower plasma pH. Potassium will counteract the adverse effects of excess phosphate by providing an exchange cation to remove excess hydrogen ion. Manganese deficiency produces reproductive disorders and pancreatic hypoplasia, and copper deficiency produces slow growth and myelination failure. Magnesium deficiency leads to poor weight gain, hair loss, and hindlimb weakness. 5.

Metastatic Calcification or Mineralization

Prevention of this disorder is one of the primary reasons that food for other species should not be fed to guinea pigs. Multifocal mineralization of skeletal and cardiac muscle fibers in guinea pigs over 1 year of age is usually asymptomatic. Gross evidence includes irregular, gray patches of mineral, which grates when cut. Histologic changes, notable in hindlimb muscles, include mononuclear cell infiltration, mineralization, and fibrosis (King and Alroy, 1996). Clinical signs, if they occur, include poor growth, muscle stiffness, bone deformities, nephrosis, and death. Mineral deposition, however, is not confined to muscles but may also occur in kidneys (collecting tubules, interstitium, convoluted tubules, Bowman's capsule); soft tissues around elbows and ribs; and in lungs, trachea, aorta, liver, stomach, uterus, and sclera. The probable cause, or one of the causes, of the various abnormalities is a primary magnesium deficiency or diets high in calcium and phosphates and low in magnesium. Salts deposited are usually calcium phosphates or carbonate combined with other minerals (Jones et al., 1996). Local tissue low pH may also be involved (Navia and Hunt, 1976).

JOHN E. HARKNESS,KATHLEENA. MURRAY,AND JOSEPH E. WAGNER

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6. Diabetes Mellitus

Diabetes mellitus is a rare condition in guinea pigs, except in colonies with a genetic predisposition or yet unidentified infectious agent. Clinical signs are evident first at 3 to 6 months of age; affect both sexes; and include loss of fertility, cataracts, variable glycemia, hyperlipemia, glycosuria (over 100 mg/dl), and rare ketonuria (Lang et al., 1977). The disease resembles type I diabetes mellitus in humans, with islet hyperplasia, degranulation of beta ([3) cells, thickening of basal membranes of peripheral capillaries, fatty infiltration of exocrine cells, and glomerulosclerosis. Spontaneous remissions occur, and injected insulin is not needed to maintain the animals. 7. Anorexia

Anorexia in guinea pigs is common, especially if feeders or waterers or food (odor, taste, texture, form) or water (flavor) is changed. Guinea pigs develop food preferences by 4 days of age and may not recognize other diets as suitable food. Other factors that may cause a guinea pig to stop eating are recovery from surgery, ketosis, illness, drafts, and water deprivation (Harkness and Wagner, 1995). Treatments include providing preferred or sweetened foods; changing feeder or waterer; treating disease; reducing crowding; reducing obesity (without fasting); or feeding a high caloric food supplement, yogurt, vitamin C, ground food and guinea pig feces, and 50% glucose solution. 8. Heat Stress

Guinea pigs are sensitive to sudden or extreme environmental changes, and such changes have long been considered a predisposing factor to respiratory disease and stress-precipitated illnesses. In addition, guinea pigs, whose ancestors lived at high, cooler altitudes, are heat-stressed easily, even at temperatures as low as 70~ when in direct sunlight. Heat-stressed guinea pigs show shallow, rapid respiration, weakness, hyperthermia, coma, and death. Timely intervention with cool water baths, corticosteroids, and parenteral fluids may prevent deaths (Schaeffer and Donnelly, 1996).

C.

Traumatic Lesions

1. Barbering and Skin Biting

Chewed hair of varying lengths may occur with or without skin bite wounds and lacerations. Self-barbering occurs caudal to the anterior shoulders, but status-associated or agonistic barbering by conspecifics occurs often on the rump, back, and ears and around the eyes (Harper, 1976; Wagner, 1976). Barbering and skin damage occur most often among adult males with or without a sow present, but they can also occur when parents

groom young (and nibble around eyes and ears) or when weanlings chew the sow's hair. Particularly severe chewing occurs with intermale competition for food, water, toys, or space; within dominant-submissive relationships; and with sows and with boars on young. Self-barbering also occurs. Biting may cause skin lacerations and deep wounds (King, 1956) or severe preputial dermatitis (Lee et al., 1978). Perineal wounds contaminated with bedding and feces may become infected, extend to the prepuce, and cause bleeding and urine retention, pain during mating, and decreased reproductive activity. Bedding adhering to the moist prepuce can cause similar signs. Prevention of barbering and chewing involves reduction of environmental stressors, early weaning, separation of boars into individual cages, and perhaps hay feeding. Treatment involves frequent, thorough cleaning of the wound and placing the guinea pig into a clean cage (Lee et al., 1978). Few topical antimicrobials are effective as a treatment and may, if ingested, facilitate an enterotoxemia. Severe ear chewing can interfere with ear notch or tag identification of guinea pigs on research projects. 2.

Other Traumatic Injuries

Traumatic injuries in guinea pigs include limbs caught and injured in wire cage walls or floors, bone fractures, diaphragmatic hernia, broken or luxated spines, fracture of the liver capsule, and broken teeth. Guinea pigs may be traumatized when dropped, when leaping from a cage, or when bitten by another animal.

D.

Iatrogenic and Management-Related Disorders

Cardiac puncture with cardiac tamponade or subsequent bacterial infection; improper bleeding techniques; percutaneous injection of various initiating substances, e.g., adjuvants, antibiotics, or antiparasitic drugs; overexposure to heat or ultraviolet radiation; use of certain antibiotics; and unintentional feeding of improper feed can cause pain, distress, and even death to guinea pigs. 1. Adjuvant-Induced Pulmonary Granulomas

Guinea pigs injected subcutaneously with Freund's complete adjuvant may develop pulmonary granulomas. These lesions may be compared with perivascular lymphoid nodules or focal pneumonia (Schiefer and Stunzi, 1979). 2. Alopecia

Because of the high metabolic demands of pregnancy in the guinea pig, and probably genetic and metabolic factors, fre-

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6. BIOLOGY AND DISEASES OF GUINEA PIGS

quently bred sows housed singly or in groups often show hair thinning (see Fig. 5), although Gerold et al. (1997) found few fur defects in group-housed breeding sows fed 15.5 % crude protein and 19.5% crude fiber supplemented with 200 g hay scattered throughout the cage floor. Hair loss in breeding groups was attributed to trichophagia. Hair regrows when breeding ceases (Wagner, 1976). Alopecia in the absence of aggressive grooming and chewing by other guinea pigs may also occur in young animals at weaning, when the hair coat changes character, and when guinea pigs are on low-protein or low-calorie diets, and within social relationships (see Section III,C,1, Barbering and Skin Biting).

3. Dystocia Dystocias occur commonly in guinea pigs, either because of preexisting pregnancy toxemia and overall weakness of the sow caused by fetuses that are too large (over 100 gm) to pass through the pelvic canal, or because the pubic symphysis failed to separate the 2.5 to 3 mm needed to allow fetal passage. Failure of the fibrocartilaginous joint to separate occurs most commonly in a sow bred for the first time over 7 months of age. Signs of dystocia include a still-narrow symphysis as gestation nears day 73, straining, depression, and vaginal discharge. Prevention involves breeding first before 7 months of age, preventing obesity and fasting while pregnant, and removing animals with a known family history of dystocias. Young guinea pigs involved in a dystocia experience hypoxia, which is a dangerous condition for newborns. Treatment involves digital removal of the fetuses from the tract, provision of 1-3 units/kg oxytocin, or cesarean section (Schaeffer and Donnelly, 1996).

E.

ports are rare, and therefore the impression given by a single report can belie the actual, long-term incidences (Manning, 1976; Harkness and Wagner, 1995).

1. Hemolymphopoietic System Hemopoietic and lymphoreticular neoplasia are seen rarely, with the noted exception of leukemia. Cavian leukemia, a B-cell neoplasm, is discussed usually in connection with viral diseases, even though the C-type retroviruses in lymphocytes and the herperviruses in other tissues in affected animals probably have no role in causation. Guinea pigs are leukemic (usually) or aleukemic, have rough hair coats, dull eyes, lethargy, anemia, icterus, enlarged lymph nodes, and perhaps hepatosplenomegaly. Leukocyte counts range between 25,000 and 250,000 per mm 3. The cells are primarily lymphoblasts, which infiltrate lymph nodes, spleen, liver, marrow, and perivascular tissues. Allgoewer et al. (1999) reported multicentric lymphosarcoma with bilateral conjunctival masses. The tumor is transplantable rather than transmissible. The disease has a course of 2 to 5 weeks.

2. Respiratory System Neoplasms of the respiratory tract include papillary adenomas of bronchogenic origin. These tumors are visible grossly as small, white nodules. Microscopically the nodules consist of papillae of loose connective tissue covered by cuboidal epithelium. A lesion that may be confused with neoplasia in the lung is epithelial hyperplasia and adenomatous changes. Such lesions are probably inflammatory responses to foreign body stimuli or to Streptococcus pneumoniae.

Neoplastic Diseases 3. Integumentary System

With the exception of sporadic high prevalences of certain neoplasms in some strains, neoplasia is rare in guinea pigs, especially the younger animals found in research colonies. Genetic predisposition must have a role in this prevalence pattern. Contributing also to the rare reports of neoplasia are the failure to examine blood and bone marrow and the suspected presence of the antineoplastic factor asparaginase in guinea pig plasma (Manning, 1976). Around 25 types of benign and malignant neoplasms are known, with fibrosarcomas, lipomas, several types of adenomas, liposarcomas, and leiomyosarcomas occurring occasionally in any of several organs or tissues. Of the hundreds of tumors reported in tens of thousands of guinea pigs necropsied, those of the hemolymphopoietic system are most common, followed by those of the respiratory system, integument, reproductive tract, mammary gland, hemopoietic system, cardiovascular system, and endocrine glands. Neoplasia does occur in guinea pigs, especially in those over 3 years of age; but even then re-

Integumentary tumor prevalence has long been distorted by a single report of 29 trichofolliculomas, which is a basal cell epithelioma containing stratified squamous epithelium with hair follicles, keratin, and sebum. Trichofolliculomas occur primarily over the lumbar area, and they can be removed surgically. The other cutaneous neoplasm noted over the past several decades is the fibrosarcoma, which may also occur in other body systems. Cutaneous (and foot pad) papillomas also occur.

4. Reproductive Tract Localized ovarian teratomas, uterine leiomyomas, and various sarcomas are the most common reproductive tract neoplasms. Testicular tumors are very rare. Teratomas may contain tissues of organ types from the three germ layers, including nervous, endocrine, skin, and muscle tissue. This neoplasm resembles the presumed nonneoplastic embryonal structures

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JOHN E. HARKNESS, KATHLEEN A. MURRAY, AND JOSEPH E. WAGNER

developing within the ovary and may grow to 10 cm in diameter. These structures are transitory and are resolved by fibrosis. 5.

Mammary Glands

a.

Mammary neoplasia is represented most often by fibroadenomas and adenocarcinomas of ductal origin, which may metastasize to the lymph nodes, viscera, and lungs (Fig. 17). Other tumor types also occur in the mammary glands. 6.

Other Neoplasms

Neoplasms of the cardiovascular, endocrine, musculoskeletal, nervous, urinary, and gastrointestinal systems are reported rarely and are represented by adrenalcortical adenomas, mesenchymal tumors, islet cell tumors, thyroid adenomas, osteosarcomas, gliomas, and amelioblastomas.

F. 1.

anus, corneal and dermoid abnormalities, conjoined twins, agnathia, and malformed uterus.

Miscellaneous Conditions

Congenital Abnormalities

Many congenital abnormalities in guinea pigs have been noted, but few are described in the literature. Some examples of such abnormalities not described below include imperforate

Malocclusion

The open-rooted, hypsodont teeth of the herbivorous guinea pig are worn continuously from abrasive materials in plants and from silicas in the feed, but when malalignment of dental surfaces occurs, teeth become malformed and overgrown. Malocclusion may occur because of genetically based shortness of the maxilla, an abnormally narrow mandibular separation (anaesognathism), a nutritional deficiency, and broken or deviated teeth because of trauma or periodontal infection (Wagner, 1976; Emily, 1991). Root abscesses of the upper molar teeth may extend into the maxillary sinus and cause exophthalmos. Affected teeth are removed, the abscess drained, and suitable antibiotic given (Grahn et al., 1995). Premolars and molars are involved most often, but incisors may also be maloccluded. Clinical signs include weight loss, drooling ("slobbers"), buccal and lingual lacerations, starvation, oral bleeding, and death. Necropsy findings include periodontal disease and overgrown teeth, often with sharp edges and points on the labial side of maxillary teeth and lingual side of mandibular teeth. Diagnosis is by clinical signs and oral examination, which is facilitated with sedation and a penlight or otoscope. Other causes of drooling include folic acid deficiency, chronic fluoro-

Fig. 17. Mammaryadenocarcinomain a guinea pig.

6. BIOLOGY AND DISEASES OF GUINEA PIGS sis, heat stress, hypovitaminosis C, and dental abscesses (Harkness and Wagner, 1995). Treatment, which provides relief for several weeks, involves cutting the overgrown teeth to 2 to 3 mm above the gingiva with a high-speed dental bur and filing sharp points. Tooth extraction via bucotomy is complicated because of fragile bones. Guinea pigs losing weight and stressed periodically by dental disease may be poor research subjects. Also, because of the probable inherited component, they are undesirable as breeders. b.

Rhabdomyomatosis

Pale, pink foci or streaks with indistinct margins are a relatively common finding in the myocardium, usually in the left ventricle, of guinea pigs. The streaks represent glycogen accumulation in myofibers and occur because of a congenital abnormality of glycogen metabolism. The lesions are seen best in alcohol-fixed specimens stained with periodic acid-Schiff stain (Manning, 1976). There is no apparent cardiac impairment caused by the lesions. 2.

Age-Related Disorders a.

Perivascular Lymphoid Nodules

Beginning at an early age and continuing as a common occurrence in older animals, perivascular lymphoid nodules, consisting of normal lymphocytes, are present in the adventitia of the pulmonary arteries and veins. In older guinea pigs, the aggregations reach 0.5 mm in diameter and are visible grossly as pinpoint-sized, subpleural foci. The primary cause for these nodules is unknown (Wagner, 1976; Percy and Barthold, 1993). b.

Nephrosclerosis

Chronic renal disease in guinea pigs has no certain cause. Autoimmune, infectious, and vascular disorders may underlie the signs, and a high-protein diet may contribute to the disease. Nephrosclerosis, seen occasionally as an incidental finding in aged guinea pigs, is characterized by weakness, anemia, dilute urine, and increased blood urea nitrogen and creatinine. Necropsy signs include a pitted subcapsular renal surface with pale streaks extending into the cortex and even into the medulla. This segmental to diffuse interstitial fibrosis causes the kidney to have an irregular surface. Most glomeruli remain normal, but immune complex deposition occurs in basement membranes (Percy and Barthold, 1993). In guinea pigs, chronic renal failure may predispose to cochlear dysfunction, especially in the hair cells (Ohashi et al., 1999). c.

Amyloidosis

Deposition of amyloid in the kidney, liver, spleen, and adrenal glands is associated with aging or chronic inflammatory condi-

239

tions, such as staphylococcal pododermatitis and osteoarthritis (Taylor et al., 1971; Borkowski et al., 1988). Amyloid is an extracellular deposition of polymerized protein subunits. Amyloid exhibits green birefringence after Congo red staining and when viewed under polarized light (Grauer and DiBartola, 1995). The condition is slowly progressive but begins in the mesangium, then moves into subendothelial portions of capillary basement membranes (Jones et al., 1996).

d.

Ovarian Cysts

In the normal guinea pig, ovaries lie caudal and lateral to the kidneys and are 6 to 8 mm long and 4 to 5 mm in diameter (Breazile and Brown, 1976) (Fig. 18). The rete ovarii are derived from mesonephric tubules and occur in the hilus of the ovary. In some colonies, cysts of the rete ovarii are common in sows between 1.5 and 5 years of age, but are most common between years 1 to 4. A certain cause of the cysts is unknown, as is the overall prevalence in laboratory-housed guinea pigs; however, both androgens and estrogens may be involved in the pathogenesis (Field et al., 1989). Incidences noted at postmortem may range from 76 to 90% in older sows. Clinical signs include anorexia; depression; abdominal distension; bilateral, symmetric hair loss over flanks and rump; and reproductive failure (Keller et al., 1987). Diagnosis is by clinical examination, radiography, or real-time ultrasonographic imaging using a 6.0 or 10.0 MHz mechanical sector transducer (Beregi et al., 1999). The cysts are up to 7 cm or more in diameter, singular or multilocular, unilateral or bilateral (right more often than left), and may be associated with leiomyomas of the uterine body or horn, cystic endometrial hyperplasia, or endometritis (Schaeffer and Donnelly, 1996). All reports of leiomyomas in guinea pigs note associated cystic rete ovarii. The larger cysts may cause pressure atrophy in adjacent ovarian tissues. Treatment involves surgical removal via median laparotomy (Beregi et al., 1999).

e.

Osteoarthrosis

Jimenez et al. (1997) reported spontaneous, progressive osteoarthritis of the stifle and other joints in male Dunkin Hartley guinea pigs. The condition was noted as early as 3 months of age and had become severe by 22 months. Changes in cartilage included increased levels of proteoglycans and decrease in collagen. Histologic abnormalities included osteophytes, calcification of collateral ligaments of the joint, and degeneration of weight-bearing, articular surfaces. In addition to genetic predisposition, joint injury, hypovitaminosis C, and obesity may contribute to joint degeneration. Wei et al. (1998) studied the pathogenesis of osteoarthrosis in depth and determined that mechanical load and stiffness are significant pathogenic mechanisms.

JOHN E. HARKNESS,KATHLEENA. MURRAY,AND JOSEPH E. WAGNER

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Fig. 18. Ovariancysts and uterus externalizedfrom a guineapig.

f

Fatty Infiltration of the Pancreas

Older guinea pigs may exhibit large foci of adipose tissue spaced among normal, functional pancreatic tissue. There is no associated effect on pancreatic function.

3.

Plant Toxicoses

A pet guinea pig that had grasses as part of its diet developed dry gangrene of its four feet. The animal was anorectic, depressed, lame, and hypothermic. The feet were contracted, dark, and necrotic. Hyphal masses (sclerotia) of Claviceps purpurea, or ergot, were formed on the feed grass (Frye, 1994). Ergot produces the alkaloids ergotamine and ergometrine, which damage the capillary epithelium and lead to thrombosis and necrosis. The fungus germinates when the grasses flower. Kirsch (1997) described a guinea pig that had eaten leaves from Nerium oleander and exhibited seizures, bloating, and cardiac dysrythmia. Following intensive care with sodium pentobarbital, heat, lactated Ringer's solution, furosamide, activated carbon, glucose, diazepam, and vitamin C, the guinea pig recovered within 24 hr of presentation. Bendele et al. (1990) reported that an SC injection of the quinolone nalidixic acid at 350 mg/kg 1 time into 6-week-old male guinea pigs caused severe degeneration of middle-zone chondrocytes in weight-bearing joints by 48 hr postdosing. Quinolones and fluoroquinolones should, therefore, be used cautiously in immature guinea pigs.

Otoconial loss in the striola region of both utricle and saccule occurred in adult, mixed-sex guinea pigs following seven IP injections of streptomycin at 250 mg/kg per injection (Takumida et al., 1997). Recovery often occurred in 8 to 10 weeks. Aminoglycosides interfere with calcium uptake into otoconia.

4.

Other Conditions

a.

Miscellaneous Gastrointestinal and Hepatic Conditions

Gastric ulcers are probably secondary to other conditions, such as uremia, ketosis, excessive stress, or perhaps Citrobacter infection (Wagner, 1976). Acute gastric volvulus and dilation were reported by Lee et aL (1977). Six breeder guinea pigs aged up to 26 months were found dead or with dyspnea, cyanosis, tachycardia, and distended stomachs containing fluid and gas and rotated 180 ~ on the mesenteric axis. The diaphragm was displaced anteriorly. The cause of the volvulus was not apparent. Wagner (1976) and Vanrobaeys et al. (1998) reported several cases of an acute, usually fatal necrotic cecitis, or typhlocolitis in guinea pigs of all ages. Strain 13 guinea pigs were involved more commonly than were other strains, and the author postulated causes to be experimental manipulation, antibiotic use, corticosteroid injection, fasting, torsion, or advanced pregnancy. There may be no associated clinical signs except death. He also observed cecal impaction by wood shavings, hair, or inspissated digesta.

241

6. BIOLOGY AND DISEASES OF GUINEA PIGS

Other intestinal conditions include a case of colonic stricture, dilation, and ulceration caused by a heterotypic pancreas (Cheeseman et al., 1997). Cecal torsion, cecal impaction, rectal prolapse, rectal impactions, and circumanal sebaceous accumulations also occur. Hepatic problems include contusions and focal hepatic necrosis, perhaps due to agonal hypoxia (Percy and Barthold, 1993). b.

Ocular Problems

The eye of the guinea pig has a paurangiotic retina, few vessels near the optic disk, and no tapetum. Pupil dilation is accomplished by using 1% tropicamide drops or, in pigmented animals, one drop each of 1% atropine and 10% phenylephrine given 3 - 4 times within a 15 min period (Kern, 1989). Examination of the eye is accomplished best using a 20-diopter (D) or 30-D indirect condensing lens. Fluorescein dye, exfoliative conjunctival examination, and culture are diagnostic methods. Perhaps the ocular disorder seen most often in guinea pigs is blepharitis with epithelial flaking, crusting, alopecia, swelling, and reddening of the lids (Kirschner, 1996). These signs constitute what is often called "dull eyes" and are usually seen in guinea pigs with marginal hypovitaminosis C; with other subclinical infections, usually of the upper respiratory tract; or with malocclusion or renal disease (Bauck, 1989). Other ocular problems discussed elsewhere in this chapter include dermatophytosis of the lids, common bacterial infections, herpesvirus conjunctivitis, and listerial keratoconjunctivitis. Conjunctivitis in guinea pigs may be caused by chlamydia, streptococci, staphylococci, Pasteurella, physical or chemical irritants, and undoubtedly, other agents. Panoophthalmitis is due usually to an infection with Streptococcus equi subsp, zooepidemicus (Kern, 1989). An upper molar root abscess may extend into a maxillary sinus and orbit, causing exophthalmos. Other causes of exophthalmos in guinea pigs are orbital trauma, foreign bodies, sialocele, lacrimal gland cysts or inflammation, and neoplasia (Grahn et al., 1995). Allgoewer et al. (1999) reported conjunctival lesions with lymphosarcoma. A nodule ("pea eye") protruding from the conjunctival sac of an adult guinea pig may be a portion of a lacrimal gland (Kern, 1989) or a yellow, subconjunctival fat deposit (Bauck, 1989). Cataracts may result from feeding a diet low in L-tryptophan (under 0.1%) (Reid and von Sallman, 1960). Cataracts have been reported (Bettelheim et al., 1997) in strain 13/N guinea pigs and are due to a single, autosomal, gene deletion of 34 residues that produces a novel ~-crystalline lens protein. They may also occur in diabetic guinea pigs (Lang et al., 1977). Homozygote lenses are opaque, and heterozygotes have a welldemarcated opaque nucleus with a normal cortex. Other ocular abnormalities include ophthalmia, microphthalmia, corneal dryness, ulceration, calcification, and an osseous choristoma of the ciliary body (Griffith et al., 1998; Bauck, 1989).

Treatment of infectious ocular disorders includes topical or systemic antibiotics known safe and effective in guinea pigs, which in most cases are fluoroquinolones, chloramphenicol, and the trimethoprim-sulfonamides.

c.

Behavioral Concerns

Behaviors in guinea pigs that may affect experimental conclusions, kill animals, or predispose to infection are often a consequence of domestication, including assembled social groups, and caging. Such concerns, some of which were described previously, include hair chewing, skin biting, ear nibbling, trampiing of young by stampeding groups in a square cage, boars climbing from pen to pen, and males being aggressive toward each other. Guinea pigs are fastidious eaters and do not adapt well to changes in many aspects of their food and water and in how each is presented. Because guinea pigs "imprint" food type (and water taste) early in life, they may not recognize other foods, including powdered diets, water additives, and vegetable supplements. Placing powder in an agar matrix or blending foods during a transition does allow food changes. Guinea pigs scatter food and dribble water from sipper tubes, which makes measuring consumption difficult (Harper, 1976; Harkness and Wagner, 1995).

d.

Incidental Findings

There are many rarely reported conditions in guinea pigs, some of which are common but seldom noticed, whereas others are truly rare. These conditions include osseous metaplasia or bony spicules (with marrow) in the interstitium of alveolar septa; degenerate thymocytes in the young; vaginal prolapse; necrotizing myopathy of the larger muscles of the hindlimb; adrenal cortical degeneration; anemia; and footpad hyperkeratosis.

REFERENCES

Aldred, E, Hill, A. C., and Young, C. (1974). The isolation of Streptobacillus moniliformis from cervical abscesses of guineapigs. Lab. Anim. 8, 213-221. Allgoewer, I., Ewingmann, A., and Pfleghaar, S. (1999). Multicentric lymphosarcoma and conjunctival manifestations. Vet. Ophthalmol. 2, 117-119. Altman, E L., and Dittmer, D. S., eds. (1974). "Biology Data Book," 2nd ed., Vol. 3. Fed. Am. Soc. Exp. Biol., Bethesda, Maryland. Anonymous (1994). Unusual sea animal may give clues to how kidney stones form. FBR News, (March-April) 8. Anonymous (1997). Saccharomyces bonlardii for the treatment of Clostridium difficile-associated colitis. Ann. Pharmacother. 31, 919-921. Assali, N. S., Longo, L. D., and Holm, L. W. (1960). Toxemia-like syndromes in animals. Obstet. Gynecol. Surv. 15, 151-181.

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JOHN E. HARKNESS, KATHLEEN A. MURRAY, AND JOSEPH E. WAGNER

Baggio, B., Plebani, M., and Gambaro, G. (1997). Pathogenesis of idiopathic calcium nephrolithiasis. Crit. Rev. Clin. Lab. Sci. 35, 153-189. Ball, R. A., Suckow, M. A., and Hawkins, E. C. (1991). Bilateral ureteral calculi in a guinea pig. J. Small Exotic Anim. Med. 1, 60-63. Barnes, D. D. (1971). "Special Anatomy of Laboratory Animals." University of California, Davis. Barzago, M. M., Bortolotti, A., Stellari, E E, Pagoni, C., Marraro, G., and Bonati, M. (1994). Respiratory and hemodynamic functions, blood-gas parameters, and acid-base balance of ketamine-xylazine anesthetized guinea pigs. Lab. Anim. Sci. 44, 648-650. Bauck, L. (1989). Ophthalmic conditions in pet rabbits and rodents. Compend. Contin. Educ. Pract. Vet. 11, 258-268. Baumann, G. (1997). Growth without a pituitary? Lessons from the guinea pig [editorial; comment]. Endocrinology 138, 3575-3576. Bendele, A. M., Hulman, J. E, Harvey, A. K., Hrubey, P. S., and Chandrasekhar, S. (1990). Passive role of articular chondrocytes in quinolone-induced arthropathy in guinea pigs. Toxicol. Pathol. 18, 304-312. Beregi, A., Zorn, S., and Felkai, E (1999). Ultrasonic diagnosis of ovarian cysts in ten guinea pigs. Vet. Radiol. Ultrasound 40, 74-76. Bergman, E. N., and Sellers, A. F. (1960). Comparison of fasting ketosis in pregnant and non-pregnant guinea pigs. Am. J. Physiol. 198, 1083-1086. Berryman, T. C. (1976). Guinea pig vocalizations: Their structure, causation, and function. Z. Tierpsychol. 41, 80-106. Bettelheim, E A., Churchill, A. C., and Zigler, J. S., Jr. (1997). On the nature of hereditary cataract in strain 13/N guinea pigs. Curr. Eye Res. 16, 917-924. Boot, R., and Walvoort, H. C. (1986). Otitis media in guineapigs: Pathology and bacteriology. Lab. Anim. 20, 242-248. Boot, R., Van Knapen, E, Kruijt, B. C., and Walvoort, H. C. (1998). Serological evidence for Encephalitozoon cuniculi infection (nosemiasis) in gnotobiotic guineapigs. Lab. Anim. 22, 337-342. Boot, R., Angulo, A. E, and Walvoort, H. C. (1989). Clostridium difficileassociated typhlitis in specific pathogen free guineapigs in the absence of antimicrobial treatment. Lab. Anim. 23, 203-207. Boot, R., Thuis, H., and Wieten, G. (1994). Multifactorial analysis of antibiotic sensitivity of Bordetella bronchiseptica isolates from guineapigs, rabbits, and rats. Lab. Anim. 29, 45-49. Boot, R., Thuis, H. D. W., and Veenema, J. L. (1999). Serological relationship of some V-factor dependent Pasteurellaceae (Haemophilus sp.) from guineapigs and rabbits. Lab. Anim. 33, 91-94. Borkowski, G. L., Griffith, J. W., and Lang, C. M. (1988). Incidence and classification of renal lesions in 240 guinea pigs. Lab. Anim. Sci. 38, 514. Bostrom, R. E., Huckins, J. G., Kroe, D. J., Lawson, N. S., Martin, J. E, Ferrell, J. E, and Whitney, R. A., Jr. (1969). Atypical fatal pulmonary botryomycosis in two guinea pigs due to Pseudomonas aeruginosa. J. Am. Vet. Med. Assoc. 155, 1195-1199. Bozeman, R. M., Humphries, T. W., and Campbell, J. M. (1968). A new group of rickettsia-like agents recovered from guinea pigs. Acta ViroL (English, ed., Prague) 12, 87-93. Breazile, J. E., and Brown, E. M. (1976). Anatomy. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 53-62. Academic Press, New York. Brewer, N. R., and Cruise, L. J. (1994). The guinea pig heartmsome comparative aspects. Contemp. Top. Lab. Anim. Sci. 33, 64-70. Brewer, N. R., and Cruise, L. J. (1997). The respiratory system of the guinea pig: Emphasis on species differences. Contemp. Top. Lab. Anim. Sci. 36, 100-108. Cao, Y., Okada, N., and Hasegawa, M. (1997). Phylogenetic position of guinea pigs revisited. Mol. Biol. Evol. 14, 461-464. Chakrabarty, S., Nandi, A., Mukhopadhyay, C. K., and Chatterjee, I. B. (1992). Protective role of ascorbic acid against lipid peroxidation and myocardial injury. Mol. Cell. Biol. 111, 41-47. Charles River Breeding Laboratories (1982). Technical Bulletin 1. Wilmington, Massachusetts: Charles River Breeding Laboratories.

Cheeseman, M. T., and Kelly, D. E, and Birnie, E. D. C. (1997). Heterotopic pancreas at a site of colon stricture and ulceration in a guineapig. Lab. Anim. 32, 219-222. Cherian, P. V., and Magee, W. E. (1990). Monoclonal antibodies to Chlamydia psittaci guinea pig inclusion conjunctivitis (GPIC) strain. Vet. Microbiol. 22, 43-51. Chiba, J., Otokawa, M., Nakagawa, M., and Egashira, Y. (1978). Serological studies on the major histocompatibility complex of new inbred strains of the guinea pig. Microbiol. Immunol. 22, 545-555. Clarke, G. L., Allen, A. M., Small, J. D., Lock, A. (1980). Subclinical scurvy in the guinea pig. Vet. Pathol. 17, 40-44. Clausen, M. R. (1998). Production and oxidation of short-chain fatty acids in the human colon: Implications for antibiotic-associated diarrhea, ulcerative colitis, colonic cancer, and hepatic encephalitis. Dan. Med. Bull. 45, 51-75. Cleary, R. K., Grossman, M. D., Fernandez, E B., Stull, T. S., Fowler, J. J., Waiters, M. R., and Lampman, R. M. (1998). Metronidazole may inhibit intestinal colonization with Clostridium difficile. Dis. Colon Rectum 41, 464-467. Clifford, C. B., and White, W. J. (1999). The guinea pig. In "The Clinical Chemistry of Laboratory Animals," 2nd ed. (W. E Loeb and E W. Quimby, eds.), pp. 65-70. Philadelphia: Taylor and Francis. Clifford, D. H. (1984). Preanesthesia, analgesia, and euthanasia. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 527562. Academic Press, Orlando, Florida. Colgin, L. M. A., Nielsen, R. E., Tucker, E S., and Okerberg, C. V. (1995). Case report of listerial keratoconjunctivitis in hairless guinea pigs. Lab. Anim. Sci. 45, 435-436. Cook, E. B., Stahl, J. L., Lilly, C. M., Haley, K. J., Sanchez, H., Luster, A. D., Graziano, E M., and Rothenberg, M. E. (1998). Epithelial cells are a major cellular source of the chemokine eotaxin in the guinea pig lung. Allergy and Asthma 19, 15-22. Proceedings, Providence, Rhode Island. Cooper, G., and Schiller, A. L. (1975). "Anatomy of the Guinea Pig." Harvard Univ. Press, Cambridge, Massachusetts. Craig, S. J., Conboy, G. A., and Hanna, P. E. (1995). Baylisascaris sp. infection in a guinea pig. Lab. Anim. Sci. 45, 312-313. Crippa, L., Giusti, A. M., Sironi, G., Cavalletti, E., and Scanziani, E. (1997). Asymptomatic adenoviral respiratory tract infection in guinea pigs. Lab. Anim. Sci. 47, 197-202. Davis, J. A. (1993). Dyspnea and diarrhea in guinea pigs. Lab. Anim. 22, 20-21. Davis, S. R., Mapham, T. B., and Lock, K. J. (1979). Relative importance of prepartum and post-partum factors in the control of milk yield in the guinea pig. J. Dairy Res. 46, 613-621. Debout, C., Taouji, S., and Izard, J. (1995). Increase of a guinea pig natural killer cell (Kurloff cell) during leukemogenesis. Cancer Lett. 97, 117-122. Deeb, B. J., DiGiacomo, R. E, and Wang, S. P. (1989). Guineapig inclusion conjunctivitis (GPIC) in a commercial colony. Lab. Anim. 23, 103-106. Dodson, A. P., and Borriello, S. P. (1996). Clostridium difficile infection of the gut. J. Clin. Pathol. 49, 529-532. Donovan, B. T., and Kopriva, P. C. (1965). Effect of removal or stimulation of the olfactory bulbs on the estrous cycle of the guinea pig. Endocrinology 77,213-217. Dorrestein, G. M., VanBronswijk, J. E. M. H. (1979). Trixacarus caviae Fain, Howell, and Hyatt. 1972 (Acari: Sarcoptidae) as a cause of mange in guinea pigs and papular urticaria in man. Vet. Parasitol. 5, 389-398. Dunham, W. B., Young, M., and Tsao, C. S. (1994). Interference by bedding materials in animal test systems involving ascorbic acid depletion. Lab. Anim. Sci. 44, 283-285. Eckhoff, G. A., Mann, P., Gaillard, E. T., Dykstra, M. J., and Swanson, G. L. (1998). Naturally developing virus-induced lethal pneumonia in two guinea pigs (Cavia porcellus). Contemp. Top. Lab. Anim. Sci. 37, 54-57. Ediger, R. D. (1976). Care and management. In "The Biology of the Guinea

6. BIOLOGY AND DISEASES OF GUINEA PIGS Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 5-12. Academic Press, New York. Elwell, M. R., Chapman, A. L., and Frenkel, J. K. (1981). Duodenal hyperplasia in a guinea pig. Vet. Pathol. 18, 136-139. Emily, P. (1991). Problems peculiar to continually erupting teeth. J. Small Exotic Anim. Med. 1, 56-59. Ernstr6m, V., and Larsson, B. (1967). Export and import of lymphocytes in the thymus during steroid-induced involution and regeneration. Acta Pathol. Microbiol. Scand. 70, 371-374. Eva, J. K., Fifield, R., and Rickett, M. (1976). Decomposition of supplementary vitamin C in diets compounded for laboratory animals. Lab. Anim. 10, 157-159. Farrar, W. E., and Kent, T. H. (1965). Enteritis and coliform bacteremia in guinea pigs given penicillin. Am. J. Pathol. 47, 629-642. Festing, M. E W. (1976a). Genetics. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 99-120. Academic Press, New York. Festing, M. E W. (1976b). The guinea-pig. In "The UFAW Handbook on the Care and Management of Laboratory Animals" (Universities Federation for Animal Welfare, ed.), 5th ed., pp. 229-247. Churchill Livingstone, Edinburgh. Field, J. J., Griffith, J. W., and Lang, C. M. (1989). Spontaneous reproductive tract leiomyomas in guinea pigs. J. Comp. Pathol. 101, 287-294. Flynn, R. J. (1973). Nematodes. In "Parasites of Laboratory Animals" (R. J. Flynn, ed.), pp. 226-228. Iowa State Univ. Press, Ames. Freund, M. (1969). Interrelationships among the characteristics of guinea pig semen collected by electro-ejaculation. J. Reprod. Fert. 19, 393-403. Frye, F. L. (1994). Apparent spontaneous ergot-induced necrotizing dermatitis in a guinea pig. J. Small Exotic Anim. Med. 2, 165-166. Ganaway, J. R. (1976). Bacterial, mycoplasma, and rickettsial diseases. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 121135. Academic Press, New York. Ganaway, J. R., and Allen, A. M. (1971). Obesity predisposes to pregnancy toxemia (ketosis) of guinea pigs. Lab. Anim. Sci. 21, 40-44. Gaschen, L., Ketz, C., Lang, J., Weber, U., Bacciarini, L., and Kohler, I. (1998). Ultrasonographic detection of adrenal gland tumor and ureterolithiasis in a guinea pig. Vet. Radiol. Ultrasound (Raleigh NC) 39, 43-46. Gerold, S., Huisinga, E., Iglauer, E, Kurzawa, A., Morankic, A., and Reimers, S. (1997). Influence of feeding hay on the alopecia of breeding guinea pigs. Zentralbl. Veterinarmed., Reihe A 44, 341-348. Gibson, S. V., and Wagner, J. E. (1986). Cryptosporidiosis in guinea pigs: A retrospective study. J. Am. Vet. Med. Assoc. 189, 1033-1034. Golden, J. G., Hughes, H. C., and Lang, C. M. (1980). Experimental toxemia in the pregnant guinea pig (Cavia porcellus). Lab. Anim. Sci. 30, 174-179. Grahn, B., Wolfer, J., and Machin, K. (1995). Diagnostic ophthalmology/Ophthalmologie diagnostique. Can. Vet. J. 36, 59. Grauer, G. E and DiBartola, S. P. (1995). Glomerular disease. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), Vol. 2, pp. 1760-1775. Saunders, Philadelphia. Green, L. E., and Morgan, K. L. (1991). Toxoplasma abortion in a guinea pig. Vet. Rec. 129, 266-267. Griffith, J. W., Brasky, K. M., and Lang, C. M. (1996). Experimental pneumonia virus of mice infection of guineapigs spontaneously infected with Bordetella bronchiseptica. Lab. Anim. 31, 52-57. Griffith, J. W., Sassani, J. W., Bowman, T. A., and Lang, C. M. (1988). Osseous choristoma of the ciliary body in guinea pigs. Vet. Pathol. 25, 100-102. Groves, M. H. (1992). Hypovitaminosis C in the guinea pig [letter]. Vet. Rec. 131, 40. Gupta, B. N., Conner, G. H., and Meyer, D. B. (1972). Osteoarthritis in guinea pigs. Lab. Anim. Sci. 22, 362-368. Hansen, A. K., Thomsen, P., and Jensen, H.-J. S. (1997). A serological indication of the existence of a guineapig poliovirus. Lab. Anim. 31, 212-218. Harkness, J. E. (1986). The guinea pig. In "Current Therapy in Theriogenology 2" (D. A. Murrow, ed.), pp. 1022-1026.

243 Harkness, J. E. (1997). "Pet rodents--A guide for practitioners." AAHA Press, Lakewood, Colorado. Harkness, J. E., and Wagner, J. E. (1995). "The Biology and Medicine of Rabbits and Rodents." Williams and Wilkins, Baltimore. Harned, M. A., and Casida, L. E. (1972). Failure to obtain group synchrony of estrus in the guinea pig. J. Mammal. 53, 223-225. Harper, L. V. (1968). The effects of isolation from birth on the social behavior of guinea pigs in adulthood. Anim. Behav. 16, 58-64. Harper, L. V. (1976). Behavior. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 31-51. Academic Press, New York. Harvey, C. (1995). Rabbit and rodent skin diseases. Seminars Avian and Exotic Pet Med. 4, 195-204. Henderson, J. D. (1973). Treatment of cutaneous acariasis in the guinea pig. J. Am. Vet. Med. Assoc. 163, 591-592. Hennessy, M. B., and Jenkins, R. (1994). A descriptive analysis of nursing behavior in the guinea pig (Cavia porcellus). J. Comp. Psychol. 108, 23-28. Hillyer, E. V., Quesenberry, K. E., and Donnelly, T. M. (1997). Biology, husbandry, and clinical techniques. In "Ferrets, Rabbits, and RodentsmClinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 243-259. Saunders, Philadelphia. Hintz, H. E (1969). Effect of coprophagy on digestion and mineral excretion in the guinea pig. J. Nutr. 99, 375-378. Hisaw, E L., Zarrow, M. X., Money, W. L., Talmage, R. V. N., and Abramowitz, A. A. (1944). Importance of the female reproductive tract in the formation of relaxin. Endocrinology 34, 122-143. Hodgson, R. J., and Funder, J. W. (1978). Glucocorticoid receptors in the guinea pig. Am. J. Physiol. 235, R115-R120. Hong, C. C., Ediger, R. D., Raetz, R., and Djurickovic, S. (1977). Measurement of guinea pig body surface area. Lab. Anim. Sci. 27, 474-476. Hsiung, G.-D., Griffith, B. P., and Bia, E J. (1986). Herpesvirus and retroviruses of guinea pigs. In "Viral and Mycoplasmal Infections of Laboratory Rodents" (P. N. Bhatt, R. O. Jacoby, H. C. Morse III, and A. E. New, eds.), pp. 451-504. Academic Press, Orlando, Florida. Hurley, R. J., Murphy, J. C., and Lipman, N. S. (1995). Diagnostic exercise: Depression and anorexia in recently shipped guinea pigs. Lab. Anim. Sci. 45, 305-308. Ishihara, C. (1980). An exfoliative skin disease in guinea pigs due to Staphylococcus aureus. Lab. Anim. Sci. 30, 552-557. Isom, H. C., and Gao, M. (1988). The pathogenecity and molecular biology of guinea pig cytomegalovirus. In "Virus Diseases in Laboratory and Captive Animals" (G. Darai, ed.), pp. 247-265. Martinus Nijhoff Publ., Boston. Jaax, G. P., Jaax, N. K., Petrali, J. P., Corcoran, K. D., and Vogel, A. P. (1990). Coronavirus-like virions associated with a wasting syndrome in guinea pigs. Lab. Anim. Sci. 40, 375-378. Jilge, B. (1980). The gastrointestinal transit time in the guinea-pig. Z. Versuchstierkd. 22, 204-210. Jimenez, P. A., Glasson, S. S., Trubetskoy, O. V., and Haimes, H. B. (1997). Spontaneous osteoarthritis in Dunkin Hartley guinea pigs: Histologic, radiologic, and biochemical changes. Lab. Anim. Sci. 47, 598-600. Jones, T. C., Hunt, R. D., and King, N. W., eds. (1996). "Veterinary Pathology." Williams and Wilkins, Baltimore. Kapel'ko, V. I., and Navikova, N. A. (1993). Comparison of functional load in rat and guinea pig hearts. News Physiol. Sci. 8, 157-160. Keller, L. S. E, Griffith, J. W., and Lang, C. M. (1987). Reproductive failure associated with cystic rete ovarii in guinea pigs. J. Vet. Pathol. 24, 335-339. Kelly, C. P., and LaMont, J. T. (1998). Clostridium diJ~cile infection. Ann. Rev. Med. 49, 375-390. Kern, T. J. (1989). Ocular disorders of rabbits, rodents, and ferrets. In "Current Veterinary Therapy XmSmall Animal Practice" (R. W. Kirk et al., eds.), pp. 681-685. Saunders, Philadelphia. Kim, J. C. S. (1977). Ultrastructural studies of vascular and muscular changes in ascorbic acid deficient guineapigs. Lab. Anim. 11, 113-117. King, J. A. (1956). Social relations of the domestic guinea pigs after living under semi-natural conditions. Ecology 37, 221-228.

244

JOHN E. HARKNESS, KATHLEEN A. MURRAY, AND JOSEPH E. WAGNER

King, N. W., and Alroy, J. (1996). Intracellular and extracellular depositions: Degenerations. In "Veterinary Pathology" (T. C. Jones, R. D. Hunt, and N. W. King, eds.), 6th ed., pp. 50-55. Williams and Wilkins, Baltimore. Kinkier, R. J., Jr., Wagner, J. E., Doyle, R. E., and Owens, D. R. (1976). Bacterial mastitis in guinea pigs. Lab. Anita. Sci. 26, 214-217. Kirchner, B. K., Lake, S. G., and Wightman, S. R. (1992). Isolation of Strepto' bacillus moniliformis from a guinea pig with granulomatous pneumonia. Lab. Anim. Sci. 42, 519-521. Kirsch, M. (1997). Akute Glykosidintoxikation durch Aufnahme von Oleanderbl/ittern (Nerium oleander) bei einem Meerschweinchen. Tierarz tl. Prax. Ausgabevk, Kleintiere/Heimtiere (Stuttgart) 25, 398-400. Kirschner, S. E. (1996). Ophthalmologic diseases in small mammals. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 339-345. Saunders, Philadelphia. Klaassen, C. D., and Doull, J. (1980). Evaluation of safety: Toxicologic evaluation. In "Casarett and Doull's Toxicology" (J. Doull, C. D. Klaassen, and M. O. Amdur, eds.). 2nd ed., pp. 11-27. Macmillan, New York. Kohn, D. F. (1974). Bacterial otitis media in the guinea pig. Lab. Anim. Sci. 24, 823-825. Kok, D. J. (1997). Intratubular crystallization events. World J. UroL 15, 219-228. Koneman, E. W. (1977). "Color Atlas and Textbook of Diagnostic Microbiology," 5th ed., p. 610. Lippincott, Philadelphia. Konig, B. (1985). Maternal activity budget during lactation in two species of Caviidae (Cavia porcellus and Galea musteloides). Z. Tierpsychol. 68, 215-230. Kummel, B. A., Estes, S. A., and Arlian, L. G. (1980). Trixacarus caviae infestation of guinea pigs. J. Am. Vet. Med. Assoc. 177, 903-908. Kunst~L I., Niculescu, E., Naumann, S., and Lippert, E. (1980). Torulopsis pintolopesii, an opportunistic pathogen in guineapigs? Lab. Anim. 14, 43-45. Laird, C. W. (1974). Clinical pathology: Blood chemistry. In "Handbook of Laboratory Animal Science" (E. C. Melby, Jr., and N. H. Altman, eds.), Vol. 2, pp. 345-436. CRC Press, Cleveland. Landemore, G., Quillec, M., Letaief, S. E., and Izard, J. (1994). The proteoglycan skeleton of the Kurloff body as evidenced by cuprolinic blue staining. Histochem. J. 26, 350-354. Lang, C. M., Munger, B. L., and Rapp, F. (1977). The guinea pig as an animal model of diabetes mellitus. Lab. Anim. Sci. 27, 789-805. Lee, K. J., Johnson, W. D., and Lang, C. M. (1977). Acute gastric dilation associated with gastric volvulus in the guinea pig. Lab. Anim. Sci. 27, 685-686. Lee, K. J., Johnson, W. D., and Lang, C. M. (1978). Preputial dermatitis in male guinea pigs (Cavia porcellus). Lab. Anim. Sci. 28, 99. Lilley, K. G., Epping, R. J., and Hafner, L. M. (1997). The guinea pig estrous cycle: Correlation of vaginal impedance measurements with vaginal cytologic findings. Lab. Anim. Sci. 47, 632-637. Lindsey, D. S. (1990). Laboratory models of cryptosporidiosis. In "Cryptosporidium and Cryptosporidiosis" (R. Fayer, ed.), pp. 209-223. CRC Press, Boca Raton, Florida. Lipton, H. L., and Rozhon, E. J. (1986). Theiler's encephalomyelitis viruses. In "Viral and Mycoplasmal Infections of Laboratory Rodents" (P. N. Bhatt, R. O. Jacoby, H. C. Morse III, and A. E. New, eds.), pp. 253-275. Academic Press, Orlando, Florida. Loeb, W. E, and Quimby, F. W., eds. (1999). "The Clinical Chemistry of Laboratory Animals." 2nd ed. Taylor and Francis, Philadelphia. Lowe, B. R., Fox, J. G., and Bartlett, J. G. (1980). Clostridium difficile-associated cecitis in guinea pigs exposed to penicillin. Am. J. Vet. Res. 41, 1277-1279. McAleer, R. (1980). An epizootic in laboratory guinea pigs due to Trichophyton mentagrophytes. Aust. Vet. J. 56, 234-236. McCormick, J. G., and Nuttall, A. L. (1976). Auditory research. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 281-303. Academic Press, New York. McKellar, Q. A., Midgley, D. V., Galbraith, E. A., Scott, E. W., and Bradley, A.

(1992). Clinical and pharmacological properties of ivermectin in rabbits and guinea pigs. Vet. Rec. 130, 71-73. McLeod, C. G., Stookey, J. U, Harrington, D. G., and White, J. D. (1977). Intestinal Tyzzer's disease and spirochetosis in a guinea pig. Vet. Pathol. 14, 229-235. Mannering, G. J. (1949). Vitamin requirements of the guinea pig. Vitam. and Horm. (San Diego) (New York) 7, 201-211. Manning, P. J. (1976). Neoplastic diseases. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 211-225. Academic Press, New York. Manning, P. J., Wagner, J. E., and Harkness, J. E. (1984). Biology and diseases of guinea pigs. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, F. M. Loew, eds.), pp. 150-181. Academic Press, Orlando, Florida. Marcus, R., and Coulston, A. M. (1990). Water soluble vitamins. In "The Pharmacological Basis of Therapeutics" (A. G. Gilman, T. W. Rail, A. S. Nies, and P. I. Taylor, eds.), 8th ed., pp. 1530-1552. Pergamon Press, New York. Markham, E S. (1937). Spontaneous toxoplasma encephalitis in the guinea pig. Am. J. Hyg. 26, 193-196. Markham, N. P., and Markham, J. G. (1966). Staphylococci in man and animals: Distribution and characteristics of strains. J. Comp. Pathol. 76, 49-56. Marshall, J. A., and Doultree, J. D. (1996). Chronic excretion of coronaviruslike particles in laboratory guinea pigs. Lab. Anim. Sci. 46, 104-106. Marshall, A. H. E., Swettenham, K. V., Vernon-Roberts, B., and Revell, P. A. (1971). Studies on the function of the Kurloff cell. Int. Arch. Allergy Appl. Immunol. 40, 137-152. Martin, J. G. (1994). Animal models of bronchial hyperresponsiveness. Rev. Mal. Respir. 11, 93-99. Matsubara, J., Kamiyama, T., Miyoshi, T., Ueda, H., Saito, M., and Nakagawa, M. (1988). Serodiagnosis of Streptococcus pneumoniae infection in guinea-pigs by an enzyme-linked immunosorbent assay. Lab. Anim. 22, 304-308. Mayora, J., Soave, O., and Doak, R. (1978). Prevention of cervical lymphadenitis in guinea pigs by vaccination. Lab. Anim. Sci. 28, 686-690. Medlean, L., and Ristic, Z. (1992). Diagnosing dermatophytosis in dogs and cats. Vet. Med. (Kansas City MO) 87, 1086-1104. Morales, E. (1995). "The Guinea Pig: Healing, Food, & Ritual in the Andes." Univ. Arizona Press, Tucson. Murphy, J. C., Ackerman, T. I., Marini, R. P., and Fox, T. G. (1991). Cervical lymphadenitis in guinea pigs: Infection via intact ocular and nasal mucosa by Streptococcus zooepidemicus. Lab. Anim. Sci. 41, 251-254. Muto, T., Noguchi, Y., Suzuki, K., and Zaw, K. H. (1983). Adenomatous intestinal hyperplasia in guinea pigs associated with Campylobacter-like bacteria. Jpn. J. Med. Sci. Biol. 36, 337-342. Nagase, T., Dallaire, M. J., and Ludwid, M. S. (1994). Airway and tissue responses during hyperpnea-induced constriction in guinea pigs. Am. J. Respir. and Crit. Care Med. (New York NY) 149, 1342-1347. Nakagawa, M., Muto, T., Yoda, H., Nakano, T., and Imaizumi, K. (1971). Experimental Bordetella bronchiseptica infection in guinea pigs. Jpn. J. Vet. Sci. 33, 53-60. National Research Council. (1979). "Animals for Research." 10th ed. NRC, Washington, D.C. National Research Council. (1996). "Guide for the Care and Use of Laboratory Animals." National Academy Press, Washington, D.C. Navia, J. M., and Hunt, C. E. (1976). Nutrition, nutritional diseases, and nutrition research applications. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 235-267. Academic Press, New York. Nelson, W. L., Kaye, A., Moore, M., Williams, H. H., and Harrington, B. L. (1951). Milking techniques and composition of guinea pig milk. J. Nutr. 44, 585 -594. Obwolo, M. J. (1977). The pathology of experimental yersiniosis in guinea pigs. J. Comp. Pathol. 87, 213-221.

6. BIOLOGY AND DISEASES OF GUINEA PIGS Ocholi, R. A., Chima, T. C., Uche, E. M., and Oyetunde, I. L. (1988). An epizootic of Citrobacterfreundii in a guineapig colony: Short communication. Lab. Anim. 1O, 119 - 142. Ohashi, T., Kenmochi, M., Kinoshita, H., Ochi, K., and Kikuchi, H. (1999). Cochlear function of guinea pigs with experimental chronic renal failure. Ann. Otol. Rhinol. Laryngol. 108, 955-962. Okewole, P. A., Odeyemi, P. S., Oyetunde, I. L., Irokanulo, E., and Chima, J. C. (1989). Abortion in guinea pigs. Vet. Rec. 124, 248. Okewole, P. A., Odeyemi, P. S., Oladunmade, M. A., Ajagbonna, B. O., Onah, J., and Spencer, T. (1991). An outbreak of Streptococcus pyogenes infection associated with calcium oxalate urolithiasis in guineapigs (Cavia porcellus). Lab. Anim. 25, 184-186. Osborn, J. G. (1987). Cytomegalovirus and other herpesviruses of mice and rats. In "Viral and Mycoplasmal Infections of Laboratory Rodents" (P. N. Bhatt, R. O. Jacoby, H. C. Morse III, and A. E. New, eds.), pp. 421-450. Academic Press, Orlando, Florida. Parker, G. A., Russell, R. J., and De Paoli, A. (1977). Extrapulmonary lesions of Streptococcus pneumoniae infection in guinea pigs. Vet. Pathol. 14, 332-337. Payne, B. J., Lewis, H. B., Murchison, T. E., and Hart, E. A. (1976). Hematology of laboratory animals. In "Handbook of Laboratory Animal Science" (E. C. Melby, Jr., and N. H. Altman, eds.), Vol. 3, pp. 383-461. CRC Press, Cleveland. Peng, X., Griffith, J. W., and Lang, C. M. (1990). Cystitis, urolithiasis, and cystic calculi in aging guineapigs. Lab. Anim. 24, 159-163. Peplow, A. M., Peplow, P. V., and Hafez, E. S. E. (1974). Parameters of reproduction. In "Handbook of Laboratory Animal Science" (E. C. Melby, Jr., and N. H. Altman, eds.), Vol. 1, pp. 105-116. CRC Press, Cleveland. Percy, D. H., and Barthold, S. W. (1993). "Pathology of Laboratory Rodents and Rabbits." Iowa State Univ. Press, Ames. Perez-Martinez, J. A., and Storz, J. (1985). Antigenic diversity of Chlamydia psittaci of mammalian origin determined by microimmunofluorescence. Infect. lmmun. 50, 905-910. Phoenix, C. H. (1970). Guinea pigs. In "Reproduction and Breeding Techniques for Laboratory Animals" (E. S. E. Hafez, ed.), pp. 244-257. Lea and Febiger, Philadelphia. Post, K., and Saunders, J. R. (1979). Topical treatment of experimental ringworm in guinea pigs with griseofulvin in dimethylsulfoxide. Can. Vet. J. 20, 45-48. Pring-/~erblom, P., Bla~ek, K., Schramlovfi, J., and Kunst37~, I, (1997). Polymerase chain reaction for detection of guinea pig adenovirus. J. Vet. Diag. Investig. (Columbia MO) 9, 232-236. Quillec, M., Debout, C., and Izard, J. (1977). Red cell and white cell counts in adult female guinea pigs. Pathol. Biol. 25, 443-446 Quinn, P. J., Carter, M. E., Markey, B., and Carter, G. R. (1994). "Clinical Veterinary Microbiology." Wolfe Publ., London. Rank, R. G., White, H. J., and Barron, A. L. (1979). Humoral immunity in the resolution of genital infection in female guinea pigs infected with the agent of guinea pig inclusion conjunctivitis. Infect. Immun. 26, 573-579. Rehg, J. E., and Pakes, S. P. (1981). Clostridium difficile antitoxin neutralization of cecal toxin(s) from guinea pigs with penicillin-associated colitis. Lab. Anim. Sci. 31, 156-160. Reid, M. E., and Bieri, J. G. (1972). Nutrient requirements of the guinea pig. In "Nutrient Requirements of Laboratory Animals" (Subcommittee on Laboratory Animal Nutrition), pp. 9-19. National Academy of Sciences, Washington, D.C. Reid, M. E., and von Sallman, L. (1960). Nutritional studies with the guinea pig. VI. Tryptophan (with ample dietary niacin). J. Nutr. 70, 329-336. Robinson, R. (1971). Guinea pig chromosomes. Guinea Pig News. 4, 15-18. Ronald, N. C., and Wagner, J. E. (1976). The arthropod parasites of the genus Cavia. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 201-209. Academic Press, Orlando, Florida. Rothwell, T. L. W., Pope, S. E., Rajczyk, Z. K., and Collins, G. H. (1991).

245 Haematological and pathological responses to experimental Trixacarus caviae infection in guinea pigs. J. Comp. Pathol. 104, 179-185. Rowlands, I. W. (1949). Postpartum breeding in the guinea pig. J. Hyg. 47, 281-287. Rowlands, I. W. (1957). Insemination of the guinea pig by intraperitoneal injection. J. Endocrinol. 16, 98-106. Sachser, N. (1998). Of domestic and wild guinea pigs: Studies in sociophysiology, domestication, and social evolution. Naturwissenschaften 85, 307-317. Samii, V. F., Dumonceaux, G., and Nyland, T. G. (1996). Radiographic diagnosismprostatitis in a guinea pig. Vet. Radiol. Ultrasound (Raleigh NC) 37, 357-358. Sanderson, J. H., and Phillips, C. G. (1981). "An Atlas of Laboratory Animal Haematology." Clarendon, Oxford. Sansone, M., and Bovet, D. (1970). Avoidance learning by guinea pigs. Queensland J. Exper. Psychol. 22, 458-461. Schaeffer, D. O., and Donnelly, T. M. (1996). Disease problems of guinea pigs and chinchillas. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 260-281. Saunders, Philadelphia. Schalm, O. W., Jain, N. C., and Carroll, E. J. (1975). "Veterinary Hematology." 3rd ed. Lea and Febiger, Philadelphia. Schermer, S. (1967). "The Blood Morphology of Laboratory Animals." Davis, Philadelphia. Schiefer, B., and Stunzi, H. (1979). Pulmonary lesions in guinea pigs and rats after subcutaneous injection of Freund's complete adjuvant on homologous pulmonary tissue. Zentralbl. Veterinarmed., Reihe A 26, 1-10. Seidl, D. C., Hughes, H. C., Bertolet, R., and Lang, C. M. (1979). True pregnancy toxemia (preeclampsia) in the guinea pig (Cavia porcellus). Lab. Anim. Sci. 29, 472-478. Senyk, G., Keflan, R., Stites, D. P., Schanzlin, D. J., Ostler, H. B., Hanna, L., Keshishyan, H., and Jawetz, E. (1981). Cell-mediated and humoral immune responses to chlamydial antigens in guinea pigs infected ocularly with the agent of guinea pig inclusion conjunctivitis. Infect. Immun. 32, 304-310. Short, D. J., and Woodnott, D. P., eds. (1969). "The Institute of Animal Technicians Manual of Laboratory Animal Practice and Techniques." Thomas, Springfield, Illinois. Sisk, D. B. (1976). Physiology. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 63-98. Academic Press, New York. Smith, H. W. (1965). The development of the flora of the alimentary tract in young animals. J. Pathol. BacterioL 90, 495-513. Sprouse, R. F. (1976). Mycoses. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 153-161. Academic Press, New York. Stalheim, O. H. V., and Matthews, P. J. (1975). Mycoplasmosis in specificpathogen-free and conventional guinea pigs. Lab. Anim. Sci. 25, 70-71. Stephenson, E. H., Trahan, C. J., Ezzell, J. W., Mitchell, W. C., Abshire, T. G., Oland, D. D., and Nelson, G. O. (1989). Efficacy of a commercial bacterin in protecting strain 13 guineapigs against Bordetella bronchiseptica pneumonia. Lab. Anim. 23, 261-269. Stockard, C. R., and Papanicolaou, G. N. (1917). The existence of a typical oestrous cycle in the guinea pig--with a study of its histological and physiological changes. Am. J. Anat. 22, 225-283. Stuppy, D. E., Douglass, P. R., and Douglass, P. J. (1979). Urolithiasis and cystotomy in a guinea pig (Cavia porcellanus). Vet. Med. Small Anim. Clin. 74, 565 -567. Sturegard, E., Sjunnesson, H., Ho, B., Willen, R., Aleljung, P., Ng, H. C., and Wadstrom, T. (1998). J. Med. Microbiol. 47, 1123-1129. Surawicz, C. M. (1998). Clostridium difficile disease: Diagnosis and treatment. Gastroenterologist 6, 60-65. Takumida, M., Zhang, D. M., Yajin, K., and Harada, Y. (1997). Effect of streptomycin on the otoconial layer of the guinea pig. J. Oto-Rhino-Laryngol. and Related Specialities (Basel) 59, 263-268. Taouji, S., Buat, M.-L., Izard, J., and Landemore, G. (1994). Kurloff cell lyso-

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somal arylsulphatases: Presence of both cationic and highly anionic isoforms of the sole B class. Biol. Cell 80, 43-48. Taylor, J. L., Wagner, T. E., Owens, D. R., and Stuhlman, R. A. (1971). Chronic pododermatitis in guinea pigs: A case report. Lab. Anim. Sci. 21,944-945. Thurnham, D. I. (1997). Micronutrients and immune function: Some recent developments. J. Clin. Pathol. 50, 887-891. Timoney, J. E, Artiushin, S. C., and Boschwitz, J. S. (1997). Comparison of the sequences and functions of Streptococcus equi pr-like proteins SeM and SzPSe. Infect. Immun. 65, 3600-3605. Trahan, C. J., Stephenson, E. H., Ezzell, J. W., and Mitchell, W. C. (1987). Airborne-induced experimental Bordetella bronchiseptica pneumonia in strain 13 guineapigs. Lab. Anim. 21, 226-232. U. S. Department of Agriculture, Animal and Plant Health Inspection ServiceAnimal Care. (2001). "Animal Welfare Report--Fiscal Year 2000." USDA, Washington, D.C. Upston, J. M., Karjalinen, A., Bygrave, E L., and Stocker, R. (1999). Efflux of hepatic ascorbate: A potential contributor to the maintenance of plasma vitamin C. Biochem. J. 342, 49-56. Valiant, M. E., and Frost, B. M. (1984). An experimental model for evaluation of antifungal agents in a Trichophyton mentagrophytes infection of guinea pigs. Chemotherapy 30, 54-60. Van Andel, R. A., Franklin, C. L., Besch-Williford, C., Riley, L. K., Hook, R. R., Jr., and Kazacos, K. R. (1995). Cerebrospinal larva migrans due to Baylisascaris procyonis in a guinea pig colony. Lab. Anim. Sci. 45, 27-30. Van Beek, E., and Peeters, L. L. H. (1998). Pathogenesis of preeclampsia: A comprehensive model. Obster Gynecol. Surv. 53, 233-239. Van Herck, H., van den Ingh, Th. S. G. A. M., van der Hage, M. H., and Zwart, P. (1988). Dermal cryptococcosis in a guineapig. Lab. Anim. 22, 88-91. Van Hoosier, G. L., Jr., and Robinette, L. R. (1976). Viral and chlamydial diseases. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 137-152. Academic Press, New York. Vanrobaeys, M. de Herdt, P., Ducatelle, R., Devrise, L. A., Charlier, G., and Haesebrouck, E (1998). Typhlitis caused by intestinal Serpulina-like bacteria in domestic guinea pigs (Cavia porcellus). J. Clin. Micro. 36, 690-694. Vetterling, J. M. (1976). Protozoan parasites. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 163-196. Academic Press, New York. Voge, M. (1973). Trematodes. In "Parasites of Laboratory Animals" (R. Flynn, ed.), pp. 120-154. Iowa State Univ. Press, Ames. Waggie, K. S., Wagner, J. E., and Kelley, S. T. (1986). Naturally occurring Bacillus piliformis infection (Tyzzer's disease) in guinea pigs. Lab. Anim. Sci. 36, 504-506. Wagner, J. E. (1976). Miscellaneous disease conditions of guinea pigs. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 227234. Academic Press, New York.

Wagner, J. E., and Foster, H. L. (1976). Germfree and specific pathogen free. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 21-30. Academic Press, New York. Wagner, J. E., and Manning, P. J., eds. (1976). "The Biology of the Guinea Pig." Academic Press, New York. Wagner, J. E., A1-Rabiai, S., and Rings, R. W. (1972). Chirodiscoides caviae infestation in guinea pigs. Lab. Anim. Sci. 22, 750-752. Wan, C.-H., Franklin, C., Riley, L. K., Hook, R. R., Jr., and Besch-Williford, C. (1996). Diagnostic exercise: Granulomatous encephalitis in guinea pigs. Lab. Anim. Sci. 46, 228-230. Wei, L., deBri, E., Lundberg, A., and Svensson, O. (1998). Mechanical load and primary guinea pig osteoarthrosis. Acta Orthop. Scand. 69, 351-357. Wescott, R. B. (1976). Helminth parasites. In "The Biology of the Guinea Pig" (J. E. Wagner and P. J. Manning, eds.), pp. 197-200. Academic Press, New York. White, W. J., and Lang, C. M. (1989). The guinea pig. In "The Clinical Chemistry of Laboratory Animals" (W. E Loeb and E W. Quimby, eds.), pp. 2730. Pergamon Press, New York. White, W. J., Balk, M. W., and Lang, C. M. (1989). Use of cage space by guinea pigs. Lab. Anim. 23, 208-214. Witt, W. M., Hubbard, G. B., and Fanton, T. (1988). Streptococcus pneumoniae arthritis and osteomyelitis with vitamin C deficiency in guinea pigs. Lab. Anim. Sci. 38, 192-194. Wolf, B., Reinecke, K., Aumann, K.-D., Brigelius-Flohe, R., and Flohe, L. (1993). Taxonomical classification of the guinea pig based on its Cu/Zn superoxide dismutase sequence. Biol. Chem. Hoppe-Seyler 374, 641-649. Wright, J. (1936). An epidemic of Pasteurella infection in guinea pig stock. J. Pathol. Bacteriol. 42, 209-212. Wullenweber, M., and Boot, R., (1994). Interlaboratory comparison of enzymelinked immunosorbent assay (ELISA) and indirect immunofluorescence for detection of BordetelIa bronchiseptica antibodies in guinea pigs. Lab. Anim. 28, 335-339. Young, W. C., Dempsey, E. W., and Myers, H. I. (1935). Cyclic reproductive behavior in the female guinea pig. J. Comp. Physiol. Psychol. 19, 313-335. Young, J. D., Hust, W. J., White, W. J., and Lang, C. M. (1987). An evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs. Lab. Anim. Sci. 37, 652-656. Zajac, A., Williams, J. E, and Williams, C. S. E (1980). Mange caused by Trixacarus caviae in guinea pigs. J. Am. Vet. Med. Assoc. 177, 900-903. Zarrow, M. X. (1947). Relaxin content of blood, urine, and other tissues of pregnant and postpartum guinea pigs. Proc. Soc. Exp. Biol. Med. 66, 488-491. Zloch, Z., and Ginter, E. (1995). Moderate alcohol consumption and vitamin C status in the guinea-pig and the rat. Physiol. Res. 44, 173-176. Zwicker, G. M., Dagle, G. E., and Adee, R. R. (1978). Naturally occurring Tyzzer's disease and intestinal spirochetosis in guinea pigs. Lab. Anim. Sci. 28, 193-198.

Chapter 7 Biology and Diseases of Other Rodents Thomas M. Donnelly and Fred W. Quimby

I. II.

III.

IV.

V.

VI.

VII.

VIII.

LABORATORY ANIMALMEDICINE, 2nd edition

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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G r o u n d Squirrels or Susliks: S p e r m o p h i l u s

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Pocket Gophers: G e o m y i d a e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

263

B.

Biology

264

C.

Husbandry

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

................................................ ..............................................

W h i t e - F o o t e d M i c e or D e e r Mice: P e r o m y s c u s

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264 264 265

A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

265

B.

Biology

266

C.

Husbandry

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

................................................ ..............................................

267 267

Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

248

THOMAS M. DONNELLY AND FRED W. QUIMBY IX.

X.

XI.

XII.

XIII.

XIV.

XV.

XVI.

Rice Rats:

Oryzomys

..........................................

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

268

B.

Biology

268

C.

Husbandry ..............................................

268

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

268

Cane Mice:

................................................

Zygodontomys

.....................................

268

A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

269

B.

Biology

269

C.

Husbandry ..............................................

269

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

270

Cotton Rats:

................................................

Sigmodon

........................................

270

A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

270

B.

Biology

271

C.

Husbandry ..............................................

271

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

271

................................................

White-Tailed Rats:

..................................

272

A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

272

B.

Biology

273

C.

Husbandry

D.

Diseases . . . . .

Mystromys

................................................

Gerbils and Jirds:

.............................................. ...........................................

Meriones

.....................................

274 274 275

A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

275

B.

Biology

276

C.

Husbandry

..............................................

276

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

277

................................................

Voles and M e a d o w Mice:

Microtus

...............................

279

A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

279

B.

Biology

280

C.

Husbandry

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

................................................ ..............................................

M u l t i m a m m a t e Rats:

Mastomys

.................................

A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B.

Biology

C.

Husbandry

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

................................................ ..............................................

D e g u s or T r u m p e t - T a i l e d Rats:

Octodon

..........................

A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B.

Biology

C.

Husbandry

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

X V I I . Chinchillas:

................................................ ..............................................

Chinchilla

........................................

281 281 281 281 283 283 283 284 284 284 285 285 286

A.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

286

B.

Biology

286

C.

Husbandry

D.

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

................................................ ..............................................

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

267

A.

Introduction

The order Rodentia, with more than approximately 1814 living species placed in 29 families (approximately half of all mammalian species), is the largest order of mammals (Luckett and Hartenberger, 1985). Rodents range in size from pygmy mice weighing 5 gm to capybaras that weigh more than 70 kg. They are found worldwide except in Antarctica, in New Zealand, and on some oceanic islands. Ecologically, they are remarkably

286 287 291

diverse. Some species spend their entire lives above the ground in the canopy of rain forests; others rarely emerge from beneath the ground. Some species are highly aquatic, while others are equally specialized for life in deserts. Many rodents are to some degree omnivorous; others are highly specialized, eating, for example, only a few species of invertebrates or fungi. Despite their morphological and ecological diversity, all rodents share one characteristic: a highly specialized dentition for gnawing (the term rodent is derived from the Latin rodens, meaning "gnawing"). Rodents have a single pair of upper and a

7. BIOLOGY AND DISEASES OF OTHER RODENTS

single pair of lower incisors. Between each incisor and the first cheek tooth is a toothless interval called the diastema. The incisors are rootless and grow continuously. Enamel is deposited on the anterior and lateral incisor surfaces; the posterior incisor surface is dentin. During gnawing, as the incisors grind against each other, they wear away the softer dentin, leaving a chisellike enamel edge. This "self-sharpening" system is very effective and is one of the keys to the enormous ecological success of rodents. Bringing the incisors together or using them to chisel away at a surface requires muscle that forcefully brings the lower jaw forward. In rodents, this is done primarily by the masseter muscle. The masseter can be divided into three parts: the superficial masseter, the lateral masseter, and the medial masseter. By moving the skeletal attachment or origin of the masseter rostrally, rodents gain both a mechanical advantage and an additional range of lower jaw movement. Mammalogists divide rodents into three groups based on how the masseter attachments evolved to move forward. The Animal Diversity Web of the University of Michigan Museum of Zoology describes rodent jaws with excellent images of the arrangement of the massetei" muscles. We have summarized much of the following text from this website (Myers, 1999). In the sciuromorphous condition (from Latin sciurus, meaning "squirrel"), the origin of the lateral masseter moves forward and attaches to the front of the zygomatic arch where it meets the rostrum. The origin of the superficial masseter is also shifted forward, but the origin of the medial masseter does not change much. The front of the zygomatic arch has developed into a large, distinctive zygomatic plate in sciuromorph rodents such as squirrels, beavers, geomyids, and heteromyids. In the hystricomorphous condition (from Latin hystrix, meaning "porcupine"), the infraorbital foramen becomes very large. Through it, part of a much-expanded medial masseter passes to originate on the side of the rostrum rostrally to the zygomatic arch. This condition is found in porcupines (both New and Old World families), guinea pigs, and jerboas. The third, myomorphous condition (from Greek mys, meaning "mouse"), probably arose from a sciuromorphous ancestral state. It includes the development of a zygomatic plate and rostral shifting of the lateral masseter, as in sciuromorphs. The infraorbital foramen is also moderately enlarged, and a slip of medial masseter passes through it. The myomorphous condition is found in true rats and mice (New and Old World families), hamsters, gerbils, and voles. How these three conditions evolved is widely debated. It is accepted, however, that more than one group of rodents achieved some of them independently. The morphology of the insertion of the masseter on the lower jaw also differs among groups of rodents. In the sciurognathous jaw, the angular process, which receives most of the masseter, arises almost in a line with the rest of the jaw; that is, it originates in the same vertical plane that also includes the socket of

249

the incisors. In the hystricognathous jaw, the origin of the angular process is distinctly lateral to this plane, and the angular process is often flared laterally. The coronoid process is usually reduced in hystricognathous forms. Based on a large body of morphological evidence, Carleton (1984) recognized two rodent suborders, the Sciurognathi and Hystricognathi. The Sciurognathi include true rats, mice, hamsters, and squirrel-like rodents; the Hystricognathi, also representing a group of Old and New World rodent families, are now restricted primarily to Africa and South America. Examples of this diverse order include guinea pigs, chinchillas, and porcupines. The current best guess about the phylogeny of rodents, however, suggests that the difference in pattern of insertion of the masseter (sciurognathy vs. hystricognathy) reflects a deep phylogenetic division. Rodent phylogeny and classification have been the focus of many disagreements, some of which continue. Most researchers now generally agree on the two suborder classifications proposed by Carleton (1984). However, controversies still arise based on resolving anatomical data, paleontological data, and molecular data (Luckett and Hartenberger, 1993). For example, Graur et al. (1991) analyzed amino acid sequence data and suggested that the order Rodentia is polyphyletic, in contrast to most morphological data, which support rodent monophyly. At issue was whether the hystricognath rodents, such as the guinea pig, represent an independent evolutionary lineage within mammals, separate from the sciurognath rodents. Alternative methods of molecular analysis using repetitive sequences, mitochondrial DNA sequences, nuclear protein coding genes, internal gene duplications, newly arisen genes, and gene loss or inactivation contradicted Graur et al.'s theory (Frye and Hedges, 1995; Martignetti and Brosius, 1993; Nedbal et al., 1994; Serdobova and Kramerov, 1998). The strong molecular evidence for rodent monophyly demonstrated the need to choose appropriate methods for phylogenetic inference. The discovery of nearly complete dental remains of a primitive rodent (Tribosphenomys minutus) from strata of the transitional PaleoceneEocene in Inner Mongolia, China, has provided fresh insight on rodent phylogeny, reinforcing the rodent monophylic theory (Meng et al., 1994). However, this paleontological discovery substantially modified previous ideas about the ancestral rodent morphotype, which in turn have important implications for understanding the origin of rodents and their relationship to other eutherian mammals (Novacek, 1992). In summary, rodent phylogeny and classification are more complex than first proposed. The suborder Sciurognathi is fairly stable; and it is generally agreed that some families, such as murid rodents, are not closely related to other families in this suborder. Consequently, it is considered polyphyletic. The suborder Hystricognathi remains monophyletic, but problematic because New World families assigned to this group appear to have evolved independently of Old World families. In one sense, the Hystricognathi are used as a catch-group to place

250

THOMAS M. DONNELLY AND FRED W. QUIMBY Table I Taxonomy of Rodents Family Suborder Sciurognathi Aplodontidae Sciuridae Geomyidae Heteromyidae Castoridae Anomaluridae Pedetidae Muridae Gliridae Seleviniidae Zapodidae Dipodidae Suborder Hystricognathi Hystricidae Erethizontidae Caviidae Hydrochaeridae Dinomyidae Dasyproctidae Chinchillidae Capromyidae Octodontidae Ctenomyidae Abrocomidae Echimyidae Thryonomyidae Petromuridae Bathyergidae Ctenodactylidae

Common names

Number of species

Mountain beaver or sewellel Squirrels, chipmunks, marmots, and prairie dogs Pocket gophers Pocket mice, kangaroo rats, and mice Beavers Scaly-tailed squirrels Springhare or springhaas Rats, mice, hamsters, voles, lemmings, and gerbils Dormice Desert dormouse Birch mice and jumping mice Jerboas

1 260 37 60 2 7 1 1138 20 1 17 32

Old World porcupines New World porcupines Cavies and Patagonian "hares" Capybara Pacarana Agoutis and pacas Chinchillas and visachas Hutias and nutria Degus and octodonts Tuco-tucos Chinchilla rats or chinchillones Spiny rats Cane rats or grasscutters Rock rat or dassie African mole rats or blesmols Gundis

groups of rodents whose phylogeny and relationship to other rodents are uncertain at this time. The taxonomic relationships of these rodents may be found in Table I. Although laboratory rats and mice constitute over 90% of all vertebrates used in research, other species, such as hamsters, guinea pigs, and more recently woodchucks, have also been widely used and have contributed significantly to innovations in the biomedical sciences (see Animal Models in Biomedical Research). Each of these species has a dedicated chapter in the text. All other rodent species account for less than 0.5% of rodents used annually; however, they too have been responsible for insights into biological processes and disease pathogenesis. In this chapter we will discuss gerbils, white-footed mice, cane mice, grasshopper mice, squirrels and gophers, degus, multimammate rats, white-tailed rats, cotton rats, rice rats, wood rats, voles, kangaroo rats, and chinchillas. Although sources have been identified for many species listed in this chapter, others may be sought directly from investigators using these animals in studies. A current listing of sources for each of the species discussed may be found in the Institute for Animal Research (National Research Council) Animal Models database <www.nationalacademies.org/ILAR>.

II.

11 10 17 1 1 15 6 33 9 44 2 69 2 1 8 5

Ground Squirrels or Susliks:

Spermophilus

Included in this classification are Spermophilus richardsonii (Richardson's ground squirrel), S. armatus (Uinta ground squirrel), S. tridecemlineatus (thirteen-lined ground squirrel), S. lateralis (golden-mantled ground squirrel), and S. beecheyi (California ground squirrel; Beechey ground squirrel; rock squirrel). (See Figs. 1 and 2).

A. 1.

Introduction

Description

The head and body length of ground squirrels is 1 3 0 - 4 0 6 mm, the well-furred tail is 3 8 - 2 5 4 m m long, and weight range is 85-1000 gm (Nowak, 1999). Their fur is grizzled brownish or yellowish gray, often with fine light spots on the upper parts, and the underparts are whitish or yellow. Ground squirrels have large internal cheek pouches to carry food. In S. tridecemlineatus there is a series of alternating dark and light longitudinal

7. BIOLOGY AND DISEASES OF OTHER RODENTS

251

Fig. 1. Spermophilusbeecheyi, Californiaor Beechey ground squirrel. Also known as rock squirrel. Side view. (Photographby the Audio Visual Office, U.S. Fish and Wildlife Service and the Mammal Images Library of the American Societyof Mammalogists.)

stripes with a row of light spots on each of the dark stripes (Streubel and Fitzgerald, 1978). The name Citellus has often been used for this genus, but is not now in general use. 2.

Distribution

Ground squirrels inhabit prairies and steppes, tundra, rocky country, open woodlands, or desert mountain ranges. They are not found in areas with a dense forest cover. Spermophilus richardsonii inhabits the prairies of central Alberta and western Montana to western Minnesota; S. armatus inhabits southwestern Montana, southeastern Idaho, western Wyoming, and northern Utah; S. tridecemlineatus occupies central Alberta to Ohio and southern Texas; S. lateralis is found in southwestern Canada and from North Dakota to Washington; S. beecheyi extends from southern Washington to northern Baja California (Nowak, 1999). 3.

0.06 hectares, which cover the burrows of three to five females. Within a week after mating, the females establish their own territories, averaging 0.016 hectares, within the male territories (Michener and Koeppl, 1985). Thirteen-lined ground squirrels live in small, scattered groups. They are not highly social and lack territorial activity; although the occupied burrow is defended, the area around it is not (Streubel and Fitzgerald, 1978).

Habitat

Most ground squirrels construct burrows. Spermophilus richardsonii constructs mazelike burrows consisting of galleries, about 3.5-15.0 meters long and 0.75-2.00 meters deep; there are several chambers and an average of eight entrances, the main one having a mound (Michener and Koeppl, 1985). Spermophilus tridecemlineatus constructs burrows about 5 - 6 m long and 0.3 m deep; there is no mound (Streubel and Fitzgerald, 1978). During warm months Richardson's ground squirrels live in extensive colonies. Males establish territories of about

Fig. 2. Spermophilus elegans, Wyoming ground squirrel. Side view at burrow entrance, Rocky Mountain National Park, CO. (Photograph by B. J. Bergstrom and the Mammal Images Library of the American Society of Mammalogists.)

THOMASM. DONNELLYAND FRED W. QUIMBY

252 4.

Use in Research

Ground squirrels have been used extensively to investigate the phenomenon of hibernation (Barnes, 1989; Bintz, 1969; Storey, 1997). Despite the virtual arrest of physiologic functions and minimal delivery of glucose and oxygen, homeostatic control is maintained. Many researchers have investigated the adaptive hibernation strategies of ground squirrels because they might reveal new mechanisms of tolerance, with potential clinical usefulness. Thus, the effect hibernation has on infection, radiation exposure, immunity, and inflammation has been studied (Anderson, 1982; Frank, 1992; Frerichs, 1999; Storey, 1997). Ground squirrels have also been used to investigate hepatitis B infections and hepatocellular carcinoma (Marion et al., 1986; Tennant et al., 1991). The cholesterol-fed Richardson's ground squirrel is an effective animal model in which to study factors that influence cholesterol gallstone formation and associated alterations in the gallbladder epithelium (MacPherson and Pemsingh, 1997; MacPherson et al., 1987).

1979; Davis, 1984; Frank, 1992; Reiter et al., 1983). Ground squirrels survive well on diets commercially prepared for rats. However, Anderson (1982) reports that the fur of ground squirrels raised on commercial rodent diets lacks the luster of that of wild squirrels and they are more prone to develop dental problems. Ground squirrels can be maintained in a laboratory at 18~176 and induced to hibernate in an unlighted room maintained at 5~176 (Reiter et al., 1983). Ground squirrels will lose considerable weight during hibernation and may die if forced to hibernate much beyond the end of their natural hibernating season (Davis, 1984). Emaciated squirrels should be removed from hibernation at any time to reduce mortality. Enrichment activities for thirteen-lined ground squirrels during captivity and rehabilitation have been described (CookBabcook, 1996).

D.

Diseases

1. Infectious Diseases 5.

Sources

a.

Thirteen-lined ground squirrels may be obtained from Steven Gruningen (5N 301 Ringneck Lane, Bartlett, IL 60103-6332; phone: 630-351-0991). B.

Biology

All ground squirrels are diurnal. Spermophilus richardsonii is reported to have three daily activity periods: during the first two hours after dawn, from 10 AM to 2 PM, and from 4 PM to sunset (Michener and Koeppl, 1985). For about 7 months (September or October to April or May) Richardson's ground squirrels hibernate in their burrows. Spermophilus tridecemlineatus doubles its weight by September and hibernates from October to early April (Streubel and Fitzgerald, 1978). Female ground squirrels are monestrous and normally bear one litter per year; mating takes place shortly after emergence from hibernation. The gestation period is 23-31 days, litter size averages 2-15 (7..5 in S. richardsonii), and females wean their young at 4 6 weeks. By 11 months juveniles have attained full size and sexual maturity (Michener, 1973). C.

Husbandry

In the wild the diet of ground squirrels consists of seeds, nuts, grains, roots, bulbs, mushrooms, green vegetation, insects and other small invertebrates, and birds' eggs (Schitoskey and Woodmansee, 1978; Tomich, 1982). Although they may store food in their burrows, they do not appear to use it until awakening in spring. Many laboratory colonies of ground squirrels are wild-adapted (Anderson, 1982; Barnes, 1989; Bintz et al.,

Bacterial Infections

Gangrenous dermatitis caused by Corynebacterium ulcerans has been reported in wild Richardson's ground squirrels (Olson et al., 1988). Six squirrels died of toxemia, but 57 responded to topical and parenteral administration of antibiotics. The epizootic was believed to be associated with fighting; infected and carrier ground squirrels most likely transmitted the C. ulcerans through bite wounds. Purulent cutaneous and visceral lesions associated with Staphylococcus aureus were observed in a colony of goldenmantled ground squirrels used in a hibernation study (Campbell et al., 1981). The squirrels had been purchased from a commercial supplier. Three weeks after their purchase and during the following five weeks, one-third of the squirrels died. The predominant gross and histologic findings consisted of multifocal suppurative lesions involving the skin, brain, and numerous visceral organs. Infection of wild California ground squirrels with Yersinia pestis occurs frequently in plague-endemic areas such as southern California and Alaska (Mian et al., 1996; Spano, 1994; Townzen et al., 1996). Transmission of the bacillus is associated with the fleas Hoplopsyllus anomalus and Oropsylla montana. Ground squirrels become infected with plague following hibernation and again when reoccupying colonial burrows (Davis, 1999). b.

Viral Infections

Ground squirrels infected for up to 10 years with ground squirrel hepatitis virus (GSHV) develop a mild, nonprogressive persistent hepatitis (Cullen and Marion, 1996). Histologically there is a mild to moderate chronic portal hepatitis composed of a

253

7. BIOLOGY AND DISEASES OF OTHER RODENTS lymphocytic and plasmacytic infiltrate with occasional individual necrotic hepatocytes and small aggregates of mononuclear inflammatory cells in the parenchyma. Serum levels of aspartate and alanine transaminases (AST and ALT) are mildly elevated. Adult Richardson's ground squirrels have been infected experimentally with Western equine encephalomyelitis virus by intranasal instillation (Leung et al., 1978). Mortality followed the instillation of a minimum threshold of 4.7 logs of virus; infection was produced by a dosage of 2.3 logs. The incubation period was from 4 to 7 days, being preceded by a viremic phase. Signs were depression, ataxia, and paralysis of the limbs. Highest titers of virus were recovered from the brain, and histopathological changes involving the central nervous system included meningitis, vasculitis, perivascular cuffing, gliosis, neuronophagia, and neuronal degeneration. The virus was also found in a variety of extraneural tissues. Lesions in extraneural tissues included necrosis of brown fat and an apparent increase in number of Kupffer's cells in the liver. The lymphoid tissue was involved, indicating a possible source for viremia. The duration and magnitude of viremia were sufficient to provide virus source for arthropods. Ground squirrels are susceptible to infection with rabies virus, and Russian scientists have shown a prolonged incubation period of rabies virus during hibernation (Botvinkin et al., 1985). In the United States most cases of rabies are in woodchucks (Marmota monax), primarily from the eastern United States, which are temporally and spatially associated with reports of raccoon rabies (Childs et al., 1997). Antigenic or genetic characterization of variants of rabies viruses from rodents and woodchucks corresponds to the variants associated with the major terrestrial wildlife reservoir within the geographic region of specimen origin (Childs et al., 1997). Yellow susliks (S. fulvus) are the natural host of a poxvirus in Turkmenia that resembles cowpox virus and is different from infectious ectromelia virus. The authors found that the virus is apparently identical to other poxvirus isolates made from white rats and Felidae in the Moscow Zoo. Experimental inoculation produced a severe infection with a high mortality rate. Transmission of virus to uninoculated cagemates was shown to occur. Virus persisted in convalescent animals and was present in urine 3 weeks after inoculation and in kidney and testis for at least 5 weeks after inoculation (Marennikova et al., 1978).

beginning on day 9. Meronts were most numerous in the lung; none were found in liver or spleen. Four of 10 squirrels infected died between days 11 and 13. There were petechial hemorrhages in skeletal muscle, lung, serosal membranes, and brain in these animals, with microscopic evidence of pulmonary, myocardial, and brain injury. Foci of inflammation were visible in the myocardium and brain of animals killed to 64 days. The most prevalent intestinal parasites in a large survey of wild Wyoming ground squirrels (Spermophilus elegans) were coccidia of the genus Eimeria (Stanton et al., 1992). Most ground squirrels harbored 2 or more species. There were no differences in the prevalence of infection among different ages and populations or between sexes. The presence or absence of helminths was independent of the presence or absence of Eimeria. Capillaria hepatica has been recorded as a cause of of liver disease in a Richardson's ground squirrel (Rocke, 1992). An undescribed species of Demodex mite was described in the hair follicles and ducts of sebaceous glands in the ear canals of 7 California ground squirrels from California (Waggie and Marion, 1997). Similar mites were observed in the lumens of hair follicles and ducts of Meibomian glands in the eyelids of two of these squirrels. Microscopic changes in the epithelium and surrounding dermis, when present, were minimal. No associated clinical signs of disease or macroscopic lesions were observed. Lethal myiasis of wild Richardson's ground squirrels by the sarcophagid fly Neobellieria citellivora has been reported (Michener, 1993).

d.

Lungs from three species of ground squirrels collected in south-central Saskatchewan were examined for adiaspores of Emmonsia crescens (Leighton and Wobeser, 1978). Two of 81 (2.5%) S. richardsonii, 3 of 17 (17.6%) S. tridecemlineatus, and 35 of 44 (79.5%) S. franklini were infected. Infection was more common in adults than in young born that year. 2.

Parasitic Infections

Richardson's ground squirrels have been infected experimentally with sporocysts of Sarcocystis campestris from badgers (Wobeser et al., 1983). No lesions were found in animals killed 1 to 3 days postinfection. Hepatitis and phlebitis of hepatic veins were present in animals killed between 4 and 8 days. No meronts were detected in these animals, but the lesions suggested that a generation of merogony occurred in the hepatic veins. Meronts were found in endothelial cells in many tissues

Metabolic and Nutritional Diseases

Spontaneous diabetes mellitus is described in a captive golden-mantled ground squirrel (Heidt et al., 1984) 3.

c.

Fungal Infections

Neoplastic Diseases

Hepatocellular carcinoma has developed in California ground squirrels infected with ground squirrel hepatitis virus (GSHV) (Marion et al., 1986). Hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV), a virus closely related to HBV, have been associated with the development of hepatocellular carcinoma in their respective hosts. Liver carcinoma appeared in 2 of 28 GSHV-bearing animals studied and in 1 of 23 squirrels with antibody to the virus. The animals were observed for a minimum of 2.4 years, and each animal was 4-years or older when

THOMAS M. DONNELLYAND FRED W. QUIMBY

254

the tumor was detected. Integrated GSHV DNA was found in the hepatocellular carcinoma tissue of the 1 carrier animal examined, paralleling the frequent findings of integrated hepatitis B and woodchuck hepatitis viral DNA in human and woodchuck hepatocellular carcinoma. During studies of seasonal obesity, hepatocellular carcinoma was observed in 6 (50%) of 12 Richardson's ground squirrels examined thoroughly (Tennant et al., 1991). Serological tests for GSHV of California ground squirrels were uniformly negative. Southern blot analyses of liver cell DNA demonstrated infection with a hepadnavirus related to GSHV. Single case reports of exophthalmos associated with a Harderian gland adenocarcinoma and a squamous cell skin carcinoma have been reported in 2 male California ground squirrels (Morrow and Day-Lollini, 1990; Trigo and Riser, 1981). 4.

Miscellaneous

Supernumerary teeth are described in Richardson's ground squirrel and hybrid individuals (Goodwin, 1998).

Ill.

PRAIRIE DOGS: Cynomys

passages, and prairie dogs build a volcano-shaped cone of soil to keep surface water running down the burrows (Hoogland, 1996a). The black-tailed prairie dog shows a high degree of social organization. It is found in large colonies, or "towns," that usually cover about 100 hectares. Towns are divided into "wards," which are made up of several "coteries." Containing an average of 8.5 individuals, the coterie is a discrete social unit typically consisting of a single adult male, 3 or 4 adult females, and several yearlings and 1- to 2-year-old juveniles (Hoogland, 1996a). All members of a coterie are socially integrated and display territorial defense toward outsiders (Hoogland, 1979, 1996b). 4.

Use in Research

The black-tailed prairie dog is a widely used animal model for investigations into biliary physiology and the pathophysiology of gallstone formation (Chen et al., 1997). The prairie dog has an accessible gallbladder that lies on the ventral surface of the right lobule of the median lobe. The common bile duct, a union of the cystic and the hepatic ducts, enters the duodenum approximately 5 mm distal to the pylorus. The distal portion of the common duct dilates to form an ampulla surrounded proximally

The most common example is Cynomys ludovicianus (blacktailed prairie dog) (Fig. 3).

A. 1.

Introduction

Description

Black-tailed prairie dogs are stout, short-tailed, short-legged squirrels. Their head and body length is 280-330 mm, tail length is 30-115 mm, and weight is 0.7-1.4 kg. Their coat is a grizzled yellow-gray on top, and the underparts are lighter. The tail of C. ludovicianus has a black tip and accounts for the common name of this prairie dog (Hoogland, 1996a). 2.

Distribution

C. ludovicianus inhabits open plains and plateaus and is found on the Great Plains from Montana and southern Saskatchewan to extreme northern Mexico (Hoogland, 1996a). 3.

Habitat

Black-tailed prairie dogs excavate elaborate burrows for shelter and protection from predators. Their tunnels are about 100150 mm wide, 4 - 3 4 meters long, and 1-5 meters deep. They connect with grass-lined nest chambers about 300-450 mm wide. The burrows reach ground level by vertically ascending

Fig. 3. Cynomys ludovicianus, Black-tailed prairie dog. Side view, Wind Cave, SD. (Photographby G. L. Twiestand the MammalImagesLibraryof the American Society of Mammalogists.)

255

7. BIOLOGY AND DISEASES OF OTHER RODENTS

by the muscular choledochal sphincter. Opening separately into the duodenum approximately 80 mm from the pylorus is the pancreatic duct. Earlier physiologic studies showed that the choledochal sphincter has intrinsic motility distinct from that of the duodenum (Grace et al., 1988). The cholesterol-fed blacktailed prairie dog has been especially useful as an animal model of the effects of diet on gallstone formation (Holzbach, 1984). Cohen and Mosbach (1993) reviewed the historical perspective and recent advances in this animal model of cholelithiasis. The black-tailed prairie dog has been described as an animal model for the study of antibiotic-induced diarrhea (Muller et al., 1987). In prairie dogs, cefoxitin induces Clostridium difficile cecitis. However, the disease in prairie dogs has a more chronic course than in other animal models of C. di~cileinduced diarrhea.

B.

Biology

Black-tailed prairie dogs are diurnal and terrestrial. They become dormant during severe weather but are not deep hibernators, and may be seen above ground at times, even in midwinter (Nowak, 1999). Prairie dogs are very social and reinforce their relationship within a coterie by activities such as nuzzling, grooming, playing together, and vocal communication. Waring (1970) listed nine vocalizations for C. ludovicianus. Female prairie dogs are monestrous and in the wild do not start breeding until 2 years of age (Hoogland, 1998). Mating takes place from early March to late April and gestation lasts 34-37 days (Hoogland, 1997). Each coterie is a cooperative breeding unit in which breedirig occurs at the same time, and each adult female produces one litter per year. Offsetting the nepotism and potential detrimental inbreeding, lactating females will then kill offspring of close kin (Hoogland, 1994). Black-tailed prairie dogs have good low-frequency hearing, can hear frequencies as low as 4 Hz, and are more sensitive than any other rodent yet tested at frequencies below 63 Hz (Heffner et al., 1994). In contrast, they are relatively insensitive in their midrange and have poor high-frequency hearing. Heffner suggested that the reduced midrange sensitivity and high-frequency hearing are related to their adaptation to an underground lifestyle with its reduced selective pressure for sound localization. In this respect they seem intermediate between the more exclusively subterranean rodents (such as gophers and mole rats) and surface dwellers (such as chinchillas and kangaroo rats). Broughton (1992) reported a range of normal hematology and blood chemistry values for the prairie dog. He examined 45 adult prairie dogs and reported the mean values for a complete blood cell count, electrolytes, blood urea nitrogen, creatinine, calcium, phosphorus, liver enzymes, total bilirubin, protein, albumin, cholesterol, triglycerides, and lipids. He found that the prairie dog has normochromic, microcytic blood with an increased number of red blood cells and a high concentration of

small platelets. Compared to humans, the prairie dog has a higher CO2 concentration with a slightly increased potassium concentration; the anion gap is 12 with a calculated serum osmolality of 316; the BUN concentration is elevated and serum AST is increased threefold. The prairie dog also has lower serum values for cholesterol, very low density lipoprotein (VLDL), and low density lipoprotein (LDL) cholesterol than humans. However, the high density lipoprotein (HDL) cholesterol consists of 67% of the total cholesterol concentration, and the LDL and HDL ratio is 0.3. Hematologic values may be found in Table II.

C.

Husbandry

In the wild, black-tailed prairie dogs feed on herbs and grasses, maintaining a "rotatingpasture" that causes fast-growing plants to predominate around colonies. They do not store food in their burrows (Hoogland, 1996a). Many laboratory colonies of prairie dogs are wild-caught. Ozaki (1993) described the care and breeding techniques used for a colony of prairie dogs in a Japanese zoo.

D.

Diseases

There are few reports on naturally developing diseases in prairie dogs, and reports of neoplasia consist predominantly of hepatocellular carcinoma and odontoma. 1.

Infectious Diseases

Tularemia was diagnosed in an adult, wild-caught prairie dog found dehydrated and ataxic, with severe diarrhea (La Regina et al., 1986). Gross necropsy revealed scattered white pinpoint hepatosplenic lesions and massive, purulent bronchopneumonia. Diagnosis was confirmed by direct fluorescent antibody tests and culture of spleen and liver samples. Feces collected from live-trapped black-tailed prairie dogs in Wyoming showed five species of the intestinal coccidia Eimeria (Seville, 1997). It appears that the prairie dogs and ground squirrels have the same group of Eimeria parasites, and host switching is common. Baylisascaris spp. larvae caused central nervous system disease in three prairie dogs maintained at a research facility (Dixon et al., 1988). The infection was transmitted from Baylisascaris eggs on the cages. Before introduction of the prairie dogs, the research facility housed racoons. The cages were washed with water at 82.2~ (180~ in a large passthrough cage washer. Despite these measures and storage of the cages for up to six months, some eggs survived and infected the prairie dogs. Clinical signs consisted of ataxia, stumbling, and head tilt, which progressed to severe torticollis, paddling in

Table II Comparative Hematological Values of Selected Exotic Rodents a

Family and species

Red blood cells (X 106/mm 3)

PCV (1111%)

Hb (gm/dl)

MCV (i~m3)

MCH (mg)

MCHC (%)

White blood cells (X 103/mm 3)

Neutro phils (%)

Lymphocytes (%)

MONO (%)

Eosinophils (%)

Basophils (%)

0

Sciurognath rodents Family Sciuridae Prairie dogs Cynomys ludovicianus

6.6

36.5

3.2-8.0

14.8-46.8

55.1

18.5

6.4-14.8

12.0

44.2-68.2

15.5-27.9

31.4-44.0

33.5

19.0

29.3

2.3

63.3

33.3

0.8

0.2

0.1

m

1.5-6.7

9-99

4-79

0-6

0-4

0-3

6.3

3.9

2.2

0.1

0.1

3.3-10.5

0.8-7.9

1.0-5.6

0-0.4

0-0.5

(black-tailed prairie dog) Tree squirrels Sciurus carolinensis

6.6

43

12.6

65.1

4.0-10.0

31-56

8.3-17.7

m

56.4

~

(gray squirrel)

Family Muridae Subfamily Gerbillinae Gerbils Meriones unguiculatus

Subfamily Mierotinae Voles Microtus californicus

(field vole) Subfamily Murinae Multimammate rats Mastomys natalensis

8.5

48

15

7. 0 - 1 0 . 0

41-52

1 2 . 1 - 16.9

13.3

47

14.9

10.7-16.0

42-52

13.5-16.3

7.5

40

13.0

~

.

17.6

~

~

35.3

11.2

~

~

53.3 .

17.3 .

31.2 ~

31.7 ~

32.5

.

11.0 4.3-21.6

4.3 2.4-6.2

19.0 3-41

78.0

3.0

32-97

0-9

1.0 0-4

0.6 0-2

13.0

78.0

5.0

3.0

0.2

5-23

64-92

0-10

0-7

0-1

7.5

20.0

75.0

0

1.5

0.6

2.8-13.0

8-48

48-93

0-1

0-9

0-1

Hystricognath rodents Family Chinchillidae Chinchillas Chinchilla laniger

6.9

38

11.7

55.0

16.9

30.7

7.8

43.0

54.0

5.2-10.3

27-54

8.0-15.4

32.1-69.2

10.4-19.8

20.0-38.5

4.0-25.0

9-78

19-98

a Values shown are the mean, with the range in italics underneath the mean.

1.0 0-6

0.7

0.6

0-9

0-11

257

7. BIOLOGY AND DISEASES OF OTHER RODENTS lateral recumbency, and loss of the righting reflex. Gross lesions were not found at necropsy. Microscopically, the cerebellar white matter and medulla oblongata were most severely affected and had many sections of large ascarid larvae. Hepatic infestation with Taenia sp. has been described in a wild-caught black-tailed prairie dog (Banks et al., 1995). A 2 mm white nodule on the left lobe of the liver was identified at necropsy. Histologically, the lesion was a fluid-filled cyst in which a larval cestode was encapsulated. Surrounding liver parenchyma appeared normal. The cysticercus was identified as a member of the genus Taenia, but further speciation was not possible. Wild-caught white-tailed prairie dogs (C. leucurus) were identified as new mammalian hosts for Taenia mustelae (Rockett et al., 1990). The liver contained many clear, spherical cysts approximately 1 mm in diameter. On dissection, most of the cysts exhibited a dense, white area with four muscular suckers at a single point on the bladder. Based on the size of the organisms, the characteristically tiny hooks on the scolex, and the location of the cysts in this host, the parasites were identified as cysticerci of T. mustelae. Histologically, the cysts were surrounded by an intense inflammatory response consisting primarily of lymphocytes mixed with some eosinophils and early deposition of fibrous connective tissue. Dermatophyte infection causing hair loss was reported in 3 captive Mexican prairie dogs (C. mexicanus) in a Texas zoo. The causative agent was Microsporum gypseum (Porter, 1979). 2.

Neoplastic Diseases

Of 11 hepatocellular carcinomas reported, 7 were found in a series of 60 prairie dogs necropsied, and all were associated with hepatitis (Snyder, 1979). In another report, 2 black-tailed prairie dogs were found to have hepatocellular carcinoma with metastasis to the lung, coexisting chronic active hepatitis, and hyperplasia of hepatocytes in the nonneoplastic areas of the liver tissue (Une et al., 1996). The hyperplastic hepatocytes had many eosinophilic inclusion bodies positive for orcein stain in their cytoplasm. Electron microscopy revealed areas of a specific mail-like mesh structure in the location of the cytoplasmic inclusion bodies. Ultrastructure of inclusion bodies differed from that of the hepadnavirus usually associated with hepatocellular carcinoma in other species of Sciuridae (e.g., woodchucks and ground squirrels), and hepadnavirus-like particles were not observed. Primary hepatocellular carcinomas have been described in a case report of a female black-tailed prairie dog and an adult prairie dog in a survey of zoo animals (Hubbard et al., 1983; Woolf et al., 1982). Odontoma is a common cause of upper respiratory symptoms in captive black-tailed prairie dogs. Ten black-tailed prairie dogs (5 females and 5 males) presenting with upper respiratory symptoms of varying degree had radiographic lesions consistent with hypercementosis, complex odontomas, and obstruction of nasal air passage (Wagner, 1999). The animals were pri-

vately owned, originating from different breeders, and were 2.5 to 6 years of age. Medical treatment with antibiotics, decongestants, and steroids was palliative in the prairie dogs studied. Attempted extraction of the incisors often resulted in fractured incisors and incomplete removal and did not alleviate clinical signs. Tracheostomy eliminated the signs of upper airway obstruction, but patency was difficult to maintain. The author suspects that as odontogenic tumors are hamartomous growths, they may develop in reaction to mechanical trauma of the upper incisors. A large firm lipoma was found in the cranial mediastinum of a 3-year-old female prairie dog. It encompassed the carotid arteries, jugular veins, and thymus and compressed the esophagus and trachea. Localized compression caused dysphagia, weight loss, and dyspnea, which eventually resulted in death (Rogers and Chrisp, 1998). A case report of an epiglottal fibrosarcoma in an adult prairie dog described the clinical signs, management, and treatment (Suedmeyer and Pace, 1994). 3.

Miscellaneous

Respiratory disease is a common problem in captive prairie dogs. It is not a problem in wild prairie dogs or in zoo prairie dogs. Although many etiologies are proposed, it is believed that diet and environmental conditions are the cause of the problems seen in captive prairie dogs. Affected animals present with labored open-mouth breathing that improves slightly with oxygen. Griner (1983) found that prairie dogs in the San Diego Zoo become obese and develop severe dermatitis after 2 to 3 years in captivity.

IV.

POCKET GOPHERS: Geomyidae

Geomys (Eastern pocket gophers) includes eight species and Thomomys (Western pocket gophers), nine species.

A.

Introduction

1. Taxonomy

Specific boundaries in the genera Geomys and Thomomys are poorly defined. Researchers have described complex relationships for several definable geographic units of pocket gophers (Patton and Smith, 1989). Their work is based on a variety of contact zone analyses that have employed morphological, karyological, allozyme, and/or mitochondrial and nuclear DNA biochemical analyses. Varying degrees of hybridization exist between geographically differentiated pocket gophers, and authors vary in their recognition of these individual animals at the

258

THOMAS M. DONNELLYAND FRED W. QUIMBY

specific or subspecific levels. In Geomys, the traditional species (arenarius, bursarius, personatus, pinetis, and tropicalis) seem an inadequate representation of specific diversity in the genus (Block and Zimmerman, 1991). Although specific boundaries for some species are poorly defined, Patton and Smith (1989) provided an operational definition that can be applied to all pocket gophers. 2. Description

The description is adapted from Nowak (1999). Pocket gophers have stout, thickset bodies with little external evidence of a neck. The large skull, adapted for burrowing, is angular and flattened. The tail is short, naked, and very sensitive to touch. Ears and eyes are small, and well-developed lacrimal glands supply a thick fluid that cleans the cornea of dirt while the animal is burrowing. The lips can be closed behind the curved incisors, so the animal can gnaw dirt while burrowing and not get any in its mouth. Two long external fur-lined cheek pouches that can be turned inside out for cleaning extend back from the mouth to the shoulders. The legs are short with powerful forearms and five strong digging claws. Size varies widely, but males are larger than females. Head and body length is 90-300 mm, and tail length is 40-140 mm. 3. Distribution

Western pocket gophers live in deserts, prairies, open forests, and meadows. They are found west of the Rocky Mountains, ranging from southwestern Canada to southern Baja California and central Mexico. Eastern pocket gophers prefer loose, sandy soil in open and sparsely wooded areas. They are found east of the Rocky Mountains, ranging from southern Manitoba and Wisconsin to northeastern Mexico and across to Florida (Hall, 1981). 4.

Habitat

Pocket gophers are fossorial and spend most of their life underground, although at times they surface to gather food for storage in their burrows. They dig two types of tunnels: long, winding, shallow tunnels constructed to obtain food such as roots and tubers from above; and deep tunnels for shelter, with chambers for nests, food storage, and fecal deposits (Hall, 1981). 5.

Use in Research

Pocket gophers have become important animal models for molecular evolution (Hafner et al., 1994; McClellan, 2000; Smith, 1998). Phylogenetic relationships among pocket gophers are examined based on the complete sequence of selected mitochondrial genes. For example, different DNA sequences in a molecular marker for a selected mitochondrial gene reveal

asymmetry between species. Differences between animals from another genus (within a family) vary by an average of 20% uncorrected gene sequence divergence. The overall rates of nucleotide substitution and identical substitution (based on analysis of degenerate sites) reveal different rates of molecular evolution. Extensive sampling within one species of pocket gophers often reveals a geographic unit of well-differentiated animals. In some areas, the geographic unit coincides with a geographic region, based on allozyme data, but not in other areas. These patterns suggest a rapid radiation within the species, followed by geographic subdivision.

B.

Biology

Most of the information is summarized from Nowak (1999) or Hall (1981). Pocket gophers do not hibernate, although they may become inactive during winter. They are solitary animals intolerant of other conspecifics. When two adults are placed together, they usually fight viciously. The only exception to this behavior occurs during the breeding season. The mating system of pocket gophers is promiscuous, with female choice at its base (Patton and Smith, 1993). Western pocket gophers are monestrous and produce one litter per year. The gestation ranges 18-19 days, and litter size ranges 1-10 with an average of 3 - 6 , depending on the species. Species of Eastern pocket gopher in northern regions have one litter per year, but species in southern regions have two litters per year. Litter size in northern species ranges 18, and in southern species the young usually number 1-3. The young of Western and Eastern pocket gophers are generally weaned at 4 - 5 weeks, remain in their mother's burrow for about 1-2 months, and shortly after leave the nest to establish new homes. They reach adult weight at 5 - 6 months and are sexually mature the following breeding season. An exception is Geomys pinetis in which females reach sexual maturity at 4 - 6 months.

C.

Husbandry

Pocket gophers feed mainly on the underground parts of plants, especially the roots and tubers, but they also eat stems. They rarely drink and get sufficient water in the wild from moist vegetable matter.

D.

Diseases

Approximately 90% of wild pocket gophers have coccidian oocysts in their intestines when examined (Wilber et al., 1994). The parasites are Eimeria species. Filarioid nematodes of the genus Litomosoides occur in the abdominal and/or thoracic cavities of wild pocket gophers (Brant and Gardner, 2000). The two common species are L. thomomydis and L. westi. One hundred

259

7. BIOLOGY AND DISEASES OF OTHER RODENTS

twenty-two species of biting lice (order Mallophaga) are known to infest pocket gophers (Hellenthal and Price, 1994). The lice are highly host-specific and do not seem to infest other species. Using the mitochondrial cytochrome oxidase I gene, researchers study pocket gophers and their lice to determine the extent to which these groups have cospeciated through evolutionary time (Hafner and Page, 1995). Griner (1983) described malocclusion of the incisor teeth leading to malnutrition in a male pocket gopher.

V.

KANGAROO RATS: Dipodomys

The two species generally studied are Dipodomys spectabilis (Bannertail kangaroo rat) (Fig. 4) and D. merriami (Merriam's kangaroo rat) (Fig. 5).

A. 1.

Introduction

Taxonomy

The family Heteromyidae contains 6 genera and 60 species. The genus Dipodomys contains 21 species that live primarily in North America (Nowak, 1999). 2.

Description

The description is adapted from Nowak (1999). Kangaroo rats are highly modified for travel by jumping. The hindlegs are long and powerful, and the forelimbs reduced. The tail is longer than the head and body and serves as a balancing organ in locomotion and as a prop when standing. The tail is dark above and

Fig. 5. Dipodomys merriami, Merriam's kangaroo rat. Side view, feet and tail visible, Santa Rita Experimental Range, Pima County, AZ. (Photograph by T. L. Best and the Mammal Images Library of the American Society of Mammalogists.)

below and has white sides continuing from a white band running across the thigh. The body of the tail is well furred, and the end has a tuft of longer hairs. The leaping action of Dipodomys is similar to that of kangaroos (Macropus spp.), hence their common name of kangaroo rats. As in Geomyidae, all Heteromyidae rodents have external fur-lined cheek pouches. Dipodomys are unique in the Heteromyidae because their cheek teeth grow throughout life; the cheek teeth of other genera of Heteromyidae do not. The skull is papery in consistency and not modified for a fossorial life. The head and body length is 100-200 mm, tail length is 100-215 mm, and weight range is 35-180 gm. 3.

Distribution

All species of Dipodomys are found in North America, including northern Mexico. D. spectabilis is found in Arizona, New Mexico, western Texas, and northern Mexico; D. merriami is found in the southwestern United States, northern Mexico, and Baja California (Hall, 1981). 4.

Habitat

Kangaroo rats dwell in arid and semiarid country with some brush or grass (Nowak, 1999). They prefer open ground that permits an unobstructed view of the surroundings and is best for their method of locomotion. Dipodomys spp. construct burrows in well-drained, easily worked soil. D. spectabilis builds a labyrinth of tunnels within a prominent mound that is gradually built up as soil is excavated (Best, 1972). 5. Fig. 4. Dipodomys spectabilis, Banner-tailed kangaroo rat. Side view, Pedro Armendariz lava field, Socorro County, NM. (Photograph by T. L. Best and the Mammal Images Library of the American Society of Mammalogists.)

Use in Research

Kangaroo rats have been used extensively in the study of renal physiology and water conservation (Stallone and Braun, 1988) and behavior (Yoerg and Shier, 1997). They have also

THOMAS M. DONNELLYAND FRED W. QUIMBY

260

been used in investigations looking at disuse osteoporosis (Muths and Reichman, 1996) and evolutionary neuroanatomy (Jacobs and Spencer, 1994). The size and habits of the kangaroo rat have made it a good animal model for marginal decompression sickness. The average minimum no-tail-biting depth for kangaroo rats is closer to the minimum-bends depth of humans than to the equivalent depth for other rodents, and is as good an animal model as the goat (Hills and Butler, 1978).

B.

Biology

All species of Dipodomys are primarily diurnal. Dipodomys spectabilis is most active during the first 2 to 3 hr after sunset and in the last 2 hr before dawn (Schroder, 1979). Kangaroo rats seldom drink water, and utilize metabolically derived water from the breakdown of food within the body. They conserve moisture by coming out of their burrows at night when humidity is the highest and concentrating urine within highly efficient kidneys (Hall, 1981). The dorsal gland, a prominent androgenindependent, oil-secreting gland, is present on the back between the shoulders of males and females (Randall, 1986). Kangaroo rats are highly territorial, and only one adult is found per burrow. Savage battles often take place when two kangaroo rats are placed together (Eisenberg and Isaac, 1963). They are not vocal but are known to produce a thumping or drumming sound with hindfeet. The foot drumming functions as an alarm system against predation by snakes, advertises territory, and repels intruders (Randall and Stevens, 1987). Breeding may occur throughout the year. Females are seasonally polyestrous, but breeding is correlated to availability of food, decreasing after a drought and increasing following a favorable moist season. Although providing captive kangaroo rats with water for restricted periods mimics abundant seasons, Eisenberg and Isaac (1963) do not recommend ad libitum water, as some kangaroo rats become addicted to drinking and develop a diabetes insipidus-like syndrome. Gestation lasts approximately 29-33 days (Eisenberg and Isaac, 1963). Litter size ranges 1-6 in the wild, with 1-8 in D. spectabilis and an average of 2.6 in D. merriami (Eisenberg and Isaac, 1963). Weaning in Dipodomys spp. occurs between 21 and 29 days.

Daly et al., 1984). However, the methods required are time-consuming and intensive. During periods of nonreceptivity, female kangaroo rats are extremely aggressive toward males and will frequently attack and sometimes kill cagemates (Fine et al., 1986). The diet in the wild consists of seeds, some fruits, leaves, stems, buds, and insects and other invertebrates. A study of D. merriami in Arizona showed that approximately 80% of its food consisted of seeds and 15% consisted of insects (Reichman, 1975). Because of potential shortages caused by drought, kangaroo rats store food in their burrows. The diet in the laboratory is usually grains and seeds with lettuce as a source of water (Williams, 1980). Although kangaroo rats rarely drink in the wild, they will drink if offered water in captivity. Bathing in dust, like chinchillas, is necessary for the welfare of kangaroo rats. When denied dust bathing in captivity, they develop sores on the body and the fur becomes matted from oily secretions on the back (Nowak, 1999). Kangaroo rats have fragile tails that will break off if used to restrain the animal. The recommended technique of restraining a kangaroo rat is by the skin on the nape of the neck (Fine et al., 1986).

D. 1.

Husbandry

Anderson and Jones (1984) contend that Dipodomys spp. are difficult to breed in the laboratory because of their aggressive nature toward one another. However, they are not aggressive toward humans (Williams, 1980). Kangaroo rats can be maintained together in an extremely large housing area (Fine et al., 1986). Several researchers have been successful at breeding Dipodomys and have produced many litters of kangaroo rats in captivity (Butterworth, 1961; Chew, 1958; Day et al., 1956;

Infectious Diseases

Parasitic diseases have been extensively described in wildcaught kangaroo rats (Ernst and Chobotar, 1978; Fain and Lukoschus, 1978; Gummer et al., 1997; Hill and Best, 1985; Pfaffenberger et al., 1985; Stout and Duszynski, 1983; Thomas et al., 1991). However, there appear to be no cases in laboratoryreared kangaroo rats. 2.

Metabolic/Nutritional Diseases

Suckow et al. (1996) described a male, wild-caught kangaroo rat that developed anorexia and wasting due to a gastric trichobezoar. They commented on the lack of information regarding the clinical medicine of this species. 3.

C.

Diseases

latrogenic Diseases

Kangaroo rats develop spongiform degeneration of the central auditory system similar to that seen in the Mongolian gerbil (Meriones unguiculatus) (McGinn and Faddis, 1997). In D. deserti and D. merriami the spongiform lesions develop in dendrites and oligodendrocytes of the cochlear nucleus and in oligodendrocytes of the auditory nerve root. The lesions are morphologically indistinguishable from those described in the gerbil (McGinn and Faddis, 1998). The spongiform degeneration is more numerous in animals continually exposed to modest levels of low-frequency noise (< 75 dB SPL).

261

7. BIOLOGY AND DISEASES OF OTHER RODENTS 4.

2.

Neoplastic Diseases

Griner (1983) diagnosed mammary gland adenocarcinoma in a female kangaroo rat from the San Diego Zoo.

VI.

WOOD RATS OR PACK RATS: Neotoma

Wood or pack rats include Neotoma floridana (Allegheny wood rat) (Fig. 6), N. albigula (white-throated wood rat), N. mexicana (Mexican wood rat), N. cinerea (bushy-tailed wood rat), and N. fuscipes (dusky-footed wood rat).

A. 1.

Introduction

Taxonomy

There are 19 species in the genus Neotoma. While foraging wood rats gather material for their nests and carry it to their homesite, they will drop the item they are carrying and take new material if they find a more attractive object. They often pick up shining objects or silverware from camps and leave the material they had been carrying. This characteristic has given them the name "pack rat" or "trade rat" (Nowak, 1999).

The description is taken from Nowak (1999). Head and body length is 150-230 mm, tail length is 75-240 mm, and weight is 2 0 0 - 4 5 0 gm. The fur is generally soft and dense, and the color on the back ranges from pale to dark gray; the underparts range from a pure white to pale gray. The ears are large, and bare on the tips or naked. In some species the tail is sparsely haired, but in the bushy-tailed wood rat the tail is well covered. 3.

Distribution

Wood rats are found only in CentraI America (Honduras and Nicaragua) and North America. Neotomafloridana is found primarily on the eastern coast of the United States (Wiley, 1980); N. albigula is found in the southwestern United States to central Mexico; N. mexicana ranges from Colorado and southwestern Utah to western Honduras; N. cinerea ranges from northwestern Canada to North Dakota and Arizona (Escherich, 1981); and N. fuscipes ranges from western Oregon to northern Baja California. 4.

Habitat

Wood rats live in a variety of habitats ranging from low, hot, dry deserts to humid jungles and to rocky slopes above the timberline. Some species of wood rats build elaborate dens composed of twigs, stems, foliage, bones, rocks, or whatever material is available. These "houses" often rest on the ground or are placed against rocks or the base of a tree. Species inhabiting areas where spiny cactus grows build their houses almost entirely over this plant. The dens are placed so that it is almost impossible for a predator to approach without being pierced by thorns. Some species do not build large houses but use crevices among rocky outcrops. They close the openings to the crevices with sticks or other material. Much variation exists between shelters, even within the same species, depending on habitat conditions and availability of materials. Cameron and Rainey (1972) reported all three types of housing for N. lepida. 5.

Fig. 6. Neotomafloridana,Eastern or Alleghenywoodrat. Front view, Riley County,KS. (Photographby D. Postand the MammalImagesLibraryof the American Societyof Mammalogists.)

Description

Use in Research

Wood rats have been used in behavioral and neurological studies (Williams, 1980; Towe and Harrison, 1993). However, researchers have investigated them as a natural source of zoonotic or potentially zoonotic microorganisms. Worth (1950) first identified wood rats as a source of endemic murine typhus (Rickettsia typhus) and Stoenner and Lackman (1957) described natural infection with Brucella neotomae from the desert wood rat N. lepida. Galaviz-Silva and Arredondo Cantu (1992) identified N. micropus as a new reservoir host in Mexico for the causative agent of Chagas' disease, Trypanosoma cruzi, while McHugh et al. (1996) and Kerr et al. (1995) identified N.

262

THOMAS M. DONNELLY AND FRED W. QUIMBY

micropus as a reservoir in Texas for Leishmania mexicana, a causative agent of cutaneous leishmaniasis. The results of recent surveys suggest that arenaviruses are widely distributed in the southern United States and that one or more indigenous arenaviruses are associated with Neotoma spp. in North America (Kosoy et al., 1996). Bushy-tailed wood rats and dusky-footed wood rats have been identified as mammalian hosts for plague (Yersinia pestis) in British Columbia and California (Lewis, 1989; Mian et al., 1995). N. fuscipes is an important mammal reservoir of Lyme disease (Borrelia burgdorferi) in California and Oregon (Burkot et al., 1999) and of human granulocytic ehrlichiosis (Ehrlichia phagocytophila) in certain areas of northern California (Nicholson et al., 1999). Although Ixodes spp. have been identified as tick vectors of Lyme disease, the tick species involved in the transmission of the human granulocytic ehrlichiosis agent remains unknown. Sin nombre virus (hantavirus) has been identified in four species of Neotoma from Arizona and Utah (Dearing et al., 1998). Other Bunyaviridae (Jamestown Canyon virus and Morro Bay virus) were isolated from the dusky-footed wood rat in California (Fulhorst et al., 1996b). The results of recent surveys suggest that arenaviruses are widely distributed in the southern United States and that one or more indigeneous arenaviruses are associated with Neotoma spp. in North America (Kosoy et al., 1996). Whitewater Arroyo virus is the first arenavirus to be characterized and isolated from a white-throated wood rat (Fulhorst et al., 1996a). An antihemorrhagic factor in the serum of N. micropus against western rattlesnake venom has been identified and found similar to that in hispid cotton rat (Sigmodon hispidus) and in opossum (Didelphis virginiana) sera (Garcia and Perez, 1984).

B.

dependent (Clarke, 1975). Howell (1926) has made detailed anatomical descriptions of Neotoma.

C.

Researchers have maintained colonies of Eastern wood rats successfully in captivity (Dewsbury, 1974b; Kinsey, 1976; Knoch, 1968; Worth, 1950). Worth (1950) found N. floridana easy to handle, but Dewsbury (1974b) used metal mesh gloves. The diet of wood rats in the wild consists of roots, stems, leaves, seeds, and some invertebrates (Birney and Twomey, 1970; Chess and Chew, 1971; Harriman, 1974). They do not drink much water, but during the dry season they will consume fleshy stems of cacti or other plants that are well filled with water. In captivity, wood rats can be successfully maintained on standard laboratory rat diets (Williams, 1980). Neotoma spp. are neat, sanitary animals and make good pets if they can overcome their extreme timidity (Nowak, 1999). Neotoma spp. are generally solitary; rarely does more than one adult occupy a den, even when home ranges overlap. In a colony of captive Eastern wood rats, a tyrannical social organization developed in which one animal killed or wounded all others (Kinsey, 1976). In field and laboratory studies, Escherich (1981) found that male N. cinera fought ferociously for harems of one to three females. However, Williams (1980) reported that fighting in captivity is uncommon, even in overcrowded or multigenerational cages, and the males do not attack pups. Blood samples can be obtained in the field by femoral vein puncture in bushy-tailed wood rats that have been injected intramuscularly with ketamine hydrochloride (Frase and Van Vuren, 1989). Dosages ranged from 30 to 110 mg/kg.

Biology

Wood rats are nocturnal and active throughout the year. Wood rats generally do not have large litters. However, they have long or all-year breeding cycles. In the southern part of the range, female Neotoma can breed any time of the year. In the north, females begin breeding in December or January and continue producing litters until August to October. Wild N. floridana produce only two or occasionally three litters annually, and litter size ranges from 2 to 4 young. Williams (1980) reported a similar breeding profile for wood rats in captivity. The gestation period of Neotoma spp. varies from 30 to 40 days (Olsen, 1968). The mothers wean the young in about 4 weeks and the pups reach sexual maturity at 7 - 8 months. The incisor teeth of nursing pups are splayed out for the first 2 weeks of life, and the mother's nipple fits into the gap (Hamilton, 1953). The young are then dragged around attached to the mother's teat. Eastern wood rats possess a ventral marking gland that is androgen-

Husbandry

D. 1.

Diseases

Infectious Diseases

Wild-caught wood rats are infected with numerous ectoparasites, such as ticks and fleas, and oocysts of host-specific Eimeria spp. (Durden et al., i997; Lang, 1996; Marchiondo and Upton, 1987; Straneva and Gallati, 1980; Wheat and Ernst, 1974). They are also important reservoirs of human disease (see Uses in Research), and suitable precautions must be taken when handling wild-caught animals. Laboratory colonies of wood rats should be screened serologically and treated where possible. Nonzoonotic diseases include blood-borne flagellate protozoa. Trypanosoma neotomae is found in dusky-footed wood rats (Wood, 1936). The insect vector for this parasite is the wood rat flea. Upton et al. (1989) described T. kansasensis from 3 of 23 (13 %) Eastern wood rats collected from Kansas. All flagellates found in the blood of wood rats were trypomastigotes

263

7. BIOLOGY AND DISEASES OF OTHER RODENTS and larger than T. neotomae in overall dimensions. Stoenner and Lackman (1957) described natural infection with Brucella neotomae from the desert wood rat N. lepida. The filarial worm Dunnifilaria meningica has been described in N. micropus trapped in Mexico (Gutierrez-Pena, 1987). The small adult worms are found in the subarachnoid spaces along the cerebellum and the medulla oblongata. Female worms are approximately 50 mm long, males about 25 mm. Short, sheathed microfilariae that do not show periodicity are found in the peripheral blood. Echinococcus multilocularis was found in a bushy-tailed wood rat in Wyoming (Kritsky et al., 1977). This report represents a new host for the cestode. The larva had undergone active multilocular vesiculation in the liver with minimal host-tissue reaction. Protoscolices and calcareous corpuscles were absent. Naturally acquired rabies was detected in an Eastern wood rat by fluorescent antibody testing and mouse inoculation (Dowda et al., 1981). 2.

Fig. 8. Onychomysleucogaster, Northern grasshopper mouse. Side view, Portal, Chochise,AZ. (Photographby R. B. Forbes and the MammalImagesLibrary of the American Society of Mammalogists.)

Neoplastic Diseases

Montali (1980) described a uterine adenocarcinoma in a 48month-old female Eastern wood rat housed at the National Zoological Park in Washington, D.C.

A. 1.

VII.

G R A S S H O P P E R MICE: Onychomys

Onychomys torridus (Fig. 7) and O. leucogaster (Fig. 8) are commonly studied animals.

Introduction

Taxonomy

The genus Onychomys contains three species of animals commonly called grasshopper mice. Two of the three species, O. leucogaster and O. torridus, have been studied more extensively in the laboratory. 2.

Description

The head and body length is 9 0 - 1 3 0 mm, tail length is 3 0 60 mm, and weight is 3 0 - 6 0 gm. The dorsal coat of O. torridus is gray and of O. leucogaster, brown. Both species have white ventral fur and distal tail tips (McCarty, 1975, 1978). 3.

Distribution

Onychomys torridus is found in the southwestern United States and northern Mexico; O. leucogaster is found from eastern Washington and southern Manitoba to extreme northern Mexico (McCarty, 1978). 4.

Fig. 7. Onychomystorridus, Southern grasshopper mouse. Oblique front view, Portal, Cochise, AZ. (Photographby R. B. Forbes and the Mammal Images Library of the American Societyof Mammalogists.)

Habitat

Grasshopper mice inhabit short-grass prairies and desert scrub. They live in any shelter they can find at ground level, although they are good climbers. The nest may be constructed in a burrow taken over from another rodent. Like that of most predators, the population density of grasshopper mice is low

THOMAS M. DONNELLYAND FRED W. QUIMBY

264 (average of 1.8 per hectare), although male-female pairs may associate all year. Their home range is well defined and covers 2 - 3 hectares. Captive individuals of the same sex are very aggressive to one another, often fighting to the death (McCarty, 1975, 1978).

5.

Use in Research

The aggressive nature of grasshopper mice has led to their use in behavior studies. These have included studies on predatory behavior (McCarty et al., 1976); sex differences in behavior, activity, and discrimination learning (Kemble and Enger, 1984); and drug effects on aggression (Cole and Wolf, 1970). Grasshopper mice have also been used to investigate Lyme disease (Borrelia burgdorferi) transmission via urine (Czub et al., 1992) and the effect of photoperiod and melatonin on seasonal gonadal cycles (Frost and Zucker, 1983). In recent years grasshopper mice have been used to examine comparative antibody formation (Lochmiller et al., 1991), cancer induction (Taylor et al., 1993), and population dynamics using DNA and mitochondrial analysis (Riddle et al., 1993; Sullivan, 1996; Sullivan et al., 1996). Grasshopper mice have been widely used in research investigations of reproduction (Dewsbury 1974a, 1984).

B.

Biology

Both O. torridus and O. leucogaster are nocturnal and active throughout the year. They are largely carnivorous. Grasshopper mice can breed throughout the year, but most reproductive activity in the wild occurs from May to September. Females have a 5 to 7 day estrous cycle, and gestation lasts 26-37 days (Horner, 1968; Pinter, 1970; Taylor, 1968). In the wild they produce several litters per year, although in the laboratory they have been reported to produce up to 12 litters per year (Pinter, 1970). The litter size ranges from 1 to 6, averaging 3.6 in O. leucogaster and 2.6 in O. torridus (McCarty, 1978). The young are weaned at 3 weeks, and sexual maturity is reached between 2 and 5 months in O. leucogaster and as early as 6 weeks in O. torridus. The copulatory behavior of southern grasshopper mice has been described (Dewsbury and Jansen, 1972). The midventral sebaceous gland is larger in the male than in the female; castration causes an involution of the gland in both sexes. The secretions of the midventral gland are probably used for communications such as territorial marking, advertising of gonadal status, or pup identification (Pinter, 1985). In captivity the northern grasshopper mouse (like a chinchilla) requires dust baths to keep its coat from becoming too oily (Fine et al., 1986). Swindle et al. (1985) determined normal hematologic values from a colony of healthy adult O. torridus.

C.

Husbandry

Grasshopper mice eat plant material occasionally in the wild, but the main diet consists of grasshoppers, beetles, and small vertebrates, including other rodents. Onychomys torridus prey extensively on scorpions; however, in the laboratory they are reported to survive well eating fresh mouse carcasses supplemented with seeds and water ad libitum (Fine et al., 1986). Egoscue (1960) successfully maintained and bred a laboratory colony of northern grasshopper mice. The animals were fed commercial mouse ration ad libitum that was periodically supplemented with a grain mixture and canned dog food. McCarty (1978) successfully maintained O. leucogaster in outdoor pens. Matsuzaki et al. (1994) established in Japan a breeding colony of northern grasshopper mice captured in New Mexico. The rate of pregnancy for this colony was 75% when they caged males and females together for more than 30 days, and 4% when breeding pairs were caged from 1 to 7 days. Both cases were of monogamous mating. The mean litter size was 3.5 +_1.2, with a range of 1 to 6. The rate of weaning was 78.8%. The mean gestation period was 27.4 _ 2.0 days, with a range of 25 to 31. Matsuzaki et al. found it was possible to breed grasshopper mice year-round in a rearing room with fixed temperature and humidity. Southern grasshopper mice can be successfully crossspecies fostered on white-footed mice (Peromyscus leucopus) (McCarty and Southwick, 1977a,b). Harriman (1976) determined taste preferences, as measured in 48 hr, Richter-type drinking tests (test solution opposite distilled water) for northern grasshopper mice. The animals showed strong drinking preferences for-all concentrations of sugars above 0.05-0.10 M. The grasshopper mice also showed preferences for hypotonic concentrations of saline; other salts and both acids, however, were indifferently preferred at low concentrations and were rejected at the higher concentrations. Taste preferences by grasshopper mice for these chemicals were similar to those exhibited by Mongolian gerbils tested with the same items. Grasshopper mice do not hear low frequencies as well as do other desert rodents such as kangaroo rats and gerbils (Heffner and Heffner, 1985). Although grasshopper mice are aggressive predators in the wild, it is claimed that they become quite gentle in captivity and make fascinating pets (Nowak, 1999).

D.

Diseases

1. Infectious Diseases

Swindle et al. (1985) evaluated a captive colony of southern grasshopper mice for infectious diseases and parasites. They found no infectious diseases, but tropical rat mites (Ornithonyssus bacoti) parasitized the colony twice.

265

7. BIOLOGY AND DISEASES OF OTHER RODENTS

a.

Bacterial Infections

Thomas et al. (1988) challenged laboratory-born progeny from two geographically distant populations of northern grasshopper mice with Yersinia pestis to determine their relative susceptibilities to plague. One of the O. leucogaster populations was associated with a known epizootic focus of the disease and was nearly 2000 times more resistant to mortality than were members of another population from an area historically free of plague. In another study, Thomas et al. (1989) fed a mouse (Mus musculus) inoculated with Y pestis to 20 laboratory-reared O. leucogaster from a parental population naturally exposed to plague. Three of the 20 O. leucogaster died, 4 survived with antibody titers against Y pestis, and 13 survived with no titer against Y pestis. In contrast, when 20 O. leucogaster from a plague-naive parental population were fed infected prey, 7 died and 13 survived with no antibody titer against Y pestis. The resuits suggested that the carnivorous behavior of O. leucogaster appears to promote strong selection for resistance to plague in areas where they are naturally exposed. Grasshopper mice (and white-footed mice, Peromyscus leucopus) experimentally infected and naturally exposed to the Lyme disease spirochete, Borrelia burgdorferi, develop cystitis (Czub et al., 1992). The pathologic changes observed in the urinary bladder of infected mice include the presence of lymphoid aggregates; vascular changes, including an increase in the number of vessels and thickening of the vessel walls; and perivascular infiltrates. Grasshopper mice are susceptible to Coxiella burnetii although spontaneous infections have not been described under laboratory conditions (Wallach and Boever, 1983).

b.

Isolates recovered from O. leucogaster and O. torridus were inoculated into O. leucogaster and produced infections with a prepatent period of 7 days and a patent period of 7-23 days. Feral grasshopper mice have numerous parasitic infestations with the flea Monopsyllus exilis (Davis et al., 1981). Nutting et al. (1973) described an unidentified Demodex mite that infected the tongue, esophagus, and oral cavity of O. leucogaster. Hughes and Nutting (1981) named the parasite Demodex leucogasteri and described its biology and host pathogenesis.

d.

Fungal Infections

Grasshopper mice are susceptible to Coccidioides immitis although spontaneous infections have not been described under laboratory conditions (Wallach and Boever, 1983). 2.

Idiopathic Diseases

Convulsive seizures similar to that seen in Mongolian gerbils (Meriones unguiculatus) were observed to occur in 9 male (n = 29) and 23 female (n = 50) southern grasshopper mice maintained in captivity (McCarty and Southwick, 1975). The preweaning parental environment has a significant influence on the prevalence of convulsive seizures (McCarty and Southwick, 1977b). Swindle et al. (1985) observed multiple limb defects, including brachydactyly, syndactyly, and hemimelia, in offspring from a colony of O. torridus in the United States.

Viral Infections

VIII.

WHITE-FOOTED MICE OR DEER MICE:

Keis and Mitchell (1975) found inclusion bodies typical of cytomegalovirus (CMV) in the submandibular and sublingual glands of southern grasshopper mice. Serologic evaluation of wild-caught grasshopper mice suggests they can be infected with St. Louis equine encephalitis virus (Davis et al., 1981).

Peromyscus

A. I.

c.

Parasitic Infections

Pfaffenberger et al. (1985) trapped and examined 92 O. leucogaster in New Mexico for helminths. The authors found the grasshopper mice infected with two species of nematodes (Litomosoides carinii, Mastophorus muris), two species of cestodes (Hymenolepis citelli, and one unknown), and one species of acanthocephalan (Moniliformis clarki). All infections represent new host and distribution records. Eimeria onychomysis is a coccidian parasite infecting O. leucogaster (Upton et al., 1992). Hnida et al. (1998) described a new species of Eimeria infecting three species of Onychomys (0. arenicola, O. leucogaster, and O. torridus) captured in New Mexico and Arizona. Six of 59 animals (10%) were infected.

Introduction

Description

The genus Peromyscus contains 7 subgenera and 60 species. The 2 species most commonly used in research are P. maniculatus, the prairie deer mouse (Fig. 9), and P. leucopus, the white-footed mouse (Fig. 10). Peromyscus maniculatus ranges from southeastern Alaska throughout Canada and the United States to Mexico; however, it is not found regularly in moist environments (Baker, 1968). In New York, Pennsylvania, and Michigan two morphological and behavioral forms of P. maniculatus coexist but occupy different ecologic niches (Hamilton and Whitaker, 1979). In the wild, P. maniculatus builds nests of leaves and lives with a population density of 1 to 25 individuals per hectare. It is considered a social animal. Peromyscus leucopus lives in population densities

266

THOMAS M. DONNELLY AND FRED W. QUIMBY

trypanosomiasis, and tularemia (see recent references) (Anosa and Kaneko, 1984). 3.

Fig. 9. Peromyscusmaniculatus (subspecies rufinus), Prairie deer mouse. Lateral view, Socorro County,NM. (Photographby R. B. Forbes and the Mammal ImagesLibraryof the American Societyof Mammalogists.)

of 5 to 39 per hectare with males and females pair bonding and females excluding other females from their home ranges (0.1 hectare).

Sources

Colonies of P. leucopus and P. maniculatus are maintained by Dr. Wallace Dawson (Peromyscus Stock Center, Department of Biology, University of South Carolina, Columbia SC 29208; phone: 803-777-3107). Additionally, P. leucopus is maintained by Dr. Richard Hill (Department of Zoology, Michigan State University, East Lansing M148824; phone: 517-353-4603). Additional colonies of P. maniculatus are maintained by Dr. Donald Dewsbury (Department of Psychology, University of Florida, Gainesville FL 32611; phone: 904-392-0596; e-mail: [email protected]) and by Dr. Muriel Davisson (Jackson Laboratory, 600 Main Street, Bar Harbor ME 04609-1500; phone: 800-422-MICE; e-mail: [email protected]). B.

2.

Biology

Use in Research

Animals vary in color from nearly black to white, although most are dark brown to sandy with white or nearly white underparts. Adults molt once a year. Deer mice have large, haired ears and a long tail. They are active throughout the year and generally nocturnal. Peromyscus maniculatus may live 8 years under laboratory conditions (Nowak and Paradisco, 1983). Peromyscus leucopus is primarily nocturnal but may be active throughout the day. The species conserves water through urine volume reduction. Reproduction, thermoregulation, molting, and nest building all influence melatonin (Hamilton and Whitaker, 1979; Lackey et al., 1985). The dental formula is I 1/1, C 0/0, PM 2/1, M 3/3, and molars are rooted so they cease growing in adults. While Peromyscus does have a stomach with two compartments, the separation of cardiac from pyloric portions is less distinct than that of Old World rodents. Data for hematology, metabolic rates, and food and water consumption have been published (Wallach and Boever, 1983; Lackey et al., 1985). Nutrition has not been thoroughly studied. In the wild their diet consists of seeds, nuts, berries, insects, small invertebrates, and carrion. Under laboratory conditions both P. maniculatus and P. leucopus thrive on commercially available pelleted rodent feed (Dewsbury, 1974a; Dewsbury et al., 1977). The natural breeding season for P. maniculatus is March to October in the northern portions of its range. Peromyscus leucopus is also a seasonal breeder in the northern range but breeds all year in Mexico. Both species breed year-round under laboratory conditions. Peromyscus maniculatus females are polyestrous and produce 3 or 4 litters per year (in the laboratory they may have as many as 14 litters per year). Peromyscus leucopus Fig. 10. Peromyscus leucopus (subspecies tornillo), White-footed deer mouse. Obliquefrontview,BernalilloCounty,NM. (Photographby R. B. Forbes females have 6-day estrous cycles with spontaneous ovulation and the Mammal Images Library of the American Society of Mammalogists.) and a postpartum estrus. In both species litter size averages 5,

Both P. maniculatus and P. leucopus adapt readily to the laboratory environment and have been used extensively for studies in genetics, physiology, aging, cataracts, and behavior (Southwick, 1964; Dewsbury, 1974a, 1984; Horner et al., 1980; Burger and Gochfeld, 1992; Cutler, 1985; Tripathi et al., 1991). Dr. George Smith inbred both species for studies on aging (Fine et al., 1986). Renewed interest in the species has centered around their susceptibility to a variety of zoonotic diseases, including sin nombre and other hantaviruses, Lyme disease, vesicular stomatitis virus, granulocytic ehrlichia, babesiosis,

267

7. BIOLOGYAND DISEASES OF OTHER RODENTS and the gestation period is 22-23 days in length (Drickamer and Vestal, 1973). The presence of males does not accelerate the onset of puberty nor induce estrous cycle synchrony in females; however, it is critical for cycle regularity (Yasukawa et al., 1978). When males and females are housed together as preweanlings, they show delayed reproduction as adults (Hill, 1974). As with most rodents, males have twice the distance between external genitalia and anus as females, which can be used to easily sex newborns. Some physiologic and reproductive data are summarized in Table III.

C.

Husbandry

Both P. maniculatus and P. leucopus adapt readily to plastic solid-bottom cages, which are especially well suited for caging breeding pairs (Brand and Ryckman, 1968; Dewsbury, 1975). Peromyscus leucopus self-selected a higher room temperature (32.4 ~C) than did laboratory mice (Lackey et al., 1985). Cages should be equipped with bedding (wood shavings) and separate nest-building material (Dewsbury 1974a, 1975). They are maintained on 16 hr a day of light, and although they move more rapidly than other domesticated rodents, they are easy to handle.

D.

et al., 1986; Clark, 1984). Although many of these diseases are generally transmitted via an arthropod vector, e.g., Lyme disease, borreliosis, babesiosis, Rocky Mountain spotted fever, and equine encephalitis, in at least one instance Lyme disease was transmitted from infected to naive Peromyscus by direct contact (Burgess et al., 1986). In addition, many ectoparasites, including mites, fleas, and lice, have been observed on feral animals. The endoparasites Aspicularis, Syphacia, Capillaria, Nippostrongylus, Nematospiroides, Trichuris, Mastophorus, Ricturlaria coloradensis, and Acanthocephala clarki have been observed as well. Strict quarantine must be practiced to avoid introduction of these agents into the laboratory. Ectoparasites have been treated effectively with 5% methyl carbamate dust (Clark, 1984). Although adiaspiromycosis may be seen as an incidental finding, with large pulmonary spores in the pulmonary parenchyma with little or no granulomatous reaction, few spontaneous diseases have been observed under laboratory conditions. Tyzzer's disease, with its typical pathologic lesions in the intestine and liver associated with high mortality, was observed following a period of fluctuating temperatures. In addition, ringtail has been associated with low humidity (Fine et al., 1986). While they seroconvert on exposure, Peromyscus do not develop clinical disease with mouse hepatitis virus (Silverman et al., 1982).

Diseases

When Peromyscus are brought into an animal facility from the wild, special precautions must be taken to avoid introduction of several zoonotic diseases, including leptospirosis, toxoplasmosis, Hymenolepis spp., Rocky Mountain spotted fever, chlamydiosis, Q-fever, Western and Venezuelan equine encephalitis, sylvantic plague, coccidioidomycosis, and hantaviruses (Davis et al., 1981; Wallach and Boever, 1983; Fine

IX.

RICE RATS: Oryzomys

Oryzomys palustris is the most commonly studied species (Fig. 11).

Table Ill Normative Data for Peromyscus

Parameter Adult weight (gm) Male Female Puberty (days) Male Female Estrous cycle (days) Gestation (days) Birth weights (gms) Eyes open (days) Weaned (days)

P. leucopus

P. maniculatus

22 22

19- 21 19-21

42-48 42-48 4-5 22-24 1.5-2.4 12-15 21-28

35-37 35-37 4-5 23 1.3-2.2 12-16 21-28

Fig. 11. Oryzomyscapito, Rice rat. Side view, Parque Nacional Guatapo, Miranda, Venezuela. (Photographby P. V. Augustand the MammalImagesLibrary of the American Societyof Mammalogists.)

268

THOMAS M. DONNELLYAND FRED W. QUIMBY A.

Introduction

1. Taxonomy

Within the genus Oryzomys are 5 subgenera and 50 species. Oryzomys palustris is the best-known species and most widely studied. Multiple chromosomal polymorphisms within single, natural populations, more than have previously been reported in a mammal, have made many aspects of the systematics of this genus undetermined (Koop et al., 1983; Maia and Hulak, 1981). Polymorphic variation seems stable within a population and is not the result of hybridization, human disturbance, or nonspecific mutagenic agents. 2. Description

Head and body length is 100-200 mm, tail length is 75-250 mm, and the weight is usually 4 0 - 8 0 gm (Wolfe, 1982). Williams (1980) states that Oryzomys may range from 25 to 150 gm. The upperparts are grayish brown to yellow brown, mixed with black; the sides are paler, with less black; the underparts are white to pale buff; and the tail varies from brownish above and white below to uniformly dusky. Oryzomyspalustris is mouse-like; the pelage is coarse but not bristly or spiny. The tail is usually long, with annulations showing through the sparse hairs (Wolfe, 1982). Rice rats may be confused with cotton rats (Sigmodon), but the latter have longer, grizzled fur and a shorter, stouter tail.

chromosome polymorphism, characterized by a varying diploid number of 50, 49, 48, and 46, was found. All specimens showed a chromosome arm number of 56. G-banding patterns in somatic cells allowed identification of the chromosome pairs (2, 3, 5, and 7) involved in centric fusion (Maia and Hulak, 1981)

C.

Husbandry

Several papers describe the care in captivity and reproduction of different species of Oryzomys. Graeff Teixeira et al. (1998) described care in captivity and recommended providing a hollow brick to use as shelter and to reduce stress; Mello (1978) reviewed aspects of the biology, growth, and reproduction of Oryzomys eliurus under laboratory conditions. Villela and Alho (1983) described the postnatal development and growth of O. subflavus in a laboratory setting; and Worth (1967) reviewed the reproduction, development, and behavior of captive O. laticeps. Park and Nowosielski-Slepowron (1975) examined body growth using weight and length parameters following the introduction of the rice rat to laboratory conditions and described the history of breeding the animals over 15 generations. The same authors surveyed skull development of the rice rat (O. palustris natator) covering a period of 21 days to 16 months and involving equal numbers of males and females (n = 108) (Park and Nowosielski-Slepowron, 1976).

3. Use in Research

The rice rat spontaneously develops periodontal disease (Leonard, 1979), and placing the animal on a high-sucrose diet accelerates the periodontitis (Shklair and Rails, 1988). Using this animal model, researchers have investigated the effects of pharmacological drugs, dietary vitamin E, and rotational stress on periodontal bone resorption (Cohen and Meyer, 1993; Leonard et al., 1979). Park (1974a,b,c) described the anatomy and histology of the rice rat's teeth, the growth and development of the maxilla and mandible, and the relationship of these processes to tooth eruption and calcification. Edmonds and Stetson (1993) investigated the effect of photoperiod on reproduction and reproductive development in rice rats. Their work highlighted the role of the pineal gland and its secretion of melatonin on reproduction (Edmonds et al., 1995; Edmonds and Stetson, 1994). Rice rats have recently been used to study metal-pollutant uptake and its relationship to genetic damage in South Carolina (Peles and Barrett, 1997). B.

Biology

Eighty-four specimens of Oryzomys subflavus, collected in the State of Pernambuco, Brazil, were studied. A Robertsonian

D.

Diseases

The spiny rat louse, Polyplax spinulosa, was collected from a wild rice rat in Tennessee (Durden, 1988). This sucking louse is typically parasitic on domestic rats, and the author commented that as most sucking lice are normally host-specific, such crossfamilial host infestation is noteworthy. The rice rat is the predominant reservoir host for Bayou virus, a cause of hantavirus pulmonary syndrome (Torrez Martinez et al., 1998). Webster (1987) reported a rice rat found in North Carolina with vertebral column deformity. Webster believed the kyphosis was probably inherited.

X.

CANE MICE: Zygodontomys

Researchers have sometimes described Zygodontomys brevicauda (Fig. 12) as Z. microtinus in the literature. Older references refer to unrelated species (e.g., Z. lasiurus and Z. pixuna) that are properly called the genus Bolomys (e.g., lasiurus, lenguarum, and pixuna) (Voss, 1991).

269

7. BIOLOGY AND DISEASES OF OTHER RODENTS

less difficult (Voss et al., 1992). Virologists have used laboratory colonies of cane mice in studies of arboviruses, such as yellow fever virus (Bates and Weir, 1944); Nariva virus, a rodent paramyxovirus (Beare, 1975; Tikasingh et al., 1966); Venezuelan equine encephalomyelitis (Downs et al., 1962); and Cocal virus, a rhabdovirus (Jonkers et al., 1964). Zygodontomys brevicauda is a natural host of Guanarito virus, the cause of Venezuelan hemorrhagic fever (VHF) (Fulhorst et al., 1999). Fever, malaise, and a sore throat initially characterize this disease in humans. Abdominal pain, diarrhea, a variety of hemorrhagic manifestations, and convulsions usually follow. Since a VHF emergence in 1989 up until 1997, Venezuelan authorities have reported 220 cases with a fatality rate of 33% (Salas et al., 1998)

Fig. 12. Zygodontomys brevicauda, Cane mouse. Side view, captive, The Rockefeller University, NY. (Photograph by T. M. Donnelly.)

A.

Introduction

1. Description

Zygodontomys brevicauda is about 100 gm or less in body weight with a grizzled brown pelage and a tail about threefourths the length of its head and body. 2.

Distribution

Cane mice are widely distributed in the tropical lowlands of eastern Central America, northern South America, and adjacent continental islands. 3.

Habitat

Mainland populations of Zygodontomys typically inhabit savannas or weedy areas around human settlements where the species is sometimes an agricultural pest. Cane mice are nocturnal, omnivorous, and terrestrial, seldom or never climbing trees (Voss, 1991). 4.

Use in Research

Cane mice are used in comparative studies of mammalian circannual reproductive cycles since this is one of the few tropical species known to lack a reproductive response to photoperiod (Bronson and Heideman, 1992; Heideman and Bronson, 1990). Zygodontomys brevicauda is also a good animal model for evolutionary quantitative genetics. Its short generation time, high diploid chromosome numbers, and the availability of F1 and F2 hybrids from crosses between geographically isolated populations make experimental analyses of evolutionary divergence

B.

Biology

Sexual maturation of cane mice is rapid, and litter sizes are moderately large in comparison with those of other New World muroids such as Peromyscus. The work of Voss et al. (1992) and Aguilera (1985) are in close accord. Females become sexually mature at 21-26 days and males at 4 0 - 6 0 days. Gestation lasts 25 days, with litters usually consisting of 4 or 5 young but ranging from 1 to 11. Larger litters consist of smaller neonates, and the young open their eyes at 6 - 8 days. By day 16 females wean their young. Ovulation in females is spontaneous. Pairing females with males in cages divided by wire partitions that permit limited physical contact, but not copulation, does not accelerate the occurrence of estrus or ovulation. Estrous cycling does not differ between single-housed females and females housed with males in divided cages. Copulatory plugs are formed when animals mate.

C.

Husbandry

Voss et al. (1992) maintained their colony according to recommendations in the "Guide" for temperature and humidity. However, cages were cleaned only once every 1 or 2 weeks because cane mice urinate and defecate sparingly compared with laboratory mice (Mus musculus.) Food consisted of commercial mouse chow, supplemented by dry cat food and chopped frozen vegetables. Lactating females were provided double rations. Cane mice are strong and agile and jump abruptly with slight provocation (Voss et aL, 1992; Worth, 1967). Cages should be placed at the bottom of a deep (at least 2.5 feet) box before removing the wire lid to prevent escape. Voss et aL (1992) found that animals of all ages in their colony were aggressive and leather gloves were necessary for handling. Females became aggressive shortly before parturition and males were removed to prevent injuries. Males rarely injured females, and pairing younger females with older males reduced female aggression.

270

THOMAS M. DONNELLYAND FRED W. QUIMBY

Williams (1980) reported that researchers maintaining 2 different colonies of Zygodontomys found the animals nonaggressive to handlers.

D. 1.

Diseases

Infectious Diseases

Voss et al. (1992) were unable to isolate known bacterial pathogens from 7 of 8 animals randomly selected for microbiological screening. Yersinia pseudotuberculosis was isolated from the nasal cavity of one animal. The major concern with cane mice is their zoonotic potential as the natural host of Guanarito virus (family Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever (Fulhorst et al., 1999). Animals developed chronic viremic infections characterized by persistent shedding of infectious virus in oropharyngeal secretions and urine. Pinworms (Syphacia sp.) and the trichomonad intraduodenal protozoan Hexamita were found in the colony of Voss et al. (1992). Fifty-five species of arthropod ectoparasites are known to infest Z. brevicauda in nature, but only the tropical rat mite (Ornithonyssus bacoti) appears to persist in laboratory-bred, wild-derived animals (Voss et al., 1992).

Xl.

mals. However, S. hispidus is used far more often than S. fulviventer. The term cotton rat as used in the text refers to S. hispidus.

A. 1.

Introduction

Description

Sigmodon hispidus is a robust, stocky rodent that weighs 8 0 130 gm with a head-to-body length of 125-200 mm. The tail length is 75-166 mm (Cameron and Spencer, 1981). The fur is coarse, dark brown to black interspersed with yellow or light tan hairs over the back and sides, while the underparts are usually pale to dark gray (Cameron and Spencer, 1981). The ears are small, and the three central digits of each paw are larger than the the other two. All species of cotton rats are similar in appearance although there is wide variation in chromosome number among species (Zimmerman, 1970). Voss (1992) has revised the South American species of Sigmodon, but further a-taxonomic studies are needed. 2.

Distribution

Sigmodon hispidus occurs in the southern United States to northern Venezuela and northwestern Peru. Sigmodonfulviventer occurs in southeastern Arizona and central New Mexico to central Mexico (Nowak, 1999).

C O T T O N RATS" Sigmodon 3.

There are eight species of cotton rats. Of these, Sigmodon hispidus (Fig. 13) and S. fulviventer are used as laboratory ani-

Cotton rats prefer grassy and shrubby areas (Nowak, 1999). They are the most abundant rodents in the southeastern United States, Mexico, and Central America. 4.

Fig. 13. Sigmodonhispidus(subspeciesberlandieri),Hispidcottonrat. Side view, LunaCounty,NM. (Photographby R. B. Forbesand the MammalImages Library of the American Societyof Mammalogists.)

Habitat

Use in Research

Cotton rats are an important animal model for viral respiratory tract disease caused by paramyxoviruses. Respiratory syncytial virus, a leading cause of respiratory tract infections in human infants (Piazza et al., 1995; Prince et al., 1999); parainfluenza virus 3, the second leading cause of pediatric respiratory disease (Ottolini et al., 1996; Prince and Porter, 1996); and measles virus (Niewiesk, 1999; Niewiesk et al., 1997) replicate well in cotton rats. Cotton rats can also be infected with human adenovirus. Adenovirus pneumonia in infected animals is similar to that in humans (Prince et al., 1993), and ocular adenovirus infection in cotton rats is the only animal model of epidemic keratoconjunctivitis (Tsai et al., 1992). The susceptibility of cotton rats to human adenovirus replication has lead to their use in toxicity studies of adenoviral mediated gene therapy. In this research replicate-defective adenoviruses are used as vectors to deliver foreign genes. Researchers

271

7. BIOLOGY AND DISEASES OF OTHER RODENTS

have used cotton rats to investigate gene therapy for cystic fibrosis (Yei et al., 1994), erythropoiesis stimulation (Setoguchi et al., 1994), gene transfer to the ocular surface epithelium (Tsubota et al., 1998), cancer treatment of certain human tumors such as cervical carcinoma (Bischoff et al., 1996), and malignant tumors of the central nervous system (Shine et al., 1997). Many viral, protozoan, metazoan, and bacterial pathogens can be transmitted to cotton rats. Rickettsia tsutsumagushi, the causative agent of scrub typhus, has been studied extensively in the cotton rat (Gage et al., 1990; Ignatovich et al., 1983; Ridgway et al., 1986). Cotton rats are naturally infected with the filarial nematode Litmosoides carinii and are popular animal models to screen novel antifilarial drugs (Chatterjee et al., 1989; Kershaw and Storey, 1976). Leishmania donovani and Echinococcus multilocularis have been studied in cotton rats (Azazy et al., 1994; Kroeze and Tanner, 1985). Researchers have investigated the role of the wild cotton rat as a natural reservoir of Lyme disease, the hantavirus Black Creek Canal virus, and Venezuelan equine encephalitis (Oliver et al., 1995; Rollin et al., 1995; Zarate and Scherer, 1968). Elangbam et al. (1989) have proposed the cotton rat as a biomonitor to study the impact of environmental pollutants on exposed wildlife.

5.

1996). Itoh et al. (1989) have characterized the gastrointestinal flora of cotton rats.

C.

Husbandry

Faith et al. (1997) reviewed the husbandry of cotton rats used in biomedical research. They found that cotton rats will attempt to bite and escape when picked up. Leather garden gloves simplify handling. Cotton rats can be kept housed in standard polycarbonate cages with hardwood-chip or corncob bedding. Animal caretakers should change cages twice a week. Kawase and Satoh (1978) described breeding cotton rats in the laboratory. Faith et al. (1997) noted that reproductive performance is poor when few animals are kept in the room, but good when it is fully occupied. Cotton rats are omnivorous and feed on vegetation, insects, and other small animals. They will eat eggs and chicks of bobtail quail and become pests by eating sugarcane and sweet potatoes (Nowak, 1999). Nutritional requirements for cotton rats are similar to those of laboratory rats (Rattus norvegicus), and standard rodent diets appear suitable for maintenance and reproduction (Cameron and Eshelman, 1996).

Sources

Cotton rats may be obtained from Adam Goedde (Harlan Sprague Dawley, Inc., PO Box 29176, 298 South Carroll Road, Indianapolis IN 46229-0176; phone: 317-894-7521).

B.

Biology

Cotton rats are active night and day. However, their activity patterns may change in the wild or in the laboratory, and nocturnal animals may become diurnal (Johnston and Zucker, 1983). A relative dominance system in which males dominate females and adults dominate juveniles characterizes cotton rat behavior. Cotton rats are solitary animals, with the only prolonged social contact occurring between males and females for reproduction. Aggressive behavior is common, and caged animals will inflict severe bite wounds on cagemates and sometimes fight to the death (Faith et al., 1997). Gestation in cotton rats is 27 days, and litter size is 1-12 young with an average of 5-7. The young weigh 6.5-8.0 gm and open their eyes at 24 hr. Although they may be weaned at 5 days, young cotton rats occasionally remain with their mother for 7 or more days. Sexual maturity occurs at 4 0 - 6 0 days (Nowak, 1999). Hematological data have been reported for wild and laboratory cotton rats of different ages and sexes (Dotson et al., 1987; Katahira and Ohwada, 1993; McMurry et al., 1995; Robel et al.,

D.

Diseases

There are few incidents of clinical disease in cotton rats. Most lesions in laboratories result from fighting. 1.

Infectious Diseases

All literature citations describing parasitic disease in the cotton rat are taken from field surveys in the United States or Costa Rica. Parasites have not been described in laboratory-maintained cotton rats. a.

Protozoa

Barnard et al. (1974) isolated Eimeria spp. and Isopora spp. coccidia from the feces of wild cotton rats in Alabama. Castro et al. (1998) identified E. sigmodontis, E. tuskegeensis, E. roperi, and E. webbae in the feces of wild cotton rats in Costa Rica. Elangbam et al. (1993) observed cryptosporidia in 1 of 9 cotton rats from Oklahoma. Infection was confined to the large intestine. Dubey and Sheffield (1988) found protozoan muscle cysts in 3 of 4 cotton rats from Georgia. The protozoan cysts were several centimeters long and not infectious for dogs and cats. Based on morphology, the authors named the parasite Sarcocystis sigmodontis because it differed from all known rodent sarcocysts.

2 72

b.

THOMAS M. DONNELLY AND FRED W. QUIMBY

Nematodes

Boggs et al. (1991) collected 113 adult wild cotton rats (68 male and 45 female) from Oklahoma and recovered five species of gastrointestinal helminths: Longistriata adunca, Syphacia sigmodontis, Strongyloides sp., Protospirura muris, and Raillietina sp. Elangbam et al. (1990) collected 40 cotton rats from central Oklahoma and found Strongyloides sp. in 31 (78%) animals. Rhabditiform or filariform Strongyloides larvae were not demonstrable in intestinal contents and scrapings. Cotton rats infected with Strongyloides sp. were indistinguishable clinically from noninfected hosts. Holliman and Meade (1980) found encapsulated larvae of Trichinella spiralis in wildtrapped cotton rats in Virginia.

c.

Mites/Lice~Ticks

Durden et al. (1993) found eight species of parasitic arthropods on 28 wild cotton rats collected in Florida. The most prevalent parasites were the flea Polygenis gwyni; the American dog tick, Dermacentor variabilis; and the tropical rat mite, Ornithonyssus bacoti. 2.

Metabolic/Nutritional Disease

Iglauer et al. (1993) reported ringtail in a colony of cotton rats in Scandinavia. 3.

Traumatic Injuries

Faith et al. (1997) reported that the main lesions they saw in a laboratory colony in Texas resulted from fighting among animals. Cotton rats should not be picked up by the tail, as they spin when picked up and the tail skin easily degloves. 4.

latrogenic Diseases

Constant and Phillips (1952a) described a calcinosis syndrome in cotton rats consuming a partially purified diet. Lesions were present in the heart, skeletal muscles, liver, adipose tissues, kidneys, and urinary bladder (Constant and Phillips, 1952b). Chandra et al. (1993) observed cystic epithelial calcification in 37 of 60 wild cotton rat urinary bladders. They trapped the rats in Oklahoma near petrochemical-contaminated sites and noncontaminated sites and found that the animals did not differ significantly in serum calcium and phosphorus levels.

were characterized by adenomatous hyperplasia affecting fundic but not antral mucosa. In half the rats the gastric serosal surface was also involved. Faith et al. (1997) in Texas saw no evidence of neoplasia in their colony of cotton rats. 6.

Miscellaneous

a.

Congenital Disorders

Sorden and Watts (1996) reported sporadic exophthalmos and heart failure in 3- to 12-month-old cotton rats in a colony originally derived from 3 male and 4 female littermates. The exophthalmos resulted from orbital venous sinus thrombosis caused by stasis of venous blood secondary to right heart failure. The authors attributed the heart failure to a heritable cardiomyopathy. Cardiac lesions included right ventricular dilatation and unilateral or bilateral atrial thrombosis. Microscopically, there was multifocal cardiac myocyte necrosis, mineralization, and mononuclear inflammatory cell infiltration; cotton rats more than 5 months of age also had foci of interstitial fibrosis and myocyte atrophy. Faith et al. (1997) also reported a similar form of degenerative cardiomyopathy in their colony. However, they observed skeletal muscle lesions concurrent with myocardial degeneration. Affected animals had skeletal muscle degeneration, necrosis, regeneration, chronic inflammation, and mineralization with the formation of multinucleated giant cells. Swensen and Telford (1973) described similar skeletal and myocardial lesions in the experimentally vitamin E-deficient cotton rat.

b.

Age-Related Disorders

Faith et al. (1997) necropsied 18 laboratory-maintained adult cotton rats (9 males and 9 females) to define the morphological profile of their colony. In one-third of the animals they found a condition similar to chronic nephropathy (chronic progressive nephrosis) in laboratory rats (Rattus norvegicus).

XII.

WHITE-TAILED RATS: Mystromys

Mystromys albicaudatus (Fig. 14) is the species principally studied.

A. 5.

Introduction

Neoplastic Diseases

Kawase and Ishikura (1995) described a colony of 258 female and 283 male cotton rats in Japan in which 61 females and 2 males spontaneously developed gastric tubular adenocarcinoma. Lesions developed in rats more than 2 months of age and

1.

Description

The white-tailed rat is the only species in the genus Mystromys. Mystromys is in the subfamily Cricetinae (hamsters), which includes Calomyscus, Phodopus, Cricetus, Cricetulus,

7. BIOLOGYAND DISEASESOF OTHER RODENTS

273 Mystromys albicaudatus is an excellent model of American cutaneous leishmaniasis (Leishmania braziliensis) and has also been used as an experimental host for L. donovani and L. mexicani (McKinney and Hendricks, 1980; Mikhail and Mansour, 1973; Sayles et al., 1981). It has been used for vaccination studies with L. braziliensis (Beacham et al., 1982; Franke et al., 1985). White-tailed rats have been infected experimentally with Crimean-Congo hemorrhagic fever virus (Shepherd et al., 1989). They have also been infected with human Streptococcus spp. to investigate caries development (Larson and Fitzgerald, 1968). Other studies using Mystromys include chemically induced carcinogenicity (Roebuck and Longnecker, 1979; Yamamoto et al., 1972), thermoregulation (Downs and Perrin, 1995), and digestion (Mahida and Perrin, 1994; Perrin, 1987). B.

Fig. 14. Mystromysalbicaudatus, White-tailedrat. (FromLaboratory Animal Medicine, 1984.)

and Mesocricetus (Nowak, 1999). It is thick-bodied, is relatively large (13.6-18.4 cm long), has a long white tail (5-8 cm), and weighs 75-185 gm. The fur is gray-brown and smooth and the belly is white (Clark, 1984). The white-tailed rat has ungrooved, yellow incisor teeth; sharp-tipped claws; and no cheek pouches. In addition, it has a two-compartment stomach and a large, ventral sebaceous gland (Fine et al., 1986). Females have a rudimentary prostate gland (Hall et al., 1967). The ears of M. albicaudatus are erect; the eyes are dark; and the animal is alert, inquisitive, and quick. 2.

Distribution

This animal is found on the grassy flats and dry, sandy areas of South Africa: Cape Province, Natal, Free State, and Swaziland (Skinner and Smithers, 1990). 3.

Habitat

Mystromys albicaudatus lives underground, often in burrows made by other animals, and is nocturnal (Nowak, 1999). It eats seeds and other vegetable matter. 4.

Use in Research

White-tailed rats are used in diabetes mellitus research where they have been demonstrated to spontaneously develop hyperglycemia, polyuria, glycosuria, ketonuria, and degenerative changes in the islets of Langerhans. The disease is more common in males and is not associated with obesity (Clark, 1984; Riley et al., 1975; Little et al., 1982). The animals have been used to study diabetic angiopathy and hepatic mitochondrial function in diabetes (Schmidt et al., 1974, 1980).

1.

Biology

Physiological Characteristics

Although found in a temperate climate, M. albicaudatus has been shown to have thermal characteristics typical of a rodent adapted to a cold temperature regime and consequently has a higher metabolic rate than anticipated (Downs and Perrin, 1995). The growth and development of the stomach, gastric epithelia, and associated microflora have been well documented. The neonatal monogastric stomach with distinct separations into glandular and cornified regions gives way in the infantile period to fornical papillae, which provide microhabitats for colonization by symbiotic anaerobic bacilli. As development continues and ingestion of solid food begins, papillae become colonized by bacilli, which increase in abundance without epithelial damage, suggesting that the bacilli are autochthonous and symbiotic and aid in digestion processes (Maddock and Perrin 1981; 1983; Perrin and Curtis, 1980). The details of chemical reactions mediated by stomach bacteria during ingestion of food have been documented (Perrin and Maddock, 1983, 1985; Perrin and Kokkinn, 1986). Females have two pairs of inguinal mammae, and males have an os penis. 2.

Normal Values

The normal weights of spleen, kidneys, liver, heart, lung, pancreas, brain, and gonads for 53 adult female and 51 adult male white-tailed rats have been published (Becker et al., 1979). Serum chemistry and electrolyte determinations and nonfasted urinalysis results have been reported (Becker and Middleton, 1979; Street and Highman, 1971). Serum chloride and serum glucose levels were greater and serum sodium levels lower for female rats. An unusually high physiological level of urine protein was detected, and it was determined that standard dipstick methods for determining urine protein levels in this species gave artificially high results. Cantrell and Padovan (1987)

THOMAS M. DONNELLYAND FRED W. QUIMBY

274

determined blood values from 90 Mystromys, 3 - 2 4 months of age, to establish normal values and to evaluate the influence of age and gender (see Table II). Males over 6 months of age had higher red blood cell counts, packed cell volumes, and hemoglobin levels than females of the same age. Age and gender did not cause detectable differences in leukocyte numbers in animals over 6 months old. Intraperitoneal pentobarbital anesthesia (3 mg/50 gm) has been recommended (Padovan, 1985). A technique for repeat blood sampling has been described (Stuhlman et al., 1972b). 3.

Biology of Reproduction

2.

Females become sexually mature at 146 days and have a gestation period of 38 days (Hallett and Meester, 1971). Breeding pairs should be established at a young age to avoid aggressiveness among adults, and males are removed at parturition. Females give birth to small litters (3) with high survival to weaning (80%). Neonatal survival can be increased to 95% by selecting as breeders dams that do not cannibalize their young (Fine et al., 1986). Newborns attach firmly to the nipples of the dams and are dragged about for 3 weeks (Hall et al., 1967). The life span of white-tailed rats is 6 years (Dean, 1978).

C.

Husbandry

Wild Mystromys eat seeds, other vegetable matter, and insects (Perrin, 1987). In the laboratory, commercial rodent diet and water are offered ad libitum. White-tailed rats are housed as breeding pairs in solid-bottom rodent cages lined with corncob bedding. Standard environmental conditions for temperature (22~176 humidity (40-70%) and light (12 hr cycles) are satisfactory. Animals are maintained as monogamous lifetime mates (Fine et al., 1986). White-tailed rats should be picked up around the thorax and never by the tail because it is fragile. Perrin described a system of activity monitor for M. albicaudatus (Perrin, 1981). Stuhlman et al. (1972b) used heat-dilated tail veins to obtain repeated blood samplings for glucose measurements in white-tailed rats. Intraperitoneal pentobarbital (3 mg/50 gm) has been used for anesthesia (Padovan, 1985).

D.

munosuppressed weanling M. albicauditus by oral inoculation with Clostridium piliformis spores. Focal necrosis was observed in the tunica muscularis of the intestine, periportal region of the liver, the ventricular myocardium, and the brain stem (Waggie et al., 1986). LaRegina et al. (1978) described a fatal enteric syndrome with high mortality (59%) in adult Mystromys, associated with ingestion of a topically applied antibiotic of bacitracin, neomycin, and polymixin. Clinical signs included anorexia, depression, and rough hair coat. Predominant necropsy findings were hemorrhagic typhlitis and colitis.

Diseases

Metabolic Diseases

Spontaneous diabetes mellitus in the white-tailed rat is common (Hallett and Politzer, 1972; Packer et al., 1970). The disease is more common in males. Hyperglycemia (> 170 mg/dl) is a consistent finding, whereas polyuria, polydipsia, glycosuria, and ketonuria are less commonly found; obesity is not a characteristic (Stuhlman et al., 1972a, 1975, 1974). The primary lesion is in the islets of Langerhans (Goeken et al., 1972). Little et al. (1982) measured glycosylated hemoglobin in normal and diabetic white-tailed rats. The mean glycosylated hemoglobin for nondiabetic Mystromys (n = 321) was 14.8 nmol hydroxymethylfurfural/10 mg hemoglobin (similar to that of nondiabetic humans), and animals with a glycosylated hemoglobin value greater than 19 nmol hydroxymethylfurfural/ 10 mg hemoglobin were characterized as diabetics. In a study of 175 nondiabetic and diabetic white-tailed rats, it was found that glomerulosclerosis is associated with diabetes in this species (Riley et al., 1975). Skeletal muscle capillary basement membrane thickness determined in age- and sex-matched normal and spontaneously diabetic M. albicaudatus indicates that the average basement membrane thickness is 482.6 ___48.7 A compared with 779.0 ___ 319.9 A in diabetic animals (Yesus et al., 1976). 3.

Iatrogenic Diseases

A case report of ringtail in suckling white-tailed rats (< 7 days of age) was described by Stuhlman and Wagner (1971). Tail lesions ranged from slight reddening with minimal discoloration to annulation and swelling. In severely affected animals, necrosis and subsequent autoamputation of part or all of the tail occurred. The relative humidity in the room ranged from 12-38%. When the relative humidity was maintained at 50% no more cases occurred.

1. Infectious Diseases

Clark (1984) observed that little information is available about specific diseases of white-tailed rats. He reported lesions and causes of death in one colony as pneumonitis, ulcerative enteritis, septicemia, gastric ulcer, cataracts, keratitis, scleritis, hepatitis, nephritis, and otitis externa and media. Tyzzer's disease has been induced experimentally in im-

4.

Neoplastic Diseases

Rantanen and Highman (1970) reported spontaneous tumors in a colony of M. albicaudatus. The neoplasms listed were perianal squamous cell carcinoma, adnexal tumor of the skin, osteosarcoma of the scapula, leiomyosarcoma of the uterus, adenocarcinoma of the liver, hepatoma, and pituitary adenoma.

275

7. BIOLOGY AND DISEASES OF OTHER RODENTS

5.

Miscellaneous

a.

Congenital Disorders

Rodrigues et al. (1972) described an inherited condition of partial oculocutaneous albinism with ophthalmic pathology in white-tailed rats. The condition appeared similar to ChediakHigashi syndrome, a phagocyte bactericidal disorder. Prievr et al. (1979) studied tissues from affected M. albicaudatus and found no evidence of cytoplasmic granule enlargement. They concluded that the inherited partially albinic disease is different from the Chediak-Higashi syndrome (Prieur et al., 1979).

and Adams (1972a) found weight to average 50-55 gm in laboratory-raised females and 60 gm in males. The covering of fur on the tail is short near the base and progressively longer toward the tip so that it is slightly bushy. Coloration of upper parts varies from pale, clear yellowish through sandy and gray. The sides of the body are generally lighter than the back. 2.

Wild M. unguiculatus are found in Mongolia, adjacent parts of southern Siberia and northern China, and Manchuria. 3.

XIll.

GERBILS AND JIRDS: Meriones

Gerbils generally studied include Meriones unguiculatus (Mongolian gerbil) (Fig. 15), M. libycus (Libyan jird, red-tailed jird), M. crassus (desert gerbil), M. hurrianae (Indian desert gerbil), and M. vinagradovi (grapevine gerbil). There are 14 species in 4 subgenera. The term gerbil as used in the text refers to the Mongolian gerbil, Meriones unguiculatus. All Mongolian gerbils available for research were derived from 20 pairs trapped in eastern Mongolia in 1935. The animals were taken to the Kitsato Institute, Japan, and later a subcolony was established at Central Laboratories for Experimental Animals, Tokyo. Schwenker imported 11 pairs to the United States from the Tokyo subcolony in 1954 (Marston, 1976). A. 1.

Introduction

Description

Externally, gerbils are quite rat-like. The head and body length is 95-180 mm, and tail length is 100-193 mm. Norris

Fig. 15. Meriones unguiculatus, Mongolian gerbil orjird. Side view, Prague Zoo, Czechoslovakia. (Photograph by M. Andera and the Mammal Images Library of the American Society of Mammalogists.)

Distribution

Habitat

Gerbils inhabit clay and sandy deserts, bush country, and arid steppes. They are terrestrial, and wild M. unguiculatus construct simple burrows in soft soil where they spend most of their time. The tunnels are underground, about 2-3 feet in length and 1.3 inches in diameter. 4.

Use in Research

The gerbil is highly susceptible to cerebral infarction following unilateral ligation of one common carotid artery and is used in studies of the pathogenesis of stroke (Akai et al., 1995; Baskaya et al., 1999; Hall et al., 1991; Pelliccioli et al., 1995; Somova et al., 2000). Gerbils are used as an animal model for the study and treatment of epilepsy (Bartoszyk and Hamer, 1987; Buckmaster et al., 2000; Fisher, 1989; Loskota and Lomax, 1975; Wolf-Dieter et al., 1989). Spontaneous epileptiform seizures mimic those of human idiopathic epilepsy, and both seizure-sensitive and resistant strains have been bred. The gerbil is also used in auditory research (Urquiza et al., 1988). Hessel et al. (1997, 1998) in Germany use deafened gerbils in ontogenetic cochlear implant research The gerbil has been used extensively in behavioral investigations, especially those relating to territoriality. A major monograph describing olfactory communication and territorial behavior in the gerbil afforded a detailed study on the neurological and physiological mechanisms that control these functions (Thiessen and Yahr, 1977). A wide variety of parasitic infections can be transmitted to gerbils. Experimental protozoal infections include Giardia lamblia (Belosevic et al., 1983; Faubert et al., 1983; Wallis and Wallis, 1986) and the cattle piroplasm Babesia divergens (Lewis and Williams, 1979; Lewis et al., 1981; Liddell et al., 1982). Researchers frequently use gerbils to maintain nematode parasites in the laboratory, study the pathogenesis of nematode infections, and investigate anthelmintic drug resistance. Examples of nematodes infecting gerbils are Strongyloides stercoralis (Nolan et al., 1993), Ostertagia circumcincta (Court et al., 1988), Haemonchus contortus (Conder et al., 1991), Nematospiroides dubius (Hannah and Behnke, 1982; Jenkins, 1977), Trichostrongylus colubriformis (Maclean et al., 1987),

276 Heligmosomoides kurilensis (Asakawa, 1987), Capillaria philippinensis (Cross et al., 1978), and Acanthocheilonema viteae (Maki and Weinstein, 1991). Gerbils are excellent animal models for studying diseases caused by filarial nematodes (Klei et al., 1997; Nasarre et al., 1997). They have been used extensively to investigate serious human diseases such as lymphatic filariasis caused by Wuchereria bancrofii (Cross et al., 1981; Zielke, 1979) and Brugia malayi (Deng et al., 1994; Li et al., 1991; Partono and Purnomo, 1987; Trpis, 1994). Wuchereria bancrofii and B. malayi are responsible for 90% and 10%, respectively, of the 90 million infections worldwide in Latin America, sub-Saharan Africa, and Southeast Asia. Researchers also use gerbils to study the filarial parasite Onchocerca volvulus, a major cause of human blindness in equatorial Africa where the parasite infects more than 20 million persons (Bianco et al., 1989). Other filarial nematodes used in gerbils include B. pahangi (Nasarre et al., 1997) and Loa loa, the African eye worm (Mackenzie et al., 1982; Vincent et al., 1979a). Hydatid disease has been investigated in gerbils. Larvae of the tapeworms Echinococcus granulosus and E. multilocularis are found as cysts in the liver and other organs of rodents, ruminants, and humans. The cysts, though slow-growing, are often proliferative and large, and result in fatal infections. E. multilocularis infection is an important zoonotic disease in Asia. Echinococcal infections in gerbils are used to investigate larval parasite proliferation and metastasis (Eckert et al., 1983; Kamiya et al., 1987; Kanazawa et al., 1995; Ohnishi and Kutsumi, 1995). Gerbils are also used as animal models of cysticercosis and infected with taenid tapeworms such as Taenia polyacantha (Fujita et al., 1991) T. crassiceps (Saitoh, 1987), and T. solium (Maravilla et al., 1998). Gerbils are infected with digenetic flukes to study diseases caused by parasitic trematodes, such as schistosomiasis, which affects 200 million persons and kills 250,000 annually. Trematodes experimentally infecting gerbils include Schistosoma japonicum (Yingrui et al., 1983); S. haematobium (Bayssade Dufour et al., 1994); Paragonimus heterotremus, a cause of pulmonary dis tomiasis (Asavisanu et al., 1985); and the avian schistosome Austrobilharzia variglandis, a cause of marine cercarial dermatitis, or "swimmer's itch" (Bacha et al., 1982). The young gerbil is an animal model in which uniformly fatal Rift Valley fever virus-induced encephalitis is produced without significant extraneural lesions (Anderson et al., 1988). Gerbils have been used as a vertebrate host to study the enhancement of arbovirus transmission by concurrent host infection with microfilariae (Vaughan and Turell, 1996).

5. Sources Mongolian gerbils are available from Adam Goedde (see Section XI, A, 5, Cotton Rats); from B&K Universal Ltd. (The Field Station, Grimson, Aldbrough, Hull, N. Humberside, Hull

THOMAS M. DONNELLYAND FRED W. QUIMBY 4QE, UK; (phone:0964-527-555); and from Charles River Laboratories (251 Ballardvale Street, Wilmington MA 01887; (phone:800-522-7287; email: comments @criver.com).

B.

Biology

Gerbils have a large, ventral, abdominal marking gland that is androgen-dependent. It attains greater size in the male and develops at an earlier age (Thiessen and Yahr, 1977). The adrenal cortex produces nearly equal amounts of corticosterone and 19hydroxycorticosterone (Drummond et al., 1988). When the adrenal gland weight is compared with the body weight, the gerbil adrenal gland is approximately 3 times the size of the adrenal in the rat (Cullen et al., 1971). Mays (1969) and Gattermann (1979) reported normal reference ranges for hematological and clinical biochemical parameters in the gerbil (see Table II). General data on organ weights are found in the monograph by Thiessen and Yahr (1977). Norris and Adams (1972b, 1974, 1981, 1982) have published extensively on reproduction in the gerbil. Male gerbils attain sexual maturity by 70-84 days. Vaginal opening in females occurs at 4 0 - 6 0 days, 30 days before sexual maturity occurs. Gerbils tend to pair-bond, and when older females lose their mate, getting them to accept another is often impossible. The gestation period of nonlactating gerbils is 24-26 days, but lactating females always have a prolonged gestation of 27 days. If females are bred in the postpartum period, they delay implantation, and gestation can be as long as 48 days. Mean litter size ranges from 3 to 7. Young gerbils suckle for about 21 days and begin to eat solid foods at 16 days. In general, researchers consider day 25 to be most suitable for weaning.

C.

Husbandry

Gerbils of other species are often used in other countries as research animals. Several papers describe the breeding and care in captivity of these species: Meriones vinagradovi (Ismailov and Ismailov, 1981); M. crassus (Marafie et al., 1978); M. hurrianae (Saibaba et al., 1988); and M. libycus (Wisniewski, 1985). The diet of wild Meriones consists of green vegetation, roots, bulb seeds, cereals, fruits, and insects. Zeman (1967) described a semipurified diet for gerbils, and in the laboratory, gerbils thrive on commercially available pelleted rodent diets. However, because of the fat metabolism of gerbils, they develop high blood cholesterol concentrations on diets containing more than 4% fat (Leach and Holub, 1984; Nicolosi et al., 1981). Gerbils excrete little urine, and fecal pellets are hard and dry. Their cages require less frequent cleaning than those of other laboratory rodents. Gerbils adapt to a wide range of ambient temperatures but are generally maintained in laboratories at 700-72 ~E Due to their propensity to develop nasal dermatitis at relative humidities above 50%, a low humidity is advisable.

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7. BIOLOGY AND DISEASES OF OTHER RODENTS

Gerbils require sandbathing to keep their coats from becoming oily. Tortora et al. (1974) described the effect of sand deprivation on behavior in gerbils. Gerbils often stand erect on their hindlimbs, so it is important that cages have a solid bottom and that the floor-to-lid height is tall enough to allow for this behavior.

D. 1.

Diseases

Infectious Diseases

a.

Bacterial/Mycoplasmal/Rickettsial Diseases

Facial eczema, "sore nose," and nasal dermatitis all describe a common skin condition seen in gerbils. Clinical lesions next to the external nares appear erythematous initially, progress to localized alopecia, and develop into an extensive moist dermatitis. The cause is believed to be increased Harderian gland secretion of porphyrins (similar to chromodaccryorhea in rats), which act as a primary skin irritant. Experimental Harderian gland-adenectomized gerbils do not develop nasal or facial lesions (Farrar et al., 1988). Various staphylococcal species (Staphylococcus aureus and S. xylosus) may act synergistically to produce the dermatitis (Bresnahan et al., 1983; Solomon et al., 1990). Stress factors such as environmental humidity above 50% or overcrowding cause excessive Harderian gland secretion (Farrar et al., 1988). Bacterial maxillary sinusitis can be detected clinically in the gerbil using magnetic resonance imaging (Allen et al., 1993). Naturally occurring Tyzzer's disease, an enterohepatic disease caused by the obligately intracellular bacterium Clostridium piliforme, is the most frequently described fatal infectious disease of gerbils (Koopman et al., 1980; Port et al., 1971; Vincent et al., 1975; Motzel and Gibson, 1990; White and Waldron, 1969). Common clinical and pathological findings were sudden death or death after a short period of disease, and the presence of multiple foci of hepatic necrosis. Diarrhea and necrotic lesions in the intestinal tracts were variably present. Experimentally induced Tyzzer's disease has confirmed that gerbils are extremely susceptibile to infection (Waggie et al., 1984). The probable route of infection in naturally occurring infection is by mouth, as gerbils exposed to infected bedding will contract Tyzzer's disease (Yokomori et al., 1989). Strittmatter (1972) eliminated Tyzzer's disease in the gerbil by fostering offspring to mice. De La Puente-Redondo et al. (1999) describe an epidemic of Citrobacter rodentium colitis in Mongolian gerbils. The disease occurred acutely in 9 gerbils, 6 dying in less than 48 hr after the onset of diarrhea. Microscopically, these animals had a thickening of the colon and rectum with extension into the small intestine. There was intestinal ulceration and goblet cell hyperplasia in the colon. The lesions were similar in appearance to those of transmissible murine colinic hyperplasia (Barthold, 1980). Cultures from abdominal masses and kidney grew pure cultures of

C. rodentium, and the addition of oxytetracycline (800 mg/ liter) to the drinking water prevented further disease (De La Puente-Redondo et al., 1999). The Mongolian gerbil is also susceptible to infection by Helicobacter pylori, which causes severe gastritis, gastric ulceration, and intestinal metaplasia (Wang and Fox, 1998). Watanabe et al. (1998) have described the development of gastric adenocarcinoma in 37% of infected gerbils maintained for 62 weeks. The histological progression of H. pylori infection in the gerbil was found to closely resemble that observed in human patients, where early intestinal metaplasia and gastric ulceration are replaced by antral gastric adenocarcinoma. b.

Viral/Chlamydial Diseases

Orthopoxvirus bovis (cowpox virus) has been isolated from M. libycus in the Eastern European republic of Georgia (Tsanava Sh et al., 1989). c.

Parasitic Diseases

i. Protozoa. Vincent et al. (1975) recovered Tritichomonas caviae and a species of entamoeba from experimental animals. ii. Nematodes. Wightman et al. (1978a,b) have found Syphacia obvelata, the mouse pinworm, and Dentostomella translucida, an oxyurid in Mongolian gerbils. An adult gerbil from a research colony and a litter of 5-week-old gerbils from a pet store were found to have pinworms identified as S. obvelata. Wightman et al. (1978b) caged infected gerbils with uninfected gerbils and uninfected mice, and infected mice with uninfected gerbils. The results of these studies showed that S. obvelata can be transmitted from gerbil to gerbil, gerbil to mouse, and mouse to gerbil. Dentostomella translucida was found in the small intestine of 39 out of 43 gerbils from a research colony and in 5 pet gerbils, establishing the gerbil as a new host for the parasite. Wightman et al. (1978a) found an average of 4 parasites per animal and no clinical manifestations ot~disease associated with the infection. The prepatent period of infection was between 25 and 29 days. iii. Cestodes. Vincent et al. (1975) recovered the cestode Hymenolepis diminuta from laboratory gerbils, and Lussier and Loew (1970) reported a case of natural H. nana infection in gerbils. iv. Mites. Levine and Lage (1984) described Liponyssoides sanguineus, the primary vector of Rickettsia akari, infesting Mongolian gerbils, mice (Mus musculus), and laboratory-reared Egyptian gerbils (Meriones libycus) but did not observe any manifestations of disease. They found only a few mites were present on each animal although numerous mites were present in the bedding. Vincent et al. (1975) recovered the forage mite

THOMAS M. DONNELLYAND FRED W. QUIMBY

278 Tyrophagus castellani from a colony of laboratory gerbils. (T. castellani is a pest of stored food products and causes "copra itch" among workers handling copra and a dermatitis in people who handle cheese.) Schwarzbrott et al. (1974) reported a case of demodicosis in a male laboratory gerbil. d.

Fungal Infections

There have been no reports of naturally occurring or experimental dermatophyte infections in the Mongolian gerbil (Donnelly et al., 2000). Other fungal infections in Meriones spp. are exceedingly rare. Cryptococcus neoformans was reported in a captive M. libycus at the Zoological Society of London (Parsons et al., 1987). 2.

Metabolic/Nutritional Diseases

Vincent et al. (1979b) found that research gerbils develop spontaneous, insidious periodontal disease after 6 months on standard laboratory rodent diets. On the same diets about 10% of the animals became obese, and some showed decreased glucose tolerance, elevated serum immunoreactive insulin, and diabetic changes in the pancreas and other organs. The authors noted that some breeder gerbils exhibited hyperactivity of the adrenal cortex associated with hyperglycemia, hyperlipidemia, and degenerative vascular disease. 3.

Traumatic Disorders

Thin skin covers the tail of the gerbil. Unlike rats or mice, if a gerbil is picked up by the tip of its tail, the skin will often slip off, leaving a raw, exposed tail that eventually becomes necrotic and will shed (Donnelly, 1997). If the tail skin is lost, the bare tail must be surgically amputated where the skin ends. 4.

Iatrogenic Diseases

A fatal syndrome of acute toxicity was produced in Mongolian gerbils following the injection of a penicillin-dihydrostreptomycin-procaine combination. The toxicity was determined to be due to the dihydrostreptomycin component. Fifty milligrams of dihydrostreptomycin produced 80-100% mortality in 55-65 gm gerbils (Wightman et al., 1980). Approximately 2 0 - 4 0 % of gerbils develop reflex, stereotypic, epileptiform (clonic-tonic) seizures from around two months of age (Kaplan, 1975; Kaplan and Miezejeski, 1972). Animals seize in response to sensory stimulation and forced exploratory behavior, but the incidence and severity of their seizures are variable; the seizures generally pass in a few minutes, may be mild or severe, and have no lasting effects. Although the incidence and severity of seizures often decrease with age, certain subsets of adult gerbils can be induced to seize following prolonged test regimens with progressive severity

(Scotti et al., 1998). The susceptibility is seen in selectively bred lines, inherited and related to a deficiency in cerebral glutamine synthetase (Laming et al., 1989). Spongiform lesions arise in dendrites and glia in the brain stem of domestic Mongolian gerbils and M. libycus (McGinn and Faddis, 1998). The lesions are characterized by the microcysts and vacuolar neuronal degeneration in the absence of astrocytosis. Axonal, dendritic, and neuronal perikarya degeneration accompanied by phagocytosis is often seen (Ostapoff and Morest, 1989). The lesions are bilateral, most pronounced within the cochlear nucleus, and increase in number, size, and extent with age. These spongiform lesions either cause or are associated with significant neural degeneration and appear to be the result of a common excitotoxic mechanism such as lowfrequency noise (McGinn and Faddis, 1997). In contrast, feral Mongolian gerbils and their offspring show few spongiform lesions. Cystic ovaries occur frequently in Mongolian gerbils (Clark, 1978; Norris and Adams, 1972b). Removal of affected ovaries does not significantly affect reproductive performance. Females with one ovary are slightly inferior in fertility compared with normal females; a general decline in fertility may be evident in older females. The mean number of litters, young born, and age at last parturition were 8 _+ 4, 37 ___20, and 462 _+ 96 days (two ovaries) compared with 7 + 4, 30 ___ 16, and 417 _+ 142 days (one ovary), respectively (Norris and Adams, 1982). Clark et al. (1986) found they could enhance the productivity of gerbil breeding colonies by using only the 40% of females that exhibit vaginal opening before 25 days of age as breeder females. Earlymaturing females are more likely to breed successfully on first pairing, and the lifetime fecundity of early-maturing females is more than twice that of their late-maturing littermates. Twothirds of the early-maturing females that fail to reproduce following a first pairing became pregnant following a second, but only 11% of late-maturing females do so. Mighell and Baker (1990) have reported cesarean section for successful treatment of dystocia in a gerbil. 5.

Neoplastic Diseases

Major surveys of spontaneous neoplasia in laboratory colonies of Mongolian gerbils are reported (Benitz and Kramer, 1965; Matsuoka and Suzuki, 1995; Ringler et al., 1972; Rowe et al., 1974; Vincent and Ash, 1978; Vincent et al., 1975, 1979b). A 24-39% incidence of neoplasia in gerbils usually occurs after 2 - 3 years of age (Matsuoka and Suzuki, 1995; Vincent et al., 1979b). Squamous cell carcinoma of the sebaceous ventral marking gland in males and ovarian granulosa cell tumor in females account for 80% of tumors seen in animals greater than 3 years of age. The ventral marking gland tumors invade locally and can metastasize to lymph nodes and lung (Ratio and Diamond, 1980). Adrenocortical tumors, cutaneous squamous cell carcinoma, malignant melanoma, and renal and

7. BIOLOGYAND DISEASESOF OTHER RODENTS

279

splenic hemangiomas are the next most commonly reported tumors. Numerous other tumors, including duodenal and cecal adenocarcinoma, hepatic lymphangioma, hemangioma and choleangiocarcinoma, splenic and renal hemangioma, uterine leiomyoma and hemangiopericytoma, ovarian teratoma, testicular teratoma, and malignant melanoma were reported. However, the total incidence of these tumors was less than 5% (Matsuoka and Suzuki, 1995; Meckley and Zwicker, 1979; Vincent and Ash, 1978; Vincent et al., 1975, 1979b). Case reports of spontaneously occurring tumors in gerbils include infiltrative craniopharyngioma (Guzman-Silva et al., 1988), histiocytic sarcoma (Chen et al., 1992), systemic mastocytosis (GuzmanSilva, 1997), malignant melanoma (Cramlet et al., 1974), and astrocytoma (Kroh et al., 1987).

6.

Miscellaneous

a.

Congenital Disorders

Shakibi and Weiss (1969) found a prevalence of ventricular septal heart disease in newborn gerbils. In a group of 12- to 30week-old male gerbils, Ninomiya and Nakamura (1987) reported spontaneous hyperplasia in both seminiferous and epididymal tubules. They considered the hyperplasia in young animals as congenital.

b.

Fig. 16. Microtusochrogaster, Prairie vole. Side view,in simulatedgrassland environment,IL. (Photographby R. B. Forbesand the MammalImagesLibrary of the AmericanSocietyof Mammalogists.)

Age-Related Disorders

In two separate reviews, Bingel (1995) and Vincent et al. (1975) reported the pathologic findings in aging Mongolian gerbil colonies. Besides neoplasia, they found a high incidence of chronic interstitial nephritis. Other nonneoplastic lesions included renal cortical retention cysts and liver disease. Calcinosis cutis was also observed in two older male gerbils (Vincent and Ash, 1978). Mongolian gerbils have a remarkable propensity for the development of aural cholesteatoma; canal cholesteatomas develop spontaneously in aged animals (Fulghum and Chole, 1985; Kim and Chole, 1998).

XIV.

A. 1.

Introduction

Description and Distribution

With the exception of M. pinetorum, which belongs to the subgenus Pitymys, the species mentioned above belong to the subgenus Microtus. Individuals in the subgenus Pitymys are adapted to semifossorial life, showing a reduction of eyes, external ears, and tail and a close, velvety pelage (Nowak and Paradiso, 1983). With the exception of M. arvalis, which is found

VOLES AND MEADOW MICE: Microtus

Within this genus there are 8 subgenera and 67 species commonly referred to as voles or meadow mice; those most widely used in research include Microtus californicus (field or California vole), Microtus ochrogaster (prairie vole) (Fig. 16), Microtus pennsylvanicus (common field vole, Eastern meadow vole) (Fig. 17), Microtus montanus (montane vole), Microtus oeconomus (tundra vole), Microtus pinetorum (pine vole), and Microtus arvalis (common vole).

Fig. 17. Microtuspennsylvanicus, Easternmeadowvole. (FromLaboratory Animal Medicine, 1984.)

280

THOMAS M. DONNELLY AND FRED W. QUIMBY

extensively throughout Europe, all others listed above are distributed in North America. In addition, M. oeconomus is distributed throughout northern Europe, Siberia, and north-central China. Microtus pennsylvanicus is found in the northern United States, Canada, and Alaska; it is widely used in research, and adults weigh about 40 gm with pelage of varying shades of brown (Clark, 1984).

tains M. ochrogaster and M. montanus. Don Hartbauer (Institute of Arctic Biology, University of Alaska, Fairbanks AK 99775; phone: 907-474-7020) provides several species of vole, including M. pennsylvanicus and M. oeconomus. B. 1.

2.

Use in Research

Weanling meadow voles have been used in nutrition studies where they are good bioassay animals for protein content of feeds, the digestibility of forages, and the presence of toxins (Jackson, 1997; Clark, 1984; Shore and Douben, 1994; Talmage and Walton, 1991; Talmage et al., 1999). Handling and environmental stress predispose some meadow voles to prolonged tonic-clonic seizures following a period of head shaking and stilted gait. Propensity to seizures appears to be inherited, and affected animals are excellent models of epileptiform seizures in humans (Bronson and DeLaRosa, 1994). Microtus pennsylvanicus has also been used in studies of the influence of sexually dimorphic spatial learning (Galea et al., 1996), as well as in studies on experimental infection with borrelia and babesia (Anderson et al., 1986; Campbell et al., 1994). Prairie voles have been used extensively to study the physiology of the vomeronasal organ and the ways in which chemosensory cues affect courtship, territorial marking, aggression, and reproduction (Wyscoki and Lepri, 1991; Taylor, 1997). In addition, prairie voles are monogamous, and studies have focused on the neuropeptides oxytocin and vasopressin and their control over such complex behaviors as pair-bonding, paternal care, maternal care, and mate-guarding (Young et al., 1998, 1997; Insel and Shapiro, 1992). Often the polygamous and somewhat antisocial M. pennsylvanicus serves as a contrasting species in these studies. Microtus montanus has been developed as an experimental model for the study of African trypanosomiasis (Seed and Hall, 1980). They have also proven sensitive to the effects of plantderived 6-methoxybenzoxazolinone, which alters the sex ratio of litters (Berger et al., 1987). The common vole, M. arvalis, has been used as an herbivorous model of chemically induced diabetes mellitus (Sasaki et al., 1991a,b) and for the evaluation of sex chromosome abnormalities (Zima et al., 1992). The research uses of the tundra vole have been previously reviewed (Dieterich and Preston, 1977c), and M. oeconomus has been used to study the effect of cholesterol with atherogenic diets (Dieterich and Preston, 1979). 3.

Sources

Donald Dewsbury (Department of Psychology, University of Florida, Gainesville FL 32611; phone: 904-392-0596) main-

Biology

Unique Physiologic Characteristics and Attributes

Voles are among the most prolific mammals, with reports of female M. pennsylvanicus producing as many as 17 litters in a single year (Hamilton and Whitaker, 1979). The common vole, M. arvalis, is sexually mature at 2 - 3 weeks, and some females mate and conceive at 13-14 days of age. Young grow very fast, reaching 30 gm by 40 days of age. Litter size is 4 - 7 with females capable of having a new litter every 3 weeks (Clark, 1984). The prairie vole lives in a monogamous relationship, and the male assists the female in raising offspring. Young remain part of a family group until they are ready to establish their own relationship. Both female and male offspring remain nonproductive into adulthood if maintained in the presence of the dominant male. In contrast, M. pennsylvanicus males are promiscuous, and the sexes nest separately. Pine voles display a cooperative system of breeding in social groups of 2 - 9 animals in which only 1 female reproduces but all members of the group participate in the care of newborns. Presence of prairie vole fathers within the group accelerates pup development, whereas the presence of meadow vole fathers hinders development (Jackson, 1997). Pregnancy is often terminated in the pregnant female if urine from an unknown male is introduced. Young from M. pinetorum and M. ochrogaster cling tenaciously to the teats of the females and can be dragged for considerable distances if the female is frightened. Other species of Microtus display this clinging behavior with varying degrees of tenacity. 2.

Normal Values

Normal hematologic values for the field vole and meadow vole are presented in Table II. Physiologic and clinical pathologic values have also been published (Dieterich and Preston, 1977a,b,c; Clark et al., 1978; Wallach and Boever, 1983). 3.

Nutrition

Voles have a high metabolic rate and are adapted to herbivory. Meadow voles have evolved such that they subsist on a lowcalorie diet and must be fed frequently. This species of vole relies heavily on the breakdown of carbohydrates during fasting. The carbohydrate catabolism results in profound hypoglycemia after only 6 hr of fasting (Nagy and Pistole, 1988). In voles on high-fiber diets, cellulolytic bacteria are isolated from their esophageal sac and gastric fermentation takes place, leading to higher pH and volatile fatty acids compared to those in the fundic or pyloric regions of the stomach (Kudo and Oki, 1993).

281

7. BIOLOGY AND DISEASES OF OTHER RODENTS

Voles can detect the presence of various solutes added to drinking water, which could complicate oral dosing (Jackson, 1997). A complete discussion of the nutrient requirement of voles has been published (National Research Council, 1978). 4.

Reproduction

Female voles are polyestrous and usually have multiple litters each year. Microtus pennsylvanicus has, like most Microtus females, 8 mammae and produces 3-10 immature young per litter that are weaned by 12 days of age. Female M. ochrogaster have 6 mammae and produce 1-7 young per litter (average 3.5). Microtus pinetorum females have only 4 mammae and produce 1-4 young per litter (average 2-6). The success of females of a particular species becoming pregnant during the postpartum estrus varies from 40 to 70% (Morrison et al., 1976). Neonatal mortality may be high, especially if the litter is disturbed during the first week. At least for the bank vole, Clethrionomys glareous, litter size had no effect on infant mortality (Mappes et al., 1995). Female M. pennsylvanicus are induced ovulators and can be bred any time. Ovulation occurs 12-18 hr after copulation. Species of voles vary in the ability to detect definitive estrous cycle patterns. Despite attempts to elucidate breeding cycles by vaginal cytology, the Japanese field vole (M. montebelli) shows no clear pattern; the common vole (M. arvalis) shows 6-18 day cycles. C.

Husbandry

All species of voles can be bred successfully in solid-bottom cages of varying sizes (Fine et al., 1986; Solomon and Vandenbergh, 1994), although a minimum floor space of 1 ft 2 was recommended for housing a breeding pair (Richmond and Conaway, 1969). Both unsupplemented commercial rabbit feed and supplemented (wheat germ and oats) commercial rodent feed have been used successfully. Water should be offered ad libitum, and special care must be taken to provide sufficient water with the diet (Solomon and Vandenbergh, 1994). Voles are generally housed in a controlled environment: temperature 23 ~ 25~ relative humidity 60-70%, and a 12 hr light cycle for maintenance or 14 hr light cycle for breeding (Fine et al., 1986). Care must be taken to avoid complete cage cleaning during the first week following parturition since this commonly results in cannibalism of an entire litter. Likewise, leaving a small amount of dirty litter in an otherwise cleaned cage helps maintain normal reproductive cycles in female voles. D. 1.

Diseases

Infectious Diseases

Wild voles have been hosts for hantaviruses; cowpox virus; Leptospira sp.; Emmonsia sp.; Toxoplasma gondii; the larval

stages of Echinococcus multilocularis; the mites Echinolaelaps sp. and Psorergates simplex; and the blood parasites Babesia microti, Haemobartonella microti, and Trypanosoma microti. The latter parasites are not generally associated with clinical signs (Clark, 1984; Fine et al., 1986; Bennett et al., 1997). In addition, various dermatomycoses, rabies virus, and Yersinia pseudotuberculosis have been isolated from voles (Davis et al., 1981). The Acanthocephalan parasites Moniliformis clarki and Cochliomyia hominivorax have been found to parasitize meadow voles (Wallach and Boever, 1983). Bordetella bronchiseptica has been found associated with fatal pulmonary infection of Microtus montanus (Jensen and Duncan, 1980). 2.

Neoplastic Diseases

Adenocarcinoma of the mammary gland has been diagnosed infrequently in colony-raised M. ochrogaster, however, lacrimal adenocarcinoma was a common cause of death among colonyreared adult M. montanus (Fine et al., 1986). 3.

Other Diseases

Peridontitis and malocculsion have been diagnosed in montane and prairie voles. Chronic interstitial nephritis is not uncommon in older colony-bred meadow voles. The etiology is unknown, but the kidneys have histologic evidence of increased interstitial connective tissue with hyaline casts in many tubules (Fine et al., 1986; Clark, 1984).

XV.

M U L T I M A M M A T E RATS: Mastomys

A. 1.

Introduction

Taxonomy

The common name describes the high number of mammary glands, usually 8-12 pairs, but there may be as many as 18 pairs. The genus Mastomys requires careful taxonomic revision. Definition of species from some regions by chromosomal and biochemical traits has proceeded faster than definitions based on morphology (Duplantier et al., 1990). The result is a new view of species diversity in the genus, but also an ignorance of morphological limits of those species and their real geographic distributions. Robbins and van der Straeten (1989) analyzed all the taxa associated with Mastomys but did not allocate any of them to species. At present, M. natalensis (Fig. 18) is probably the correct term for colonies of Mastomys used in research (Dettman et al., 1987; Kruppa et al., 1990). The following genus and species names have been used in the past to describe this animal: M.

282

THOMAS M. DONNELLY AND FRED W. QUIMBY

Fig. 18. Mastomys natalensis, Multimammate rat. (From Laboratory Animal Medicine, 1984.)

coucha, Rattus (Praomys) natalensis, R. (Praomys) coucha, and R. (Mastomys) natalensis. 2.

Description

The description is taken from Nowak (1999). The head and body length is 60-170 mm, tail length is 60-150 mm, and weight is 2 0 - 8 0 gm. The tail is approximately equal in length to the body. Animals have a light gray to brown dorsum and light gray underside. Mastomys superficially resembles Rattus. 3.

Distribution

Originally believed to range in sub-Saharan Africa, animals with identical chromosomal features have been found in Tanzania and Senegal in West Africa (Duplantier et al., 1990). Given the confusion with taxonomic status, it probably suffices to state that the multimammate rat is one of the most widely distributed and abundant rodents in Africa. 4.

Habitat

Multimammate rats occur in many types of habitat. They may once have been restricted to savannas but can now be found throughout much of sub-Saharan Africa, chiefly in association with people (Nowak, 1999). They do not occupy large towns probably because of competition with Rattus. The diet and ecology of M. natalensis in their native habitat have been published (Oguge, 1995). 5.

Use in Research

Multimammate rats (which have also been called multimammate mice) serve as hosts for a wide variety of infectious

agents, including some important zoonotic agents. Due to their susceptibility to Yersinia pestis, they have been used extensively for research into the pathogenesis of plague as well as in vaccine trials and disease diagnosis (Isaacson et al., 1981; Williams et al., 1982). Mastomys natalensis has been used as a model for studying bacterial infection with Mycobacterium ulcerans (Singh et al., 1984). Multimammate rats are natural reservoirs for leptospirosis, leishmaniasis, and Lassa virus, and they have been the subjects of research on these agents as well as on Crimean-Congo hemorrhagic fever virus (Shepherd et al., 1989; Green et al., 1978; Machang'u et al., 1998). Mastomys natalensis has been used extensively to investigate the pathogenesis and treatment of parasitic diseases, including the protozoans Leishmania donovani, L. major, Plasmodium berghei, Entamoeba histolytica, and Trypanosoma brucei rhodensiense (Nolan and Farrell, 1987; Khare et al., 1984; Srivastava and Gupta, 1985; Rickman and Kanyangala, 1990); the nematodes Nippostrongylus brasiliensis, Dipetalonema viteae, Brugia malayi, B. pahangi, Litomosoides carinii, Acanthocheilonema viteae, Wuchereria bancrofii, and Capillaria hepatica (Murthy, 1997; Zahner and Rudolph, 1980; Zielke, 1980; Zahner et al., 1989; Sangvaranond and Zahner, 1989; Chatterjee et al., 1988; Redl and Kassai, 1979); and the trematode Schistosoma mansoni (Coehlo et al., 1980). Mastomys natalensis spontaneously develops gastric carcinoid and serves as a model for Zollinger Ellison syndrome in humans (Nilsson et al., 1992; Kumazawa et al., 1989). The role of hypergastrinemia in tumor production, as well as CCK-A and CCK-B/gastrin receptors on enterochromaffin-like carcinoid cells and their relationship to histamine secretion have been well studied (Kolby et al., 1998). Mastomys natalensis has an endogenous papillomavirus which has been sequenced and shown to produce cutaneous tumors on activation (Tan et al., 1994). The morphology of experimentally induced keratoacanthamous and squamous cell carcinomas has been published (Rudolph et al., 1981). This animal has also been the subject of many studies demonstrating the oncogenic potential of chemicals (Hoch-Ligeti et aL, 1985; Wayss et al., 1981; Tajima, 1981), as well as noncarcinogenic toxicology (Holmes et al., 1995, 1996, 1997). Multimammate rats have been used to study the role of mucosal anaphylaxis on gastric ulcer formation (Andre and Andre, 1981), and some individuals spontaneously develop autoimmune thyroiditis (Solleveld et al., 1985).

6.

Sources

Colonies of M. natalensis have been established by many of the scientists that use them, and inbred lines varying in susceptibility to gastric carcinoids have been developed (Randeria, 1978).

7. BIOLOGY AND DISEASES OF OTHER RODENTS B. 1.

C.

Biology

Physiological Characteristics

The submaxillary salivary glands ofM. natalensis are the richest available source of nerve growth factor, and these glands have been the focus of several investigations (Aloe et al., 1981; Sirigu et al., 1985; Matsushima et al., 1990; Sirigu et al., 1988). Both males and females have a prostate gland, and this organ has been the subject of several investigations (Gross and Didio, 1987, 1988). As a result of work involved with the immune response to various infectious agents, the immune response and the nature of cellular immunity have been characterized in normal animals (Zahner et al., 1987; Beucher and Charreire, 1983). Mammary gland growth and response to reproductive hormones have also been investigated and compared to those in laboratory mice (Nagasawa et al., 1989). The multimammate rat does not have a gallbladder. 2.

283

Normal Values

The multimammate rat has a life span of 3 years, its weight at birth is 2 - 3 gm, its eyes open at 13-17 days, and it is weaned at 19-21 days. Selected hematologic data have been published (Clark, 1984; see Table II).

Under field conditions M. natalensis eats mostly grass and other seeds (Nowak, 1999). In suitable habitats multimammate rats will eat insects. Near human populations M. natalensis eats nearly everything humans do (Nowak, 1999). In the laboratory, commercial laboratory rat food is satisfactory (Redl and Gyorffy, 1978; Williams, 1980). Multimammate rats thrive in the laboratory environment when housed in standard solid-bottom rodent cages with wood-chip bedding and fed commercial laboratory rodent feed with ad libitum water. D. 1.

Nutrition

Although this animal survives mainly on a diet of seeds in its natural habitat, it thrives on commercial rodent ration. 4.

Reproduction

Multimammate rats reproduce well as monogamous pairs and have litters of 6 - 1 2 young, although numbers as high as 22 are reported (Neal, 1977). Birth weight is 2 - 3 gm, young open their eyes at 13-17 days, and they are weaned at 19-21 days. Males and females reach puberty at 55-75 days of age. They breed year-round and will mate at the postparturient estrus. Estrous cycles average 6 - 8 days, and females have a 23-day gestation period. The multimammate rat has a life span of 3 years (Williams, 1980). 5.

Behavior

While these animals appear to be almost tame in the wild, Veenstra (1958) claims they are very aggressive when held under laboratory conditions, and care must be taken to avoid being bitten. They keep their bodies well groomed and their cages clean and will attempt to dispose of waste by pushing it through a hole in the cage (Clark, 1984). In captivity, stereotypic behaviors similar to those of rats, characterized by repetitive sniffing, rearing, licking, exhibiting head movements, and biting have been described (Gulati et al., 1986, 1988).

Diseases

Infectious Diseases

A number of infectious agents have been associated with wild-caught multimammate rats (see above); however, these agents should not be problematic in laboratory colonies. Mastomys natalensis does carry a papillomavirus that is responsible for cutaneous tumors (Tan et al., 1994; Pruthi et al., 1983). Psorergatic mange has also been described as a disease in which mites cause raised, crusty cutaneous nodules (Clark, 1984). 2.

3.

Husbandry

Neoplastic Diseases

Lymphosarcomas, parathyroid adenomas, prostatic tumors, reticulum cell sarcomas, adenomas of the glandular stomach, and gastric carcinoids are relatively common (Holland, 1970; Kozima et al., 1970; Kumazawa et al., 1989; Kurokawa et al., 1968; Saito et al., 1977; Snell and Stewart, 1967, 1969, 1975; Stewart and Snell, 1968, 1975; Tielemans et al., 1987). Lymphoepithelial thymoma is common in animals over 2 years of age and is often associated with myositis, atrophy of skeletal muscle, and myocarditis (Kurokawa et al., 1968; Stewart and Snell, 1968). Other less common tumors reported include granulosa cell tumors of ovaries, testicular tumors, adrenal gland adenomas, pituitary adenomas, hepatomas, nephroblastomas, and adenomas of the pancreas (Hosoda et al., 1976; Jobard et al., 1974; Snell and Hollander, 1972; Snell and Stewart, 1975; Stewart and Snell, 1975). 3.

Miscellaneous a.

Aging Lesions

In animals over 2 years of age, many multimammate rats develop osteoarthritis and are reluctant to ambulate. Degenerative joint disease affects the diarthroses and intervertebral discs, with many peripheral joints involved. Diarthrodial joints show extensive erosion of articular cartilage and sclerosis of epiphyseal bone. Degenerated discs protrude into the vertebral canal at multiple sites along the vertebral column (Snell and Stewart,

THOMAS M. DONNELLYAND FRED W. QUIMBY

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1975). A histopathological survey of lesions in aged Mastomys has been published (Solleveld et al., 1982). b.

Autoimmune Disease

Autoimmune thyroiditis occurs spontaneously in many individuals (Solleveld et al., 1985). In addition, as the animals age, increasing numbers of autoantibodies are found (Coolen et al., 1981). Membranous glomerulonephritis with hyaline thickening of the glomerular loops and basement membrane of Bowman's capsule is a frequent lesion. In the proliferative form of the disease, glomeruli have, in addition to the above findings, marked hypercellularity. In more chronic cases, tubules are involved, displaying tubular atrophy, flattened epithelia, and the presence of casts. The interstitial tissue is thickened and is filtrated with lymphocytes, plasma cells, and granulocytes. These kidney lesions are thought to have an autoimmune pathogenesis (Snell and Stewart, 1975). c.

2.

Distribution

Degus occur naturally in northern and western Chile, on the west slope of the Andes up to 1200 m (Woods and Boraker, 1975).

3.

Habitat

Degus are found in open areas near thickets, rocks, or stone walls. They construct an elaborate communal burrow system, with the main section under rocks or shrubs. A complex network of tunnels and surface paths leads out to feeding sites.

Other Conditions

A relatively high incidence of duodenitis and duodenal ulcer occurs in multimammate rats, and afflicted rats have been used to study cellular regeneration (Smedley et al., 1990).

XVI.

DEGUS OR TRUMPET-TAILED RATS: Octodon

A. 1.

length is 125-195 mm, and tail length is 105-165 mm. Weight varies between 170 and 300 gm for adults. The upper parts are grayish to brown, and underparts are creamy yellow. A black brush at the tip of the tail is prominent.

Introduction

Description

The description of Octodon degus (Fig. 19) is adapted from the review by Woods and Boraker (1975). The head and body

4.

Use in Research

The unique characteristics of the degu have made it increasingly popular as a laboratory animal. The precocious neonates are animal models used to study neurobiological developmental patterns (Braun et al., 2000; Poeggel et al., 1999). The diurnal sleep pattern of the degu, in contrast to that of nocturnal rats and mice, makes it an excellent model to investigate human sleep/ wake and circadian behavior (Goel and Lee, 1997; Kas and Edgar, 1999). Researchers have also used the degu to investigate how herbivorous animals match their foraging and digestion to seasonal changes in availability and quality of food (Bozinovic et al., 1997; Kenagy et al., 1999), drug tolerance (Gaule et al., 1990; Letelier et al., 1985), and diabetes development and cataract formation (Barker et al., 1983; Datiles and Fukui, 1989; Nishi and Steiner, 1990; Spear et al., 1984).

B.

Fig. 19. Octodon degus, Degu. Obliqueview,PragueZoo, Czechoslovakia. (Photograph by M. Andera and the MammalImages Library of the American Society of Mammalogists.)

Biology

The first four digits of degus are well developed with sharp claws for burrowing. The fifth digit is shorter. Females have four pairs of mammary glands, and the testicles of males are intra-abdominal (Contreras and Bustos-Obregon, 1980; Weir, 1970). The adrenal glands are large compared with those of other rodents on a per body weight basis (Galli and Marusic, 1976). The degu spleen is unusual, with sinusoids lined by endothelial cells having cuboidal morphology that gives the spleen a glandular appearance (Murphy et al., 1980). The hematologic and serum protein values for degus are similar to values reported for guinea pigs and rats. Reference hematologic and serum protein values from normal degus that ranged

7. BIOLOGY AND DISEASES OF OTHER RODENTS in age from 3 to 48 months were similar for both sexes (Murphy et al., 1978).

C.

Husbandry

Octodon degus live in small colonies with a strong social organization based on group territoriality. The burrow is the center of the defended territory. Females of the same social group often rear their young in a common burrow (White et al., 1982). Captive animals exhibit considerable social tolerance, and strange degus may be housed together without fighting (Altmann et al., 1994; Davis, 1975). Laboratory colonies breed throughout the year, and females may have more than one annual litter. Females are induced ovulators and require the presence of a male to induce ovulation (Weir, 1970). The gestation period averages 90 days (Weir, 1970). Litters contain 1-10 young with an average of 7 pups. The young weigh about 14 gm at birth, ranging in size from 5 6.5 cm from nose to base of tail, and are born fully furred with their eyes open (Reynolds and Wright, 1979). Pups must nurse for at least 14 days, usually 28 days, and almost double their size in 3 weeks. Researchers report the age of sexual maturity to be as early as 45 days and as late as 20 months, but the average is about 6 months (Altmann et al., 1994; Weir, 1970). Successful breeding programs report maintaining breeding females at a weight of 250 gm (Najecki and Tate, 1999). When holding a degu by the tail, the handler must take care or the animal will spin like a top and leave the person holding only the skin (Woods and Boraker, 1975). Degus familiar with the handler do not show this behavior, and tail shedding is unusual (Altmann et aL, 1994). Some laboratory colonies use nylon fishnets to transfer and handle degus (Najecki and Tate, 1999). In the wild, degus take dust baths to keep their coats free of oil. Like chinchillas, captive degus must be provided with a dustbath twice a week (Altmann et al., 1994). A commercial rodent diet with vegetable supplements is suitable for degus. However, caretakers should not feed the animals ad libitum because obesity is likely to occur (Weir, 1970).

D. 1.

Diseases

Infectious Diseases

Degus do not drink much water. When degus are supplied with water bottles, Najecki and Tate (1999) emphasize that unless husbandry practices provide treated water (acidified) and frequent changes (at least 3 times a week) Pseudomonas infection is likely to occur. In the same colony, Najecki and Tate (1999) described fatal diarrhea in adults and pups. Enzymelinked immunosorbent assay (ELISA) on fecal samples of the affected animals showed the presence of Giardia spp. Acute

285

suppurative bronchopneumonia, caused by Klebsiella pneumoniae, was described in a degu (Murphy et al., 1980). Babero et al. (1975) described helminth parasites from O. degus in Chile, with descriptions of three new species, including a whipworm, Trichuris bradleyi (Babero and Cattan, 1975). 2.

Metabolic/Nutritional Diseases

The degu develops spontaneous diabetes mellitus and has been found to have islet amyloidosis (Nishi and Steiner, 1990). Cytomegalovirus-inducing insulitis, a-cell crystals with a herpes-type virus presence, and foods such as guinea pig chow or fresh fruit that elevate blood sugar levels are associated with the development of diabetes in the degu (Fox and Murphy, 1979; Najecki and Tate, 1999; Spear et al., 1984). A diabetic degu will develop cataracts within four weeks (Datiles and Fukui, 1989). 3.

Traumatic Disorders

A tibial fracture in a 3 month-old degu was repaired by medullary fixation (Beregi et al., 1994). 4.

Neoplastic Diseases

Anderson et al. (1990) diagnosed a primary bronchioloalveolar carcinoma with renal and hepatic metastases in a mature male degu that was found dead in a zoological exhibit. At necropsy they saw a discrete 0.5 cm diameter nodule in the lung. Smaller but similar nodules were scattered in the liver and kidneys. Histologically, nests and sheets of an infiltrating population of cuboidal to low-columnar neoplastic epithelial cells partially effaced pulmonary architecture. Vascular invasion was evident. Similar nests and sheets of neoplastic cells were present within the renal cortex and medulla, and a small nest was present within the hepatic parenchyma. Murphy et al. (1980) describe pathologic changes in 189 degus ranging in age from 2 to 60 months. One reticulum cell sarcoma involving a cervical lymph node resulted in death due to tracheal compression. Hepatocellular carcinoma was diagnosed in 2 degus; one highly anaplastic tumor appeared to be composed of a combination of hepatic and biliary elements. This tumor had metastasized to the lung and kidney. A second carcinoma was characterized by multifocal accumulations of pleiomorphic cells compressing adjacent hepatic parenchyma. This tumor appeared to metastasize to the right side of the heart. Other tumors encountered included a splenic hemangioma and a lipoma in the mesentery. 5.

Miscellaneous

Naturally occurring congenital cataracts were found in a colony of degus (Worgul and Rothstein, 1975). Histological examination of the lens epithelium revealed that a marked

THOMAS M. DONNELLYAND FRED W. QUIMBY

286

disorganization of the meridional rows was associated with the opacity.

throughout the year. They are gregarious, living in groups of several hundred (Barber and Thompson, 1990). 4.

XVII.

CHINCHILLAS: Chinchilla

A.

Introduction

1. Description

The family Chinchillidae contains three recent genera and six species of New World animals commonly called viscachas and chinchillas. These are slender-bodied, medium-size rodents with short forelimbs and long muscular hindlimbs that give the animal a rabbit-like appearance. The head, eyes, and ears are relatively large, and the bullae are greatly expanded on members of the genus Chinchilla. Members of this family have long gestation periods and deliver fully furred young with open eyes (Nowak and Paradiso, 1983; Anderson and Jones, 1984). 2.

Distribution

The genus Chinchilla consists of two species; however, only C. laniger (Fig. 20) has been widely used in research. In the wild, C. laniger lives in relatively barren areas of the Andes of northern Chile at elevations of 3000-5000 m. All domestic chinchillas are descendants of 13 individuals brought to the United States in 1927 (Anderson and Jones, 1984). 3.

Habitat

Chinchilla laniger live in burrows or rock crevices and are cursorial. They dustbathe, are vegetarians, and are active

Use in Research

Chinchillas are good laboratory animals because of their small size, ease of handling, and long life span (12-20 years). Because of their large accessible bullae and freedom from middle ear infections, chinchillas have been used for auditory research (Ding et al., 1999; Javel and Mott, 1988; Siegel, 1986; Dancer, 1995; Goldberg et al., 1985), including the pathogenesis, treatment, and prevention of otitis media (Morizono and Tono, 1991; Juhn et al., 1991; Giebink, 1999; DeMaria, 1989). Chinchillas have also been used to study the pathogenesis of Chagas' disease and atherosclerosis (Clark, 1984; Stehbens, 1986), endocrinology of digestion (Fan et al., 1987; Shinomura et al., 1987), thermoregulation (Wang et al., 1985), cerebral blood flow (Jablonski and Brudnicki, 1984; Gielecki et al., 1996), behavior (Bowe et al., 1987), and reproduction (Ponce et al., 1998; Johnson et al., 1987; Gromadzka-Ostrowska et al., 1985). 5.

Sources

Chinchillas are available through Harlan Sprague Dawley, Inc. (see Section XI, A, 5, Cotton Rats) and Moulton Chinchilla Ranch, 976 14 Avenue SW, Rochester MN 55902 (phone: 507288-334; email: [email protected]).

B.

The female chinchilla has an estrous cycle of 38 days and a vaginal closure membrane. Females are seasonally polyestrous, and the breeding season for captive colonies is November to May in the Northern hemisphere. The gestation period averages 110.8 days. Generally females will have 2 litters a year with 1 to 6 young (average 2) per litter. Young become sexually mature at 8 months of age. Reproductive data have been thoroughly reported (Weir, 1966; 1973, 1976; Rowlands and Weir, 1974; Kuroiwa and Imamichi, 1977). References range from hematology and clinical chemistry, and selected physiologic parameters have been published (Juhn et al., 1974; Clark et al., 1978; Clark, 1984; Jakubow et al., 1984; Spannl, 1988; Strike, 1970; see Table II).

C.

Fig. 20. Chinchilla laniger, Chinchilla. Front view of captive. (Photograph by R. Altig and the Mammal Images Library of the American Society of Mammalogists.)

Biology

Husbandry

Chinchillas are very tolerant of cold but sensitive to heat. A constant temperature of 20 ~ ___2 ~C is optimum. Light should be provided 14 hr each day. In the laboratory, chinchillas are easily housed in either wire-mesh (standard rabbit cage) or solid-bottom cages, although solid-bottom cages are recommended for

7. BIOLOGY AND DISEASES OF O T H E R RODENTS

pregnant females about to have young. Because chinchillas habitually dustbathe, a box containing a mixture of silver sand and Fuller's earth should be placed in the cage daily (Clark, 1984). Polygamous breeding colonies are common among chinchilla ranchers, and a system of individual female housing has been devised that allows a single male to serve 12 females (Weir, 1976). A variety of breeding techniques have been used successfully, and mating is facilitated by observing changes in the vaginal closure membrane and performing vaginal cytology. Pregnant females do not make a nest (Kitchen and Kitchen, 1967; Weir, 1967, 1973, 1976). A pelleted chinchilla ration is commercially available and suitable; however, chinchillas have also been raised on guinea pig or rabbit rations (Kitchen and Kitchen, 1967; Weir, 1976; Zeinert, 1983; Clark, 1984). Water should be provided ad libitum.

D. 1.

Diseases

Infectious Diseases

Nearly all significant reports on infectious diseases of chinchillas over the past 50 years come from colonies of chinchillas raised for fur, and most reports of bacterial disease in colonies are 20 years of age or older. Opportunistic infections by normal bacterial residents of chinchillas will cause frank disease, either localized to one organ (e.g., Streptococcus, Pseudomonas, and colibacillosis) or as septicemia. Affected animals may be immunocompromised through age, nutritional status, or husbandry-related stress. a.

Bacterial/Mycoplasmal/Rickettsial Diseases

Worthington and Fulghum (1988) from the Virginia Polytechnic Institute Anaerobe Laboratory studied the predominant anaerobic bacterial flora of the chinchilla cecum and fecal pellets. They isolated species of Bacteroides, Lactobacillus, Bifidobacterium, and Eubacterium and found the flora did not differ significantly from that of other rodents (Worthington and Fulghum, 1988). Their findings are similar to earlier work by Mathieu et al. (1982). In Germany, chinchillas on fur farms are vaccinated for Pseudomonas infection, Escherichia coli infection, and Yersinia infection (Matthes, 1985). Within the genus Yersinia, 11 species are recognized, but 3, Y. pestis, Y. pseudotuberculosis, and certain serovars of Y. enterocolitica (although some authors delineate "Y. enterocoliticalike" species), are important infectious organisms for humans and warm-blooded animals. The causative agents of yersiniosis, Y. pseudotuberculosis (previously called Pasteurella pseudotuberculosis) and Y. enterocolitica, occur worldwide in areas of moderate and subtropical climate, and outbreaks in chinchillas are commonly described (Gueraud, 1988; Raevuori et al., 1979;

287

Wuthe and Aleksic, 1992). Asymptomatically infected warmblooded animals that cause environmental contamination are the most important factors for the epidemiology of yersiniosis. Apathogenic Yersinia species and serovars are adapted to environmental conditions and independent from warm- or coldblooded host organisms (Aleksic and Bockemuhl, 1990). Classically, Y. pseudotuberculosis is of interest in rabbits and rodents (causing classical pseudotuberculosis or mesenteric adenitis in guinea pigs) and has been routinely isolated from many species of rodents. Yersinia enterocolitica is now the species of Yersinia most frequently isolated from human and animal infections. The species includes both pathogens and ubiquitous strains. Among the human pathogens, those isolated in America are more virulent than those isolated elsewhere, especially in Europe and Japan, and these isolates differ biochemically and serologically (Cornelis et al., 1987). Molecular biology has contributed significantly to the understanding of the pathogenicity of enteric Yersinia. All human pathogenic Yersinia harbor a 70-kilobase plasmid that is essential for virulence expression. Microbiologists have identified 13 plasmid-encoded polypeptides. Plasmid-mediated pathogenic functions are survival in serum, resistance to phagocytosis, cell adhesion, and cytotoxicity (Heesemann, 1990). Virulence in Y. enterocolitica depends on chromosomal genes: the endocytosis in intestinal epithelial cells seems not to be encoded by a plasmid. Studies of Y. pseudotuberculosis suggest that this property depends on a single chromosomal locus (Cornelis et al., 1987). Plasmid-encoded proteins are more suitable antigens for serologic diagnosis of yersiniosis (immunoblotting and indirect immunofluorescence) than whole bacterial antigens (Heesemann, 1990). Wuthe and Aleksic (1992) described a "chinchilla-type" strain of Y. enterocolitica (biovar 3, antigens, or serovar 1, 2a, 3) that appears to persist enzootically among chinchilla stock worldwide. Hubbert (1972) in the United States and Langford (1972) in Canada have reviewed cases of yersiniosis reported in mammals (including chinchillas) and birds, and Kageruka (1970) reviewed the incidence of yersiniosis in mammals and birds in Antwerp Zoo. Guerard (1988) recorded a chinchilla epizootic of Y. enterocolitica in France and described the pathological effects and distribution of the organism in chinchillas, and microbiological isolation methods. Raevuori et al. (1979) investigated the virulence and experimental pathogenicity of Y. enterocolitica in chinchillas. Yersiniosis is an enteric disease that damages epithelium of the ileum, cecum, and colon, resulting in mucosal hemorrhage and ulceration. Lymphoid infiltration resuits in Peyer's patches and mesenteric lymph node hypertrophy with necrotizing granulomas. Systemic spread results in granulomatous lesions in the lungs, spleen, and liver. Opportunistic Pseudomonas aeruginosa systemic infections are described in chinchillas (Lusis and Soltys, 1971b; Menchaca et al., 1980). Doerning et al. (1993) described a case of P. aeruginosa infection in a laboratory chinchilla. The affected animal displayed a variety of clinical signs, including scrotal

288

swelling, conjunctivitis, anorexia, weight loss, and corneal and oral ulcerations. The authors treated the animal with chloramphenicol therapeutically and butorphanol for analgesia, and the chinchilla developed unusual intradermal pustules 8 days after recovery from the infection. A vaccine against P. aeruginosa has been developed for attempted immunization and is used in fur-ranched chinchillas (Lusis and Soltys, 1971a); (Matthes, 1985). Klebsiella infection associated with K. pneumoniae, including treatment and immunization, was described in chinchillas in Poland (Bartoszcze et al., 1990). Clostridial enterotoxemia is reported in chinchillas. Moore and Greenlee (1975) described an enterotoxemia associated with Clostridium spp.; Nowakowska et al. (1991) described enterotoxemia caused by C. perfringens enterotoxin; and Bartoszcze et al. (1990) described chinchilla deaths due to C. perfringens A enterotoxin. There are two case reports of Salmonella infection in companion chinchillas. Mountain (1989) reported a case of Salmonella arizona septicemia in the United Kingdom in a chinchilla, and Yamagishi et al. (1997) reported a case of septic infection in a companion chinchilla with Salmonella enteritidis in Japan. Listeriosis in chinchillas was first reported by MacKay et al. (1949). It is still common in fur-ranched chinchillas but not in laboratory chinchillas (MacDonald et al., 1972; Wilkerson et al., 1997; Nilsson and Soderlind, 1974; Finley and Long, 1977). Although Gray and Killinger (1966) claimed that chinchillas appear to be the most susceptible animal to infection with Listeria monocytogenes, this has not been proven. Case reports of listeriosis in chinchillas describe nonlaboratory animals in high northern latitudes such as Canada, Washington state, or the United Kingdom, where the animals are fed silage or substitute roughage in pellets, such as beet pulp, during the winter (Cavill, 1967; Finley and Long, 1977; Wilkerson et al., 1997). Listeriosis is also recorded in group-housed animals such as cattle, sheep, goats, deer, swine, rabbits, rodents, birds, and turtles in similar situations (MacDonald et al., 1972; Nilsson and Soderlind, 1974). Knowledge of L. monocytogenes and the various forms of disease that it causes has been extremely limited, but advances in taxonomy, isolation methods, bacterial typing, molecular biology, and cell biology have extended our understanding (Low and Donachie, 1997; Rouquette and Berche, 1996; Schlech, 1996). Listeria monocytogenes is a highly adaptable environmental bacterium that can exist both as an animal pathogen and plant saprophyte with a powerful array of regulated virulence factors. It is a resident of normal microbial flora in healthy ruminants and is found in environmental sources such as decaying vegetation. Most animal and human cases of listeriosis arise from the ingestion of contaminated food, and the disease is particularly common in animals fed on silage (Low and Donachie, 1997). Unlike most food-borne pathogens that primarily cause gastrointestinal disease, L. monocytogenes causes a number of easily recognized invasive syndromes, such as encephalitis, abortion, and septicemia.

THOMAS M. DONNELLY AND FRED W. QUIMBY

However, the epidemiological aspects and pathogenesis of infection remain poorly understood. Although 16 serotypes of L. monocytogenes are identified, 3 serotypes cause about 30% of human cases (Low and Donachie, 1997; Rouquette and Berche, 1996). Serotyping is based on somatic (O) and flagellar antigens (H); molecular subtyping techniques such as multilocus enzyme electrophoresis, phage typing, and ribosomal DNA analysis improve the ability to discriminate among strains of Listeria (Low and Donachie, 1997). These techniques are not used commonly in infectious outbreaks of listeriosis in animals. In chinchillas, listeriosis is a cecal disease with blood-borne dissemination. The main target organ is the liver, where the bacteria multiply inside hepatocytes. Early recruitment of polymorphonuclear cells leads to hepatocyte lysis, bacterial release, septicemia, and in surviving hosts the development of lung, brain, spleen, lymph node, and liver abscesses. The invasion of peripheral nerve cells and rapid entry into the brain, giving the classic histopathological lesion of monocytic perivascular cuffing, is believed to be a unique characteristic of its virulence. b.

Viral/Chlamydial Diseases

There are no species-specific viral diseases described for chinchillas. However, Goudas and Giltoy (1970) described a spontaneous herpeslike viral infection in a female chinchilla. c.

Parasitic Diseases

i. Protozoa. Toxoplasmosis was commonly found in furranched chinchillas but is rarely seen now (Keagy, 1949). Necropsy lesions include hemorrhagic lungs and enlarged spleens and mesenteric lymph nodes. Turner (1978) reviewed the wide pathological manifestations of toxoplasmosis, with or without necrosis, and tissue cyst formation as it occurs in sheep, cattle, pigs, horses, dogs, cats, chinchillas, and humans. Meingassner and Burtscher (1977) described 2 chinchillas with focal necrotic meningoencephalitis due to Toxoplasma gondii. The authors found several lobulated Frenkelia cysts up to 0.6 mm diameter in the brain of the 2 animals, independent of and remote from the Toxoplasma inflammatory reaction. The cysts resembled both morphologically and in staining Frenkelia in various Microtus species and Ondatra zibethica. The authors considered that chinchillas may be susceptible to a Frenkelia occurring in other free-living species. In the past, group-housed chinchillas, such as in fur ranches and research colonies, often had a high prevalence of Giardia infection (Newberne, 1953; Shelton, 1954a,b). Forty years later, Eidmann (1992) examined feces from chinchillas and found that they normally harbor Giardia species in large numbers. However, Eidmann did not perform histological examination of the intestines. Stress and poor husbandry are believed to cause an increase in Giardia and predispose animals to opportunistic gastroenteric bacterial infections, resulting in severe diarrhea

289

7. BIOLOGY AND DISEASES OF OTHER RODENTS

and death. Case reports of Giardia infection in chinchillas are described in Taiwan (Chang et al., 1986). Vos (1970) and Vos and Dobson (1970) studied the etiology, pathology, and host range of Eimeria chinchillae in chinchillas, guinea pigs, hamsters, mice, rats, and rabbits in South Africa. Individual case reports of acute hepatic sarcocystosis in a pet female chinchilla (Rakich et al., 1992) and gastroenteritis associated with a Cryptosporidium sp. in a pet chinchilla (Yamini and Raju, 1986) are described.

ii. Nematodes. Sanford (1989, 1991) reported disease outbreaks of cerebral nematodiasis caused by the raccoon ascarid (Baylisascaris procyonis) in chinchillas in western Canada. d.

Fungal

There are two reports of Histoplasma capsulatum infection in chinchillas. Burtscher and Otte (1962) described histoplasmosis in a chinchilla exported from the United States to Switzerland. Owens et al. (1975) diagnosed histoplasmosis in a female chinchilla originating from a commercial chinchilla ranch in central Missouri. Necropsy lesions revealed multiple foci of pulmonary hemorrhage, consolidation, and bronchopneumonia, with the organism in numerous giant cells; and multifocal pyogranulomatous splenitis and hepatitis, with H. capsulatum in giant cells. Histoplasma capsulatum was cultured from timothy hay used for food. Dermatophytosis is uncommon in chinchillas. Trichophyton mentagrophytes is the dermatophyte most commonly isolated although Microsporum canis and M. gypseum have been incriminated in outbreaks of spontaneously occurring dermatophytosis (Bohm and Loliger, 1969; Gimesi, 1980; Hagen and Gorham, 1972; Horvath and Keri, 1980; Male and Fritsch, 1966; Morganti and Gomez Portugal, 1970). Infected chinchillas show small scaly patches of alopecia on the nose, behind the ears, or on the forefeet. Lesions may appear on any part of the body, and in advanced cases a large circumscribed area of inflammation with scab formation occurs. While most mycological studies of chinchillas are based on animals with clinical signs, fungal cultures of fur-ranched chinchillas show a 5% incidence of T. mentagrophytes in animals with normal skin and a 30% incidence in animals with fur damage (Graham, 1961; Hagen and Gorham, 1972; Male and Fritsch, 1966). 2.

Metabolic/Nutritional Diseases

In the early 1960s the Chinchilla Fur-Breeders Association of England showed that approximately half of all deaths in adult chinchillas were due to disorders of the digestive tract; malocclusion accounted for one-quarter of those deaths (Cousens, 1963; Dall, 1963). Husbandry-related disorders of the digestive tract still remain one of the most frequent problems seen. Lethargy and anorexia are typical clinical signs. Cheek tooth crown

and root abnormalities are common in chinchillas, and breeders call dental malocclusion "slobbers" (Crossley, 1997; Crossley et al., 1997). Crossley has used computerized tomographic (CT) scanning to investigate tooth structure in chinchillas and has shown CT to be a useful tool in the early diagnosis of malocclusion (Crossley et al., 1998). Complications of malocclusion include periodontitis, alveolar periostitis, and alveolar abscessation of maxillary and mandibular cheek teeth (Emily, 1991; Griner, 1983). Chinchillas cannot vomit, and esophageal choke has been described in chinchillas of all ages (Cousens, 1963). Affected animals show drooling, retching, dyspnea, and anorexia. Choke is more common in animals that eat their bedding and in postparturient females that eat their placentas. Bloat or gastric tympany is a problem of lactating females and generally associated with overeating. Affected females are distended and lie on their sides, lethargic and dyspneic. Gastric trichobezoars are often associated with the vice of fur chewing and may be associated with anorexia. In the chinchilla, constipation occurs more often than diarrhea (Hartmann, 1993). Chinchillas with constipation strain to defecate, and the few pellets passed are thin, short, hard, and occasionally bloodstained. The usual cause of constipation is insufficient roughage or fiber (Cousens, 1963). Other causes of constipation may include obesity, intestinal obstruction, and intestinal compression due to large fetuses (Hartmann, 1993). In chronic cases of constipation or gastroenteritis, intestinal torsion and impaction of the cecum or colonic flexure may occur (Bowden, 1959; McGreevy and Carn, 1988). Rectal prolapse may also occur in severe cases of constipation or diarrhea. Among 91 chinchillas necropsied over a 4 year period in a Japanese laboratory, the major causes of death were malocclusion (10%) and prolapse of the rectum (6%) (Kuroiwa and Imamichi, 1977). Pathologists often see fatty liver histopathology without clinical signs or other histopathology in routine necropsies (Egri et al., 1994). Kruckenberg et al. (1975) described clinical toxicities, including adverse effects of alfalfa, in chinchillas (and other rodents and rabbits). Marlow (1995) described a case of diabetes mellitus in a 5-year-old overweight female chinchilla. The animal had a 3 week history of poor appetite, lethargy, and weight loss. Diagnostic workup revealed polydypsia, polyuria, and bilateral cataracts; blood and urine analysis indicated a hyperglycemia greater than 400 mg/dl, a heavy glucosuria, and ketonuria. The chinchilla was euthanatized, and microscopic examination of pancreatic islets showed prominent vacuolation consistent with diagnosis of diabetes mellitus. 3.

Traumatic Disorders

Chinchillas possess a predator-avoidance mechanism known as fur slip. When the animal is fighting or roughly handled, it

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THOMAS M. DONNELLY AND FRED W. QUIMBY

can release a large patch of fur, thus enabling it to escape. A clean, smooth area of skin is left; hair may require several months to regrow. Fur slip should not be confused with the vice of fur chewing seen when chinchillas chew each other's fur, resulting in a moth-eaten coat. A current popular theory suggests that fur chewing is a behavioral disorder. Mothers frequently transmit the vice to offspring. Breeders often suggest that the higher incidence of fur chewing in commercial herds is evidence for maladapted displacement behavior. Eidmann (1992) suggested that affected animals suffer from malnutrition and chew their fur for dietary requirements. Multiple food factors are probably involved in this type of malnutrition, and the exact etiology requires further dietary studies. Previous theories for fur chewing have suggested that fur chewers might have abnormal endocrine activity, as there is increased thyroidal and adrenocortical activity (Vanjonack and Johnson, 1973) and a yet-to-be-discovered fur-breakage fungus (Shaull, 1988). Eidmann (1992) showed that thyroid hyperplasia correlated to the size of chewed fur over the body and interpreted it as a reactive response of the thyroids due to insulation loss following fur removal. Eidmann also concluded that an infectious etiology of fur chewing was unlikely after she compared mycological and bacterial culture of skin and fur from 39 fur chewers with that of 19 healthy chinchillas. During breeding, bite wounds that abscess occur frequently in group-housed animals. Culture of the abscesses often yields Staphylococcus species (Jenkins, 1992). Female chinchillas are larger than males and more aggressive. They are highly selective in their choice of males for mating and will keep "unsuitable" males at bay by urinating, kicking, and biting (Bignami and Beach, 1968; Weir, 1973). Often, bite wounds result in the loss of pieces of ears and toes. Killing a young male housed in the same cage is common for older females (Weir, 1970). Traumatic fractures of the tibia are commonly seen (Hoefer, 1994) and are associated with the animal catching its hindlimb in a cage bar. The tibia is a straight bone longer than the femur and with little soft tissue covering; the fibula is virtually nonexistent. Tibial fractures are either transverse or short spiral and generally are associated with bony fragments. Other traumatic lesions seen in chinchillas include a report by Dall (1967) of a diaphragmatic hernia. 4.

Iatrogenic and Reproductive Disorders

In chinchillas the fine structure of the interhemal membrane of the placental labyrinth is hemomonochorial, consisting of a single layer of syncytial trophoblast. In this respect, the placental labyrinth is similar to that of another caviomorph rodent, the guinea pig (King and Tibbitts, 1976). Tvedten and Langham (1974) described an unusual puerpal disorder of trophoblastic emboli in a chinchilla, resulting in pulmonary embolism. Chinchillas usually give birth early in the morning and only

rarely after midnight (Weir, 1970). Dystocia is usually associated with the presentation of a single, oversized fetus or malpresentation of one or more kits (Cousens, 1963). Uterine inertia has also been reported as a cause of dystocia (Prior, 1986). Chinchillas repond well to cesarian section (Jones, 1990; Prior, 1986; Stephenson, 1990; Caspari, 1990; Sims, 1990). Gitlin and Adler (1969) described an unusual case report of coexisting intrauterine and intraperitoneal pregnancy due to superfetation in a chinchilla. Kuroiwa and Imamichi (1977) followed birthto-weaning mortality in a Japanese laboratory colony of chinchillas. Among 91 animals that died, the cause of death in 23 (25.3%) was neonatal-related. Thirty-seven chinchillas gave birth to 71 kits with an average litter size of 1.9, and 59 kits were successfully weaned (1.6 per litter). Of these kits, 50 ( 1.3 per litter) attained 240 days of age. Male chinchillas that groom excessively, frequently produce small amounts of urine or strain to urinate, and repeatedly clean their penis may have a fur ring (Schaeffer and Donnelly, 1997). This is a ring of hair around the penis and under the prepuce that eventually stops the penis from going back into the prepuce. In severe cases, an engorged penis is seen protruding 4 - 5 cm from the prepuce, resulting in paraphimosis. The condition is painful and may cause urethral constriction and acute urinary retention. Chronic paraphimosis may culminate in infection and severe damage to the penis, affecting the breeding ability of the animal. Getting fur from a female during copulation is the most common cause of fur ring. However, the fur may come from other males or the same animal as the condition is also seen in grouphoused and single-housed males not exposed to females. Males should be examined for fur rings at least 4 times a year; active stud males should be examined every few days. In some male chinchillas the penis will hang out of the prepuce all the time and is not engorged. In these males the cause of this condition is not associated with fur ring but is due to overexcitement brought on by separation from their mates or overexhaustion due to too many females in the same cage. Fur rings can be cut or gently rolled off the penis after applying a sterile lubricant. Occasionally, sedation or anesthesia of the male may be required to remove the fur ring. Six spontaneous cases of oxalate nephrosis have been described in adult female chinchillas (Goudas and Lusis, 1970), and three cases of urinary calculi and urolithiasis in male chinchillas are reported (Jones et al., 1995; Newberne, 1952; Spence and Skae, 1995). The bladder calculi are composed of calcium carbonate (Jones et al., 1995; Spence and Skae, 1995). 5.

Neoplastic Diseases

Despite a life span reported up to 20 years, references on neoplasia in chinchillas are rare. Postmortem examinations on 1005 chinchillas before 1949 and another 1000 chinchillas between 1949 and 1952, ranging in age between less than 6 months and

7. BIOLOGY AND DISEASES OF OTHER RODENTS

11 years, did not list neoplasia as a cause of death (Brenon, 1953, 1955). In contrast, during the 1950s the Annual Reports of the San Diego County Livestock Department listed tumors such as neuroblastoma, carcinoma, lipoma, and hemangioma as occurring in chinchillas. Newberne and Robinson (1957) described a malignant lymphoma in a chinchilla. Schaeffer and Donnelly 1997 reported a uterine leiomyosarcoma in a 1-yearold female chinchilla as an incidental finding at necropsy. 6.

Miscellaneous

a.

Congenital Disorders

DeNooij (1985) described a case of abnormal embryological development in a chinchilla, resulting in a schistosomus reflexus fetus. b.

Age-Related Disorders

Pfeiffer examined the eyes of 14 aged chinchillas. He found that the mean intraocular pressure was 18.5 ___5.75 mm/Hg. He observed bilateral posterior cortical cataracts and asteroid hyalosis in two animals (Peiffer and Johnson, 1980). c.

Husbandry-Related Disorders

Bathing in dust is necessary for the welfare of chinchillas. When chinchillas in captivity are denied dustbathing, the fur becomes matted from oily secretions on the back (Hillyer et al., 1997). Dustbathing often causes irritation of the eyes, resulting in conjunctivitis without clinical signs of upper respiratory infection (Schaeffer and Donnelly, 1997). Trautwein and Helmboldt (1967) described experimental pulmonary talcum granuloma and epithelial hyperplasia in the chinchilla associated with excessive dustbathing. d.

Other

Led and Brandetti (1974) described a case of subcutaneous myiasis by Cuterebra larvae in Argentina.

References

Aguilera, M. (1985). Growth and reproduction in Zygodontomys microtinus (Rodentia, cricetidae) from Venezuela in a laboratory bred colony. Mammalia 49, 75-83. Akai, F., Maeda, M., Hashimoto, S., Taneda, M., and Takagi, H. (1995). A new animal model of cerebral infarction: Magnetic embolization with carbonyl iron particles. Neurosci. Lett. 194, 139-141. Aleksic, S., and Bockemuhl, J. (1990). Microbiology and epidemiology of Yersinia infections. Immun. Infekt. 18, 178-185. Allen, K. L., van Bruggen, N., and Cooper, J. E. (1993). Detection of bacterial sinusitis in the Mongolian gerbil (Meriones unguiculatus) using magnetic resonance imaging. Vet. Rec. 132, 633-635.

291 Aloe, L., Cozzari, C., and Levi-Montalcini, R. (1981). The submaxillary salivary glands of the African rodent Praomys (Mastomys) natalensis as the richest available source of the nerve growth factor. Exp. Cell Res. 133, 475-480. Altmann, D., Schwendenwein, I., and Wagner, K. (1994). Zu Biologie, Haltung, Ernahrung und Erkrankungen des Degus (Octodon degus). Erkrank. Zoot. 36, 277-292. Anderson, L. C. (1982). The capture and laboratory maintenance of hibernating ground squirrels. Lab. Anim. 11, 64-65. Anderson, S., and Jones, J. K., eds. (1984). "Orders and Families of Recent Mammals of the World." Wiley, New York. Anderson, J. E, Johnson, R. C., Magnarelli, L. A., Hyde, E W. and Myers, J. E. (1986). Peromyscus leucopus and Microtus pennsylvanicus simultaneously infected with Borrelia burgdorferi and Babesia microti. J. Clin. Microbiol. 23, 135-137. Anderson, G. W. Jr., Slone, T. W. Jr., and Peters, C. J. (1988). The gerbil, Meriones unguiculatus, a model for Rift Valley fever viral encephalitis. Arch. Virol. 102, 187-196. Anderson, W. I., Steinberg, H., and King, J. M. (1990). Bronchioalveolar carcinoma with renal and hepatic metastases in a degu (Octodon degus). J. Wildl. Dis. 26, 129-131. Andre, E, and Andre, C. (1981). Gastric ulcer disease: Gastric ulcer induced by mucosal anaphylaxis in ovalbumin-sensitized Praomys (Mastomys) natalensis. Am. J. Pathol. 102, 133-135. Anosa, V. O., and Kaneko, J. J. (1984). Pathogenesis of Trypanosoma brucei infection in deer mice (Peromyscus maniculatus). Light and electron microscopic study of testicular lesions. Vet. Pathol. 21, 238-246. Asakawa, M. (1987). Genus Heligmosomoides Hall, 1916 (Heligmosomidae: Nematoda) from the Japanese wood mice, Apodemus spp. 3. The life cycle of Heligmosomoides kurilensis kobayashii (Nadtochii, 1966) in ICR mice and preliminary experimental infection to jirds. J. Coll. Dairying Nat. Sci. (Japan) 12, 131-140. Asavisanu, R., Setasuban, P., and Komalamisra, C. (1985). Experimental infection of Paragonimus heterotremus metacercariae to the Mongolian gerbils (Meriones unguiculatus). Southeast Asian J. Trop. Med. Public Health 16, 344-345. Azazy, A. A., Devaney, E., and Chance, M. L. (1994). A PEG-ELISA for the detection of Leishmania donovani antigen in circulating immune complexes. Trans. R. Soc. Trop. Med. Hyg. 88, 62-66. Babero, B. B., and Cattan, P. E. (1975). Helmintofauna de Chile: III. Parasitos del roedor degu, Octodon degus Molina, 1782, con la descripcion de tres nuevas especies. Bol. Chil. Parasitol. 30, 68-76. Babero, B. B., Cattan, P. E., and Cabello, C. (1975). Trichuris bradleyi sp. n., a whipworm from Octodon degus in Chile. J. Parasitol. 61, 1061-1063. Bacha, W. J. Jr., Roush, R. Jr., and lcardi, S. (1982). Infection of the gerbil by the avian schistosome Austrobilharzia variglandis (Miller and Northrup 1926; Penner 1953). J. Parasitol. 68, 505-507. Baker, R. H. (1968). Habitats and distribution. In "Biology of Peromyscus (Rodentia)" (J. A. King, ed.), pp. 98-121. American Society of Mammalogists, Stillwater, Oklahoma. Banks, R. E., Coviello, G. M., Bowers, T. M., and Sherman, K. E. (1995). Cysticercosis in prairie dogs. Contemp. Top. Lab. Anim. Sci. 34, 96-98. Barber, N., and Thompson, R. L. (1990). Sandbathing reduces for lipids of chinchillas, Chinchilla laniger. Anim. Behav. 39, 403-405. Barker, S. A., Fish, E P., Tomana, M., Garner, L. C., Settine, R. L., and Prchal, J. (1983). Gas chromatographic/mass spectrometric evidence for the identification of a heptitol and an octitol in human and Octodon degus eye lenses. Biochem. Biophys. Res. Commun. 116, 988-993. Barnard, W. P., Ernst, J. V., and Dixon, C. E (1974). Coccidia of the cotton rat, Sigmodon hispidus, from Alabama. J. Parasitol. 60, 406-414. Barnes, B. M. (1989). Freeze avoidance in a mammal: Body temperatures below 0 degree C in an Arctic hibernator. Science 244, 1593-1595. Barthold, S. W. (1980). The microbiology of transmissible colonic hyperplasia. Lab. Anim. Sci. 30, 167-173.

292 Bartoszcze, M., Nowakowska, M., Roszkowski, J., Matras, J., Palec, S., and Wystup, E. (1990). Chinchilla deaths due to Clostridium perfringens A enterotoxin [letter]. Vet. Rec. 126, 341-342. Bartoszcze, M., Matras, J., Palec, S., Roskowski, J., and Wystup, E. (1990). Klebsiella pneumoniae infection in chinchillas [letter]. Vet. Rec. 127, 119. Bartoszyk, G. D., and Hamer, M. (1987). The genetic animal model of reflex epilepsy in the Mongolian gerbil: Differential efficacy of new anticonvulsive drugs and prototype antiepileptics. Pharmacol. Res. Commun. 19, 429-440. Baskaya, M. K., Dogan, A., and Dempsey, R. J. (1999). Application of endovascular suture occlusion of middle cerebral artery in gerbils to obtain consistent infarction. Neurol. Res. 21, 574-578. Bates, M., and Weir, J. M. (1944). The adaptation of a cane rat (Zygodontomys) to the laboratory and its susceptibility to the virus of yellow fever. Am. J. Trop. Med. Hyg. 24, 35-37. Bayssade Dufour, C., Vuong, P. N., Farhati, K., Picot, H., and Albaret, J. L. (1994). Speed of skin penetration and initial migration route of infective larvae of Schistosoma haematobium in Meriones unguiculatus. Comptes Rendus Acad. Sci. Serie lii Sci. Vie 317, 529-533. Beacham, B. E., Romito, R. and Kay, H. D. (1982). Vaccination of the African white-tailed rat, Mystromys albacaudatus, with sonicated Leishmania braziliensis panamensis promastigotes. Am. J. Trop. Med. Hyg. 31, 252-258. Beare, A. S. (1975). Myxoviruses. Dev. Biol. Stand. 28, 3-17. Becker, S. V. and Middleton, C. C. (1979). Organ weights and organ:body weight ratios of the African white-tailed rat (Mystromys albicaudatus). Lab. Anim. Sci. 29, 44-47. Becker, S. V., Schmidt, D. A. and Middleton, C. C. (1979). Selected biological values of the African white-tailed sand rat. Lab. Anim. Sci. 29, 479-481. Belosevic, M., Faubert G. M., MacLean, J. D., Law, C., and Croll, N. A. (1983). Giardia lamblia infections in Mongolian gerbils: An animal model. J. Infect. Dis. 147, 222-226. Benitz, K. F., and Kramer, A. W. Jr. (1965). Spontaneous tumors in the Mongolian gerbil. Lab. Anim. Care 15, 281-294. Bennett, M., Crouch A. J., Begon, M., Duffy, B., Feore, S., Gaskell, R. M., Kelly, D. F., McCraken, C. M., Vicary, L., and Baxby, D. (1997). Cowpox in British voles and mice. J. Comp. Pathol. 116, 35-44. Beregi, A., Felkai, E, Seregi, J., and Sarosi, L. (1994). Medullary fixation of a tibial fracture in a three-month-old degu (Octodon degus). Vet. Rec. 134, 652-653. Berger, P. J., Negus, N. C., and Rowsemitt, C. N. (1987). Effect of 6-methoxybenzoxazolinone on sex ratio and breeding performance in Microtus montanus. Biol. Reprod. 36, 255-260. Best, T. L. (1972). Mound development by a pioneer population of the bannertailed kangaroo rat, Dipodomys spectabilis baileyi Goldman in eastern New Mexico. Am. Midl. Nat. 87, 201-206. Beucher, E, and Charreire, J. (1983). Lymphoid cells from the rodent Praomys (Mastomys) natalensis express antigenic determinants present on mouse and rat lymphocytes. Immunol. Lett. 7, 99-106. Bianco, A. E., Mustafa, M. B., and Ham, P. J. (1989). Fate of developing larvae of Onchocerca linealis and O. volvulus in micropore chambers implanted into laboratory hosts. J. Helminthol. 63, 218-226. Bignami, G., and Beach, E A. (1968). Mating behaviour in the chinchilla. Anim. Behav. 16, 45-53. Bingel, S. A. (1995). Pathologic findings in an aging Mongolian gerbil (Meriones unguiculatus) colony. Lab. Anim. Sci. 45, 597-600. Bintz, G. L. (1969). Tissue catabolism by laboratory rats and Spermophilus lateralis during acute negative water balance. J. Mammal. 50, 355-356. Bintz, G. L., Rosebery, H. W., and Bintz, L. B. (1979). Glycogen levels in field and laboratory-acclimated Richardson ground squirrels. Comp. Biochem. Physiol. A Comp. Physiol. 62, 339-342. Birney, E. C., and Twomey, S. L. (1970). Effects of sodium chloride on water consumption, weight, and survival in the woodrats, Neotoma micropus and Neotoma floridana. J. Mammal. 51, 372-375. Bischoff, J. R., Kirn, D. H., Williams, A., Heise, C., Horn, S., Muna, M., Ng, L., Nye, J. A., Sampson-Johannes, A., Fattaey, A., and McCormick, E

THOMAS M. DONNELLY AND FRED W. QUIMBY

(1996). An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 274, 373-376. Block, S. B., and Zimmerman, E. G. (1991). Allozymic variation and systematics of plains pocket gophers (Geomys) of south-central Texas. Southwest. Nat. 36, 29-36. Boggs, J. F., McMurry, S. T., Leslie, D. M. Jr., Engle, D. M., and Lochmiller, R. L. (1991). Influence of habitat modification on the community of gastrointestinal helminths of cotton rats. J. Wildl. Dis. 27, 584-593. Bohm, K. H., and Loliger, C. (1969). The distribution of dermatophytes in fur animals (mink and chinchilla). Zentralbl. Veterinarmed. [B] 16, 775-783. Botvinkin, A. D., Nikforova, T. A., and Sidorov, G. N. (1985). Experimental rabies in hibernator rodents. Acta Virol. 29, 44-50. Bowden, R. S. T. (1959). Diseases of chinchillas. Vet. Rec. 71, 1033-1039. Bowe, C. A., Miller, J. D., and Green, L. (1987). Qualities and locations of stimuli and responses affecting discrimination learning of chinchillas and pigeons. J. Comp. Psychol. 101, 132-138. Bozinovic, E, Novoa, E E, and Sabat, P. (1997). Feeding and digesting fiber and tannins by an herbivorous rodent, Octodon degus (Rodentia: Caviomorpha). Comp. Biochem. Physiol. A Physiol. 118, 625-630. Brahd, L. R., and Ryckman, R. E. (1968). Laboratory life histories of Peromyscus eremicus and Peromyscus interparietalis. J. Mammal. 49, 495501. Brant, S. V., and Gardner, S. L. (2000). Phylogeny of species of the genus Litomosoides (Nematatoda: Onchocercidae): Evidence of rampant host switching. J. ParasitoL 86, 545-554. Braun, K., Lange, E., Metzger, M., and Poeggel, G. (2000). Maternal separation followed by early social deprivation affects the development of monoaminergic fiber systems in the medial prefrontal cortex of Octodon degus. Neuroscience 95, 309-318. Brenon, H. C. (1953). Postmortem examination of chinchillas. J. Am. Vet. Med. Assoc. 121, 310. Brenon, H. C. (1955). Postmortem examination of chinchillas. J. Am. Vet. Med. Assoc. 123, 222-223. Bresnahan, J. E, Smith, G. D., Lentsch, R. H., Barnes, W. G., and Wagner, J. E. (1983). Nasal dermatitis in the Mongolian gerbil. Lab. Anim. Sci. 33, 258263. Bronson, E H., and DeLa Rosa, J. (1994). Tonic- clonic convulsions in meadow voles. Physiol. Behav. 56, 683-685. Bronson, E H., and Heideman, P. D. (1992). Lack of reproductive photoresponsiveness and correlative failure to respond to melatonin in a tropical rodent, the cane mouse. Biol. Reprod. 46, 246-250. Broughton, G. D. (1992). Hematologic and blood chemistry data for the prairie dog (Cynomys ludovicianus). Comp. Biochem. Physiol. Comp. PhysioL 101, 807-812. Buckmaster, P. S., Jongen-Relo, A. L., Davari, S. B., and Wong, E. H. (2000). Testing the disinhibition hypothesis of epileptogenesis in vivo and during spontaneous seizures. J. Neurosci. 20, 6232-6240. Burger, J., and Gochfeld, M. (1992). Survival and reproduction in Peromyscus leucopus in the laboratory: Viable model for aging studies. Growth Dev. Aging 56, 17-22. Burgess, E. C., Amundson, T. E., Davis, J. P., Kaslow, R. A., and Edelman, R. (1986). Experimental inoculation of Peromyscus spp. with Borrelia burgdorferi evidence of contact transmission. Am. J. Trop. Med. Hyg. 35, 355-359. Burkot, T. R., Clover, J. R., Happ, C. M., DeBess, E., and Maupin, G. O. (1999). Isolation of Borrelia burgdorferi from Neotoma fuscipes, Peromyscus maniculatus, Peromyscus boylii, and Ixodes pacificus in Oregon. Am. J. Trop. Med. Hyg. 60, 453-457. Burtscher, H., and Otte, E. (1962). Histoplasme beim chinchilla (Histoplasma in the chinchilla). Dtsch. Tierartzl. Wochenschr. 69, 303-307. Butterworth, B. B. (1961). The breeding of Dipodomys deserti in the laboratory. J. Mammal. 42, 413-414. Cameron, G. N., and Eshelman, B. D. (1996). Growth and reproduction of hispid cotton rats (Sigmodon hispidus) in response to naturally occurring levels of dietary protein. J. mammal. 77, 220-231.

7. BIOLOGY AND DISEASES OF OTHER RODENTS

Cameron, G. N., and Rainey, D. G. (1972). Habitat utilization by Neotoma lepida in the Mohave Desert. J. Mammal. 53, 251-266. Cameron, G. N., and Spencer, S. R. (1981). Sigmodon hispidus. Mammal. Species 158, 1-9. Campbell, G. A., Kosanke, S. D., Toth, D. M., and White, G. L. (1981). Disseminated staphylococcal infection in a colony of captive ground squirrels (Citellus lateralis). J. Wildl. Dis. 17, 177-181. Campbell, G. D., Barker, I. K., Johnson, R. P., Shewen, P. E., McEwen, S. A., and Surgeoner, G. A. (1994). Response of the meadow vole (Microtus pennsylvanicus) to experimental inoculation with Borrelia burgdorferi. J. Wildl. Dis. 30, 408-416. Cantrell, C., and Padovan, D. (1987). Hematology of Mystromys albicaudatus. Lab. Anim. Sci. 21, 326-329. Carleton, M. D. (1984). Introduction to rodents. In "Orders and Families of Recent Mammals of the World" (S. Anderson and J. K. Jones, eds.), pp. 255265. Wiley, New York. Caspari, E. L. (1990). Caesarean section in a chinchilla [letter]. Vet. Rec. 126, 490. Castro, A., Chinchilla, M., Guerrero, O. M., and Gonzalez, R. (1998). Eimeria species (Eucoccidida: Eimeriidae) found in the cotton rat Sigmodon hispidus in Costa Rica. Rev. Biol. Trop. 46, 339-340. Cavill, J. P. (1967). Listeriosis in chinchillas (Chinchilla laniger). Vet. Rec. 80, 592-594. Chandra, A. M., Paranjpe, M. G., Quails, C. W. Jr., and Lochmiller, R. L. (1993). Calcification of the urinary bladder in the wild cotton rat (Sigmodon hispidus). J. Comp. PathoL 109, 197-201. Chang, W. E, Liu, C. H., and Du, S. J. (1986). Giardiasis of chinchillama case report. J. Chin. Soc. Vet. Sci. 12, 149-152. Chatterjee, R. K., Singh, D. P., and Misra, S. (1988). Dipetalonema viteae in Mastomys natalensis: Effect on pregnancy and lactation on establishment and course of infection. Trop. Med. Parasitol. 39, 29-30. Chatterjee, R. K., Fatma, N., Agarwal, V. K., Sharma, S., and Anand, N. (1989). Comparative antifilarial efficacy of the N-oxides of diethylcarbamazine and two of its analogues. Trop. Med. Parasitol. 40, 474-475. Chen, H. C., Slone, T. W. Jr., and Frith, C. H. (1992). Histiocytic sarcoma in an aging gerbil. Toxicol. Pathol. 20, 260-263. Chen, Q., De Petris, G., Yu, P., Amaral, J., Biancani, P., and Behar, J. (1997). Different pathways mediate cholecystokinin actions in cholelithiasis. Am. J. Physiol. 272, G838-G844. Chess, T., and Chew, R. M. (1971). Weight maintenance of the desert woodrat (Neotoma lepida) on some natural foods. J. Mammal. 52, 193-195. Chew, R. M. (1958). Reproduction by Dipodomys merriami in captivity. J. Mammal. 39, 597-598. Childs, J. E., Colby, L., Krebs, J. W., Strine, T., Feller, M., Noah, D., Drenzek, C., Smith, J. S., and Rupprecht, C. E. (1997). Surveillance and spatiotemporal associations of rabies in rodents and lagomorphs in the United States, 1985-1994. J. Wildl. Dis. 33, 20-27. Clark, G. R. (1978). Hydrovarium in a Mongolian gerbil (Meriones unguiculatus). Vet. Med. Small Anim. Clin. 73, 792-793. Clark, J. D. (1984). Biology and diseases of other rodents. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), 1st ed., pp. 183-206. Academic Press, San Diego. Clark, J. D., Loew, E M., and Olfert, E. D. (1978). Rodents. In "Zoo and Wild Animal Medicine" (M. Fowler, ed.), Saunders, Philadelphia. Clark, M. M., Spencer, C. A., and Galef, B. G. Jr. (1986). Improving the productivity of breeding colonies of Mongolian gerbils (Meriones unguiculams). Lab. Anim. 20, 313-315. Clarke, J. W. (1975). Androgen control of the ventral scent gland in Neotoma floridana. J. Endocrinol. 64, 393-394. Coehlo, P. M. Z., Gassinelli, G., and Pellegrino, J. (1980). Schistosoma mansoni: Host antigen occurrence in worms recovered from laboratory vertebrate animals. J. Parasitol. 81, 349-354. Cohen, M. E., and Meyer, D. M. (1993). Effect of dietary vitamin E supplementation and rotational stress on alveolar bone loss in rice rats. Arch. Oral Biol. 38, 601-606.

293 Cohen, B. I., and Mosbach, E. H. (1993). Cholesterol cholelithiasis. Adv. Vet. Sci. Comp. Med. 37, 289- 312. Cole, H. F., and Wolf, H. H. (1970). Laboratory evaluation of aggressive behavior of the grasshopper mouse (Onychomys). J. Pharm. Sci. 59, 969, 971. Conder, G. A., Johnson, S. S., Guimond, P. M., Geary, T. G., Lee, B. L., Winterrowd, C. A., Lee, B. H., and DiRoma, P. J. (1991). Utility of a Haemonchus contortus/jird (Meriones unguiculatus) model for studying resistance to levamisole. J. Parasitol. 77, 83-86. Constant, M. A., and Phillips, P. H. (1952a). The occurrence of a calcinosis syndrome in the cotton rat. I. The effect of diet on the ash content of the heart. J. Nutr. 47, 317-326. Constant, M. A., and Phillips, P. H. (1952b). The occurrence of a calcinosis syndrome in the cotton rat. II. Pathology. J. Nutr. 47, 327-339. Contreras, L., and Bustos-Obregon, E. (1980). Anatomy of reproductive tract in Octodon degus Molina: A nonscrotal rodent. Arch. Androl. 4, 115-124. Cook-Babcook, M.-A. (1996). Enrichment activities for thirteen-lined ground squirrels (Spermophilus tridecemlineatus). Wildl. Rehab. 14, 143 - 154. Coolen, J., Solleveld, H. A., van Rossum, A. L., and Zurcher, C. (1981). Naturally occurring autoantibodies in aged Praomys (Mastomys) natalensis. Clin. Immunol. Immunopathol. 19, 238-249. Cornelis, G., Laroche, Y., Balligand, G., Sory, M. P., and Wauters, G. (1987). Yersinia enterocolitica, a primary model for bacterial invasiveness. Rev. Infect. Dis. 9, 64-87. Court, J. P., Lees, G. M., Coop, R. L., Angus, K. W., and Beesley, J. E. (1988). An attempt to produce Ostertagia circumcincta infections in Mongolian gerbils. Vet. Parasitol. 28, 79-91. Cousens, P. J. (1963). The chinchilla in veterinary practice. J. Small. Anim. Pract. 4, 199-205. Cramlet, S. H., Toft, J. D. D., and Olsen, N. W. (1974). Malignant melanoma in a black gerbil (Meriones unguiculatus). Lab. Anim. Sci. 24, 545-547. Cross, J. H., Banzon, T., and Singson, C. (1978). Further studies on Capillaria philippinensis: Development of the parasite in the Mongolian gerbil. J. ParasitoL 64, 208-213. Cross, J. H., Partono, E, Hsu, M. Y. K., Ash, L. R., and Oemijati, S. (1981). Further studies on the development of Wuchereria bancrofti in laboratory animals. Southeast Asian J. Trop. Med. Public Health 12, 114-122. Crossley, D. A. (1997). Dental disease in chinchillas [letter]. Vet. Rec. 140, 512. Crossley, D. A., Dubielzig, R. R., and Benson, K. G. (1997). Caries and odontoclastic resorptive lesions in a chinchilla (Chinchilla laniger). Vet. Rec. 141, 337-339. Crossley, D. A., Jackson, A., Yates, J., and Boydell, I. P. (1998). Use of computed tomography to investigate cheek tooth abnormalities in chinchillas (Chinchilla laniger). J. Small Anim. Pract. 39, 385-389. Cullen, J. M., and Marion, P. L. (1996). Non-neoplastic liver disease associated with chronic ground squirrel hepatitis virus infection. Hepatology 23, 1324-1329. Cullen, J. W., Pare, W. P., and Mooney, A. L. (1971). Adrenal weight to body weight ratios in the Mongolian gerbil (Meriones unguiculatus). Growth 35, 169-176. Cutler, R. G. (1985). Peroxidase producing potential of tissues. Inverse correlation with longevity of mammalian species. Proc. Natl. Acad. Sci. USA 82, 4798-4802. Czub, S., Duray, P. H., Thomas, R. E., and Schwan, T. G. (1992). Cystitis induced by infection with the Lyme disease spirochete, Borrelia burgdorferi, in mice. Am. J. Pathol. 141, 1173-1179. Dall, J. (1963). Diseases of the chinchilla. J. Small Anim. Prac. 4, 207212. Dall, J. A. (1967) Diaphragmatic hernia in a chinchilla. Vet Rec. 81, 599. Daly, M. M., Wilson, I., and Behrends, P. (1984). Breeding of captive kangaroo rats, Dipodomys merriami and D. microps. J. Mammal. 65, 338-341. Dancer, A. L. (1995). Use of animal models in the study of the effects of noise on hearing. Occup. Med. 10, 535-544. Datiles, M. B. D., and Fukui, H. (1989). Cataract prevention in diabetic Octodon degus with Pfizer's sorbinil. Curr. Eye Res. 8, 233-237.

294 Davis, D. E. (1984). Pitfalls in the use of ground squirrels for research on hibernation. Can. J. Zool. 62, 1656-1658. Davis, R. M. (1999). Use of orally administered chitin inhibitor (lufenuron) to control flea vectors of plague on ground squirrels in California. J. Med. Entomol. 36, 562-567. Davis, T. M. (1975). Effects of familiarity on agonistic encounter behavior in male degus (Octodon degus). Behav. Biol. 14, 511-517. Davis, J. W., Karstad, L. H., and Trainer, D. O. (1981). "Infectious Diseases of Wild Animals," 2nd ed. Iowa State Univ. Press, Ames. Day, B. N., Egoscue, H. J., and Woodbury, A. M. (1956). Ord kangaroo rat in captivity. Science 124, 485-486. Dean, W. R. J. (1978). Conservation of the white-tailed rat in South Africa. Biol. Conserv. 13, 133-140. Dearing, M. D., Mangione, A. M., Karasov, W. H., Morzunov, S., Otteson, E., and St. Jeor, S. (1998). Prevalence of hantavirus in four species of Neotoma from Arizona and Utah. J. Mammal. 79, 1254-1259. De La Puente-Redondo, V. A., Gutierrez-Martin, C. B., Perez-Martinez, C., Garcia Del Blanco, N., Garcia-Iglesias, M. J., Perez-Garcia, C. C., and Rodriguez-Ferri, E. E (1999). Epidemic infection caused by Citrobacter rodentium in a gerbil colony. Vet. Rec. 145, 400-403. DeMaria, T. E (1989). Animal models for nontypical Haemophilus influenza otitis media. Pediatr. Infect. Dis. J. 8, $40-$42. Deng, S., Wang, P., Yan, X., and Song, C. (1994). Studies on specific diagnostic antigen for filariasis. Chin. J. Parasitol. Paras#. Dis. 12, 210-213. DeNooij, P. P. (1985). Schistosomus reflexus in a chinchilla kit. Mod. Vet. Pract. 66, 23-27. Dettman, C. D., Higgins-Opitz, S. B., and Bronner, G. N. (1987). A comment on the taxonomic status and correct nomenclature of laboratory colonies of Mastomys. S. Afr. J. Sci. 83, 395. Dewsbury, D. A. (1974a). The use of muroid rodents in the psychology laboratory. Behav. Res. Meth. Instr. 6, 301-308. Dewsbury, D. A. (1974b). Copulatory behavior of Neotoma floridana. J. Mammal. 55, 864-866. Dewsbury, D. A. (1975). Copulatory behavior of white-footed mice (Peromyscus leucopus). J. Mammal. 56, 420-428. Dewsbury, D. A. (1984). Muroid rodents as research animals. ILAR News 28, 8-15. Dewsbury, D. A., and Jansen, P. E. (1972). Copulatory behavior of southern grasshopper mice (Onychomys torridus). J. Mammal. 53, 267-278. Dewsbury, D. A., Estep, D. Q., and Lanier, D. L. (1977). Estrous cycles of nine species of muroid rodents. J. Mammal. 58, 89-92. Dieterich, R. A., and Preston, D. J. (1977a). The meadow vole (Microtus pennsylvanicus) as a laboratory animal. Lab. Anim. Sci. 27, 494-499. Dieterich, R. A., and Preston, D. J. (1977b). The red-backed vole (Clethrionomys rutilus) as a laboratory animal. Lab. Anim. Sci. 27, 507-511. Dieterich, R. A., and Preston, D. J. (1977c). The tundra vole (Microtus oeconomus) as a laboratory animal. Lab. Anim. Sci. 27, 500-506. Dieterich, R. A., and Preston, D. J. (1979). Atherosclerosis in lemmings and voles fed a high fat, high cholesterol diet. Atherosclerosis 33, 181189. Ding, D. L., Wang, J., Salvi, R., Henderson, D., Hu, B. H., McFadden, S. L., and Mueller, M. (1999). Selective loss of inner hair cells and type-I ganglion neuron. Ann. N.Y. Acad. Sci. 884, 152-170. Dixon, D., Reinhard, G. R., Kazacos, K. R., and Arriaga, C. (1988). Cerebrospinal nematodiasis in prairie dogs from a research facility. J. Am. Vet. Med. Assoc. 193, 251-253. Doerning, B. J., Brammer, D. W., and Rush, H. G. (1993). Pseudomonas aeruginosa infection in a Chinchilla laniger. Lab. Anim. 27, 131-133. Donnelly, T. M. (1997). Disease problems of small rodents. In "Ferrets, Rabbits and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 307-327. Saunders, Philadelphia. Donnelly, T. M., Rush, E. M., and Lackner, P. A. (2000). Ringworm in small exotic pets. Semin. Avian Exotic Pet Med. 9, 82-93. Dotson, R. L., Leveson, J. E., Marengo-Rowe, A. J., and Ubelaker, J. E. (1987).

THOMAS M. DONNELLY AND FRED W. QUIMBY Hematologic and coagulation studies on cotton rats, Sigmodon hispidus. Comp. Biochem. Physiol. A 88, 553-556. Dowda, H., DiSalvo, A. E, and Redden, S. (1981). Naturally acquired rabies in an Eastern wood rat (Neotomafloridana). J. Clin. Microbiol. 13, 238-239. Downs, C. T., and Perrin, M. R. (1995). The thermal biology of the white-tailed rat Mystromys albicaudatus, a cricetine relic in southern African grassland. Comp. Biochem. Physiol. A Comp. Physiol. A 110, 65-69. Downs, W. G., Spence, L., and Aitken, T. H. (1962). Studies on the virus of Venezuelan equine encephalomyelitis in Trindidad, W. I. III. Reisolation of virus. Am. J. Trop. Med. Hyg. 11, 841-843. Drickamer, L. C., and Vestal, B. M. (1973). Patterns of reproduction in a laboratory colony of Peromyscus. J. Mammal. 54, 523-528. Drummond, T. D., Mason, J. I., and McCarthy, J. L. (1988). Gerbil adrenal 11 beta- and 19-hydroxylating activities respond similarly to inhibitory or stimulatory agents: Two activities of a single enzyme. J. Steroid Biochem. 29, 641-648. Dubey, J. P., and Sheffield, H. G. (1988). Sarcocystis sigmodontis n. sp. from the cotton rat (Sigmodon hispidus). J. Parasitol. 74, 889-891. Duplantier, J. M., Britton-Davidian, J., and Granjon, L. (1990). Chromosomal characterization of three species of the genus Mastomys in Senegal. Z. ZooI. Syst. Evol. 28, 289-298. Durden, L. A. (1988). The spiny rat louse, Polyplax spinulosa, as a parasite of the rice rat, Oryzomys palustris, in North America. J. Parasitol. 74, 900901. Durden, L. A., Klompen, J. S., and Keirans, J. E. (1993). Parasitic arthropods of sympatric opossums, cotton rats, and cotton mice from Merritt Island, Florida. J. Parasitol. 79, 283-286. Durden, L. A., Banks, C. W., Clark, K. L., Belbey, B. V., and Oliver, J. H. Jr. (1997). Ectoparasite fauna of the Eastern woodrat, Neotoma floridana: Composition, origin, and comparison with ectoparasite faunas of Western woodrat species. J. Parasitol. 83, 374-381. Eckert, J., Thompson, R. C. A., and Mehlhorn, H. (1983). Proliferation and metastases formation of larval Echinococcus multilocularis. 1. Animal model, macroscopical and histological findings. Z. Parasitenkd. 69, 737748. Edmonds, K. E., and Stetson, M. H. (1993). Effect of photoperiod on gonadal maintenance and development in the marsh rice rat (Oryzomys palustris). Gen. Comp. Endocrinol. 92, 281-291. Edmonds, K. E., and Stetson, M. H. (1994). Photoperiod and melatonin affect testicular growth in the marsh rice rat (Oryzomys palustris). J. Pineal Res. 17, 86-93. Edmonds, K. E., Rollag, M. D., and Stetson, M. H. (1995). Effects of photoperiod on pineal melatonin in the marsh rice rat (Oryzomys palustris). J. Pineal Res. 18, 148-153. Egoscue, H. J. (1960). Laboratory and field studies of the northern grasshopper mouse. J. Mammal. 4, 99-110. Egri, B., Egri, J., and Hajnovics, B. (1994). Uber Fettinfiltration der Leber beim Chinchillabockchen (Chinchilla velligera). Tierarztl. Umsch. 49, 45-47. Eidmann, S. (1992). Untersuchungen zur Atiologie und Pathogenese von Fellscaden beim Chinchilla (Studies on etiology and pathogenesis of fur damages in the chinchilla). Thesis (D.V.M.), Institut fur Pathologie der Tierarzlichen Hochschule, Hannover, Germany. Eisenberg, J. E, and Isaac, D. E. (1963). The reproduction of heteromyid rodents in captivity. J. Mammal. 44, 61-67. Elangbam, C. S., Quails, C. W. Jr., Lochmiller, R. L., and Novak, J. (1989). Development of the cotton rat (Sigmodon hispidus) as a biomonitor of environmental contamination with emphasis on hepatic cytochrome P-450 induction and population characteristics. Bull. Environ. Contam. Toxicol. 42, 482-488. Elangbam, C. S., Quails, C. W. Jr., Lochmiller, R. L., and Boggs, J. E (1990). Strongyloidiasis in cotton rats (Sigmodon hispidus) from central Oklahoma. J. Wildl. Dis. 26, 398-402. Elangbam, C. S., Quails, C. W. Jr., Ewing, S. A., and Lochmiller, R. L. (1993).

7. BIOLOGY AND DISEASES OF OTHER RODENTS

Cryptosporidiosis in a cotton rat (Sigmodon hispidus). J. Wildl. Dis. 29, 161-164. Emily, P. (1991). Problems peculiar to continually erupting teeth. J. Small Exotic Anim. Med. 1, 56-59. Ernst, J. V., and Chobotar, B. (1978). The endogenous stages of Emeria utahensis (Protozoa: Eimeriidae) in the kangaroo rat, Dipodomys ordii. J. Parasitol. 64, 27-34. Escherich, P. C. (1981). "Social Biology of the Bushy-Tailed Woodrat, Neotoma cinerea." Univ. of California Press, Berkeley. Fain, A., and Lukoschus, E S. (1978). Dipodomydectes americanus gen. et sp. n. (Acari: Hypoderidae) from the kangaroo rat. J. Parasitol. 64, 137138. Faith, R. E., Montgomergy, C. A., Durfee, W. J., Aguilar-Cordova, E., and Wyde, P. R. (1997). The cotton rat in biomedical research. Lab. Anim. Sci. 47, 337-345. Fan, Z. W., Eng, J., Miedel, M., Hulmes, J. D., Pan, Y., Yalow, R. S. (1987). Cholecystokinin octapeptides purified from chinchilla and chicken brains. Brain Res. Bull. 18, 757-760. Farrar, P. L., Opsomer, M. J., Kocen, J. A., and Wagner, J. E. (1988). Experimental nasal dermatitis in the Mongolian gerbil: Effect of bilateral harderian gland adenectomy on development of facial lesions. Lab. Anim. Sci. 38, 72-76. Faubert, G. M., Belosevic, M., Walker, T. S., MacLean, J. D., and Meerovitch, E. (1983). Comparative studies on the pattern of infection with Giardia spp. in Mongolian gerbils. J. Parasitol. 69, 802-805. Fine, J., Quimby, E W., and Greenhouse, D. D. (1986). Annotated bibliography on uncommonly used laboratory animals: Mammals. ILAR News 24, 3-38. Finley, G. G., and Long, J. R. (1977). An epizootic of listeriosis in chinchillas. Can. Vet. J. 18, 164-167. Fisher, R. S. (1989). Animal models of the epilepsies. Brain Res. Rev. 14, 245278. Fox, J. G., and Murphy, J. C. (1979). Cytomegalic virus-associated insulitis in diabetic Octodon degus. Vet. Pathol. 16(5), 625-628. Frank, C. L. (1992). The influence of dietary fatty acids on hibernation by golden-mantled ground squirrels (Spermophilus lateralis). Physiol. ZooL 65, 906-920. Franke, E. D., McGreevy, P. B., Katz, S. P., and Sacks, D. L. (1985). Growth cycle- dependent generation of complement-resistant Leishmania promastigotes. J. Immunol. 134, 2713-2718. Frase, B. A., and Van Vuren, D. (1989). Techniques for immobilizing and bleeding marmots and woodrats. J. Wildl. Dis. 25, 444-445. Frerichs, K. U. (1999). Neuroprotective strategies in nature--novel clues for the treatment of stroke and trauma. Acta Neurochir. Suppl. 73, 57-61. Frost, D., and Zucker, I. (1983). Photoperiod and melatonin influence seasonal gonadal cycles in the grasshopper mouse (Onychomys leucogaster). J. Reprod. Fertil. 69, 237-244. Frye, M. S., and Hedges, S. B. (1995). Monophyly of the order Rodentia inferred from mitochondrial DNA sequences of the genes for 12S rRNA, 16S rRNA, and tRNA-valine. Mol. Biol. Evol. 12, 168-176. Fujita, O., Oku, Y., Okamoto, M., Sato, H., Ooi, H. K., Kamiya, M., and Rausch, R. L. (1991). Early development of larval Taenia polyacantha in experimental intermediate hosts. J. Helminthol. Soc. Wash. 58, 100-109. Fulghum, R. S., and Chole, R. A. (1985). Bacterial flora in spontaneously occurring aural cholesteatomas in Mongolian gerbils. Infect. Immun. 50, 678-681. Fulhorst, C. E, Bowen, M. D., Ksiazek, T. G., Rollin, P. E. Nichol, S. T., Kosoy, M. Y., and Peters, C. J. (1996a). Isolation and characterization of Whitewater Arroyo virus, a novel North American arenavirus. Virology 224, 114-120. Fulhorst, C. E, Hardy, J. L., Eldridge, B. E, Chiles, R. E., and Reeves, W. C. (1996b). Ecology of Jamestown canyon virus (Bunyaviridae: California serogroup) in coastal California. Am. J. Trop. Med. Hyg. 55, 185-189. Fulhorst, C. E, Ksiazek, T. G., Peters, C. J., and Tesh, R. B. (1999). Experimental infection of the cane mouse Zygodontomys brevicauda (family

295 Muridae) with Guanarito virus (Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever. J. Infect. Dis. 180, 966-969. Gage, K. L., Burgdorfer, W., and Hopla, C. E. (1990). Hispid cotton rats (Sigmodon hispidus) as a source for infecting immature Dermacentor variabilis (Acari: Ixodidae) with Rickettsia rickettsii. J. Med. Entomol. 27, 615-619. Galaviz-Silva, L., and Arredondo Cantu, J. M. (1992). First report on Neotoma micropus (Rodentia) as a reservoir of Trypanosoma cruzi in Mexico. Bol. Chil. Parasitol. 47, 54-57. (In Spanish.) Galea, L. A., Kavaliers, M., and Ossenkopp, K. P. (1996). Sexually dimorphic spatial learning in meadow voles Microtus pennsylvanicus and deer mice, Peromyscus maniculatus. J. Exp. Biol. 199, 195-200. Galli, S. M., and Marusic, E. T. (1976). Adrenal steroid biosynthesis by two species of South American rodents: Octodon degus and Abrocoma benetti. Gen. Comp. Endocrinol. 28, 10-16. Garcia, V. E., and Perez, J. C. (1984). The purification and characterization of an antihemorrhagic factor in woodrat (Neotoma micropus) serum. Toxicon 22, 129-138. Gattermann, R. (1979). Hematologic and clinical chemical normal ranges of the Mongolian gerbil (Meriones unguiculatus). Z. Versuchstierkunde 21, 273275. (In German.) Gaule, C., Vega, P., Sanchez, E., and Del Villar, E. (1990). Drug metabolism in Octodon degus: Low inductive effect of phenobarbital. Comp. Biochem. PhysioL C 96, 217-222. Giebink, G. S. (1999). Otitis media: The chinchilla model. Microb. Drug Resist. 5,57-72. Gielecki, J. S., Brudnicki, W., and Nowaki, M. R. (1996). Digital image analysis of the brain-base arteries in chinchilla: Chinchilla laniger. Anat. Histol. Embryol. 25, 117-119. Gimesi, A. (1980). Isolation of pathogenic dermatophytes from chinchillas, guinea pigs and calves (preliminary report). Magy. Allatorvosok Lapja 35, 591. Gitlin, G., and Adler, J. H. (1969). Coexisting intrauterine and abdominal (intraperitoneal) pregnancy with possible superfoetation (superfecundation) and with adhesion of placenta to foetus in a Chinchilla (Chinchilla laniger). Acta Zool. Pathol. Antverp. 49, 65-76. Goeken, J. A., Packer, J. T., Rose, S. D., and Stuhlman, R. A. (1972). Structure of the islets of Langerhans. Pathological studies in normal and diabetic Mystromys albicaudatus. Arch. PathoL 93, 123-129. Goel, N., and Lee, T. M. (1997). Social cues modulate free-running circadian activity rhythms in the diurnal rodent, Octodon degus. Am. J. Physiol. 273, R797-R804. Goldberg, J. M., Baird, R. A., and Fernandez, C. (1985). Morphophysiological studies of the mammalian vestibular labyrinth. Prog. Clin. Biol. Res. 176, 231-245. Goodwin, H. T. (1998). Supernumerary teeth in Pleistocene, recent, and hybrid individuals of the Spermophilus richardsonii complex (Sciuridae). J. Mammal. 79, 1161-1169. Goudas, P., and Giltoy, J. S. (1970). Spontaneous herpes-like viral infection in a chinchilla (Chinchilla laniger). J. Wildl. Dis. 6, 175-179. Goudas, P., and Lusis, P. (1970). Case report. Oxalate nephrosis in chinchilla (Chinchilla laniger). Can. Vet. J. 11, 256-257. Grace, P. A., McShane, J., and Pitt, H. A. (1988). Gross anatomy of the liver, biliary tree, and pancreas in the black-tailed prairie dog (Cynomys ludovicianus). Lab. Anim. 22, 326-329. Graeff Teixeira, C., Garrido, C. T., Santos, E T., and Aguiar, L. E S. (1998). The using of hollow bricks as a shelter for adaptation of wild rodents' colonies in captivity. Rev. Soc. Bras. Med. Trop. 31, 319-322. Graham, I. C. (1961). Study of chinchilla fur chewing. Vet. Bull. (London) 31, 699. Graur, D., Hide, W. A., and Li, W. H. (1991). Is the guinea-pig a rodent? Nature 351, 649-652. Gray, M. L., and Killinger, A. H. (1966). Listeria monocytogenes and listeric infections. Bact. Rev. 30, 309-382.

296 Green, C. A., Gordon, D. H, and Lyons, N. F. (1978). Biological species in Praomys (Mastomys) natalensis (Smith), a rodent carrier of Lassa virus and bubonic plague in Africa. Am. J. Trop. Med. Hyg. 27, 627-629. Griner, L. A. (1983). "Pathology of Zoo Animals," 1st ed. Zoological Society of San Diego, San Diego. Gromadzka-Ostrowska, J., Zalewska, B., Szylarska-Gozdz, E. (1985). Peripheral plasma progesterone concentration and hematological indices during normal pregnancy of chinchillas. Comp. Biochem. Physiol. A 82, 661665. Gross, S. A., and Didio, L. J. (1987). Comparative morphology of the prostate in adult male and female Praomys (Mastomys) natalensis studied with electron microscopy. J. Submicrosc. Cytol. 19, 77-84. Gross, S. A., and Didio, L. J. (1988). The prostate in pregnant and non-pregnant Praomys (Mastomys) natalensis at the subcellular level. J. Submicrosc. Cytol. Pathol. 20, 101-107. Gueraud, J. M. (1988). To an epizooty of yersiniosis in chinchilla. Bull. Acad. Vet. France 61, 95 - 98. Gulati, A., Srimal, R. C., and Dhawan, B. N. (1986). Stereotyped behaviour and striatal dopamine receptors in albino rat and the desert rat (Mastomys natelensis): A comparative study. Indian J. Exp. Biol. 24, 248-251. Gulati, A., Srimal, R. C. and Dhawan, B. N. (1988). An analysis of stereotyped behavior in Mastomys natalensis. Naunyn Schmiedebergs Arch. Pharmakol. 337, 575-575. Gummer, D. L., Forbes, M. R., Bender, D. J., and Barclay, R. M. (1997). Botfly (Diptera: Oestridae) parasitism of Ord's kangaroo rats (Dipodomys ordii) at Suffield National Wildlife Area, Alberta, Canada. J. Parasitol. 83, 601604. Gutierrez-Pena, E. J. (1987). Dunnifilaria meningica sp. n. (Filarioidea: Onchocercidae) from the central nervous system of the wood-rat (Neotoma micropus) in Mexico. Trop. Med. Parasitol. 38, 294-298. Guzman-Silva, M. A. (1997). Systemic mast cell disease in the Mongolian gerbil, Meriones unguiculatus: Case report. Lab. Anim. 31, 373-378. Guzman-Silva, M. A., Rossi, M. I., and Guimaraes, J. S. (1988). Craniopharyngioma in the Mongolian gerbil (Meriones unguiculatus): A case report. Lab. Anim. 22, 365-368. Hafner, M. S., and Page, R' D. (1995). Molecular phylogenies and host-parasite cospeciation: Gophers and lice as a model system. Philos. Trans. R. Soc. Lond. B Biol. Sci. 349, 77-83. Hafner, M. S., Sudman, P. D., Villablanca, E X., Spradling, T. A., Demastes, J. W., and Nadler, S. A. (1994). Disparate rates of molecular evolution in cospeciating hosts and parasites. Science 265, 1087-1090. Hagen, K. W., and Gorham, J. R. (1972). Dermatomycoses in fur animals: Chinchilla, ferret, mink, and rabbit. Vet. Med. Small Anim. Clin. 67, 43-48. Hall, E. R. (1981). "The Mammals of North America," 2nd ed. Wiley, New York. Hall, A. D., Persing, R. L., White, D. C. and Ricketts, R. T. Jr. (1967). Mystromys albicaudatus (the African white-tailed rat) as a laboratory species. Lab. Anim. Care 17, 180-188. Hall, E. D., Pazara, K. E., and Linseman, K. L. (1991). Sex differences in postischemic neuronal necrosis in gerbils. J. Cereb. Blood Flow Metab. 11, 292-298. Hallett, A. E, and Meester, J. (1971). Early postnatal development of the South African hamster. Zool. Afr. 6, 221-228. Hallett, A. E, and Politzer, W. M. (1972). Diabetes mellitus in Mystromys albicaudatus. Arch. PathoL 93, 178. Hamilton, W. J. Jr. (1953). Reproduction and young of the Florida wood rat Neotoma f. floridana (Ord). J. Mammal. 34, 180-189. Hamilton, W. J. Jr., and Whitaker, J. O. (1979). "Mammals of the Eastern United States," 2nd ed. Cornell Univ. Press, Ithaca, New York. Hannah, J., and Behnke, J. M. (1982). Nematospiroides dubius in the jird, Meriones unguiculatus: Factors affecting the course of a primary infection. J. Helminthol. 56, 329-338. Harriman, A. E. (1974). Self-selection of diet by Southern Plains wood rats (Neotoma micropus). J. Gen. Psychol. 90, 53-61.

THOMAS M. DONNELLY AND FRED W. QUIMBY

Harriman, A. E. (1976). Preferences by northern grasshopper mice for solutions of sugars, acids, and salts in Richter-type drinking tests. J. Gen. Psychol. 95, 85-92. Hartmann, K. (1993). Haltungsbedingte Erkrankungen beim Chinchilla [Husbandry-related diseases in the chinchilla]. Tierarztl. Prax. 21, 574-580. Heesemann, J. (1990). Enteropathogenic yersinias: Pathogenicity factors and new diagnostic methods. Immun. Infekt. 18, 186-191. (In German.) Heffner, H. E., and Heffner, R. S. (1985). Hearing in two cricetid rodents: Wood rat (Neotoma floridana) and grasshopper mouse (Onychomys leucogaster). J. Comp. Psychol. 99, 275-288. Heffner, R. S., Heffner, H. E., Contos, C., and Kearns, D. (1994). Hearing in prairie dogs: Transition between surface and subterranean rodents. Hear. Res. 73, 185-189. Heideman, P. D., and Bronson, E H. (1990). Photoperiod, melatonin secretion, and sexual maturation in a tropical rodent. Biol. Reprod. 43, 745750. Heidt, G. A., Conaway, H. H., Frith, C., and Farris, H. E. Jr. (1984). Spontaneous diabetes mellitus in a captive golden-mantled ground squirrel, Spermophilus lateralis (Say). J. Wildl. Dis. 20, 253-255. Hellenthal, R. A., and Price, R. D. (1994). Two new subgenera of chewing lice (Phthiraptera: Trichodectidae) from pocket gophers (Rodentia: Geomyidae), with a key to all included taxa. J. Med. Entomol. 31,450-466. Hessel, H., Ernst, L. S., Walger, M., von Wedel, H., Dybed, A., and Schmidt, U. (1997). Meriones unguiculatus (Gerbil) as an animal model for the ontogenetic cochlear implant research. Am. J. Otol. 18, $21. Hessel, H., Walger, M., Ernst, S., Foerst, A., von Wedel, H., Klunter, H. D., and Walkowiak, W. (1998). A method for the induction of a cochlea-specific auditory deprivation in the gerbil (Meriones unguiculatus). ORL J. Otorhinolaryngol. Relat. Spec. 60, 61-66. Hill, J. L. (1974). Peromyscus: Effect of early pairing on reproduction. Science 186, 1042-1044. Hill, T. P., and Best, T. L. (1985). Coccidia from California kangaroo rats (Dipodomys spp.). J. Parasitol. 71, 682-683. Hills, B. A., and Butler, B. D. (1978). The kangaroo rat as a model for type I decompression sickness. Undersea Biomed. Res. 5, 309-321. Hillyer, E. V., Quesenberry, K. E., and Donnelly, T. M. (1997). Biology, husbandry, and clinical techniques of guinea-pigs and chinchillas. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 243-259. Saunders, Philadelphia. Hnida, J. A., Wilson, W. D., and Duszynski, D. W. (1998). A new Eimeria species (Apicomplexa: Eimeriidae) infecting Onychomys species (Rodentia: Muridae) in New Mexico and Arizona. J. Parasitol. 84, 1207-1209. Hoch-Ligeti, C., Wagner, B. P., Deringer, M. K., and Stewart, H. L. (1985). Tumor induction in Praomys (Mastomys) natalensis by N, N-2, 7fluorenylenebisacetamide. J. Natl. Cancer Inst. 74, 909-915. Hoefer, H. L. (1994). Chinchillas. Vet. Clin. North Am. Small Anim. Pract. 24, 103-111. Holland, J. M. (1970). Prostatic hyperplasia and neoplasia in female Praomys (Mastomys) natalensis. J. Natl. Cancer Inst. 45, 1229-1236. Holliman, R. B., and Meade, B. J. (1980). Native trichinosis in wild rodents in Henrico County, Virginia. J. Wildl. Dis. 16, 205-207. Holmes, E., Bonner, E W., and Nicholson, J. K. (1995). Comparative studies on the nephrotoxicity of 2-bromoethanamine hydrobromide in the F344 rat and the multimammate desert mouse (Mastomys natalensis). Arch. Toxicol. 70, 89-95. Holmes, E., Bonner, E W., and Nicholson, J. K. (1996). Comparative biochemical effects of low doses of mercury II chloride in the F344 rat and the multimammate mouse (Mastomys natalensis). Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 114, 7-15. Holmes, E., Bonner, E W., and Nicholson, J. K. (1997). 1H NMR spectroscopic and histopathological studies on propyleneimine-induced renal papillary necrosis in the rat and the multimammate desert mouse (Mastomys natalensis). Comp. Biochem. Physiol. Pharmacol. Toxicol. Endocrinol. 2, 125134.

7. BIOLOGY AND DISEASES OF OTHER RODENTS

Holzbach, R. T. (1984). Animal models of cholesterol gallstone disease. Hepatology 4, 191S-198S. Hoogland, J. L. (1979). Aggression, ectoparasitism, and other possible costs of prairie dog (Sciuridae, Cynomys spp.) coloniality. Behaviour 69, 1-35. Hoogland, J. L. (1994). Nepotism and infanticide among prairie dogs. Ettore Majorana Int. Life Sci. Series 13, 321-337. Hoogland, J. L. (1996a). Cynomys ludovicianus. Mammal. Species 535, 1-10. Hoogland, J. L. (1996b). Why do Gunnison's prairie dogs give anti-predator calls? Anim. Behav. 51, 871-880. Hoogland, J. L. (1997). Duration of gestation and lactation for Gunnison's prairie dogs. J. Mammal. 78, 173-180. Hoogland, J. L. (1998). Estrus and copulation of Gunnison's prairie dogs. J. Mammal. 79, 887-897. Homer, B. E. (1968). Gestation period and early development in Onychomys leucogaster brevicaudus. J. Mammal. 49, 513-515. Horner, B. E., Potter, G. L., and Van Ooteghem, S. (1980). A new black coatcolor mutation in Peromyscus. J. Hered. 71, 49-51. Horvath, Z., and Keri, M. (1980). Dermatomycosis of chinchilla caused by Trichophyton mentagrophytes. Magy. Allatorvosok Lapja 35, 592-601. Hosoda, S., Suzuki, H., and Suzuki, M. (1976). Spontaneous tumors and atypical proliferation of pancreatic acinar cells in Mastomys (Praomys) natalensis. J. Natl. Cancer Inst. 57, 1341-1346. Howell, A. B. (1926). "Anatomy of the Wood Rat," Williams & Wilkins, Baltimore. Hubbard, G. B., Schmidt, R. E., and Fletcher, K. C. (1983). Neoplasia in zoo animals. J. Zoo Anim. Med. 14, 33-40. Hubbert, W. T. (1972). Yersioniosis in mammals and birds in the United States: Case reports and review. Am. J. Trop. Med. Hyg. 21,458-463. Hughes, S. E., and Nutting, W. B. (1981). Demodex leucogasteri n. sp. from Onychomys leucogastermwith notes on its biology and host pathogenesis. Acarologia 22, 181-186. Iglauer, E, Schuler, T., Holub, S., and Sachs, R. (1993). Ringtail disorder observed in cotton rats (Sigmodon hispidus). Scand. J. Lab. Anim. Sci. 20, 119-121. Ignatovich, V. E, Barkhatova, O. I., and Rydkina, E. B. (1983). Persistence of Rickettsia prowazekii with different initial biological properties in infected cotton rats. Acta Virol. 27, 528-532. Insel, T. R., and Shapiro, L. E. (1992). Oxytocin receptors and maternal behavior. Ann. N. Y. Acad. Sci. 652, 122-141. Isaacson, M., Arntzen, L. and Taylor, P. (1981). Susceptibility of members of the Mastomys natalensis species complex to experimental infection with Yersinia pestis. J. Infect. Dis. 144, 80-84. Ismailov, S. G., and Ismailov, M. N. (1981). The breeding of grapevine gerbils in laboratory conditions. Izvestiya Akad. Nauk Azerbaidzhanskoi Ssr Seriya Biolog. Nauk 1, 69-72. Itoh, K., Tamura, H., and Mitsuoka, T. (1989). Gastrointestinal flora of cotton rats. Lab. Anim. 23, 62-65. Jablonski, R., and Brudnicki, W. (1984). The effect of blood distribution to the brain on the structure and variability of the cerebral arterial circle in muskrat and in chinchilla. Folia Morphol. 43, 109-114. Jackson, R. K. (1997). Unusual laboratory rodent species: Research uses, care and associated biohazards. ILAR J. 38, 13-21. Jacobs, L. E, and Spencer, W. D. (1994). Natural space-use patterns and hippocampal size in kangaroo rats. Brain Behav. EvoL 44, 125-132. Jakubow, K., Gromadska-Ostrowska, J., and Zalewska, B. (1984). Seasonal changes in the haematological indices in peripheral blood of chinchilla. Comp. Biochem. Physiol. A Comp. Physiol. 78, 845-853. Javel, E., and Mott, J. B. (1988). Physiological and psychophysical correlates of temporal processes in hearing. Hear. Res. 34, 275-294. Jenkins, D. C. (1977). Nematospiroides dubius: The course of primary and challenge infections in the jird, Meriones unguiculatus. Exp. Parasitol. 41, 335-340. Jenkins, J. R. (1992). Husbandry and common diseases of the chinchilla (Chinchilla laniger). J. Small Exotic Anim. Med. 2, 15-17.

297 Jensen, W. I., and Duncan, R. M. (1980). Bordetella bronchoseptica associated with pulmonary disease in mountain voles (Microtus montanus). J. Wildl. Dis. 16, 11-14. Jobard, P., Delbarre, G., Delbarre, B., Aron, E., and Ghaly, A. E (1974). A renal tumour of the nephroblastoma type in Praomys (Mastomys) natalensis. Experientia 30, 416 - 418. Johnson, L. A., Flook, J. P., Look, M. V., and Pinkel, D. (1987). Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. Gamete Res. 16, 1-9. Johnston, P. G., and Zucker, I. (1983). Lability and diversity of circadian rhythms of cotton rats Sigmodon hispidus. Am. J. Physiol. 244, R338-346. Jones, A. K. (1990). Caesarean section in a chinchilla [letter]. Vet. Rec. 126, 441. Jones, R. J., Stephenson, R., Fountain, D., and Hooker, R. (1995). Urolithiasis in a chinchilla [letter]. Vet. Rec. 136, 400. Jonkers, A. H., Spence, L., Downs, W. G., and Worth, C. B. (1964). Laboratory studies with wild rodents and viruses native to Trinidad. II. Studies with the Trinidad Caraparu-like agent TRVL 34053-1. Am. J. Trop. Med. Hyg. 13, 728-733. Juhn, S. K., Haugen, J., and Steven, L. (1974). Some chemical parameters of serum, cerebrospinal fluid, perilymph, and aqueous humor of the chinchilla. Lab. Anim. Sci. 24, 691-695. Juhn, S. K., Tolan, C. J., Antonelli, P. J., Giebink, G. S., and Goycoolea, M. V. (1991). The significance of experimental animal studies in otitis media. Otolaryngol. Clin. North Am. 24, 813-827. Kageruka, P. (1970). Infections caused by the Malassez and Vignal bacillus (Y. pseudotuberculosis) in the animals of the Antwerp Zoo. Acta Zool. Pathol. Antverp. 51, 3-16. Kamiya, M., Ooi, H. K., Oku, Y., Okamoto, M., Ohbayashi, M., and Seki, N. (1987). Isolation of Echinococcus multilocularis from the liver of swine in Hokkaido, Japan. Jpn. J. Vet. Res. 35, 99-107. Kanazawa, T., Asahi, H., and Mochida, K. (1995). Simple techniques for preparation of small vesicles from Echinococcus multilocularis metacestodes and colorimetric quantitation of the viability of germinal cells. Jpn. J. Parasitol. 44, 441-446. Kaplan, H. (1975). What triggers seizures in the gerbil, Meriones unguiculatus? Life Sci. 17, 693-698. Kaplan, H., and Miezejeski, C. (1972). Development of seizures in the Mongolian gerbil (Meriones unguiculatus). J. Comp. Physiol. PsychoL 81, 267273. Kas, M. J., and Edgar, D. M. (1999). Circadian timed wakefulness at dawn opposes compensatory sleep responses after sleep deprivation in Octodon degus. Sleep 22, 1045-1053. Katahira, K., and Ohwada, K. (1993). Hematological standard values in the cotton rat (Sigmodon hispidus). Jikken Dobutsu 42, 653-656. Kawase, S., and Ishikura, H. (1995). Female-predominant occurrence of spontaneous gastric adenocarcinoma in cotton rats. Lab. Anim. Sci. 45, 244-248. Kawase, S., and Satoh, N. (1978). Breeding of cotton rat in laboratory. Rep. Hokkaido Inst. Public Health 28, 90-91. Keagy, H. E (1949). Toxoplasma in the chinchilla. J. Am. Vet. Med. Assoc. 94, 15. Keis, A. E, and Mitchell, O. G. (1975). Cytomegalovirus in the submandibular and sublingual glands of the southern grasshopper mouse (Onychomys torridus torridus). J. Dent. Res. 54, 626-628. Kemble, E. D., and Enger, J. M. (1984). Sex differences in shock motivated behaviors, activity, and discrimination learning of northern grasshopper mice (Onychomys leucogaster). Physiol. Behav. 32, 375-380. Kenagy, G. J., Veloso, C., and Bozinovic, F. (1999). Daily rhythms of food intake and feces reingestion in the degu, an herbivorous Chilean rodent: Optimizing digestion through coprophagy. Physiol. Biochem. Zool. 72, 7886. Kennedy, A. H. (1952). "Chinchilla Diseases and Ailments." Clay Publ. Co., Bewdley, Ontario. Kerr, S. E, McHugh, C. P., and Dronen, N. O. Jr. (1995). Leishmaniasis in

298 Texas: Prevalence and seasonal transmission of Leishmania mexicana in Neotoma micropus. Am. J. Trop. Med. Hyg. 53, 73-77. Kershaw, W. E., and Storey, D. M. (1976). Host-parasite relations in cotton rat filariasis. I: The quantitative transmission and subsequent development of Litomosoides carinii infections in cotton rats and other laboratory animals. Ann. Trop. Med. Parasitol. 70, 303-312. Khare, S., Saxena, J. K., Sen, A. B., and Ghatak, S. (1984). Erythrocyte membrane-bound enzymes in Mastomys natalensis during Plasmodium berghei infection. Aust. J. Exp. Biol. Med. Sci. 62, 137-143. Kim, H. J., and Chole, R. A. (1998). Experimental models of aural cholesteatomas in Mongolian gerbils. Ann. Otol. Rhinol. Laryngol. 107, 129-134. King, B. E, and Tibbitts, E D. (1976). The fine structure of the chinchilla placenta. Am. J. Anat. 145, 33-56. Kinsey, K. P. (1976). Social behavior in confined populations of the Allegheny woodrat, Neotoma floridana magister. Anim. Behav. 24, 181-187. Kitchen, H., and Kitchen, Y. (1967). Reproduction, breeding and management of chinchillas. Lab. Anim. Dig. 3, 3-7. Klei, T. R., McVay, C. S., Coleman, S. U., Enright, E M., and Rao, U. R. (1997). Adoptive transfer of granulomatous inflammation to Brugia antigens in jirds. J. Parasitol. 83, 626-629. Knoch, H. W. (1968). The Eastern wood rat, Neotoma floridana osagensis: A laboratory colony. Trans. Kans. Acad. Sci. 71, 361-372. Kolby, L., Wangberg, B., Ahlman, H., Modlin, I. M., and Nilsson, O. (1998). Histamine metabolism of the gastric carcinoid in Mastomys natalensis. Yale J. Biol. Med. 71, 207-215. Koop, B. E, Baker, R. J., and Genoways, H. H. (1983). Numerous chromosomal polymorphisms in a natural population of rice rats (Oryzomys, Cricetidae). Cytogenet. Cell Genet. 35, 131-135. Koopman, J. P., Mullink, J. W., Kennis, H. M., and van der Logt, J. T. (1980). An outbreak of Tyzzer's disease in Mongolian gerbils (Meriones unguiculatus). Z. Versuchstierkunde 22, 336-341. Kosoy, M. Y., Elliott, L. H., Ksiazek, T. G., Fulhorst, C. E, Rollin, P. E., Childs, J. E., Mills, J. N., Maupin, G. O., and Peters, C. J. (1996). Prevalence of antibodies to arenaviruses in rodents from the southern and western United States: Evidence for an arenavirus associated with the genus Neotoma. Am. J. Trop. Med. Hyg. 54, 570-576. Kozima, K., Muroashi, T., Soga, J., and Tazawa, K. (1970). Histological and immunohistochemical studies on spontaneous renal lesions of Praomys (Mastomys) natalensis. Acta Pathol. Jpn. 20, 311-325. Kritsky, D. C., Leiby, P. D., and Miller, G. E. (1977). The natural occurrence of Echinococcus multilocularis in the bushy-tailed woodrat, Neotoma cinerea rupicola, in Wyoming. Am. J. Trop. Med. Hyg. 26, 1046-1047. Kroeze, W. K., and Tanner, C. E. (1985). Echinococcus multilocularis: Responses to infection in cotton rats (Sigmodon hispidus). Int. J. Parasitol. 15, 233-238. Kroh, H., Walencik, S., Mossakowski, M. J., and Weinrauder, H. (1987). Spontaneous astrocytoma in the Mongolian gerbil (Meriones unguiculatus). Neuropatol. Pol. 25, 329-336. Kruckenberg, S. M., Cook, J. E., and Feldman, B. E (1975). Clinical toxicities of pet and caged rodents and rabbits. Vet. Clin. North Am. 5, 675-684. Kruppa, T. E, Iglauer, E, Ihnen, E., Miller, K., and Kunstyr, I. (1990). Mastomys natalensis or Mastomys coucha. Correct species designation in animal experiments. Trop. Med. Parasitol. 41, 219-220. Kudo, H., and Oki, Y. (1993). Microtus species as new herbivorous laboratory animals: Reproduction, bacterial flora and fermentation in the digestive tract and nutritional physiology. Jikken Dobutsu 42, 615-618. Kumazawa, H., Takagi, H., Sudo, K., Nakamura, W., and Hosoda, S. (1989). Adenocarcinoma and carcinoid developing spontaneously in the stomach of mutant strains of Mastomys natalensis. Virchows Arch. A Pathol. Anat. Histopathol. 416, 141-151. Kuroiwa, J., and Imamichi, T. (1977). Growth and reproduction of the chinchillamage at vaginal opening, oestrous cycle, gestation period, litter size, sex ratio, and diseases frequently encountered. Exp. Anim. 26, 213-222.

THOMAS M. DONNELLY AND FRED W. QUIMBY

Kurokawa, Y., Fuji, K., Suzuki, M., and Sato, H. (1968). Spontaneous tumors of the thymus in Mastomys (Rattus natalensis). Gann 59, 145 - 150. Lackey, J. A., Huckaby, D. G., and Ormiston, B. G. (1985). Peromyscus leucopus. Mammal. Species 247, 1-10. Laming, P. R., Cosby, S. L., and O'Neill, J. K. (1989). Seizures in the Mongolian gerbil are related to a deficiency in cerebral glutamine synthetase. Comp. Biochem. Physiol. C 94, 399-404. Lang, J. D. (1996). Factors affecting the seasonal abundance of ground squirrel and wood rat fleas (Siphonaptera) in San Diego County, California. J. Med. Entomol. 33, 790-804. Langford, E. V. (1972). Pasteurella pseudotuberculosis infections in Western Canada. Can. Vet. J. 13, 85-87. LaRegina, M., Kier, A. B., and Wagner, J. E. (1978). A fatal enteric syndrome in Mystromys albicaudatus (white-tailed rat) following topical antibiotic treatment. Lab. Anim. Sci. 28, 587-590. La Regina, M., Lonigro, J., and Wallace, M. (1986). Francisella tularensis infection in captive, wild caught prairie dogs. Lab. Anim. Sci. 36, 178180. Larson, R. H., and Fitzgerald, R. J. (1968). Caries development in the African white-tailed rat (Mystromys albicaudatus) infected with a streptococcus of human origin. J. Dent. Res. 47, 746-749. Leach, A. B., and Holub, B. J. (1984). The effect of dietary lipid on the lipoprotein status of the Mongolian gerbil. Lipids 19, 25-33. Led, J. E., and Brandetti, E. (1974). Miiasis por Cuterebra sp., Clark 1815 (Diptera Cuterebridae) en una chinchilla (Chinchilla lanigera). Analecta veterinaria 8, 29-33. (In Spanish.) Leighton, E A., and Wobeser, G. (1978). The prevalence of adiaspiromycosis in three sympatric species of ground squirrels. J. Wildl. Dis. 14, 362-365. Leonard, E. P. (1979). Periodontitis. Animal model: Periodontitis in the rice rat (Oryzomys palustris). Am. J. Pathol. 96, 643-646. Leonard, E. P., Reese, W. V., and Mandel, E. J. (1979). Comparison of the effects of ethane-l-hydroxy-1, 1-diphosphonate, and dichloromethylene diphosphonate upon periodontal bone resorption in rice rats (Oryzomys palustris). Arch. Oral. Biol. 24, 707-708. Letelier, M. E., Del Villar, E., and Sanchez, E. (1985). Drug tolerance and detoxicating enzymes in Octodon degus and Wistar rats. A comparative study. Comp. Biochem. Physiol. C 80, 195-198. Leung, M. K., McLintock, J., and Iversen, J. (1978). Intranasal exposure of the Richardson's ground squirrel to Western equine encephalomyelitis virus. Can. J. Comp. Med. 42, 184-191. Levine, J. E, and Lage, A. L. (1984). House mouse mites infesting laboratory rodents. Lab. Anim. Sci. 34, 393-394. Lewis, R. J. (1989). Plague in bushy-tailed woodrats. Can. Vet. J. 30, 596-597. Lewis, D., and Williams, H. (1979). Infection of the Mongolian gerbil with the cattle piroplasm Babesia divergens. Nature 278, 170-171. Lewis, D., Young, E. R., Baggott, D. G., and Osborn, G. D. (1981). Babesia divergens infection of the Mongolian gerbil: Titration of infective dose and preliminary observations on the disease produced. J. Comp. Pathol. 91, 565 -572. Li, B. W., Chandrashekar, R., Alvarez, R. M., Liftis, E, and Weil, G. J. (1991). Identification of paramyosin as a potential protective antigen against Brugia malayi infection in jirds. Mol. Biochem. Parasitol. 49, 315-323. Liddell, K. G., Joss, A. W. L., and Williams, H. (1982). Serological response of the Mongolian gerbil to Babesia divergens (human strain) infection. Ann. Trop. Med. Parasitol. 76, 527-538. Little, R. R., Parker, K. M., England, J. D., and Goldstein, D. E. (1982). Glycosylated hemoglobin in Mystromys albicaudatus: A diabetic animal model. Lab. Anim. Sci. 32, 44-47. Lochmiller, R. L., Vestey, M. R., and McMurry, S. T. (1991). Primary immune responses of selected small mammal species to heterologous erythrocytes. Comp. Biochem. Physiol. A 100, 139-143. Loskota, W. J., and Lomax, P. (1975). The Mongolian gerbil (Meriones unguiculatus) as a model for the study of the epilepsies: EEG records of seizures. Etectroencephalogr. Clin. Neurophysiol. 38, 597-604.

7. BIOLOGY AND DISEASES OF OTHER RODENTS

Low, J. C., and Donachie, W. (1997). A review of Listeria monocytogenes and listerioris. Vet. J. 153, 9-29. Luckett, W. P., and Hartenberger, J. L. (1985). Evolutionary relationships among rodents: A multidisciplinary analysis. In "NATO ASI Series (Series A, Life Sciences)," Vol. 92. Plenum Press, New York. Luckett, W. P., and Hartenberger, J. L. (1993). Monophyly or polyphyly of the order Rodentia: Possible conflict between morphological and molecular interpretations. J. Mammal. Evol. 1, 127-131. Lusis, P. I., and Soltys, M. A. (1971a). Immunization of mice and chinchillas against Pseudomonas aeruginosa. Can. J. Comp. Med. Vet. Sci. 35, 60-66. Lusis, P. I., and Soltys, M. A. (1971b). Pseudomonas infections in man and animals. J. Am. Vet. Med. Assoc. 159, 416. Lussier, G., and Loew, E M. (1970). Case report. Natural Hymenolepis nana infection in Mongolian gerbils (Meriones unguiculatus). Can. Vet. J. 11, 105-107. MacDonald, D. W., Wilton, G. S., Howell, J., and Klavano, G. G. (1972). Listeria monocytogenes isolations in Alberta 1951-1970. Can. Vet. J. 13, 69-71. Machang'u, R.S., Mgode, G., and Mpanduji, D. (1997). Leptospirosis in animals and humans in selected areas of Tanzania. Belg. J. Zool. 127, 97-104. MacKay, K. A., Kennedy, A. H., Smith, D. L. T., and Bain, A. E (1949). Listeria monocytogenes infection in chinchillas. Ann. Rep. Ont. Vet. Coll., 137145. Mackenzie, C. D., Suswillo, R. R., and Denham, D. A. (1982). Survival of Loa loa following transplantation from drills (Mandrillus leucophaeus) into jirds (Meriones unguiculatus): Parasitology and pathology. Trans. R. Soc. Trop. Med. Hyg. 76, 778-782. Maclean, J. M., Lewis, D., and Holmes, P. H. (1987). Studies on the pathogenesis of Trichostrongylus colubriformis in Mongolian gerbils (Meriones unguiculatus). J. Comp. Pathol. 97, 645-652. MacPherson, B. R., and Pemsingh, R. S. (1997). Ground squirrel model for cholelithiasis: Role of epithelial glycoproteins. Microsc. Res. Tech. 39, 3955. MacPherson, B. R., Pemsingh, R. S., and Scott, G. W. (1987). Experimental cholelithiasis in the ground squirrel. Lab. Invest. 56, 138-145. Maddock, A. H., and Perrin, M. R. (1981). A microscopical examination of the gastric morphology of the white-tailed rat Mystromys albicaudatus (Smith 1834). S. Afr. J. ZooL 16, 237-247. Maddock, A. H., and Perrin, M. R. (1983). Development of the gastric morphology and fornical bacterial/epithelial association in the white-tailed rat Mystromys albicaudatus. S. Afr. J. Zool. 18, 115-127. Mahida, H., and Perrin, M. R. (1994). The effect of different diets on the amount of organic acid produced in the digestive tract of Mystromys albicaudatus. Acta Theriol. 39, 21-27. Maia, V., and Hulak, A. (1981). Robertsonian polymorphism in chromosomes of Oryzomys subflavus (Rodentia, Cricetidae). Cytogenet. Cell Genet. 31, 33-39. Maki, J., and Weinstein, P. P. (1991). Transplantation into jirds as a method of assessing the viability and reproductive integrity of adult Acanthocheilonema viteae from culture. J. Parasitol. 77, 749-754. Male, O., and Fritsch, P. (1966). Trichophyton mentagrophytes-caused epidemic and enzootic disease in a chinchilla farm. Mykosen 4, 74-84. Mappes, T., Koskela, E., and Ylonen, H. (1995). Reproductive costs and litter size in the bank vole. Proc. R. Soc. Lond. B. Biol. Sci. 261, 19-24. Marafie, E., Nayak, R., and A1Zaid, N. (1978). Breeding and reproductive physiology of the desert gerbil, Meriones crasses. Lab. Anim. Sci. 28, 397-401. Maravilla, P., Avila, G., Gabrera, V., Aguilar, L., Flisser, A. (1998). Comparative development of Taenia solium in experimental models. J. Parasitol. 84, 882-886. Marchiondo, A. A., and Upton, S. J. (1987). Eimeria albigulae and E. ladronesis: New host records from the bushy-tailed woodrat, Neotoma cinerea, from Utah. J. Parasitol. 73, 421-422. Marennikova, S. S., Ladnyj, I. D., Ogorodinikova, Z. I., Shelukhina, E. M., and Maltseva, N. N. (1978). Identification and study of a poxvirus isolated from wild rodents in Turkmenia. Arch. Virol. 56, 7-14.

299 Marion, E L., Van Davelaar, M. J., Knight, S. S., Salazar, E H., Garcia, G., Popper, H., and Robinson, W. S. (1986). Hepatocellular carcinoma in ground squirrels persistently infected with ground squirrel hepatitis virus. Proc. Natl. Acad. Sci. USA 83, 4543-4546. Marlow, C. (1995). Diabetes in a chinchilla [letter]. Vet. Rec. 136, 595-596. Marston, J. H. (1976). The Mongolian gerbil. In "The UFAW Handbook on the Care and Management of Laboratory Animals," 5th ed., pp. 236-274. Churchill Livingstone, Edinburgh. Martignetti, J. A., and Brosius, J. (1993). Neural BC1 RNA as an evolutionary marker: Guinea pig remains a rodent. Proc. Natl. Acad. Sci. USA 90, 96989702. Mathieu, X., Duran, J. C., and Rivas, M. (1982). Estudio de la flora bacteriana normal de Chinchilla laniger Silvestre [Normal bacterial flora of the wild Chinchilla laniger]. Rev. Latinoam. Microbiol. 24, 77-82. Matsuoka, K., and Suzuki, J. (1995). Spontaneous tumors in the Mongolian gerbil (Meriones unguiculatus). Exp. Anim. 43, 755-760. Matsushima, Y., Ikeno, T., Ikeno, K., and Tanaka, S. (1990). An electrophoretic polymorphism in salivary amylases (Amy-l) of Mastomys (Praomys coucha). Lab. Anim. 24, 308-312. Matsuzaki, T., Matsuzaki, K., Yokohata, Y., Ohtsubo, R., Kamiya, M., and Yates, T. L. (1994). Breeding of the northern grasshopper mouse (Onychomys leucogaster) as a laboratory animal. Jikken Dobutsu 43, 395-401. Matthes, S. (1985). Vaccination of rabbits and fur animals. Tierarztl. Prax. 43, 107-112. Mays, A. Jr. (1969). Baseline hematological and blood biochemical parameters of the Mongolian gerbil (Meriones unguiculatus). Lab. Anim. Care 19, 838-842. McCarty, R. (1975). Onychomys torridus. Mammal. Species 59, 1-5. McCarty, R. (1978). Onychomys leucogaster. Mammal. Species. 87, 1-6. McCarty, R., and Southwick, C. H. (1975). The development of convulsive seizures in the grasshopper mouse (Onychomys torridus). Dev. Psychobiol. 8, 547-552. McCarty, R., and Southwick, C. H. (1977a). Cross-species fostering: Effects on the olfactory preference of Onychomys torridus and Peromyscus leucopus. Behav. Biol. 19, 255-260. McCarty, R., and Southwick, C. H. (1977b). Effects of parental environment on the prevalence of convulsive seizures in Onychomys torridus. Dev. Psychobiol. 10, 359-364. McCarty, R. C., Whitesides, G. H., and Tomosky, T. K. (1976). Effects of pchlorophenylalanine on the predatory behavior of Onychomys torridus. Pharmacol. Biochem. Behav. 4, 217-220. McClellan, D. A. (2000). The codon-degeneracy model of molecular evolution. J. Mol. Evol. 50, 131-140. McGinn, M. D., and Faddis, B. T. (1997). Kangaroo rats exhibit spongiform degeneration of the central auditory system similar to that found in gerbils. Hear. Res. 104, 90-100. McGinn, M. D., and Faddis, B. T. (1998). Neuronal degeneration in the gerbil brainstem is associated with spongiform lesions. Microsc. Res. Tech. 41, 187-204. McGreevy, P. D., and Carn, V. M. (1988). Intestinal torsion in a chinchilla [letter]. Vet. Rec. 122, 287. McHugh, C. P., Melby, P. C., and LaFon, S. G. (1996). Leishmaniasis in Texas: Epidemiology and clinical aspects of human cases. Am. J. Trop. Med. Hyg. 55, 547-555. McKinney, L. A., and Hendricks, L. D. (1980). Experimental infection of Mystromys albicaudatus with Leishmania braziliensis: Pathology. Am. J. Trop. Med. Hyg. 29, 753-760. McMurry, S. T., Lochmiller, R. L., Chandra, S. A., and Quails, C. W. Jr. (1995). Sensitivity of selected immunological, hematological, and reproductive parameters in the cotton rat (Sigmodon hispidus) to subchronic lead exposure. J. Wildl. Dis. 31, 193-204. Meckley, P. E., and Zwicker, G. M. (1979). Naturally-occurring neoplasms in the Mongolian gerbil, Meriones unguiculatus. Lab. Anim. 13, 203-206. Meingassner, J. G., and Burtscher, H. (1977). Doppelinfektion des Gihirns mit

300 Frenkelia species und Toxoplasma gondii bei Chinchilla laniger [Double infection of the brain with Frenkelia species and Toxoplasma gondii in Chinchilla laniger]. Vet. Pathol. 14, 146-153. Mello, D. A. (1978). Some aspects of the biology of Oryzomys eliurus (Wagner, 1845) under laboratory conditions (Rodentia: Cricetidae). Rev. Bras. Biol. 38, 293-295. Menchaca, E. S., Moras, E. V., Martin, A. M., and Canevaro, L. (1980). Enfermedades infecciosas de la chinchilla (Chinchilla laniger). 4. "Pseudomona aeruginosa." Gac. Vet. 42, 96-102. Meng, J., Wyss, A. R., Dawson, M. R., and Zhai, R. (1994). Primitive fossil rodent from Inner Mongolia and its implications for mammalian phylogeny. Nature 370, 134-136. Mian, L. S., Nwadike, C. N., Hitchcock, J. C., and Madon, M. B. (1995). Plague activity in San Bernardino County during 1994. Proc. Pap. Ann. Conf. Calif. Mosq. Control Assoc. 63, 35-38. Mian, L. S., Nwadike, C. N., Hitchcock, J. C., Madon, M. B., and Myers, C. M. (1996). Plague activity in San Bernardino County during 1995. Proc. Pap. Ann. Conf. Calif. Mosq. Control Assoc. 64, 80-84. Michener, G. R. (1973). Climatic conditions and breeding in Richardson's ground squirrel. J. Mammal. 54, 499-503. Michener, G. R. (1993). Lethal myiasis of Richardson's ground squirrels by the sarcophagid fly Neobellieria citellivora. J. Mammal. 74, 148-155. Michener, G. R., and Koeppl, J. W. (1985). Spermophilus richardsonii. Mammal. Species 243, 1-8. Mighell, J. S., and Baker, A. E. (1990). Caesarean section in a gerbil [letter]. Vet. Rec. 126, 441. Mikhail, J. W., and Mansour, N. S. (1973). Mystromys albicaudatus, the African white-tailed rat, as an experimental host for Leishmania donovani. J. Parasitol. 59, 1085-1087. Montali, R. J. (1980). An overview of tumors in zoo animals. In "The Comparative Pathology of Zoo Animals" (R. J. Montali and G. Migaki, eds.), pp. 531-542. Smithsonian Institution Press, Washington, D.C. Moore, R. W. and Greenlee, H. H. (1975). Enterotoxemia in chinchillas. Lab. Anim. 9, 153-154. Morganti, L., and Gomez Portugal, E. A. (1970). Microsporum gypseum infection in chinchillas. Sabouraudia 8, 39-40. Morizono, T., and Tono, T. (1991). Middle ear inflammatory mediators and cochlear function. Otolaryngol. Clin. North Am. 24, 835-843. Morrison, P., Dieterich, R., and Preston, D. (1976). Breeding and reproduction of fifteen wild rodents maintained as laboratory colonies. Lab. Anim. Sci. 25, 237-243. Morrow, G. W., and Day-Lollini, P. A. (1990). Diagnostic exercise: Exophthalmos in a ground squirrel. Lab. Anim. Sci. 40, 411-412. Motzel, S. L., and Gibson, S. V. (1990). Tyzzer's disease in hamsters and gerbils from a pet store supplier. J. Am. Vet. Med. Assoc. 197, 1176-1178. Mountain, A. (1989). Salmonella arizona in a chinchilla [letter]. Vet. Rec. 125, 25. Muller, E. L., Pitt, H. A., and George, W. L. (1987). Prairie dog model for antimicrobial agent-induced Clostridium difficile diarrhea. Infect. Immun. 55, 198-200. Murphy, J. C., Niemi, S. M., Hewes, K. M., Zink, M., and Fox, J. G. (1978). Hematologic and serum protein reference values of the Octodon degus. Am. J. Vet. Res. 39, 713-715. Murphy, J. C., Crowell, T. P., Hewes, C. M., and Fox, J. G. (1980). Spontaneous lesions in the degu. In "The Comparative Pathology of Zoo Animals (R. J. Montali and G. Migaki, eds.), pp. 437-444. Smithsonian Institution Press, Washington, D.C. Murthy, P. K., Murthy, P. S., Tyagi, K., and Chatterjee, R. K. (1997). Fate of infective larvae of Brugia malayi in the peritoneal cavity of Mastomys natalensis and Meriones unguiculatus. Folia Parasitol. 44, 302-304. Muths, E., and Reichman, O. J. (1996). Kangaroo rat bone compared to white rat bone after short-term disuse and exercise. Comp. Biochem. Physiol. A Physiol. 114, 355- 361.

THOMAS M. DONNELLY AND FRED W. QUIMBY

Myers, E (1999). Rodent jaws. In "Animal Diversity Web" (E Myers, ed.). Univ. of Michigan Museum of Zoology . Nagasawa, H., Koshimizu, U., Watanabe, M., Tokuda, K., Sumita, H., and Kano, Y. (1989). Mammary gland growth and response to hormones in Mastomys compared with mice. Lab. Anim. Sci. 39, 313-317. Nagy, T. R., and Pistole, D. H. (1988). The effects of fasting on some physiological parameters in the meadow vole, Microtus pennsylvanicus. Comp. Biochem. Physiol. 91, 679-684. Najecki, D., and Tate, B. (1999). Husbandry and management of the degu. Lab Anim. 28, 54-62. Nasarre, C., Coleman, S. U., Rao, U. R., and Klei, T. R. (1997). Brugia pahangi: Differential induction and regulation of jird inflammatory responses by life-cycle stages. Exp. Parasitol. 87, 20-29. National Research Council (NRC) (1978). "Nutrient Requirements of Laboratory Animals." 3rd rev. ed. National Academy Press, Washington, D.C. Neal, B. R. (1977). Reproduction of the multimammate rat, Praomys (Mastomys) natalensis (Smith) in Uganda. Z. Siiugetierk. 42, 221-231. Nedbal, M. A., Allard, M. W., and Honeycutt, R. L. (1994). Molecular systematics of hystricognath rodents: Evidence from the mitochondrial 12S rRNA gene. Mol. Phylogenet. Evol. 3, 206-220. Newberne, P. M. (1952). Urinary calculus in a chinchilla. N. Am. Vet. 33, 334. Newberne, P. M. (1953). An outbreak of bacterial gastro-enteritis in the South American chinchilla. N. Am. Vet. 34, 187-188, 191. Newberne, J. W., and Robinson, V. B. (1957). Malignant lymphoma in the South American chinchilla. N. Am. Vet. 38, 362, 372. Nicholson, W. L., Castro, M. B., Kramer, V. L., Sumner, J. W., and Childs, J. E. (1999). Dusky-footed wood rats (Neotomafuscipes) as reservoirs of granulocytic Ehrlichiae (Rickettsiales: Ehrlichieae) in northern California [published erratum appears in J. Clin. MicrobioL (Dec. 1999) 37(12), 4206]. J. Clin. Microbiol. 37, 3323-3327. Nicolosi, R. J., Marlett, J. A., Morello, A. M., Flanagan, S. A., and Hegsted, D. M. (1981). Influence of dietary unsaturated and saturated fat on the plasma lipoproteins of Mongolian gerbils. Atherosclerosis 38, 359-371. Niewiesk, S. (1999). Cotton rats (Sigmodon hispidus): An animal model to study the pathogenesis of measles virus infection, lmmunol. Lett. 65, 4750. Niewiesk, S., Eisenhuth, I., Fooks, A., Clegg, J. C., Schnorr, J. J., SchneiderSchaulies, S., and ter Meulen, V. (1997). Meastes virus-induced immune suppression in the cotton rat (Sigmodon hispidus) model depends on viral glycoproteins. J. Virol. 71, 7214-7219. Nilsson, O., and Soderlind, O. (1974). Listeria monocytogenes isolated from animals in Sweden during 1958-1972. Nord. Vet. Med. 26, 248-255. Nilsson, O., Wangberg, B., Johansson, L., Modlin, I. M., and Ahlman, H. (1992). Praomys (Mastomys) natalensis: A model for gastric carcinoid formation. Yale J. Biol. Med. 65, 741-751. Ninomiya, H., and Nakamura, T. (1987). Benign testicular hyperplasia in Mongolian gerbils (Meriones unguiculatus). Jikken Dobutsu 36, 191-194. Nishi, M., and Steiner, D. E (1990). Cloning of complementary DNAs encoding islet amyloid polypeptide, insulin, and glucagon precursors from a New World rodent, the degu, Octodon degus. Mol. Endocrinol. 4, 1192-1198. Nolan, T. J. and Farrell, J. E (1987). Experimental infection of the multimammate rat (Mastomys natalensis) with Leishmania donovani and Leishmania major Am. J. Trop. Med. Hyg. 36, 264-269. Nolan, T. J., Megyeri, Z., Bhopale, V. M., and Schad, G. A. (1993). Strongyloides stercoralis: The first rodent model for uncomplicated and hyperinfective strongyloidiasis, the Mongolian gerbil (Meriones unguiculatus). J. Infect. Dis. 168, 1479-1484. Norris, M. L., and Adams, C. E. (1972a). The growth of the Mongolian gerbil, Meriones unguiculatus, from birth to maturity. J. Zool. 166, 277-282. Norris, M. L., and Adams, C. E. (1972b). Incidence of cystic ovaries and reproductive performance in the Mongolian gerbil, Meriones unguiculatus. Lab. Anim. 6, 337-342.

7. BIOLOGY AND DISEASES OF OTHER RODENTS

Norris, M. L., and Adams, C. E. (1974). Sexual development in the Mongolian gerbil, Meriones unguiculatus, with particular reference to the ovary. J. Reprod. FertiL 36, 245-248. Norris, M. L., and Adams, C. E. (1981). Pregnancy concurrent with lactation in the Mongolian gerbil (Meriones unguiculatus). Lab. Anim. 15, 21-23. Norris, M. L., and Adams, C. E. (1982). Lifetime reproductive performance of Mongolian gerbils (Meriones unguiculatus) with 1 or 2 ovaries. Lab. Anim. 16, 146-150. Novacek, M. J. (1992). Mammalian phylogeny: Shaking the tree. Nature 356, 121-125. Nowak, R. M. (1999). "Walker's Mammals of the World," 6th ed. Johns Hopkins Univ. Press, Baltimore. Nowak, R. M., and Paradico, J. L. (1983). "Walker's Mammals of the World," 4th ed, Vols. 1 and 2. Johns Hopkins Univ. Press, Baltimore. Nowakowska, M., Matras, J., Bartoscze, M., and Palec, S. (1991). Alimentary intoxication of chinchillas caused by Clostridium perfringens enterotoxin. Med. Weter. 47, 156-157. Nutting, W. B., Satterfield, L. C., and Cosgrove, G. E. (1973). Demodex sp. infesting tongue, esophagus, and oral cavity of Onychomys leucogaster, the grasshopper mouse. J. Parasitol. 59, 893-896. Oguge, N. O. (1995). Diet, seasonal abundance, and microhabitats of Praomys (Mastomys) natalensis (Rodentia: Muridae) and other small rodents in Kenyan sub-humid grassland community. Afr. J. Ecol. (UK) 33, 211-223. Ohnishi, K., and Kutsumi, H. (1995). Possible formation of new brood capsule by the previously formed brood capsule in Echinococcus multilocularis metacestodes. Southeast Asian J. Trop. Med. Public Health 26, 319-321. Oliver, J. H. Jr., Chandler, E W. Jr., James, A. M., Sanders, E H. Jr., Hutcheson, H. J., Huey, L. O., McGuire, B. S., and Lane, R. S. (1995). Natural occurrence and characterization of the Lyme disease spirochete, Borrelia burgdorferi, in cotton rats (Sigmodon hispidus) from Georgia and Florida. J. Parasitol. 81, 30-36. Olsen, R. W. (1968). Gestation period in Neotoma mexicana. J. Mammal. 49, 533-534. Olson, M. E., Goemans, I., Bolingbroke, D., and Lundberg, S. (1988). Gangrenous dermatitis caused by Corynebacterium ulcerans in Richardson ground squirrels. J. Am. Vet. Med. Assoc. 193, 367-368. Ostapoff, E. M., and Morest, D. K. (1989). A degenerative disorder of the central auditory system of the gerbil. Hear. Res. 37, 141-162. Ottolini, M. G., Porter, D. D., Hemming, V. G., Hensen, S. A., Sami, I. R., and Prince, G. A. (1996). Semi-permissive replication and functional aspects of the immune response in a cotton rat model of human parainfluenza virus type 3 infection. J. Gen. Virol. 77, 1739-1743. Owens, D. R., Menges, R. W., Sprouse, R. E, Stewart, W., and Hooper, B. E. (1975). Naturally occurring histoplasmosis in the chinchilla (Chinchilla laniger). J. Clin. Microbiol. 1, 486-488. Ozaki, R. (1993). Reproduction and social life of the black-tailed prairie dog. Anim. Zoos 45, 324-327. Packer, J. T., Kraner, K. L., Rose, S. D., Stuhlman, R. A., and Nelson, L. R. (1970). Diabetes mellitus in Mystomys albicaudatus. Arch. Pathol. 89, 410-415. Padovan, D. (1985). Intraperitoneal pentobarbitone anesthesia of Mystromys albicaudatus. Lab. Anim. Sci. 19, 16-18. Park, A. W. (1974a). Biology of the rice rat (Oryzomys palustris natator) in a laboratory environment. 3. Morphology of dentition. Acta Anat. (Basel) 87, 45-56. Park, A. W. (1974b). Biology of the rice rat (Oryzomys palustris natator) in a laboratory environment. 4. Mineralisation of the molar teeth. Acta Anat. (Basel) 87, 433-446. Park, A. W. (1974c). Biology of the rice rat (Oryzomys palustris natator) in a laboratory environment. 5. The maxillary and mandibular osteodental fissures. Acta Anat. (Basel) 88, 217-230. Park, A. W., and Nowosielski-Slepowron, B. J. (1975). Biology of the rice rat (Oryzomys palustris natator) in a laboratory environment. 7. Pre-weaning growth of the body. Acta Anat. (Basel) 91, 500-509.

301 Park, A. W., and Nowosielski-Slepowron, B. J. (1976). Biology of the rice rat (Oryzomys palustris natator) in a laborabory environment. 10. Postweaning growth of the skull. Acta Anat. (Basel) 94, 356-368. Parsons, R. C., Spratt, D. M. J., and Kirkwood, J. K. (1987). Cryptococcus neoformans in six species of small mammals at the Zoological Society of London. Erkrank. Zoot. 29, 137-141. Partono, E, and Purnomo. (1987). Periodicity studies of Brugia malayi in Indonesia: Recent findings and a modified classification of the parasite. Trans. R. Soc. Trop. Med. Hyg. 81, 657-662. Patton, J. L., and Smith, M. E (1989). Genetic structure and the genetic and morphologic divergence among pocket gopher species (genus Thomomys). In "Speciation and Its Consequences" (D. Otte and J. A. Endler, eds.), pp. 284-304. Sinauer Assoc., Sunderland, Massachusetts. Patton, J. L., and Smith, M. E (1993). Molecular evidence for mating asymmetry and female choice in a pocket gopher (Thomomys) hybrid zone. Mol. Ecol. 2, 3-8. Peiffer, R. L., and Johnson, P. T. (1980). Clinical ocular findings in a colony of chinchillas (Chinchilla laniger). Lab. Anim. 14, 331-335. Peles, J. D., and Barrett, G. W. (1997). Assessment of metal uptake and genetic damage in small mammals inhabiting a fly ash basin. Bull. Environ. Contam. Toxicol. 59, 279-284. Pelliccioli, G. P., Gambelunghe, C., Ottaviano, P. E, Iannaccone, S., and Ambrosini, M. V. (1995). Variable response of the Mongolian gerbil to unilateral carotid occlusion: Magnetic resonance imaging and neuropathological characterization. Ital. J. Neurol. Sci. 16, 517-526. Perrin, M. R. (1981). Notes on the activity patterns of 12 species of southern African rodents and a new design of activity monitor. S. Afr. J. Zool. 16, 248-258. Perrin, M. R. (1987). Effects of diet on the gastric papillae and microflora of the rodents Mystromys albicaudatus and Cricetomys gambianus. S. Afr. J. Zool. 22, 67-76. Perrin, M. R., and Curtis, B. A. (1980). Comparative morphology of the digestive system of 19 species of southern African myomorph rodents in relation to diet and evolution. S. Afr. J. Zool. 15, 22-23. Perrin, M. R., and Kokkinn, M. J. (1986). Comparative gastric anatomy of Cricetomys gambianus and Saccostomus campestris (Cricetomyinae) in relation to Mystromys albicaudatus (Cricetinae). S. Afr. J. Zool. 21, 202210. Perrin, M. R., and Maddock, A. H. (1983). Preliminary investigations of the digestive process of the white-tailed rat Mystromys albicaudatus (Smith 1834). S. Afr. J. Zool. 18, 128-133. Perrin, M. R., and Maddock, A. H. (1985). Comparative gastric morphology of some African rodents. Fortschr. Zool. 30, 317-324. Pfaffenberger, G. S., Kemether, K., and de Bruin, D. (1985). Helminths of sympatric populations of kangaroo rats (Dipodomys ordii) and grasshopper mice (Onychomys leucogaster) from the high plains of eastern New Mexico. J. Parasitol. 71, 592-595. Piazza, E M., Schmidt, H. J., Johnson, S. A., Dotson, D. L., Darnell, M. E., Ottolini, M. G., Porter, D. D., and Prince, G. A. (1995). A cotton rat model of effectors of immunity to respiratory syncytial virus other than serum antibody. Pediatr. Pulmonol. 19, 355-359. Pinter, A. J. (1970). Reproduction and growth for two species of grasshopper mice (Onychomys) in the laboratory. J. Mammal. 51, 236-243. Pinter, A. J. (1985). Effects of hormones and gonadal status on the midventral gland of the grasshopper mouse Onychomys leucogaster. Anat. Rec. 211, 318-322. Poeggel, G., Lange, E., Hase, C., Metzger, M., Gulyaeva, N., and Braun, K. (1999). Maternal separation and early social deprivation in Octodon degus: Quantitative changes of nicotinamide adenine dinucleotide phosphate-diaphorase-reactive neurons in the prefrontal cortex and nucleus accumbens. Neuroscience 94, 497-504. Ponce, A. A., Carrascosa, R. E., Aires, V. A., Fiol de Cuneo, M., Ruiz, D., Ponzio, M. E, and Lacuara, J. L. (1998). Activity of Chinchilla laniger

302 spermatozoa collected by electroejaculation and cryopreserved. Theriogenology 50, 1239-1249. Port, C. D., Richter, W. R., and Moize, S. M. (1971). An ultrastructural study of Tyzzer's disease in the Mongolian gerbil (Meriones unguiculatus). Lab. Invest. 25, 81-87. Porter, S. L. (1979). Microsporum gypseum infection in three Mexican prairie dogs. Vet. Med. Small Anim. Clin. 74, 71-73. Prieur, D. J., Olson, H. M., and Young, D. M. (1979). Partial oculocutaneous albinism in Mystromys albicaudatus: Nonhomology with the Chediak-Higashi syndrome. Lab. Anim. Sci. 29, 40-43. Prince, G. A., and Porter, D. D. (1996). Treatment of paraintiuenza virus type 3 bronchiolitis and pneumonia in a cotton rat model using topical antibody and glucocorticosteroid. J. Infect. Dis. 173, 598-608. Prince, G. A., Porter, D. D., Jenson, A. B., Horswood, R. L., Chanock, R. M., and Ginsberg, H. S. (1993). Pathogenesis of adenovirus type 5 pneumonia in cotton rats (Sigmodon hispidus). J. Virol. 67, 101-111. Prince, G. A., Prieels, J. P., Slaoui, M., and Porter, D. D. (1999). Pulmonary lesions in primary respiratory syncytial virus infection, reinfection, and vaccine-enhanced disease in the cotton rat (Sigmodon hispidus). Lab. Invest. 79, 1385-1392. Prior, J. E. (1986). Caesarean section in the chinchilla. Vet. Rec. 119, 408. Pruthi, A. K., Kharole, M. U., Gupta, R. K. P., and Kumar, B. B. (1983). Occurrence and pathology of intracutaneous cornifying epitheliomas in Mastomys natalensis, the multimammate mouse. Res. Vet. Sci. 35, 127-129. Raevuori, M., Harvey, S. M., and Pickett, M. J. (1979). Yersinia enterocolitica. Experimental pathogenicity for chinchilla. Acta Vet. Scand. 20, 82-91. Ratio, C. P., and Diamond, S. S. (1980). Metastatic squamous-cell carcinoma in a gerbil (Meriones unguiculatus). Lab. Anim. 14, 237-239. Rakich, P. M., Dubey, J. P., and Contarino, J. K. (1992). Acute hepatic sarcocystosis in a chinchilla. J. Vet. Diagn. Invest. 4, 484-486. Randall, J. A. (1986). Lack of gonadal control of the dorsal gland and sandbathing in male and female bannertail kangaroo rats (Dipodomys spectabilis). Horm. Behav. 20, 95-105. Randall, J. A., and Stevens, C. M. (1987). Footdrumming and other anti-predator responses in the bannertail kangaroo rat (Dipodomys spectabilis). Behav. Ecol. Sociobiol. 20, 187-194. Randeria, J. D. (1978). The inbreeding of the Y and the Z strains of Praomys natalensis with special reference to the laboratory uses of the Mastomys. J. S. Afr. Vet. Assoc. 49, 197-199. Rantanen, N. W., and Highman, B. (1970). Spontaneous tumors in a colony of Mystromys albicaudatus (African white-tailed rat). Lab. Anim. Care 20, 114-119. Redl, P., and Gyorffy, G. (1978). Multimammate rat (Mastomys natalensis) as a laboratory animal. Magy. Allatorvosok Lapja 33, 844-848. Redl, P., and Kassai, T. (1979). The course of primary and secondary Nippostrongylus brasiliensis infection in the multimammate rat (Mastomys natalensis) and albino rat. Parasitolog. Hungarica 12, 71-78. Reichman, O. J. (1975). Relation of desert rodent diets to available resources. J. Mammal. 56, 731-751. Reiter, R. J., Steinlechner, S., Richardson, B. A., and King, T. S. (1983). Differential response of pineal melatonin levels to light at night in laboratory-raised and wild-captured 13-lined ground squirrels (Spermophilus tridecemlineatus). Life Sci. 32, 2625-2629. Reynolds, T. J., and Wright, J. W. (1979). Early postnatal physical and behavioural development of degus (Octodon degus). Lab. Anim. 13, 93-99. Richmond, M., and Conaway, C. H. (1969). Management, breeding, and reproductive performance of the vole, Microtus ochrogaster, in a laboratory colony. Lab. Anim. Care 19, 80-87. Rickman, R., and Kanyangala, S. (1990). A comparative study of the infectivity of T.b. rhodesiense isolates from man to 3 species of small laboratory rodents. E. Afr. Med. J. 67, 413-419. Riddle, B. R., Honeycutt, R. L., and Lee, P. L. (1993). Mitochondrial DNA phylogeography in northern grasshopper mice (Onychomys leucogaster)wthe

THOMAS M. DONNELLY AND FRED W. QUIMBY

influence of Quaternary climatic oscillations on population dispersion and divergence. Mol. Ecol. 2, 183-193. Ridgway, R. L., Oaks, S. C. Jr., and LaBarre, D. D. (1986). Laboratory animal models for human scrub typhus. Lab. Anim. Sci. 36, 481-485. Riley, T., Stuhlman, R. A., VanPeenan, H. J., Esterly, J. A., and Townsend, J. E (1975). Glomerular lesions of diabetes mellitus in Mystromys albicaudatus. Arch. Pathol. 99, 167-169. Ringler, D. H., Lay, D. M., and Abrams, G. D. (1972). Spontaneous neoplasms in aging gerbillinae. Lab. Anim. Sci. 22, 407-414. Robbins, C. B., and van der Straeten, E. (1989). Comments on the systematics of Mastomys Thomas 1915 with the description of a new West African species. Senckenbergiana Biol. 69, 1-14. Robel, G. L., Lochmiller, R. L., McMurry, S. T., and Quails, C. W. Jr. (1996). Environmental, age, and sex effects on cotton rat (Sigmodon hispidus) hematology. J. Wildl. Dis. 32, 390-394. Rocke, T. E. (1992). Diagnostic riddles (Capillaria hepatica). J. Wildl. Dis. 28. Rockett, J., Seville, R. S., Kingston, N., Williams, E. S., and Thorne, E. T. (1990). A cestode, Taenia mustelae, in the black-footed ferret (Mustela nigripes) and the white-tailed prairie dog (Cynomys leucurus) in Wyoming. J. Helminthol. Soc. Wash. 57, 160-162. Rodrigues, M., Fine, B. S., Highman, B., and Streett, R. P. Jr. (1972). Partial ocular albinism in Mystromys albicaudatus (the African white-tailed rat). An electron microscope study. Arch. Ophthalmol. 87, 337-346. Roebuck, B. D., and Longnecker, D. S. (1979). Response of two rodents, Mastomys natalensis and Mystromys albicaudatus, to the pancreatic carcinogen azaserine. J. Natl. Cancer Inst. 62, 1269-1271. Rogers, K. L., and Chrisp, C. E. (1998). Lipoma in the mediastinum of a prairie dog (Cynomys ludovicianus). Contemp. Topics Lab. Anim. Sci. 37, 7 4 76. Rollin, P. E., Ksiazek, T. G., Elliott, L. H., Ravkov, E. V., Martin, M. L., Morzunov, S., Livingstone, W., Monroe, M., Glass, G., and Ruo, S. (1995). Isolation of Black Creek Canal virus, a new hantavirus from Sigmodon hispidus in Florida. J. Med. Virol. 46, 35-39. Rouquette, C., and Berche, P. (1996). The pathogenesis of infection by Listeria monocytogenes. Microbiologia 12, 245-258. Rowe, S. E., Simmons, J. L., Ringler, D. H., and Lay, D. M. (1974). Spontaneous neoplasms in aging Gerbillinae. A summary of forty-four neoplasms. Vet. Pathol. 11, 38-51. Rowlands, I. W., and Weir, B. J. (1974). "The Biology of Hystricomorph Rodents." Academic Press, New York. Rudolph, R. L., Muller, H., Reinacher, M., and Theil, W. (1981). Morphology of experimentally induced so-called keratoacanthomas and squamous cell carcinomas in 2 inbred-lines of Mastomys natalensis. J. Comp. Pathol. 91, 123-134. Saibaba, P., Urs, Y. L., Desai, B. L. M., and Sanjeevarayappa, K. V. (1988). Breeding and management of Indian desert gerbil (Meriones hurrianae Jerdon) under captivity and seminatural conditions. Indian J. Exp. Biol. 26, 481-482. Saito, S., Kurokawa, Y., and Sato, H. (1977). Effects of various diets on growth, longevity, and incidence of spontaneous tumors of Praomys (Mastomys) natalensis. Sci. Rep. Res. Inst. Tohoku Univ. [Med.] 24, 33-42. Saitoh, R. (1987). Fate of cysticerci of Taenia crassiceps following oral infection in small mammals. Jpn. J. Vet. Res. 35, 145. Salas, R. A., de Manzione, N., and Tesh, R. (1998). Venezuelan hemorrhagic fever: Eight years of observation. Acta Cient. Venez. 49, 46-51. (In Spanish.) Sanford, S. E. (1989). Cerebral nematodiasis caused by the racoon ascarid (Baylisascaris procyonis) in chinchillas. Can. Vet. J. 30, 902. Sanford, S. E. (1991). Cerebrospinal nematodiasis caused by Baylisascaris procyonis in chinchillas. J. Vet. Diagn. Invest. 3, 77-79. Sangvaranond, A., and Zahner, H. (1989). 19s and 7s antibody response of Mastomys natalensis in experimental filarial (Litmosoides carinii, Acantho-

7. BIOLOGY AND DISEASES OF OTHER RODENTS cheilonema viteae, Brugia malayi, B. pahangi) infections. Zentralbl veterinarmed [B] 36, 374-384. Sasaki, M., Arai, T., Usui, T., and Oki, Y. (1991a). Immunohistochemical, ultrastructural, and hormonal studies on the endocrine pancreas of voles (Microtus arvalis) with monosodium aspartate-induced diabetes. Vet. Pathol. 28, 497-505. Sasaki, M., Arai, T., Oki, Y., and Komeda, K. (1991b). The ultrastructural study on the pancreatic islet B cell in diabetic herbivorous voles. Vet. Res. Commun. 15, 205-210. Sayles, P. C., Hunter, K. W., Stafford, E. E., and Hendricks, L. D. (1981). Antibody response to Leishmania mexicana in African white-tailed rats (Mystromys albicaudatus). J. Parasitol. 67, 585-586. Schaeffer, D. O., and Donnelly, T. M. (1997). Disease problems of guinea-pigs and chinchillas. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 260-283. Saunders, Philadelphia. Schitoskey, E Jr., and Woodmansee, S. R. (1978). Energy requirements and diet of the California ground squirrel. J. Wildl. Manag. 42, 373-382. Schlech, W. F. (1996). Pathogenesis and immunology of Listeria monocytogenes. Pathol. Biol. (Paris) 44, 775-782. Schmidt, G., Martin, A. P., Stuhlman, R. A., Townsend, J. E, Lucas, E V., and Vorbeck, M. L. (1974). Evaluation Of hepatic mitochondrial function in the spontaneous diabetic Mystromys albicaudatus. Lab. Invest. 30, 451-457. Schmidt, G. E., Martin, A. P., Townsend, J. E, and Vorbeck, M. L. (1980). Basement membrane synthesis in spontaneously diabetic Mystromys albicaudatus. Lab. Invest. 43, 217-224. Schroder, G. D. (1979). Foraging behavior of the banner tail kangaroo rat (Dipodomys spectabilis). Ecology 60, 657-665. Schwarzbrott, S. S., Wagner, J. E., and Frisk, C. S. (1974). Demodicosis in the Mongolian gerbil (Meriones unguiculatus): A case report. Lab. Anim. Sci. 24, 666-668. Scotti, A. L., Bollag, O., and Nitsch, C. (1998). Seizure patterns of Mongolian gerbils subjected to a prolonged weekly test schedule: Evidence for a kindling-like phenomenon in the adult population. Epilepsia 39, 567-576. Seed, J. R., and Hall, J. E. (1980). A review on the use of Microtus montanus as an applicable experimental model for the study of African trypanosomiasis. Ann. Soc. Belg. Med. Trop. 60, 341-348. Serdobova, I. M., and Kramerov, D. A. (1998). Short retroposons of the B2 superfamily: Evolution and application for the study of rodent phylogeny. J. Mol. Evol. 46, 202-214. Setoguchi, Y., Danel, C., and Crystal, R. G. (1994). Stimulation of erythropoiesis by in vivo gene therapy: Physiologic consequences of transfer of the human erythropoietin gene to experimental animals using an adenovirus vector. Blood 84, 2946-2953. Seville, R. S. (1997). Eimeria spp. (Apicomplexa: Eimeriidae) from black- and white-tailed prairie dogs (Cynomys ludovicianus and Cynomys leucurus) in central and southeast Wyoming. J. Parasitol. 83, 166-168. Shakibi, J. G., and Weiss, L. (1969). The prevalence rate of congenital heart disease in newborn gerbils (Meriones unguiculatus). A preliminary report. Henry Ford Hosp. Med. J. 17, 241-246. Shaull, E. M. (1988). Fur quality and fur breakage in the chinchilla. Chin. World 37, 9. Shelton, G. C. (1954a). Giardiasis in the chinchilla. I. Observations on morphology, location in the intestinal tract, and host specificity. Am. J. Vet. Res. 15, 71-74. Shelton, G. C. (1954b). Giardiasis in the chinchilla. II. Incidence of the disease and results of experimental infections. Am. J. Vet. Res. 15, 75-78. Shepherd, A. J., Leman, P. A. and Swanepoel, R. (1989). Viremia and antibody response of small African and laboratory animals to Crimean-Congo hemorrhagic fever virus infection. Am. J. Trop. Med. Hyg. 40, 541-547. Shine, H. D., Wyde, P. R., Aguilar-Cordova, E., Chen, S. H., Woo, S. L., Grossman, R. G., and Goodman, J. C. (1997). Neurotoxicity of intracerebral in-

303 jection of a replication-defective adenoviral vector in a semipermissive species (cotton rat). Gene Ther. 4, 275-279. Shinomura, Y., Eng, J. and Yalow, R. S. (1987). Chinchilla "big" and "little" gastrins. Biochem. Biophys. Res. Commun. 143, 7-14. Shklair, I. L., and Rails, S. A. (1988). Periodontopathic micro-organisms in the rice rat (Oryzomys palustris). Microbios 55, 25-31. Shore, R. E, and Douben, P. E. (1994). The ecological significance of cadmium uptake and residues in terrestrial small mammals. Ecotoxicol. Environ. Saf. 29, 101-112. Siegel, J. H. (1986). Cochlear potentials in the study of cochlear physiology. Scand. Audiol. 25, 35-47. Silverman, J., Paturzo, E, and Smith, A. L. (1982). Effects of experimental infection of the deer mouse (Peromyscus maniculatus) with mouse hepatitis virus. Lab. Anim. Sci. 32, 273-274. Sims, E. (1990). Caesarean section in a chinchilla [letter]. Vet. Rec. 126, 490. Singh, N. B., Srivastava, A., Verma, V. K., Kumar, A., and Gupta, S. K. (1984). Mastomys natalensis: A new animal model for Mycobacterium ulcerans research. Indian J. Exp. Biol. 22, 393-394. Sirigu, P., Didio, L. J., Gross, S. A., and Perra, M. T. (1985). Morphological and histochemical study of the submandibular gland in Praomys (Mastomys) natalensis. Acta Anat. (Basel) 121, 81-83. Sirigu, P., Gross, S. A., Didio, L. J., and Lantini, M. S. (1988). Histochemical localization of prostaglandin synthetase in the salivary glands of Praomys (Mastomys) natalensis. Basic Appl. Histochem. 32, 321-325. Skinner, J. D., and Smithers, R. H. N. (1990). '~ Mammals of the Southern African Subregion," 2nd ed. Republic of South Africa, Univ. of Pretoria. Smedley, E H., Rimmer, J., Samanidis, A., and Wastell, C. (1990). Cell renewal in non-specific duodenitis, ulcer-related duodenitis, and duodenal ulcer. Experiments in Mastomys (Praomys natalensis). Digestion 45, 72-79. Smith, M. E (1998). Phylogenetic relationships and geographic structure in pocket gophers in the genus Thomomys. Mol. Phylogenet. Evol. 9, 1-14. Snell, K. C., and Hollander, C. E (1972). Tumors of the testes and seminal vesicles in Myomys (Praomys) natalensis. J. Natl. Cancer Inst. 49, 1381-1393. Snell, K. C., and Stewart, H. L. (1967). Neoplastic and non-neoplastic renal disease in Praomys (Mastomys) natalensis. J. Natl. Cancer Inst. 39, 95-117. Snell, K. C., and Stewart, H. L. (1969). Hematopoietic neoplasms and related conditions in Praomys (Mastomys) natalensis. J. Natl. Cancer Inst. 42, 175-202. Snell, K. C., and Stewart, H. L. (1975). Spontaneous diseases in a closed colony of Praomys (Mastomys) natalensis. Bull. World Health Organ. 52, 645650. Snyder, R. L. (1979). Hepatitis and hepatocellular carcinoma in captive prairie dogs (Cynomys ludovicianus). Erkrank. Zoot. 13, 325-334. Solleveld, H. A., van Zwieten, M. J., Zurcher, C., and Hollander, C. E (1982). A histopathological survey of aged Praomys (Mastomys) natalensis. J. Gerontol. 37, 656-665. Solleveld, H. A., Coolen, J., Haaijman, J. J., Hollander, C. E, and Zurcher, C. (1985). Autoimmune thyroiditis. Spontaneous autoimmune thyroiditis in Praomys (Mastomys) coucha. Am. J. Pathol. 119, 345-349. Solomon, N. G., and Vandenbergh, J. G. (1994). Management, breeding, and reproductive performance of pine voles. Lab. Anim. Sci. 44, 613-617. Solomon, H. E, Dixon, D. M., and Pouch, W. (1990). A survey of staphylococci isolated from the laboratory gerbil. Lab. Anim. Sci. 40, 316-318. Somova, L. I., Gregory, M. A., and Nxumalo, E. N. (2000). Mongolian gerbil (Meriones unguiculatus) as a model of cerebral infarction for testing new therapeutic agents. Methods Find. Exp. Clin. Pharmacol. 22, 203-210. Sorden, S. D., and Watts, T. C. (1996). Spontaneous cardiomyopathy and exophthalmos in cotton rats (Sigmodon hispidus). Vet. Pathol. 33, 375382. Southwick, C. H. (1964). Peromyscus leucopus: An interesting subject for studies of socially-induced stress responses. Science 143, 55-56. Spannl, M., and Kraft, H. (1988). [Blood values of chinchilla]. Berl. Munch. Tierarztl. Wochenschr. 101, 344-347. (In German.)

304 Spano, R. K. (1994). Ground squirrel management in the Angeles National Forest. Pro. Verteb. Pest Conf. 16, 68-71. Spear, G. S., Caple, M. V., and Sutherland, L. R. (1984). The pancreas in the degu. Exp. Mol. Pathol. 40(3), 295-310. Spence, S., and Skae, K. (1995). Urolithiasis in a chinchilla [letter]. Vet. Rec. 136, 524. Srivastava, R., and Gupta, S. K. (1985). Effect of lanosterol on the susceptibility of Mastomys natalensis to Entamoeba histolytica infection. Trans. R. Soc. Trop. Med. Hyg. 79, 422-429. Stallone, J. N., and Braun, E. J. (1988). Regulation of plasma antidiuretic hormone in the dehydrated kangaroo rat (Dipodomys spectabilis M.). Gen. Comp. Endocrinol. 69, 119-127. Stanton, N. L., Shults, L. M., Parker, M., and Seville, R. S. (1992). Coccidian assemblages in the Wyoming ground squirrel, Spermophilus elegans elegans. J. Parasitol. 78, 323-328. Stehbens, W. E. (1986). An appraisal of cholesterol feeding in experimental atherogenesis. Prog. Cardiovasc. Dis. 29, 107-128. Stephenson, R. S. (1990). Caesarean section in a chinchilla [letter]. Vet. Rec. 126, 370. Stewart, H. L., and Snell, K. C. (1968). Thymomas and thymic hyperplasia in Praomys (Mastomys) natalensis. Concomitant myositis, myocarditis, and sialodacryoadenitis. J. Natl. Cancer Inst. 40, 1135-1159. Stewart, H. L., and Snell, K. C. (1975). Patterns of neoplastic and nonneoplastic diseases of Praomys (Mastomys) natalensis. Recent Results Cancer Res. 52, 139-144. Stoenner, H. G., and Lackman, D. B. (1957). A new species of Brucella isolated from the desert wood rat, Neotoma lepida Thomas. Am. J. Vet. Res. 18, 947-951. Storey, K. B. (1997). Metabolic regulation in mammalian hibernation: Enzyme and protein adaptations. Comp. Biochem. Physiol. A Physiol. 118, 11151124. Stout, C. A., and Duszynski, D. W. (1983). Coccidia from kangaroo rats (Dipodomys spp.) in the western United States, Baja California, and northern Mexico with descriptions of Eimeria merriami sp. n. and Isospora sp. J. Parasitol. 69, 209-214. Straneva, J. E., and Gallati, W. W. (1980). Eimeria strangfordensis sp. n., Eimeria barleyi sp. n., and Eimeria antonellii sp. n. from the Eastern woodrat (Neotomafloridana) from Pennyslvania. J. Parasitol. 66, 329-332. Street, R. P. Jr., and Highman, B. (1971). Blood chemistry values in normal MystromYs albicaudatus and Osborne-Mendel rats. Lab. Anim. Sci. 21, 394-398. Streubel, D. P., and Fitzgerald, J. P. (1978). Spermophilus tridecemlineatus. Mammal. Species 1t)3, 1-5. Strike, T. A. (1970). Hemogram and bone marrow differential of the chinchilla. Lab. Anim. Care 20, 33-38. Strittmatter, J. (1972). Elimination of Tyzzer's disease in the Mongolian gerbil (Meriones unguiculatus) by fostering to mice. Z. Versuchstierkunde 14, 209 - 214. (In German.) Stuhlman, R. A., and Wagner, J. E. (1971). Ringtail in Mystromys albicaudatus: A case report. Lab. Anim. Sci. 21, 585-587. Stuhlman, R. A., Packer, J. T., and Doyle, R. E. (1972a). Spontaneous diabetes mellitus in Mystromys albicaudatus. Repeated glucose values from 620 animals. Diabetes 21, 715-721. Stuhlman, R. A., Packer, J. T., and Rose, S. D. (1972b). Repeated blood sampiing of Mystromys albicaudatus (white-tailed rat). Lab Anim. Sci. 22, 268-270. Stuhlman, R. A., Srivastava, P. K., Schmidt, G., Vorbedk, M. L., and Townsend, J. E (1974). Characterization of diabetes mellitus in South African hamsters (Mystromys albicaudatus). Diabetologia 10, (Suppl.), 685-690. Stuhlman, R. A., Packer, J. T., Doyle, R. E., Brown, R. V., and Townsend, J. E (1975). Relationship between pancreatic lesions and serum glucose values in Mystromys albicaudatus. Lab. Anim. Sci. 25, 168-174.

THOMAS M. DONNELLY AND FRED W. QUIMBY Suckow, M. A., Terril-Robb, L. A., and Grigdesby, C. E (1996). Gastric trichobezoar in a banner-tailed kangaroo rat (Dipodomys spectabilis). Lab. Anim. 30, 383-385. Suedmeyer, W. K., and Pace, L. (1994). Management of an epiglottal fibrosarcoma in a black-tailed prairie dog (Cynomys ludovicianus). J. Small Exotic Anim. Med. 2, 163-164. Sullivan, J. (1996). Combining data with different distributions of among-site rate variation. System. Biol. 45, 375-380. Sullivan, J., Holsinger, K. E., and Simon, C. (1996). The effect of topology on estimates of among-site rate variation. J. MoI. Evol. 42, 308-312. Swensen, S. R., and Telford, I. R. (1973). Lipofuscin distribution and histological lesions in the vitamin E deficient cotton rat (Sigmodon hispidus hispidus). Arch. Histol. Jpn. 35, 327-341. Swindle, M. M., Hulebak, K. L., and Yarbrough, B. A. (1985). Haematology and pathology of captive southern grasshopper mice (Onychomys torridus). Lab. Anim. 19, 195-199. Tajima, K. (1981). A light- and electron-microscopic study on BNUR esophageal carcinomas of Mastomys and rats. Acta Med. Biol. 28, 85-117. Talmage, S. S., and Walton, B. T. (1991). Small mammals as monitors of environmental contaminants. Rev. Environ. Contam. Toxicol. 119, 47-145. Talmage, S. S., Opresko, D. M., Maxwell, C. J., Welsh, C. J., Cretella, E M., Reno, P. H., Daniel, E B. (1999). Nitroaromatic munition compounds: Environmental effects and screening values. Rev. Environ. Contam. Toxicol. 161, 1-156. Tan, C. H., Tachezy, R., Van Ranst, M., Chen, S.-Y., Bernard, H., and Burk, R. D. (1994). The Mastomys natalensis papillomavirus: Nucleotide sequence, genome organization, and phylogenetic relationship of a rodent papillomavirus involved in tumorigenesis of cutaneous epithelia. Virology 198, 534-541. Taylor, J. M. (1968). Reproductive mechanisms of the female southern grasshopper mouse, Onychomys torridus longicaudus. J. Mammal. 49, 303-309. Taylor, R. (1997). The sixth sense. New Sci. 153, 36-40. Taylor, G. N., Lloyd, R. D., and Mays, C. W. (1993). Liver cancer induction by 239Pu, 241Am, and thorotrast in the grasshopper mouse, Onychomys leucogaster. Health Phys. 64, 141-146. Tennant, B. C., Mrosovsky, N., McLean, K., Cote, P. J., Korba, B. E., Engle, R. E., Gerin, J. L., Wright, J., Michener, G. R., Uhl, E., et al. (1991). Hepatocellular carcinoma in Richardson's ground squirrels (Spermophilus richardsonii): Evidence for association with hepatitis B-like virus infection. Hepatology 13, 1215-1221. Thiessen, D. D., and Yahr, P. (1977). "The Gerbil in Behavioral Investigations: Mechanisms of Territoriality and Olfactory Communication." Univ. of Texas Press, Austin. Thomas, R. E., Barnes, A. M., Quan, T. J., Beard, M. L., Carter, L. G., and Hopla, C. E. (1988). Susceptibility to Yersinia pestis in the northern grasshopper mouse (Onychomys leucogaster). J. Wildl. Dis. 24, 327-333. Thomas, R. E., Beard, M. L. Quan, T. J., Carter, L. G., Barnes, A. M., and Hopla, C. E. (1989). Experimentally induced plague infection in the northern grasshopper mouse (Onychomys leucogaster) acquired by consumption of infected prey. J. Wildl. Dis. 25, 477-480. Thomas, H. H., Best, T. L., and Lydeard, C. (1991). Parasitic and phoretic arthropods of the elephant-eared and the Santa Cruz kangaroo rats. J. Wildl. Dis. 27, 358-360. Tielemans, Y., Vierendeels, T., and Willems, G. (1987). Histochemical and proliferative changes preceding the onset of spontaneous gastric adenocarcinoma in Mastomys natalensis. Virchows Arch. A Pathol. Anat. Histopathol. 411, 275-281. Tikasingh, E. S., Jonkers, A. H., Spence, L., and Aitken, T. H. (1966). Nariva virus, a hitherto undescribed agent isolated from the Trinidadian rat, Zygodontomys b. brevicauda (J. A. Allen & Chapman). Am. J. Trop. Med. Hyg. 15, 235-238. Tomich, P. Q. (1982). Ground squirrels--Spermophilus beecheyi and allies. In

7. BIOLOGY AND DISEASES OF OTHER RODENTS "Wild Mammals of North America" (J. A. Chapman and G. A. Feldhamer, eds.). Johns Hopkins Univ. Press, Baltimore. Torrez Martinez, N., Bharadwaj, M., Goade, D., Delury, J., Moran, P., Hicks, B., Nix, B., Davis, J. L., and Hjelle, B. (1998). Bayou virus-associated hantavirus pulmonary syndrome in Eastern Texas: Identification of the rice rat, Oryzomys palustris, as reservoir host. Emerg. Infect. Dis. 4, 105-111. Tortora, D. E, Eyer, J. C., and Overmann, S. R. (1974). The effect of sand deprivation on sandbathing and marking in Mongolian gerbils (Meriones unguiculatus). Behav. Biol. 11,403-407. Towe, A. L., and Harrison, T. A. (1993). Cerebral response to pyramidal tract stimulation in wood rats and its relation to laboratory rats. Exp. Brain Res. 97, 311-316. Townzen, K. R., Thompson, M. A., and Smith, C. R. (1996). Investigations and management of epizootic plague at ice house reservoir, Eldorado National Forest, California, 1994 and 1995. Pro. Verteb. Pest Conf. 17, 68-74. Trautwein, G., and Helmboldt, C. E (1967). Experimental pulmonary talcum granuloma and epithelial hyperplasia in the chinchilla. Pathol. Vet. 4, 254267. Trigo, E J., and Riser, M. (1981). A squamous cell carcinoma in a California ground squirrel (Spermophilus beecheyi). J. Wildl. Dis. 17, 405-408. Tripathi, B. J., Tripathi, R. C., Borisuth, N., Dhaliwal, R., and Dhaliwal, D. (1991). Rodent models of congenital and hereditary cataract in man. Lens Eye Tox. Res. 8, 373-413. Trpis, M. (1994). Aedes (Gymnometopa) mediovittatus (Diptera: Culicidae) as an experimental vector of Brugia pahangi and B. malayi (Spiruida: Filariidae). J. Med. Entomol. 31,442-444. Tsai, J. C., Garlinghouse, G., McDonnell, P. J., and Trousdale, M. D. (1992). An experimental animal model of adenovirus-induced ocular disease. The cotton rat. Arch. Ophthalmol. 110, 1167-1170. Tsanava Sh, A., Marennikova, S. S., Sakvarelidze, L. A., Shelkhina, E. M., and lanova, N. N. (1989). Isolation of cowpox virus from the red-tailed jird. Vopr. Virusol. 34, 95-97. (In Russian.) Tsubota, K., Inoue, H., Ando, K., Ono, M., Yoshino, K., and Saito, I. (1998). Adenovirus-mediated gene transfer to the ocular surface epithelium. Exp. Eye Res. 67, 531-538. Turner, G. V. (1978). Some aspects of the pathogenesis and comparative pathology of toxoplasmosis. J. S. Afr. Vet. Assoc. 49, 3-8. Tvedten, H. W., and Langham, R. E (1974). Trophoblastic emboli in a chinchilla. J. Am. Vet. Med. Assoc. 165, 828-829. Une, Y., Tatara, S., Nomura, Y., Takahashi, R., and Saito, Y. (1996). Hepatitis and hepatocellular carcinoma in two prairie dogs (Cynomys ludovicianus). J. Vet. Med. Sci. 58, 933-935. Upton, S. J., Fridell, R. A., and Tilley, M. (1989). Trypanosoma kansasensis sp. n. from Neotomafloridana in Kansas. J. Wildl. Dis. 25, 410-412. Upton, S. J., McAllister, C. T., Brillhart, D. B., Duszynski, D. W., and Wash, C. D. (1992). Cross-transmission studies with Eimeria arizonensis-like oocysts (Apicomplexa) in New World rodents of the Genera Baiomys, Neotoma, Onchomys, Peromyscus, and Reithrodontomys (Muridae). J. Parasitol. 78, 406-413. Urquiza, R., Rico, E, and Figuerola, J. A. (1988). The gerbil (Meriones unguiculatus) in auditory research. Acta Otorrinolaringol. Esp. 39, 227-233. (In Russian.) Vanjonack, W. J., and Johnson, H. D. (1973). Relationship of thyroid and adrenal function to "fur-chewing" in the chinchilla. Comp. Biochem. PhysioL A 45, 115-120. Vaughan, J. A., and Turell, M. J. (1996). Dual host infections: Enhanced infectivity of Eastern equine encephalitis virus to Aedes mosquitos mediated by Brugia microfilariae. Am. J. Trop. Med. Hyg. 54, 105-109. Veenstra, A. J. E (1958). Behaviour of the multimammate mouse, Rattus (Mastomys) natalensis (A. Smith). Anita. Behav. 6, 195-206. Villela, O. M. M., and Alho, C. J. R. (1983). Postnatal development and growth

305 of Oryzomys subflavus (Rodentia: Cricetidae) in laboratory setting. Rev. Bras. Biol. 43, 321-326. Vincent, A. L., and Ash, L. R. (1978). Further observations on spontaneous neoplasms in the Mongolian gerbil, Meriones unguiculatus. Lab. Anim. Sci. 28, 297-300. Vincent, A. L., Porter, D. D., and Ash, L. R. (1975). Spontaneous lesions and parasites of the Mongolian gerbil, Meriones unguiculatus. Lab. Anim. Sci. 25, 711-722. Vincent, A. L., Ash, L. R., Sodeman, W. A. Jr., and Rodrick, G. E. (1979a). Pathology of intratesticular Brugia in jirds. J. Parasitol. 65, 990. Vincent, A. L., Rodrick, G. E., and Sodeman, W. A. Jr. (1979b). The pathology of the Mongolian Gerbil (Meriones unguiculatus): A review. Lab. Anita. Sci. 29, 645-651. Vos, A. J. d. (1970). Studies on the host range of Eimeria chinchillae De Vos & amp: Van der Westhuizen, 1968. Onderstepoort J. Vet. Res. 37, 2936. Vos, A. J. d., and Dobson, L. D. (1970). Eimeria chinchillae De Vos & amp: Van der Westhuizen, 1968 and other Eimeria spp. from three South African rodent species. Onderstepoort J. Vet. Res. 37, 185-190. Voss, R. S. (1991). An introduction to the Neotropical muroid rodent genus Zygodontomys. Bull. Am. Mus. Nat. Hist. 21t), 1-113. Voss, R. S. (1992). A revision of the South American species of Sigmodon (Mammalia: Muridae) with notes on their natural history and biogeography. Am. Mus. Novit. 3t)50, 1-56. Voss, R. S., Heideman, P. D., Mayer, V. L., and Donnelly, T. M. (1992). Husbandry, reproduction, and postnatal development of the neotropical muroid rodent Zygodontomys brevicauda. Lab. Anim. 26, 38-46. Waggie, K. S., and Marion, P. L. (1997). Demodex sp. in California ground squirrels. J. Wildl. Dis. 33, 368-370. Waggie, K. S., Ganaway, J. R., Wagner, J. E., and Spencer, T. H. (1984). Experimentally induced Tyzzer's disease in Mongolian gerbils (Meriones unguiculatus). Lab. Anim. Sci. 34, 53-57. Waggie, K. S., Thornburg, L. P., and Wagner, J. E. (1986). Experimentally induced Tyzzer's disease in the African white-tailed rat. Lab. Anim. Sci. 36, 492-495. Wagner, R. A. (1999). Diagnosing odontomas in prairie dogs. Exotic DVM 1, 7-10. Wallach, J. D., and Boever, W. J. (1983). Rodents and lagomorphs. In "Diseases of Exotic Animals: Medical and Surgical Management," pp. 135-195. Saunders, Philadelphia. Wallis, P. M., and Wallis, H. M. (1986). Excystation and culturing of human and animal Giardia spp. by using gerbils and TYI-S-33 medium. Appl. Environ. Microbiol. 51, 647-651. Wang, T. C., and Fox, J. G. (1998). Helicobacter pylori and gastric cancer: Koch's postulates fulfilled? Gastroenterology 115, 780-782. Wang, P., Qian, G., Lu, H., Sheng, H., and Zhu, L. (1985). Studies on the development of thermoregulation in Chinchilla laniger. Acta Zool. Sinica. 31, 359-364. Waring, G. H. (1970). Sound communications of black-tailed, white-tailed, and Gunnison's prairie dogs. Am. Midl. Nat. 83, 167-185. Watanabe, T., Tada, M., Nagai, H., Sasaki, S., and Nakao, M. (1998). Helicobacter pylori induces gastric cancer in Mongolian gerbils. Gastroenterology 115, 642-648. Wayss, K., Reyes-Mayes, D., and Volm, M. (1981). Chemical carcinogenesis by the two-stage protocol in the skin of Mastomys natalensis (Muridae) using topical initiation with 7, 12-dimethylbenz(a) anthracene and topical promotion with 12-0-tetradecanoylphorbol-13-acetate. Virchows Arch. B Cell Pathol. Inc. MoL PathoL 38, 13-21. Webster, W. D. (1987). Kyphosis in the marsh rice rat (Oryzomys palustris). J. Wildl. Dis. 23, 171-172. Weir, B. J. (1966). Aspects of reproduction in chinchillas. J. Reprod. FertiL 12, 410-411.

306 Weir, B. J. (1967). The care and management of laboratory hystricomorph rodents. Lab. Anim. (London) 1, 95-104. Weir, B. J. (1970). The management and breeding of some more hystricomorph rodents. Lab. Anim. 4, 83-97. Weir, B. J. (1973). The induction of ovulation and oestrus in the chinchilla. J. Reprod. Fertil. 33, 61-68. Weir, B. J. (1976). The chinchilla. In "The UFAW Handbook on the Care and Management of Laboratory Animals" (Universities Federation for Animal Welfare, eds.), 5th ed. Churchill Livingstone, Edinburgh. Wheat, B. E., and Ernst, J. V. (1974). Eimeria glauceae sp. n. and Eimeria dusii sp. n. (Protozoa: Eimeriidae) from the Eastern woodrat, Neotomafloridana, from Alabama. J. Parasitol. 60, 403-405. White, D. J., and Waldron, M. M. (1969). Naturally-occurring Tyzzer's disease in the gerbil. Vet. Rec. 85, 111-114. White, P., Fischer, R., and Meunier, G. (1982). The lack of recognition of lactating females by infant Octodon degus. Physiol. Behav. 28, 623-625. Wightman, S. R., Pilitt, P. A., and Wagner, J. E. (1978a). Dentostomella translucida in the Mongolian gerbil (Meriones unguiculatus). Lab. Anim. Sci. 28, 290-296. Wightman, S. R., Wagner, J. E., and Corwin, R. M. (1978b). Syphacia obvelata in the Mongolian gerbil (Meriones unguiculatus): Natural occurrence and experimental transmission. Lab. Anim. Sci. 28, 51-54. Wightman, S. R., Mann, P. C., and Wagner, J. E. (1980). Dihydrostreptomycin toxicity in the Mongolian gerbil, Meriones unguiculatus. Lab. Anim. Sci. 30, 71-75. Wilber, P. G., McBee, K., Hafner, D. J., and Duszynski, D. W. (1994). A new coccidian (Apicomplexa: Eimeriidae) in the northern pocket gopher (Thomomys talpoides) and a comparison of oocyst survival in hosts from radonrich and radon-poor soils. J. Wildl. Dis. 30, 359-364. Wiley, R. W. (1980). Neotomafloridana. Mammal. Species 139, 1-7. Wilkerson, M. J., Melendy, A., and Stauber, E. (1997). An outbreak of listeriosis in a breeding colony of chinchillas. J. Vet. Diagn. Invest. 9, 320323. Williams, C. S. E (1980). Wild rats in research. In "The Laboratory Rat. II. Research Applications" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), Vol. 2, pp. 245-256. Academic Press, San Diego. Williams, J. E., Arntzen, L., and Isaacson, M. (1982). Advantages and use of laboratory-reared African multimammate mice (Mastomys coucha) in the diagnosis of plague. Ann. Soc. Belges Med. Trop. Parasitol. Mycol. 62, 247-251. Wisniewski, P. J. (1985). The Libyan jird in captivity. IZN 32, 16-18. Wobeser, G., Cawthorn, R. J., and Gajadhar, A. A. (1983). Pathology of Sarcocystis campestris infection in Richardson's ground squirrels (Spermophilus richardsonii). Can. J. Comp. Med. 47, 198-202. Wolf-Dieter, R., Heuschneider, G., Sperk, G., and Riederer, R. (1989). Biochemical events in spontaneous seizures in the Mongolian gerbil. Metab. Brain Dis. 4, 3-7. Wolfe, J. L. (1982). Oryzomys palustris. Mammal. Species 176, 1-5. Wood, F. D. (1936). "Trypanosoma neotomae, sp. nov., in the dusky-footed wood rat and the wood rat flea." Univ. of California Press, Berkeley. Woods, C. A., and Boraker, D. (1975). Octodon degus. Mammal. Species 67, 1-5. Woolf, A., King, J., and Tennant, B. (1982). Primary hepatocellular carcinoma in a blacktailed prairie dog (Cynomys ludovicianus). J. Wildl. Dis. 18, 517520. Worgul, B. V., and Rothstein, H. (1975). Congenital cataracts associated with disorganized meridional rows in a new laboratory animal: The degu (Octodon degus). Biomedicine 23, 1-4. Worth, C. B. (1950). Observations on the behavior and breeding of captive rice rats and wood rats. J. Mammal. 31, 421-426. Worth, C. B. (1967). Reproduction, development, and behavior of captive Oryzomys laticeps and Zygodontomys brevicauda in Trinidad. Lab. Anim. Care 17, 355-361.

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Worthington, J. M., and Fulghum, R. S. (1988). Cecal and fecal bacterial flora of the Mongolian gerbil and the chinchilla. Appl. Environ. Microbiol. 54, 1210-1215. Wuthe, H. H., and Aleksic, S. (1992). Yersinia enterocolitica serovar 1,2a, 3 biovar 3 in chinchillas. Zentralbl. BakterioL 277, 403-405. Wyscoki, C. J., and Lepri, J. J. (1991). Consequences of removing the vomeronasal organ. J. Steroid Biochem. Mol. Biol. 39, 661-669. Yamagishi, S., Watanabe, Y., Tomura, H., Sekine, T., Mimura, M., Iijima, Y., and Fujii, M. (1997). Septic infection of a companion chinchilla with Salmonella enteritidis. J. Jpn. Vet. Assoc. 50, 345-348. Yamamoto, R. S., Kroes, R., and Weisburger, J. H. (1972). Carcinogenicity of diethylnitrosamine in Mystromys albicaudatus (African white-tailed rat). Proc. Soc. Exp. Biol. Med. 140, 890-892. Yamini, B., and Raju, N. R. (1986). Gastroenteritis associated with a Cryptosporidium sp. in a chinchilla. J. Am. Vet. Med. Assoc. 189, 1158-1159. Yasukawa, N., Michael, S. D., and Christian, J. J. (1978). Estrous cycle regulation in the white footed mouse (Peromyscus leucopus) with special reference to vaginal cast formation. Lab. Anim. Sci. 28, 46-50. Yei, S., Mittereder, N., Tang, K., O'Sullivan, C., and Trapnell, B. C. (1994). Adenovirus-mediated gene transfer for cystic fibrosis: Quantitative evaluation of repeated in vivo vector administration to the lung. Gene Ther. 1, 192-200. Yesus, Y. W., Estedy, J. A., Stuhlman, R. A., and Townsend, J. E (1976). Significant muscle capillary basement membrane thickening in spontaneously diabetic Mystromys albicaudatus. Diabetes 25, 444-449. Yingrui, L., Yaxian, G., Xinyuan, Y., Xianfang, Z., Longxiang, P., and Qingshan, Z. (1983). Schistosomajaponicum: A comparison of the development of the parasite and associated pathological changes in mice and jirds (Meriones unguiculatus). Int. J. Parasitol. 13, 531-538. Yoerg, S. I., and Shier, D. M. (1997). Maternal presence and rearing condition affect responses to a live predator in kangaroo rats (Dipodomys heermanni arenae). J. Comp. Psychol. 111, 362-369. Yokomori, K., Okada, N., Murai, Y., Goto, N., and Fujiwara, K. (1989). Enterohepatitis in Mongolian gerbils (Meriones unguiculatus) inoculated perorally with Tyzzer's organism (Bacillus piliformis). Lab. Anim. Sci. 39, 16-20. Young, L. J., Winslow, J. T., Wang, Z., Gingrich, B., Guo, Q., Matzuk, M. M., and Insel, T. R. (1997). Gene targeting approaches to neuroendocrinology: Oxytocin, maternal behavior, and affiliation. Horm. Behav. 31, 221-231. Young, L. J., Wang, Z., and Insel, T. R. (1998). Neuroendocrine basis of monogamy. Trends Neurosci. 21, 71-75. Zahner, H., and Rudolph, R. (1980). Capillaria hepatica infection of Mastomys natalensis: Pathological changes in liver and spleen following infections with embryonated and x-ray attenuated eggs. Zentralbl. Veterinarmed. B. 27, 85-101. Zahner, H., Gorkow, C., Soulsby, E. J., Schutze, H. R., Geyer, E., and Reiner, G. (1987). Homocytotropic antibodies of the multimammate rat, Mastomys natalensis. Demonstration and characterization of 2 types classified as IgE and IgG3. Z. Versuchstierkunde 29, 243-256. Zahner, H., Sanger, I., Chatterjee, R. K., and Seibold, G. (1989). Altered immune response (humoral and delayed-type hypersensitivity reactions) to sheep red blood cells in the course of experimental filarial infections (Litomosoides carinii, Brugia malayi, Acanthocheilonema viteae) of Mastomys natalensis. Parasitol. Res. 75, 401-411. Zarate, M. L., and Scherer, W. E (1968). Contact-spread of Venezuelan equine encephalomyelitis virus among cotton rats via urine or feces and the nasoor oropharnyx. A possible transmission cycle in nature. Am. J. Trop. Med. Hyg. 17, 894-899. Zeinert, K. (1983). Husbandry of chinchillas. Vet. Med. Small Anim. Clin. 78, 1292-1294. Zeman, E J. (1967). A semipurified diet for the Mongolian gerbil (Meriones unguiculatus). J. Nutr. 91,415-420. Zielke, E. (1979). Attempts to infect Meriones unguiculatus and Mastomys na-

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talensis with Wuchereria bancrofii from West Africa. Tropenmed. Parasitol. 30, 466-468. Zielke, E. (1980). On the longevity and behaviour of microfilariae of Wuchereria bancrofii, Brugia pahangi, and Dirofilaria immitis transfused to laboratory rodents. Trans. R. Soc. Trop. Med. Hyg. 74, 456-458. Zima, J., Macholan, M., Misek, I., and Sterba, O. (1992). Sex chromosome ab-

307 normalities in natural populations of the common vole (Microtus arvalis). Hereditas 117, 203-207. Zimmerman, E. G. (1970). Karyology, systematics, and chromosomal evolution in the rodent genus, Sigmodon. Publ. Mus., Mich. State Univ. (Biol. Series) 4, 385-454.

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Chapter 8 Woodchucks as Laboratory Animals Christine A. Bellezza, Patrick W. Concannon, William E. Hornbuckle, Lois Roth, and Bud C. Tennant

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INTRODUCTION

The woodchuck, Marmota monax, is an important laboratory animal model for the study of the hepatitis B virus (HBV) of humans. Woodchucks are naturally infected with the woodchuck hepatitis virus (WHV), which is similar to HBV. Woodchucks have been used to study the pathogenesis of hepadnaviral infections and for the development and evaluation of antiviral compounds. The woodchuck has also been used to study food intake, obesity and energy balance, hibernation, and circannual LABORATORY ANIMAL MEDICINE, 2nd edition

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cycles. The following chapter discusses the use of woodchucks as laboratory animals and the biology of the woodchuck, as well as diseases and disorders affecting woodchucks.

A.

Taxonomic Considerations

The Eastern woodchuck, Marmota monax, is in the family Sciuridae of the order Rodentia. Common names include groundhog, whistle-pig, and chuck. Natural distribution includes the eastern and midwestern United States, southeastCopyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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ern Alaska, and southern Canada. The woodchuck is a large, burrow-digging animal with a thickset body; short legs; long claws; broad fiat head; almost no neck; small, round ears; and a short, hairy tail (Fig. 1). Adults in captivity reach an average body size of 3 - 5 kg in males and 2.5-5 kg in females. Colors vary from a grizzly gray-brown to reddish, with a darker head and black feet. Black as well as white (presumably albino) woodchucks have been reported. The fur consists of a soft dense undercoat and longer, coarse upper fur.

B.

U s e in R e s e a r c h

Woodchucks are obligate hibernators in the wild and have an annual cycle that involves large changes in food intake, body weight, and metabolic state. Body weight increases 25-100% during the spring and summer, and decreases by 15-50% during autumn and winter hibernation. There is a brief breeding season immediately following emergence from hibernation (Hamilton, 1934; Concannon et al., 1990). Similar changes occur in laboratory-maintained woodchucks, even when prevented from entering a deep hibernation state and constantly maintained in natural photoperiod and at room temperature. Captive and laboratory-reared woodchucks have been used as models to study food intake, obesity, and energy balance (Bailey, 1965; Fall, 1971; Young, 1984; Rawson et al., 1998); endogenous circannual cycles (Concannon et al., 1992); photoperiod en-

trainment of circannual cycles (Concannon et al., 1997a); seasonal breeding (Christian et al., 1972; Baldwin et al., 1985; Concannon et al., 1990); hibernation (Davis, 1977; Spurrier et al., 1987); and viral hepatitis and its sequelae, including hepatocellular carcinoma (Popper et al., 1987). There are indigenous populations of woodchucks that have a high incidence of WHV infection. The use of the laboratory woodchuck as a model for viral hepatitis has also resulted in the use of hepatitispositive animals in the evaluation of antiviral compounds. Laboratory-maintained woodchucks typically are not subjected to hibernation-inducing conditions of food withdrawal and cold room temperatures, except where the goal is to study aspects of hibernation or role of temperature in regulation of circannual cycles. In the absence of such conditions and deep hibernation, the animals nevertheless continue to undergo circannual changes in metabolic and reproductive activity. However, circannual cycles in the laboratory then tend to advance, with animals breeding 2 - 8 weeks earlier than in the wild (Concannon et al., 1997b). The use of artificial photoperiod to synchronize the circannual cycles of animals in a group or colony is important for reproduction and synchronization of metabolic cycles among animals (Concannon et al., 1996, 1997a). The possible asynchrony among animals of the circannual changes in metabolic state should be considered with any use of woodchucks as experimental animals. The biology of circannual cycles and seasonal breeding is considered in detail in subsequent sections.

Fig. 1. Woodchuck,Marmota monax.

8. WOODCHUCKSAS LABORATORYANIMALS C.

Availability and Sources

Woodchucks are easily trapped from the wild (Young and Sims, 1979). Captive animals may be used as breeding stock to form colonies of defined animals adapted to laboratory conditions and free from WHV infection and other diseases common to wild woodchucks. State laws concerning the capture of animals for research should be investigated prior to trapping woodchucks. Newly captured woodchucks should be examined for signs of illness or abnormal behavior, tattooed for permanent identification, and dusted with an insecticidal powder. They should be bled periodically, assessed for WHV status, and isolated for 6 months before being introduced to the colony. While it is difficult to accurately age adult woodchucks, it is possible to distinguish juveniles (< 1 year) and yearlings from adults (Young and Sims, 1979). The prevalence of WHV in feral woodchucks is not known. However, several studies have demonstrated a marked dichotomy between geographical areas in the percentage of WHV-positive woodchucks (Tyler et al., 1981; Wong et al., 1982; Tennant and Gerin, 1994). Woodchucks from New York and New England may be preferable to woodchucks from the Middle Atlantic region as foundation animals for breeding colonies. Woodchucks are also commercially available, and it is possible to purchase woodchucks born and bred in captivity, woodchucks infected with WHV, and woodchucks that are certifiably free of the infection.

D.

Laboratory Management and Husbandry

1. Housing

Woodchucks may be housed in metal cages designed for rabbits, dogs, or cats. Modifications need to be made because woodchucks have sharp incisors, considerable strength, and climbing ability. They can escape through any hole large enough to admit their heads, which requires reducing gaps greater than 1~2 inches. Latches may be necessary to secure the corners of the doors. Food and water dishes, as well as cage floors, must be secured so that woodchucks cannot remove them. Solid floors are preferable to slatted floors for several reasons. The large, soft feces of woodchucks do not fall through slats into collection pans, and thorough cleaning of slatted floors is difficult. Slats are also used as toeholds and may result in toenails being ripped. Woodchucks may also be housed in large pens or runs. The tops of chain-link pens must be enclosed. Woodchucks positive for WHV are housed in Horsfall cages because of the potential biohazard and transmission of WHV to WHV-negative woodchucks. Wooden or metal nest boxes provide a burrowlike environment for woodchucks. Woodchucks typically hide in nest boxes

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in response to noise or movement, making it easier to catch and handle them. The boxes facilitate cleaning cages because woodchucks will usually remain calm inside boxes. Bedding material such as wood chips should be provided. Woodchucks carry bedding into boxes for nesting and will use bedding for covering their feces. Most woodchucks are fastidious, defecating in one corner of their cage, and will use food to cover up feces if they are not provided with bedding material. Water should be supplied ad libitum; woodchucks drink large amounts, especially during periods of high food consumption. Water can be supplied in large ceramic bowls, which are difficult to tip over, or in heavy glass bottles with sipper tubes. Woodchucks can be successfully maintained at 17~176 Woodchucks are sensitive to high environmental temperatures, and air conditioners and/or fans should be used, if necessary, to keep them cool in summer or in warm climates. Humidity can be maintained at approximately 50% using humidifiers; lower humidity levels have been associated with a ringtail-like syndrome in pups. Light cycles should approximate normal day length. Light cycles can be adjusted daily, weekly, or monthly to correspond to seasonal changes in day length. 2.

Identification

As a temporary means of identification, the fur may be clipped or dyed. Tattoo ink on fur lasts about one week. Hair growth occurs rapidly between late May and October, and clipped areas should be rechecked weekly. Ear tags, ear notches, and tattoos can be used for more permanent identification of woodchucks. Tattooing is preferred because trauma to the ear can cause the tag to be removed or the notch to be altered. Tattoos are best placed on the chest or inner thigh where the hair is thin. The hair over the tattoo must be clipped periodically to maintain visibility. When tattooing, the area should first be clipped and surgically scrubbed and an antibiotic ointment applied after the tattoo. Tattoo needles should be changed between animals and gas sterilized before reuse. 3.

Handling and Restraint

Woodchucks have powerful jaws and large incisors and are capable of inflicting serious bite wounds. They are best handled with elkhide elbow-length primate handling gloves. The gloved hand is used to pin the animal's head to the ground by applying pressure to the back of the neck. The opposite hand then grasps the base of the tail. Once immobilized, most woodchucks calm down, and minor manipulations such as intramuscular injections can be performed. Aggressive woodchucks can be lifted off the ground and carried by the tail. Calmer animals can be carried with the gloved hand underneath and supporting the body. Squeeze boxes have also been used to handle woodchucks (Snyder, 1985); however, they can be awkward to use in confined spaces and require additional time and effort.

312 4.

CHRISTINE A. BELLEZZA, PATRICK W. CONCANNON, WILLIAM E. HORNBUCKLE, LOIS ROTH, AND BUD C. TENNANT Anesthesia

Chemical restraint is necessary for most manipulations of woodchucks. In the authors' experience, ketamine (50 mg/kg) in combination with xylazine (5 mg/kg) IM has proved to be safe and effective for phlebotomy and short surgical procedures. Lower doses may be effective in animals anesthetized infrequently but may result in a shorter duration of action. Anesthesia is induced in approximately 5 min and lasts up to 20 min. If adequate sedation is not achieved within 5 min of the initial injection, a second injection of one-half the initial dose can be given. For longer surgical procedures, isofluorane is administered either by face mask or by endotracheal intubation. An alternative to inhalation anesthesia is the intravenous administration of sodium pentobarbital. A dosage of 2 - 6 mg/kg is administered through a previously implanted catheter or into the sublingual vein to prolong the anesthetic state induced by ketamine-xylazine. Anesthesia lasts on average 2 0 - 4 0 min. Other investigators have used Innovar Vet, 0.35 ml/kg IM (Snyder, 1985). Innovar Vet can be reversed with naloxone. Regardless of the anesthetic used, it is important to withhold access to water until the animals are fully recovered from anesthesia to prevent accidental drowning. 5.

puncture. Following anesthesia, the venipuncture site is clipped and scrubbed with alcohol and an antiseptic. The femoral pulse is palpated in the inguinal region and is used as a reference point since the vessels are not visible. A vacutainer tube and a 22gauge, 1-inch needle are used. Direct pressure is applied following venipuncture to minimize hematoma formation. Samples can also be obtained from the maxillary or linguifacial veins, which run in close proximity to the clavicle. Here, the woodchuck is placed on its back, head toward the phlebotomist, and a 22-gauge, 1-inch needle is directed straight into the notch formed where the clavicle meets the sternum. Care must be taken to avoid entering the thorax. Cardiac puncture has been used and is the easiest and quickest method to obtain large amounts of blood, but complications such as cardiac tamponade and death can develop. Small amounts of blood can be obtained from the cephalic veins (on the medial aspect of the front legs) or tarsal veins (on the dorsal aspect of the rear feet). b.

Tablets are crushed and mixed with liquid. Liquids are given PO by dosing syringe. Woodchucks can be trained to readily accept oral medications by mixing the medication with or following it with a small amount of a liquid, such as molasses.

Physical Examination

Woodchucks should first be observed without disturbing them, keeping in mind that when woodchucks are upset, they may chatter, whistle, or make wheezing noises that may be mistaken for evidence of respiratory problems (Young and Sims, 1979). Observations that can be detected without handling the animal include ocular discharge, labored respiration, nasal discharge, discharge or foul odors from the ears, head tilt, or apparent weight loss. On closer examination, folliculitis, bite wounds, or ventral edema, etc., may be apparent. Incisors can be examined by enticing the animal to bite on something. With appropriate restraint, auscultation of the heart and lungs, and palpation of the abdomen should be performed. To complete the examination, release the woodchuck a distance from its nest box to observe its ability to ambulate. An unwillingness to move is usually a sign of a serious underlying illness. Thorough abdominal palpation generally requires anesthesia to reduce abdominal tone. In the authors' experience, palpation of the anesthetized woodchuck is useful in diagnosing pregnancy, in estimating whelping dates, and in detection of organomegaly, including hepatomegaly due to hepatocellular carcinoma (HCC). 6.

c.

Venipuncture

Woodchucks can be routinely bled from the femoral vein or artery. In most instances, general anesthesia is required for veni-

Injections

Intramuscular injections can be given in the gastrocnemius or quadriceps muscles. While it is easier for two people working together to inject woodchucks, it is possible for an experienced technician to accomplish the task alone by holding the tail and allowing the animal to grasp the edge of the cage with its front paws. Repeated injections of large volumes of irritating substances such as ketamine may cause myonecrosis. If frequent injections are unavoidable, the alternation of muscle groups and hindlegs is recommended. 7.

Research Techniques

Reported research on woodchucks has involved the use of intravenous catheters, liver biopsies (Mrozek et al., 1994), vascular access ports (Woolf et al., 1989), embryo collection (Concannon et al., 1997b), and electroejaculation (Concannon et al., 1996).

II.

A.

Clinical Techniques

a.

Oral Medications

I.

BIOLOGY

Characteristics and Comparative Physiology

Annual Metabolic Cycles

The annual cycle of changes in metabolic state and reproductive function persists in the laboratory, even if animals are not

8. WOODCHUCKSAS LABORATORYANIMALS subjected to so-called hibernation-inducing conditions of food withdrawal or cold room temperatures of 5~176 9However, circannual cycles may become shortened to 11, 10, or 9 months if woodchucks are not subjected to seasonal changes in the daily photoperiod that mimic natural changes (Concannon et al., 1992). Most woodchucks maintained at temperatures of 20 ~ 25~ and with food and water constantly available will undergo seasonal periods of reduced body temperature (unpublished observations, 1981). In autumn and early winter, woodchucks may be found with rectal temperatures of 30~ or lower. This period of physiological hypothermia presumably reflects an attempt at hibernation, which is part of the normal circannual cycle. This period is associated with low levels of free thyroid hormone despite the presence of moderate to high levels of total thyroid hormone (Concannon et al., 1999). The majority of thyroid hormone at that time appears bound to thyroxine binding globulin (TBG) and other proteins such that very little free hormone is available to promote mitochondrial activity and basal metabolism (Young, 1984; Rawson et al., 1998). Resting metabolism has been measured, and Vo2 was reported to average 4.4 _ 0.3 ml/min/ kg in early autumn (Rawson et al., 1998). Despite the low metabolic activity, the gonads slowly regain function, such that they are fully recrudescent at the end of the "hibernation period" in mid to late winter. Body weight declines in autumn, winter, and early spring as fat stores are utilized for metabolic needs. In one study, weight losses were far greater in females than in males (Snyder, 1985). In late winter and early spring, metabolic activity increases dramatically, and rectal temperatures are increased to highnormal values; food intake increases if food is available, but body weight gain is small or negligible. These changes are presumably due both to increased pituitary secretion of thyroid stimulating hormone (TSH) causing more thyroxine secretion, and to decreased amounts of TBG resulting in increased amounts of free thyroxine. There is also a seasonal increase in prolactin at this time (Concannon et al., 1999). In early spring, resting metabolism is elevated. Average Vo2 in early spring was reported to be increased from that of autumn (7.3 vs. 4.4 ml/ min/kg). After the brief breeding season and short 31-day pregnancies of late winter and early spring, there is a decline in thyroid hormones in spring and summer. As a result, the animals undergo a transition from minimal weight gain to rapid weight gain. Weight gain consists almost entirely of fat deposits in preparation for hibernation. Increases in body weight of 7 0 100% over a 3-5 month period can occur. Typical increases from nadir to peak body weight in laboratory woodchucks have been reported to be 45-100% in one study and 40-50% in another (Young and Sims, 1979; Concannon et al., 1993). In summer, food intake spontaneously declines, often rapidly, several weeks before peak body weight is reached. The signal mechanism is not known, but the result is a brief period of very positive energy balance in summer, followed by a slow, protracted decline in body weight in autumn and winter.

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Other elements of the circannual cycle that have been less well studied include fur molts in the late summer and late winter, changes in pituitary hormone secretion, and presumably, changes in adrenal function (Young and Sims, 1979). 2.

Endogenous Circannual Cycles

The annual cycle of metabolic and reproductive activity in woodchucks persists, with changes quantitatively the same, even if the animals are maintained under constant photoperiods. In males maintained in daily photoperiods of 12 hr of light and 12 hr of dark, patterns of gonadal activity had approximately 12 month intervals for 1-3 years before then "free-running" at circannual intervals of 8-10 months (Concannon et al., 1992). The majority of animals will become asynchronous and freerun after 2-3 years if photoperiod cues simulating those of the natural environment are not provided. In studies using large block changes in the photoperiod, similar to those used in other species, photoperiod was unable to modify the circannual cycles of woodchucks. 3.

Photoentrainment of Circannual Cycles to 12 Months

The endogenous cycle of woodchucks can be entrained to 12 month intervals by exposure to simulated natural photoperiods. This has been done, using weekly or twice per month or monthly changes in the daily light cycle to approximate that of the outdoors (unpublished observations, 1999). However, the preferred and best-studied method is to change the daily photophase each day, using microprocessor-controlled timers for room lights. The simulation of natural, 0 - 4 min-per-day increases and decreases in daily photophase is accomplished by changing the time of lights on and the time of lights off each day, with the most rapid changes at the times of the vernal and autumnal equinoxes (Concannon et al., 1997a). Similarly produced but Southern Hemisphere photoperiods beginning at 3 months of age or at 15 months of age reentrained the woodchuck cycle to an austral annual cycle that was 6 months out of phase with the photoentrained Northern Hemisphere cycles (Concannon et al., 1997a). 4.

Hibernation

In the wild, woodchucks immerge into winter burrows in autumn, usually between September 15 and November 1, hibernate for 3-5 months, and emerge in late winter sometime in February or March. Field data suggest that males emerge before females, and adults emerge before yearlings. Natural hibernation involves bouts of torpor (inactivity due to reduced resting metabolism) at 2~176 above ambient temperature and bouts of arousal (activity associated with transient increases in resting basal metabolism) at varying intervals. Periods of each can range from 1 to 7 days, with torpor becoming progressively longer and then shorter during the hibernation period. The

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CHRISTINE A. BELLEZZA, PATRICK W. CONCANNON, WILLIAM E. HORNBUCKLE, LOIS ROTH, AND BUD C. TENNANT

physiology underlying the associated behavior and alteration in temperature set point is not understood. In the laboratory, a period of deep hibernation can be induced by reducing room temperature to 15~ or lower (Snyder, 1985) and removing food. Others have used temperatures of 6~176 removed both food and water, and used constant darkness, but have also observed that most animals will also hibernate in the presence of food and light (Young and Sims, 1979). In the authors' experience, at hibernation-inducing temperatures of 6~176 morbidity and mortality may increase unless measures are taken to ensure appropriate preparative weight gains under natural photoperiod. Clearly, consideration must be given to correct timing, humidity, and possible unnatural disturbances. Without so-called hibernation-inducing conditions like food withdrawal, water withdrawal, temperature reduction, and darkness, most photoentrained woodchucks will nevertheless undergo periods of physiological hypothermia during the expected period of hibernation. Body temperatures may range from euthermic to only 1~176 above room temperature. In one study, of woodchucks maintained at 20~ room temperature, the average rectal temperature was 37~ in early spring and only 29~ in early autumn (Rawson et al., 1998). The physiological hypothermia presumably reflects a hypothyroid state, reduced metabolism, and the same mechanisms that promote deep hibernation at lower temperatures. As with deep hibernation, spontaneous permanent arousal occurs in late winter. During the period of hibernation, with either deep or mild hypothermia, weight losses of 15-50% are common.

B. 1.

Normal Physiological Values

Longevity

The mean age at death for wild woodchucks has been reported to be 14.9 months with 50% surviving only 8 months (Snyder, 1985). The mean age at death for woodchucks captured at 3 - 5 months of age and maintained in a zoo environment was 4.5 years (Snyder, 1977). Maximum longevity was 10 years. In the authors' laboratory, woodchucks can live for up to 14 years (unpublished data, 1999). 2.

Hematology

Blood cell parameters in woodchucks appear to fall within the range reported for other rodent species, based on published and more recent observations (Table I). Unpublished observations by the authors also suggest that some values may vary with season. However, the time course of such changes has not been determined. Hematocrit can be higher in autumn (mean, 40%) than in spring (mean, 36%) and likewise, the percent saturation of hemoglobin (40% vs. 25%). The mean values for most other parameters are fairly narrow (Table I), suggesting little seasonal influence.

3.

Biochemistry

Serum chemistry results for woodchucks (Table II) can vary among studies, although electrolytes, as expected, are rather stable parameters. Unpublished results (1999) suggest that some serum enzymes may exhibit changes in concentration related to the annual cycle, but time courses of such changes have not been well characterized. The range of observed mean values for various studies in normal woodchucks suggests potential seasonal effects on alanine aminotransferase and alkaline phosphatase, and possibly aspartate aminotransferase (Table II). Mean values of other enzymes are less variable and may be less affected by season, including amylase, lipase, and creatine kinase. Mean protein values, especially the albumin fraction, can vary considerably, perhaps seasonally. Whether the observed variations in creatinine (0.9 to 1.5 gg/dl), total bilirubin (0.12 to 0.30 mg/dl), urea nitrogen (13 to 25 mg/dl) and cholesterol (140 to 210 mg/dl) involve seasonal changes remains to be determined.

C.

Nutrition

Specific nutritional requirements of woodchucks have not been established. However, the composition of commercially available rabbit food (at least 15% protein) resembles that of the diet of free-ranging woodchucks. In the laboratory, woodchucks have been successfully fed rabbit chow formulated into larger sized blocks (approximately 5.5 cm • 1.5 cm). The large blocks require gnawing, which helps to wear down the woodchucks' continually growing incisors. This diet seems adequate in maintaining adults and supporting growth and reproduction. Brittle incisors, increased susceptibility to infection, and alopecia, all problems associated with an inadequate diet (Young and Sims, 1979), have not been observed. A rabbit "breeder's formulation" diet, which has a higher caloric and protein content (at least 17%) and smaller-sized pellets, can be successfully fed to pregnant females and pups (unpublished observation, 1997). Significant pup mortality during the periweaning period ( 3 0 80 days of age) can occur and may be caused by competition for food. One approach to prevent this is to provide two large bowls of rabbit pellets per litter and check the bowls twice daily to ensure that clean, fresh food is always available. These measures have significantly reduced periweaning mortality (unpublished observation, 1998).

D. I.

Reproduction

Overview and Breeding Season

Males and females typically have a single, brief 2 - 4 week winter breeding season in which fertile matings occur, with the

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8. W O O D C H U C K S AS L A B O R A T O R Y A N I M A L S

Table I Hematological Data for Woodchucks a Unpublished values c Parameter Erythrocytes Hematocrit % (HCT) Red cell count (RBC) (• 106/izl) Mean corpuscle volume (MCV)(fl) Mean corpuscle Hb (MCH) (pg) Mean corpuscle Hb concentration (MCHC) (gm/dl) Red cell distribution width (RDW) (%) Nucleated RBC (per 100 WBC) Hemoglobin (Hb) (gm/dl)

% Saturation White blood cells White cell count (WBC)(X 103/!~1) Segmented neutrophils (x 103/1~1) Band neutrophils (X 103/i.d) N (Eosinophils) (• 103/IM) Lymphocytes (• 103/~1) Monocytes (x 103/IM) Hemostasis Platelets (• 103/I.d) Mean platelet volume (fl) PT (sec) APTT (sec) Fibrinogen (FIB)(mg/dl)

Published values b

39 5.3 77 26 34

+__7 __. 0.8 ___7 +_ 4 _ 3 na na 13.2 _+, 2.6

Low mean d

35.5 4.7 73 24.9 33.9 14.0 1.5 12.2

_ 3.4 _ 0.4 ___4 +__2.1 ___0.7 ___ 1.5 ___0.5 ___ 1.2

High meane

40.7 ___4.9 5.2 _ 0.6 76 ___4 26.1 + 1.4 35.9 +_ 1.3 17.5 +_ 2.5 3.0 +_ 0.5 13.4 ___2.1

na

23 ___5 (n = 5)

45 ___9 (n = 5)

10.1 __+4.0 6.4 ___3.3 0.1 __.0.1 0.4 +__0.4 2.6 ___1.3 0.6 +_.0.6

8.7 +_ 2.3 5.5 ___2.1 0.01 + 0.05 0.2 _ 0.2 2.1 ___0.9 0.6 + 0.3

10.4 +_4.4 6.6 __.2.2 0.03 __-0.05 0.4 • 0.5 3.1 ___1.2 1.1 +__0.6

na na na na na

451 ___55 6.8 ___0.6 6.5 ___0.5 26 ___3 193 +__42

525 ___91 7.1 __.0.5 7.6 ___0.6 31 +__6 225 ___30

a Range of mean (___ sem) hematological values obtained in 5 - 1 0 studies each involving 1 0 - 3 0 woodchucks in comparison to published mean (___ SD) values. b Graham (1985) mean ___SD reported for 328 observations on 62 adult woodchucks over a 1-year period, na, not available.

CMean (__+sem) values for groups of 12-30 adult woodchucks. dLowest mean value observed for a study involving a group of 10-30 healthy adult woodchucks. eHighest mean value observed for a study involving a group of 10-33 healthy adult woodchucks.

female being receptive and the male sexually aggressive. They can produce one litter per year. In the wild, the breeding season begins at or shortly after the emergence from winter hibernation in late winter. Following gonadal regression in summer, gonadal recrudescence occurs during hibernation, beginning in late autumn or early winter. Peak gonadal activity occurs in late winter. These changes and the subsequent pattern of gonadal regression during late spring and summer are all part of the endogenous circannual cycle of sequential changes in endocrine function and tissue metabolism. The endogenous cycle is entrained by photoperiod and hibernation activity in the wild. In the laboratory, without efforts to provide photoperiod entrainment, the breeding seasons of individuals usually become asynchronous after 1 or 2 years, with intervals becoming shorter than 12 months (Snyder, 1985; Concannon et al., 1992). As a resuit, the breeding season may not be concurrent within breeding pairs or groups. Therefore, laboratory breeding requires considerable attention to photoentrainment of the circannual cycle, and selection of breeding pairs or breeding groups based on concomitant periods of testis enlargement and vulval swelling. Even with efforts to photoentrain cycles, the breeding season

in the laboratory typically advances by 1 - 2 months in the calendar year during the first 1-3 years. Therefore, although the breeding season in the wild is in late February or March, colonyborn animals or animals in captivity after 1 - 2 years may be more likely to breed in late January and early February. Laboratory studies have usually not involved forced hibernation. Whether low-temperature hibernation in the laboratory would have an influence on the timing of the breeding season beyond that provided by photoperiod, perhaps delaying it to the "normal" time, has not been studied. Fertility in adults 2 or more years old can approximate 100% in the wild. Pregnancy rates in yearling females are only 50%. Males typically reach sexual maturity and first breed at 22 months of age (Snyder, 1985) but undergo pubertal changes at 1 0 - 1 1 months of age (Concannon et al., 1993). Information on basic woodchuck reproduction has been gleaned from observation in the wild (Hamilton, 1934; Snyder and Christian, 1960; Christian et al., 1972) and from studies of endocrine, behavioral, gonadal, and genital changes observed in captive and laboratory-raised animals (Concannon et al., 1984; Sinha Hikim et al., 1991, 1992; Concannon et al., 1996, 1997a,b).

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C H R I S T I N E A. B E L L E Z Z A , P A T R I C K W. C O N C A N N O N , W I L L I A M E. H O R N B U C K L E , L O I S R O T H , AND BUD C. T E N N A N T Table II Range of Mean (_sem) Serum Chemistry Values in Multiple Unpublished Studies Involving 1 5 - 3 0 Woodchucks per Study in Comparison to Published Mean (_+SD) Values ~ Unpublished values b Parameter

Proteins Albumin (g/dl) A: G ratio Globulin (g/dl) Total protein (g/dl) Electrolytes and acid-base Sodium (mEq/liter) Potassium (mEq/liter) Sodium/potassium Chloride (mEq/liter) Total CO2 (mEq/liter) Bicarbonate (mEq/liter) Anion gap (mEq/liter) Calcium (mg/dl) Phosphate (mg/dl) Liver and muscle Alanine amino transferase (U/liter) Aspartate amino transferase (U/liter) Creatine kinase (U/liter) Gamma glutamyl transferase (GGT) (U/liter) Alkaline phosphatase (U/liter) Total bilirubin (mg/dl) Cholesterol (mg/dl) Glucose (mg/dl) Other serum enzymes Amylase (U/liter) Lipase (U/liter) Renal function Urea nitrogen (mg/dl) Creatinine (mg/dl)

Published values a

3.7 ___0.8 na 3.2 _+ 1.2 6.9 __+ 1.2

Low mean t

2.3 0.7 3.0 5.5

• 0.2 ___0.2 • 0.5 ___0.3

High mean a

3.4 1.1 3.6 6.9

• 0.3 _ 0.3 • 0.6 __+0.6

147 __+4 4.7 • 0.7 na 97 ___7 na na na 10.1 • 0.7 5.0 __+ 1.3

143 ___ 3 3.7 _+ 0.3 34 ___ 3 98 ___3 31 • 2 30 _+ 3 15 ___4 9.7 ___0.5 3.8 __+0.6

151 ___3 4.4 ___0.4 40 • 4 102 ___3 34 __+3 34 ___4 20 +__4 11.0 +__0.6 5.4 ___ 1.9

2.0 ___ 1.0 26 • 13 600 • 512 1.7 • 0.9 10 ___9 0.05 ___0.05 na 184 • 61

1.0 __+ 1.0 21 ___ 12 478 ___ 350 na 7.2 __+2.1 0.11 __+0.05 140 ___40 186 ___21

3.5 ___2.5 34 ___21 690 ___ 310 na 19.3 • 5.5 0.32 __+0.1 210 • 50 220 • 40

na na

2210 ___648 201 ___62

2645 • 910 361 __+75

na na

13 • 5 0.9 ___0.2

25 • 6 1.5 ___0.5

a Grahanl (1985) mean ___SD reported for 345 observations on 62 adult woodchucks over a 1-year period. bMean (___ sem) values for groups of 1 2 - 3 0 adult woodchucks. r Lowest mean value observed for a study involving a group of 1 0 - 3 0 healthy adult woodchucks. dHighest mean value observed for a study involving a group of 1 0 - 3 3 healthy adult woodchucks.

2.

Female Reproduction

During proestrus and estrus there is detectable and measurable vulval swelling (Concannon et al., 1997b). Vulval diameter increases from 5 mm to > 7 mm over a 1-3 week period, typically remains near maximal diameter of 7-12 mm for 5 - 8 days, and then recedes over the next few weeks. Proestrus, the period of swelling before first willingness to copulate, typically lasts 1-2 weeks. The period of fertile estrus following proestrus vulval enlargement has been estimated to be about 1 week. Fertility in females paired with males either before, at, or one week after vulval swelling reached 7 mm was similar (5567%). Pairing between 1 and 2 weeks after obvious swelling was reported to reduce pregnancy rates to 33%. The conclusion was that estrus lasts 5-10 days in most instances (Concannon et al., 1997b). In general, woodchucks are reflex ovulators, with ovulation

being induced by multiple copulations during peak estrus. Mating behavior is difficult to monitor because of the secretive nature of the animals. Video study suggested that estrous females will mate with a sexually aggressive male several times in one day, usually for only 1 day, but sometimes over a 2- to 3-day period (Concannon et al., 1997b). However, in woodchucks as in cats, spontaneous ovulations can also occur (Concannon et al., 1997b). The incidence of spontaneous ovulation is presumed to be slight. Following ovulation and fertilization, serum progesterone is elevated during the 31-32 days of gestation, during the 5 - 8 weeks of lactation, and in many females, for 1-8 weeks postlactation. The only exception is a transient decline in progesterone for an 8- to 24-hour period before and during parturition. Such a decline appears to be part of the normal mechanism of parturition. The corpora lutea of pregnancy are the source of progesterone in each instance, with the same corpora lutea

8. WOODCHUCKSAS LABORATORYANIMALS becoming enlarged postpartum. Litter size can range from 1 to 10, and typically is 2 to 7. The average in one study of laboratory woodchucks was 3.8 but varied with age, being 2.9 in yearlings, 3.6 in young adults, and 4.2 in 3- to 4-year-old animals (Concannon et al., 1997b). In the absence of induced ovulation or pregnancy, signs of estrus subside, and ovarian follicles spontaneously luteinize, resuiting in moderate to extensive elevations in serum progesterone for several weeks or months. This spontaneous luteinization of follicles in nonbred and in mated but nonpregnant females typically coincides with the time when cohort females are giving birth and suckling young. It is also the period when woodchucks experience a circannual increase in prolactin. The incidence of pseudopregnancy immediately following infertile matings appears to be small but has not been studied in detail. Woodchucks have a double cervix and a complete medial septum in the body of the uterus. Therefore, all conceptuses in any one horn are derived from the ipsilateral ovary (Concannon et al., 1984). There has been limited study of serum estrogen levels and vaginal cytology changes during the breeding season (Sinha Hikim et al., 1991b, 1992), of vulval swelling patterns, of embryo collection and homologous surgical transfer (Concannon et aL, 1997b), and of behavior during mating (Sinha Hikim et al., 1992; Concannon et al., 1997). Following cohousing of estrous female(s) and a fertile male, the intervals to first mating ranged from 6 min to 6 hr. Copulation typically lasted 1-12 min. Ejaculation responses of males occurred at 2 - 7 min and were followed by 2 - 8 min of inactive amplexus (Concannon et al., 1997b). Females mate 1-17 times in a 4 - 8 hr period. Fertility was associated with the number of matings lasting longer than 3 or 5 min (but not with the total number of matings, some of which were very brief). Blastocysts implant on day 5 or 6 after fertile mating (Concannon et al., 1997b). Initial embryonic attachment and early implantation occur during a decidual reaction of the uterine endometrium at sites in the lumen opposite the region of the uterine mesentery (or mesometrium), away from the mesentery and blood supply. It is therefore termed antimesometrial implantation, a phenomenon that occurs in related species. Soon thereafter, subsequent embryo development and attachment of the discoid placenta are observed to occur on the mesometrial side of the uterus, close to the mesentery and blood supply. Fetal development can be monitored by abdominal palpation from day 10-11 onward, and more readily after day 15 (Concannon et al., 1997b). Ultrasound can also be used, but details have not been reported. Parturition occurs at 31 or 32 days after fertile mating during a transient decline in serum progesterone. The young are born with eyes closed. Lactation generally lasts 6 - 7 weeks if the dam is left to determine the time for weaning. 3.

Male Reproduction

Woodchucks have abdominal testes in summer and early autumn. In the wild, testicular recrudescence, scrotal develop-

317

ment, and testis descent occur each year during hibernation, and males emerge from hibernation in late winter with peak-sized testes and with peak spermatogenesis completed. In laboratoryhoused males these changes appear to occur more rapidly in late autumn and early winter, and breeding condition is often reached by midwinter in late January or early February. Testis volumes range from a peak size of 3 to 5 cm 3 to a nadir size of less than 0.3 cm 3. The male breeding season, while often earlier in the calender year than in the wild, typically coincides with that of females if the animals are photoentrained. In one study, fertility of males with enlarged scrotal testes was 100% (Concannon et al., 1996). The breeding season of an individual male has been estimated to range from 4 to 8 weeks and averages 6 weeks. The period of peak fertility may be less, but has not been studied. The annual cycle of spermatogenesis has been studied histologically (Christian et al., 1972), and the onset of testicular recrudescence has been advanced by administration of exogenous gonadotropin (Sinha Hikim et al., 1991 a). Woodchucks have very prominent Cowper's glands (bulbourethral glands) that are the source of the urethral plugs produced during ejaculation, and are most likely the source of vaginal plugs sometimes observed in females after mating. Stimulation parameters that yield electroejaculation have been reported, as have changes in sperm numbers obtainable by electroejaculation throughout the breeding season (Concannon et al., 1996). 4. Laboratory Breeding and Female Fertility In one study (Concannon et al., 1997b), 75% of 2- to 4-yearold estrous females became pregnant when housed with males and provided ad libitum opportunity to mate. Fertility was lower in yearling females (56%), in females 5 years of age or greater (58%), and in females subjected to repeated handling and video observation (37%). a.

Housing

Housing for breeding need not be specialized, in that pregnancies have been obtained in both cages and pens, with either recently or chronically pair-housed animals, or with males housed with a harem of 2 - 5 females. However, fertility may be improved by single housing or same-sex housing until the estimated onset of the breeding season. Pair or harem housing can be initiated in a colony based on calendar date of expected breeding season, on vulval swelling monitored in individual females, on initial vulval swelling in 25% of the females for the colony, and/or on a protocol of starting 2 weeks after enlarged scrotal testes are confirmed in 50% of the males in the colony (unpublished observations, 1998). Housing in pens rather than cages seems preferable and may yield higher fertility, but data are lacking. Continuing to provide one or more nest boxes provides individual animals with a place of refuge. Harem housing of a male with 2 - 4 females in a pen is most efficient. When pair housing in cages, results tend to be better if the cage has a solid floor rather than a grate floor.

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b.

CHRISTINE A. BELLEZZA, PATRICK W. CONCANNON, WILLIAM E. HORNBUCKLE, LOIS ROTH, AND BUD C. TENNANT

Detection of Estrus

placed with its opening at the entrance to the main nest box. The lid of the main nest box is opened, and the dam is encouraged to Proestrus can be detected based on serial measurement of vulenter the refuge box. val diameters, with diameters > 7 mm indicating obvious In our experience, normal pup weight at birth typically ranges proestrus. Estrus is suggested by attainment of a peak size, from 20 to 40 gm, and averages 27 gm. At birth, pups have a which may be variable among females (8-12 mm) (Concannon reddish color, thin translucent smooth skin, and closed eyes. At et al., 1997b). Late estrus is suggested by a softening of the 1 day of age, the skin is wrinkled and less red, the eyes are still vulva or loss of apparent turgor. Vaginal smears can be obtained closed, and the teeth nonerupted. Sex can be determined by by vaginal lavage and examined, with changes reminiscent of anogenital distance. The umbilical remnant is present and is lost those in other laboratory rodents being evident to a lesser or by day 4. Milk, if present, is visible in the stomach through the greater degree depending on the individual. Smears can also be translucent skin and is one parameter that can be checked daily obtained by use of a moist cotton-tipped swab. The potential to to ensure that the dam is caring for her pup. At 4 days, mean induce ovulation by vaginal manipulation exists. Typically, a body weight is 65 gm. The skin is graying, but pinkish on the moderate degree of cornification is observed in exfoliate cytolabdomen, and hair growth begins. Transfer of pups 4 - 2 1 days ogy. While neutrophils may be present anytime, often a decrease of age to foster dams is often successful and can be used when occurs during proestrus and estrus followed by an increase in dams become sick or perform poorly. At 2 weeks of age, fur has late estrus or postestrus. Detailed examination of smears in developed. Long muzzle hairs and a grizzled appearance are woodchucks has been reported (Sinha Hikim et al., 1992). characteristic. Pinnae are more obvious, and eye slits visible. Teeth erupt at 17-24 days of age. At 3 to 4 weeks, color varies c. Pregnancy and Pregnancy Diagnosis greatly, male testes are scrotal, animals respond to sound, and Pregnancy has been estimated to range from 30 to 33 days the eyes open. At 3 weeks, weight ranges from 100 to 220 gm, from observed matings. Pregnancies in 12 animals involved and averages 160 gm. At 4 weeks, the physiognomy is more like parturitions at 31 or 32 days after mating (mean 31.9 ___ 0.1 that of the adult, upper teeth erupt, and the pups are active and days). In that study the mean parturition date was March 13 __+ ambulatory but are still suckling. 1 day in females born into a colony in which photoperiod simulated natural changes in photoperiod, and March 17 ___ 1 day e. Neonatal Mortality for captured animals bred in the laboratory and similarly mainPup survival observed by the authors is slightly lower than tained. These dates were earlier than the mean date of April 12 that reported for some laboratory animals. In the authors' expe__. 1 days for pregnant females captured in the wild (Concannon rience, neonatal deaths average 20% in the first week, with most et al., 1997b). In that study, pregnancy was diagnosed by abdeaths occurring within the first 3 days. Mortality can reach dominal palpation following anesthesia with ketamine and xy28% by 4 weeks postpartum. Adoption of a "runt" pup to anlazine. Uterine vesicle diameters were approximately 11 mm at other postpartum dam can be successful. Another 1-5% of pups day 10, 20 mm at day 15, 27 mm at day 20, and 40 mm at day alive at 4 weeks may die before 3 months of age, possibly due 25. Palpation can be used to predict parturition date with conto competition for food. It may help to provide two large bowls siderable accuracy and can be done routinely by a trained techof rabbit pellets per litter and check the bowls frequently to ennician. Fetal heartbeats are detected by ultrasound at midgestasure that clean, fresh food is always available. tion, but the time of earliest detection has not been determined. Once pregnancy is confirmed, the female can be removed to a birthing pen or cage, or the male removed and the female left in f Lactation and Weaning the same enclosure. Litters have been successfully raised with a Mother-infant contact decreases between the fifth and sixth male cohoused with the female(s), but pup survival is someweeks after birth. Pups will frequently leave the nest box at 4 times better if the male is removed following pregnancy diagweeks of age and are weaned by the end of the sixth week nosis or immediately after birth of the litter. Having 2 pregnant (Hamilton, 1934). Pups can often be reared in the pen with the and/or postpartum females in the same pen does not affect pup dam for 1 year without problems. Pups grow rapidly and reach survival or growth. Females do better if left undisturbed with a 2.5 to 3.5 kg by 4 months. Some readily eat large block pellets nest box and bedding from midgestation onward. Nest boxes can be examined daily to confirm the date of parturition and ex- after weaning, but others require softening of such food with water or access to small pellets for several months. Providing amine pups. small pellets of a breeding rabbit chow for 6 - 9 months, beginning by 5 weeks of age, works well for all juveniles. More than d. Early Development of Newborn one bowl of food should be provided to each litter and kept full. Pups can be examined most readily by "training" the dam to Competition for food has resulted in emaciation and death in move into a second box ("refuge box"). The refuge box is weanling pups. \

8. WOODCHUCKS AS LABORATORY ANIMALS

For unknown reasons, female woodchucks must be housed individually for several weeks after weaning or serious fighting will result.

g.

Nutrition

Dams do not appear to require a special diet during gestation. They can be transferred to a small-pellet, breeding rabbit chow diet, ad libitum, in late gestation. Then food is automatically available later for the weanling pups without further changes.

E.

Behavior

Seasonal changes in food intake, activity, and hormone levels result in behavioral changes that may affect research results. In cases of weight loss, body weights for experimental animals should always be compared with those of control animals since weight loss can be normal. Torporous woodchucks may shake and show lethargy and anorexia, and can be difficult to differentiate from sick animals. As a general rule, sick animals may have a slightly decreased body temperature (normal body temperature is 37~ but the body temperature of torporous animals will approach that of room temperature (as low as 8 ~ 10~ (Beaudoin et al., 1969). Torporous animals become more active within 24 hr following handling and manipulation. Handling animals daily can prevent them from entering natural hypothermia or hibernation, yet may be necessary if animals are studied intensively during the fall and winter months. Elevated testosterone levels during the breeding season are correlated with an increased incidence of fighting between male woodchucks. Adult males in proximity to females should be housed in individual cages; young males may be housed together through their second spring. Under controlled conditions, adult males can be housed together if introduced to each other outside the breeding season, given additional nest boxes, and isolated from females.

III.

A.

DISEASES

Colony Health

During the day-to-day management of colony health, 5 0 70% of those woodchucks requiring treatment have dermatologic problems, the majority of which have bacterial folliculitis. Tail-base dermatitis is observed in woodchucks housed in metal stovepipes. Bite wounds and associated cellulitis are common during the breeding season. Ectoparasites are not a problem because recently acquired wild woodchucks are treated prior to admission to the colony.

319 Diarrhea is observed in 5% of the colony and in most instances is related to stress and/or transitional changes in food and water intake. Loose stools are commonly observed in woodchucks during the breeding season and just prior to and just after hibernation. Giardiasis is a contributing factor in some individuals with diarrhea but is not believed to be a primary intestinal pathogen in the majority of woodchucks with loose stools. Primary bacterial enterocolitis is not common and salmonellosis is rare. Rectal prolapse, intussusception, and intestinal volvulus are examples of obstructive disorders that occur in some woodchucks with diarrhea. Fifteen percent of the woodchucks in the colony require diagnostic and/or medical management of ocular lesions, respiratory conditions, dental problems, ear infections, or chronic debilitation. Conjunctivitis attributed to bedding irritants and/or bacterial infections and ocular lesions acquired from fighting are common. Respiratory problems are often associated with diaphragmatic hernias, cardiomyopathy, profound obesity, and rhinitis and/or pneumonia. Rarely, neoplasms and dental disease are determined to be the cause of obstructive airway disease. Traumatic injuries of teeth are more common than congenital malocclusions. A few woodchucks with ear infections, as discussed later in this section, will develop central nervous system signs. Central nervous system signs or edema and/or ascites occur in less than 5% of the woodchucks. Cerebral nematodiasis is common in wild woodchucks, otitis media/interna in colony-born, and hepatic encephalopathy in WHV-infected animals. Cerebral hemorrhage, vitamin E-selenium deficiency, and renal encephalopathy have been implicated in a few cases. Woodchucks with edema and/or ascites invariably have cardiomyopathy or immune-mediated glomerulonephritis, the latter of which is associated with WHV infection. A wide spectrum of miscellaneous problems account for the remaining 5-10% of woodchucks requiring examination and treatment. Toe and/or nail injuries, as well as lameness associated with soft tissue and skeletal lesions, are expected due to the active and aggressive nature of woodchucks. Osteomyelitis is not an uncommon sequela to bite wounds. Traumatic fractures and dislocations are occasionally diagnosed in young woodchucks, and pathologic fractures due to neoplasia are rare. Hematuria occurs in occasional woodchucks and is usually associated with cystitis and cystic calculi. Peripheral arteriovenous fistulas rarely occur and need to be differentiated from abscesses, neoplasms, and Taenia crassiceps lesions. Parasitic infections such as T. crassiceps, Ackertia marmotae, and Baylisascaris sp. are not a problem in laboratory-reared woodchucks. Because woodchucks hide symptoms of illness as a survival mechanism, diseases are often advanced when diagnosed. Woodchucks are frequently found dead, having displayed no apparent clinical signs. The most common cause of death in WHV-positive woodchucks is hepatocellular carcinoma. The

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CHRISTINE A. BELLEZZA,PATRICKW. CONCANNON,WILLIAME. HORNBUCKLE,LOIS ROTH, AND BUD C. TENNANT

most common causes of death in woodchucks negative for WHV are cardiomyopathy with congestive heart failure, aorta rupture, nephritis, glomerulonephritis, pneumonia, and diaphragmatic hernia.

B.

Infectious Diseases

The most commonly encountered infectious diseases in captive and laboratory-reared woodchucks are bacterial folliculitis, otitis, and pneumonia.

1.

Bacterial Diseases

a.

Bacterial Folliculitis

Folliculitis is a common bacterial skin disease of captive woodchucks. A review of records in one laboratory revealed that at any given time up to 30% of colony animals had skin lesions (Panic et al., 1992). Cytology taken from intact pustules of exudative lesions usually reveals many gram-positive bacteria (cocci). Mixed cultures of Staphylococcus aureus and Streptococcus spp. group A are most commonly obtained; however, pure cultures of the above isolates as well as of Pasteurella multocida have also been obtained. The associated skin lesions, which do not seem pruritic, include erythema, papules, pustules, epidermal collarettes, ulcers, and draining tracts. These lesions are often covered by a patch of matted hair. In later stages, crust, alopecia, hyperpigmentation, and lichenification may be seen. Multiple lesions are usually present and most frequently involve the dorsal lumbosacral region, the limbs, face, and inguinal areas. Pododermatitis is usually associated with severe erythematous swellings or draining tracts. Folliculitis can be fatal to neonates and, less often, adults if they develop septicemia. Since folliculitis is rare in wild woodchucks and in woodchucks housed in Horsfall units, husbandry practices may be implicated in causing infections. Staphylococcus aureus and Streptococcus spp. group A are common cutaneous florae reported to induce infection in rats and mice in the presence of predisposing factors, such as moisture, wounds, crowding, high environmental temperatures, and low dietary protein (Panic et al., 1992). An investigation of predisposing factors for bacterial folliculitis in woodchucks suggested that skin lesions may be associated with the stress involved in transferring animals between facilities, and/or the hormonal fluctuations that occur during the breeding season (Panic et al., 1992). Nutrition does not appear to be a factor. Folliculitis appears to be contagious among woodchucks and can spread to animals in adjacent pens. Preventive measures include individual housing, limitation of movement of woodchucks between facilities, and attention to cleaning and disinfection of cages and all materials in contact with animals.

Treatment consists of clipping the hair around the lesion, cleaning the area with diluted solutions of chlorhexidene or povidone-iodine, debriding when necessary, and applying a topical bactericide. Parenteral antibiotic treatment is reserved for animals with deep cellulitis or multiple lesions. Typically, lesions resolve within 10 days, resulting in mild hyperpigmentation and alopecia until subsequent hair growth occurs during the summer.

b.

Otitis

Otitis externa, characterized by a discharge from one or both ears, is more common in older woodchucks and occasionally extends to the inner and/or middle ear. External ear disease may lead to otitis media/interna. Signs of inner ear disease include a head tilt toward the affected ear, nystagmus, circling, rolling, and/or loss of balance. Woodchucks can have otitis media/interna without an otic discharge. Otitis media/interna occasionally develops into meningitis and/or encephalitis, and prolapse of the cerebellum through the foramen magnum has been observed (Roth, 1984). No predominant bacterial organism is responsible for these infections. Organisms cultured from the ear include Morganella morganii, Proteus sp., Escherichia coli, Citrobacter koseri diversus, and Streptococcus sp. Treatment for external ear disease involves anesthetizing the woodchuck; culturing the ear canal; examining the canal for foreign bodies, polyps, or a ruptured tympanic membrane; flushing with dilute (1:40) chlorhexidene solution; and applying topical antibiotic solution pending culture and sensitivity resuits. If the tympanic membrane has ruptured or if otitis media/interna is present, the ear should be flushed with warm, sterile saline instead of chlorhexidene, a topical antibioticanti-inflammatory drug combination used, and systemic antibiotics administered. If the tympanic membrane has not already ruptured in cases of otitis media/interna, doing so is necessary to provide ventilation and drainage. c.

Pneumonia

Lobar or bronchopneumonia, and occasionally aspiration pneumonia, are important differential diagnoses for respiratory problems. Bacterial pneumonia is more common, either occurring as a primary entity or a complication to some other illness. Severe cases are associated with pulmonary abscesses, suppurative pleuritis, or septicemia. Acute aspiration is rarely a lifethreatening problem in animals that are being gavaged or dosed orally with liquid medications. Mild, chronic granulomatous reactions to inhaled or aspirated irritants are more typical, and affected animals are not usually symptomatic. Woodchucks with pneumonia can die suddenly with no premonitory signs of illness. Lethargy, inappetence, or weight loss may be nonspecific indications. Respiratory distress may become apparent when woodchucks are restrained for examina-

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8. WOODCHUCKS AS LABORATORY ANIMALS

tion, and naso-ocular discharges may be evident. Thoracic auscultation and radiography, tracheal culture, and a hemogram are indicated in many cases. The most common bacterial organism cultured from the lungs of woodchucks with pneumonia is [3hemolytic Streptococcus sp., but mixed cultures and cultures including Staphylococcus aureus, Pasteurella sp., Escherichia coli, and Bordetella bronchiseptica are also common. B. bronchiseptica is identified with a unique form of lobar pneumonia that has to be distinguished from pulmonary abscesses or neoplasia. The morbidity and mortality attending cases with suppurative pleuritis and septicemia are high. Early diagnosis and aggressive treatment are required to resolve less serious forms of pneumonia.

2.

Viral Diseases

a.

Woodchuck Hepatitis Virus

The woodchuck infected with WHV has been shown to be an excellent animal model for the study of HBV infection of hu-

mans (Tennant and Gerin, 1994). WHV and HBV are both known as hepadnaviruses (Tennant and Gerin, 1994). Infection in both species can lead to chronic hepatitis and HCC. Because of similarities between the viruses, the woodchuck has been used to study the mechanisms of hepatocellular injury in acute and chronic viral hepatitis, to assess the oncogenic role of the virus, and to examine possible interactions between hepadnaviruses, diet, and/or other environmental factors in hepatocarcinogenesis. Woodchuck studies have also been useful to develop and evaluate antiviral strategies for treating chronic liver disease and preventing HCC, as well as to investigate therapeutic treatments for HCC (Tennant and Gerin, 1994). Experimental neonatal WHV infection results in a chronic carrier rate of 50% or more. Almost all chronic carriers will develop HCC; the median time to HCC in WHV carriers is 29 months (Tennant and Gerin, 1994). Resulting liver tumors can be detected by abdominal palpation of anesthetized woodchucks or by ultrasound examination. Grossly, tumors may range from 1 mm nodules to 10 cm masses (Fig. 2). The histological features of these tumors have been described (Tennant

Fig. 2. Grossappearanceof hepatocellularcarcinomalesion in a woodchuckliver.

322

CHRISTINE A. BELLEZZA,PATRICKW. CONCANNON,WILLIAME. HORNBUCKLE,LOIS ROTH, AND BUD C. TENNANT

Fig. 3. Hepatocellularcarcinoma with tumor cells denselypacked in irregular trabecules, characterized by basophilic cytoplasm,large vesicular nuclei, and prominent nucleoli.

and Gerin, 1994) and include tumor cells densely packed in thick, irregular trabeculae, basophilic cytoplasm, large vesicular nuclei, and prominent nucleoli (Fig. 3). Elevations in serum a-fetoprotein or serum gamma glutamyl transferase (GGT) can be used as a marker of hepatocellular carcinoma (Graham, 1985; Cote et al., 1990). Woodchucks with hepatocellular carcinoma may be asymptomatic or may develop anorexia, weight loss, and/or lethargy. Ultimately, HCC will result in death, frequently preceded by an encephalopathic episode.

b.

Herpesvirus o f Marmots

Herpesvirus of marmots (HVM) has been detected in laboratory woodchucks, Persistent infection can approach 20% in laboratory colonies (unpublished observation, 1992). No deaths or clinical signs in woodchucks have been attributed to HVM, but lysis of hepatocytes after several days of tissue culture has been reported to be associated with HVM (Schecter et al., 1988). There appears to be no association between HVM status and the outcome of experimental WHV infection (i.e., the rate of chronicity) or in the development of HCC. The virus has been

isolated from blood, lymphocytes, saliva, urine, and rectal/ genital swabs. Venereal transmission from males to females and transmission from mother to offspring is significant. c.

Rabies

Although rodents are rarely infected with rabies, there has been a steady increase since the 1980s in the incidence of woodchuck rabies, particularly in the eastern United States (Childs et al., 1997). Affected woodchucks exhibit central nervous system signs, including aggression, and can initiate unprovoked attacks, which is not unlike the presentation of woodchucks with cerebrospinal nematodiasis (see Baylisascaris sp. section below). Definitive diagnosis of rabies is made by direct immunofluorescent antibody staining of brain tissue. Personnel dealing with wild-caught woodchucks should receive preexposure rabies immunizations. d.

Powassan Virus

Woodchucks are reservoirs for Powassan virus, a little-known arbovirus that causes encephalitis in humans and can be fatal,

8. WOODCHUCKS AS LABORATORYANIMALS especially to children. Powassan virus does not cause clinical disease in woodchucks. One study demonstrated that 80% of woodchucks trapped in Tompkins County, New York, had positive titers to the virus (Fleming, 1978), and the tick Ixodes cookei is an important vector in transmitting the virus.

3.

Parasitic Disease

Woodchucks in the wild are known to harbor many parasites (Fleming, 1978). Parasite burdens decline after introduction into captivity. Except for Giardia sp., Entamoeba muris, and low levels of Citellina triradiata infections, colony-born woodchucks rarely have evidence of parasitism. Common parasites of wild woodchucks include Taenia crassiceps, Baylisascaris sp., Dicrocoelium dendriticum, Ackertia marmotae, and Capillaria sp. Of these, A. marmotae and C. hepatica are associated with hepatic lesions. External parasites can be completely eradicated from laboratory colonies by dusting with insecticidal powder and manually removing ticks upon admission of woodchucks to the colony. a.

Protozoa

i. Giardia sp. Woodchucks in captivity are known to harbor Giardia duodenalis in their small intestines. However, it is unclear whether Giardia sp. causes clinical signs since woodchucks with severe diarrhea have shown neither cysts in the feces nor positive ELISA test results. Additionally, well-formed woodchuck feces may have an excess of Giardia sp. cysts. It is also unclear whether the Giardia sp. that infects woodchucks is transmissible to humans. Diagnosis is made by finding cysts in the feces using the zinc flotation method, by finding trophozoites in fresh fecal smears, or by getting a positive result on an Enzyme-Linked Immunosorbent Assay (ELISA) test for Giardia sp. (Alexon antigen capture test). Variable treatment success has been achieved with metronidazole (10-25 mg/kg PO once daily for 5 days), albendazole (25 mg/kg BID PO for 4 days), and fenbendazole (50 mg/kg PO once daily for 10 days).

323 cat feces or by transplacental migration of tachyzoites from their mothers (Georgi, 1985). In one study, 8% of wild woodchucks exhibited antibody response to T. gondii, but none demonstrated clinical signs or gross lesions (Fleming, 1978). v. Sarcocystis sp. Sarcocystis sp. bradyzoites in sarcocysts have been found in woodchuck muscle, but no clinical signs or reactions have been reported (Roth, 1984). b.

Nematodes

Wild woodchucks harbor many nematodes, including Ackertia marmotae, Baylisascaris laevis, B. columnaris, B. procyonis, .Capillaria hepatica, C. tamiasstriati, Citellina triradiata, C. bifurcatum, Obeliscoides cuniculi, Strongyloides sp., Trichostrongylus axei (Fleming, 1978). No clinical signs or pathological lesions are observed except where woodchucks serve as intermediate hosts (Fleming, 1978). i. Ackertia marmotae. Ackertia marmotae adults are present in lymphatics of the liver and gallbladder, are rarely in the lung and kidney (Fleming, 1978), and do not appear to cause any inflammation or lesions. Microfilariae in the skin cause microfilarial dermatitis (Panic et al., 1992). The intermediate host for A. marmotae is the tick Ixodes cookei. While A. marmotae microfilariae can persist in colony woodchucks for at least 39 months, A. marmotae infection can be eliminated from colonies by manually removing ticks and using insecticidal powders when woodchucks are first introduced to the colony (Cohn et al., 1986).

ii. Entamoeba muris. Entamoeba muris is a nonpathogenic amoeba of rodents found in approximately 50% of woodchuck fecal samples obtained from the colony of the authors. No clinical signs have been observed.

ii. Baylisascaris sp. Baylisascaris procyonis and B. columnaris, parasites of the raccoon and skunk, respectively, have been implicated in causing encephalitis due to larval migration in the brains of woodchucks (Roth et al., 1982). Affected woodchucks demonstrate abnormal behavior, including increased tameness or viciousness, head tilt, circling, and/or paralysis. Because the differential diagnosis includes rabies, no treatment is recommended, and immunofluorescent antibody staining of a portion of brain tissue for rabies should be performed. Some brain tissue should be saved for histological examination. Definitive diagnosis of cerebrospinal nematodiasis can be made based on the results of histologic examination of brain tissue or examination of Baermannized brain tissue (Roth et al., 1982).

iii. Eimeria sp. Four species of Eimeria (Eimeria monacis, E. os, E. perforoides, E. tuscarorensis) are found in wild woodchucks, but no gross lesions have been attributed to coccidian infections (Fleming, 1978). Low levels of Eimeria sp. infection were reported in colony-born woodchucks (Cohn et al., 1986).

iii. Citellina triradiata. The pinworm Citellina triradiata, which is found in the cecum and large intestine, has been found in colony-born woodchucks and is transmitted directly. While it is not known to cause any pruritus or clinical signs, sanitation and single housing should reduce the incidence of infection.

iv. Toxoplasma gondii. Woodchucks, like other mammals, are intermediate hosts to Toxoplasma gondii. Cats are definitive hosts. Woodchucks become infected by ingestion of oocysts in

iv. Obeliscoides cuniculi. Obeliscoides cuniculi is a common parasite of wild woodchucks, with one study showing a 92% infection rate (Cohn et al., 1986). Obeliscoides cuniculi is

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CHRISTINE A. BELLEZZA, PATRICK W. CONCANNON, WILLIAM E. HORNBUCKLE, LOIS ROTH, AND BUD C. TENNANT

found in the stomach, where it sometimes causes petechiae of the mucosa. The natural host is the wild rabbit. In captive-bred woodchucks the infection is short-lived. Fecal egg counts fall rapidly. The nematode has not been found in colony-born animals. The larvae and adult forms do not survive hibernation of the host, and in the wild the infection of woodchucks is probably perpetuated by reinfection from rabbits. c.

Cestodes

i. Taenia crassiceps. The larval form of Taenia crassiceps, a tapeworm of wild canids, causes large, fluid-filled masses containing hundreds of cysticerci (Georgi, 1985). Woodchucks ingest the infected eggs, and the embryos migrate to their site of development. Replication occurs by budding and results in many cysticerci contained in a single cyst. Masses are most commonly found in the axillary region and the adjacent thoracic wall, but have been found in other subcutaneous locations, in the abdominal and thoracic cavities, and in the liver and lung (Anderson et al., 1990b). Lesions do not seem to be pruritic or painful or to cause any systemic signs of illness. They can interfere with movement and/or eating depending on their size and location (Anderson et al., 1990b). Surgical removal of small masses is possible. The tendency for larger lesions to infiltrate the surrounding area with fingerlike projections makes excision difficult. Care must be taken to prevent cyst rupture and dissemination of cysticerci. ii. Taenia mustelae. Taenia mustelae cysticerci are found in 4 - 6 mm cysts on the surface of and in the parenchyma of the liver. The cysts may also contain hard, yellow material that is presumably calcified pus (Fleming, 1978). d.

Trematodes

i. Dicrocoelium dendriticum. Dicrocoelium dendriticum, the lancet liver fluke, is found in the bile ducts of sheep, cattle, pigs, deer, cottontail rabbits, and woodchucks (Georgi, 1985). The fluke eggs are eliminated in the feces of the definitive hosts and are ingested by terrestrial snails. Cercaria develop in the snails, are secreted in mucus, and are ingested by ants. The definitive host then ingests the ants while grazing. The metacercaria migrate up the common bile duct. Scarring of the liver and histologic lesions typical of perilobular cirrhosis are seen in sheep and woodchucks (Mapes, 1950). e.

san virus. This tick may also serve as a vector for Rocky Mountain spotted fever rickettsia and Borrelia burgdorferi, the spirochete that causes Lyme disease (Magnarelli and Swihart, 1991). External parasites can be eliminated by treating newly captured woodchucks with organophosphate-containing powders and by manually removing ticks before entry into the colony (Cohn et al., 1986).

External Parasites

Wild woodchucks are hosts to many external parasites (Whitaker and Schmeltz, 1973). Some of the most common are the mite Androlaelaps fahrenholzi, the louse Endeleinellus marmotae, the tick Ixodes cookei, and the flea Orpsylla arctomys. L cookei is a vector for Ackertia marmotae and the Powas-

C. 1.

M e t a b o l i c / N u t r i t i o n a l Diseases

Capture Myopathy

A syndrome resembling white muscle disease has been associated with the capture of wild woodchucks. In one study, up to 60% of wild woodchucks captured in traps had lesions of myopathy, while no woodchucks that were shot had any such lesions (Fleming, 1978). Grossly, discrete, pale, white muscle was observed. Histologically, there was swelling, loss of cross striation with hyalinization or fragmentation, and occasionally mineralization. The only clinical sign in any of the affected woodchucks was lethargy. The myopathy was not related to vitamin E-selenium levels and might be similar to steroid myopathy. 2.

White Muscle Disease

White muscle disease in woodchucks, as in other species, is a nutritional disease, resulting from insufficient intake or bioavailability of vitamin E and/or selenium. Since nutritional deficiencies rarely occur in the wild, capture myopathy is the suspected cause of myodegeneration in recently trapped woodchucks, and nutritional myopathy (white muscle disease) is the suspected cause of these lesions in animals that have been in captivity for at least several weeks. Lesions, indistinguishable from those of capture myopathy, most commonly involve the muscles used most often. In woodchucks, the characteristic discrete, white, pale streaks are typically seen in the muscles of the upper forelegs, hindlegs, and cranial dorsum (hypaxial and epaxial muscles), as well as in the heart. Affected woodchucks demonstrate varying degrees of weakness, dysphagia, and reluctance to move. Histological lesions include swollen, dark pinkstained muscle, occasional fragmentation, mineralization, and in chronic cases, muscle atrophy and fibrosis. Profoundly affected woodchucks usually die. Woodchucks with milder manifestations respond to vitamin E-selenium supplementation. 3.

Hepatic Lipidosis

Hepatic lipidosis resulting in death has been reported in wildcaught woodchucks and was thought to have resulted from a failure of these animals to adapt to laboratory conditions and diet (Roth, 1984). Varying degrees of hepatic lipidosis have been observed in colony woodchucks with inadequate food

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8. WOODCHUCKS AS LABORATORY ANIMALS

intake, chronic debilitating illness, or exposure to hepatotoxic medications. For example, hepatic lipidosis was a prominent necropsy finding in woodchucks treated with the antiviral drug FIAU (Tennant et al., 1998). D.

Traumatic Disorders

Bite wounds are quite common among group-housed colony animals, particularly during the breeding season. These wounds occur most frequently on the head, neck, and limbs. While fighting usually results in minor bite wounds, severe wounds, including fractured limbs, ruptured eyes, and deep lacerations, have been seen. If not treated promptly with topical disinfectants and parenteral antibiotics for deep wounds, systemic infection and/or abscessation will occur. Attention to husbandry issues can reduce serious fighting. Anesthetized woodchucks should be allowed to recover fully from anesthesia before being returned to their cages or serious bite wounds may be inflicted upon the recovering woodchuck. Traumatic injuries can also result from woodchucks jumping out of cages. Typical injuries include broken incisors and/or fractured limbs. Woodchucks are avid climbers and occasionally get a limb caught in a cage door or bottle bracket, resulting in avulsion or fracture of toes and/or limbs. Broken incisors usually grow back without any complications, but the opposite incisor may need trimming until regrowth is complete. Fractures, if not open or badly displaced, heal well with simple cage confinement. E.

F.

Hepatocellular carcinoma (HCC) is the most common neoplasm in the woodchuck because of its association with WHV. The neoplasms are usually extensive in the liver, but metastasis to other organs is rare. The neoplastic cells are usually well differentiated and arranged in sheets, cords, and trabeculae, without portal structures or regard to lobular architecture. While HCC affects virtually all woodchucks chronically infected with WHV, it has occasionally been observed in WHVnegative animals. In one study of 128 woodchucks seronegative for WHV, one woodchuck with histological evidence of HCC was reported (Roth et al., 1984). The mass was focal and composed of well-differentiated hepatic cells. There were no signs of hepatitis. Published reports of nonhepatic neoplasms have included testicular teratoma, seminoma, Sertoli cell tumor, testicular lymphosarcoma, interstitial cell tumor, and adenoma of the rete testis (Foley et al., 1993); lymphosarcoma (Foley et al., 1993); meningioma (Podell et al., 1988); fibrosarcoma (Young and Sims, 1979); and uterine leiomyoma (Foley et al., 1993). Neoplasms, as in most species, are most common in older woodchucks, and as long-term studies continue, diagnoses of neoplasms increase. In the authors' colony, up to 15% of woodchucks that die or are euthanatized have a nonhepatic neoplasm diagnosed at necropsy. The more common neoplasms are lymphoma, seminoma, and interstitial cell tumors of the testicle, and myeloproliferative disease.

Iatrogenic Diseases

Perhaps associated with the high incidence of bacterial skin problems observed in woodchucks, localized infections and septicemia commonly occur following invasive procedures such as venipuncture, intravenous catheterization, and surgery. Careful attention to sterility can minimize these infections. The skin should be Clipped and scrubbed with a disinfectant and alcohol. These measures alone are enough to prevent infections associated with venipuncture. Sterile surgical techniques and sterilized instruments, needles, and catheters should be used. Suture materials should be chosen with care. In the authors' experience, the administration of parenteral antibiotics and topical antibiotic ointments to the incision reduces the incidence of infection following surgical procedures and catheterization. Gait abnormalities can be caused by repeated intramuscular injections and subsequent fibrosing myositis. Rarely, arteriovenous fistulas occur at venipuncture sites, particularly in the inguinal area. These fistulas can result in cardiomyopathy and associated heart failure or death due to rupture and hemorrhage. Thrombus formation is a fairly common sequelae to indwelling catheterization, but clinical problems associated with thrombosis are rare.

Neoplastic Diseases

G.

Miscellaneous

1. Congenital Disorders a.

Diaphragmatic Hernia

Woodchucks appear to have a natural weakness in the dorsal portion of the diaphragm. Pressure applied to the thorax during manual restraint may enlarge this opening enough to allow omentum and/or abdominal organs to pass. Signs of diaphragmatic hernia include dyspnea, tachypnea, and muffled thoracic auscultation. Definitive diagnosis can be challenging; radiographs and ultrasound examination may or may not show abdominal contents in the chest. Clinical signs usually result from pulmonary atelectasis and can lead to death. 2. Age-Related Disorders a.

Ringtail

A ringtail-like syndrome similar to that seen in rats has been seen in neonatal woodchucks (7-28 days of age). The lesion begins as an annular constriction of the tail that progresses to edema, inflammation, necrosis, and sloughing of the tail distal

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to the constriction. As in the rat, ringtail appears to be associated with low humidity. In the authors' experience, humidity levels of 50% prevent the problem. 3.

Cardiovascular Diseases

A high incidence of arteriosclerosis, aortic rupture, and cerebrovascular and cardiovascular disease has been reported in captive woodchucks (Snyder and Ratcliffe, 1969). a.

Cardiomyopathy

Cardiomyopathy, which frequently results in congestive heart failure, is a relatively common finding in woodchucks. Clinical signs include respiratory distress, ascites, subcutaneous edema, muffled heart sounds, heart murmurs, and arrhythmias. Sudden death in the absence of these signs can occur. Thoracic radiography and ultrasonography are useful in diagnosis. At necropsy, a grossly enlarged and dilated heart is observed. In one study, enlarged hearts weighed an average of 32.3 gm ___ 5.4 gm, whereas normal hearts weighed an average of 11.6 gm _+ 2.8 gm (Roth and King, 1986). b.

Aortic Rupture

Aortic rupture is a common cause of death in wild-caught woodchucks (Snyder and Ratcliffe, 1969), as well as in colonyborn animals (unpublished observations). In nearly all cases, there are no premonitory signs prior to death. There appears to be no gender bias and the mean age at death is 2 years greater than the mean age at all other natural deaths. No relationship between aortic rupture and hepatocellular carcinoma or chronic WHV infection is detected. Young woodchucks dying from aortic rupture are more likely to have concurrent glomerulonephritis or interstitial nephritis, suggesting that systemic hypertension is a predisposing factor (Van Schoick, 1996). The contributing roles of hypertension, dietary copper and zinc, and underlying connective tissue disorders have not been fully investigated. c.

Cerebrovascular Disease

Nearly 7% of captive woodchucks were reported to have evidence of cerebral hemorrhage at death (Snyder and Ratcliffe, 1969). In some, hemorrhage was associated with arteriosclerosis of the brain. Vascular hypertension and atherogenic diets are possible causes (Snyder, 1985). In colony woodchucks fed 16.5% protein and only 2.8% fat, cerebral hemorrhage and arteriosclerosis were only infrequently seen at necropsy (unpublished observations, 1999). Only 7 of 350 woodchucks necropsied over 4 years had evidence of cerebral hemorrhage. Six died and 1 was euthanatized because it appeared to be blind. In 3 woodchucks, the cause of death was a ruptured abdominal aorta, 2 of which had degenerative vascular disease and thrombosis in their brains; 1 also had

renal vascular lesions. Cerebellar hemorrhage was the cause of death in 2 woodchucks suspected of having vascular disease, 1 of which had glomerulonephritis and interstitial nephritis. The blind woodchuck that was euthanatized had vascular lesions in its brain, heart, and kidney. Systemic hypertension was suspected, but no antemortem blood pressure studies were performed to confirm this suspicion. In summary, vascular disease, including vessel rupture, may be linked to renal disease, vasculitis, and perhaps associated hypertension. Immune complex deposition in glomerular capillaries, as seen in HBV-infected humans, may lead to direct vessel injury. Vascular disease secondary to deposition of HBV antigen-antibody complexes has been demonstrated in humans (Shoen, 1994), and similar mechanisms may lead to vascular disease of woodchucks.

4.

Renal Diseases

A 4-year necropsy study (1994-1998) involving 390 woodchucks revealed 164 lesions of the urogenital system. A little more than a third (37%) of these lesions had histopathologic features of nonsuppurative interstitial nephritis, 14% had glomerulonephritis, 4% had tubular nephrosis, and 3% had pyelonephritis (unpublished observations). The more severe and/or chronic cases sometimes had mixed lesions, varying degrees of fibrosis, and rarely, amyloid deposition. Although a few died or were euthanatized because of signs attributed to protein-losing nephropathy and/or renal failure, the cause of death was not attributed to renal disease. The majority died of concurrent illnesses such as cardiomyopathy, aortic ruptures, neoplasia, and infections of other organ systems. Histopathologic evidence of renal disease was either coincidental, contributing, or a sequela to the primary illness. Clinical signs of renal disease are not easily detected in groups of woodchucks, which necessitates isolation of animals with suspected illness for more critical observation. Lethargy, inappetence and/or weight loss, increased water consumption and excessive soiling of bedding (polydipsia and polyuria), uremic odor that can permeate cage and room, facial swelling and abdominal distension, and neurologic signs (Anderson et al., 1990a) give reason for expanded examinations. Physical examination, clinicopathologic evaluations, and ultrasonography are useful in diagnosis. Anasarca and/or ascites and clinicopathologic evidence of protein-losing nephropathy is typical of glomerulonephritis. Enlarged kidneys occur in occasional woodchucks with glomerulonephritis, tubular nephrosis, hydronephrosis, and neoplasia. Disparity in kidney size and/or loss of renal mass is often attributed to chronic fibrosing nephritis. Immune-mediated glomerulonephritis, diagnosed based on the presence of light microscopic lesions and the presence of host immunoglobulin, is associated with woodchuck hepatitis virus infection (Peters et al., 1992). In one study, three types of glomerulonephritismmembranous, mixed membranous and

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mesangial proliferative, and mesangial proliferative glomerulonephritis--were identified in 6 of 142 woodchucks with chronic WHV infection (Peters et al., 1992). Several types of glomerulonephritis are also associated with hepatitis B infection in humans. Membranous nephritis in both diseases is more common in younger individuals (Peters et al., 1992). In both humans and woodchucks it is unclear if the types of glomerulonephritis are truly distinct disease forms or represent progression from one form to another. The woodchuck may be a useful animal model for the study of glomerulonephritis associated with HBV infection, particularly for investigating the progression of renal lesions. 5.

Gastrointestinal Diseases

Nonsuppurative inflammatory bowel disease (IBD) is occasionally observed in histologic sections of the intestinal tract of woodchucks. Lymphocytic plasmacytic enteritis, enterocolitis, enterotyphlocolitis, or colitis infrequently cause overt clinical signs of disease. In a necropsy study of 24 woodchucks with intestinal disease, IBD was a primary finding in two cases, one of which had a protracted history of diarrhea and the second had ascites and lymphangiectasia (unpublished observations, 1999). Eosinophilic enterotyphlitis and lymphoplasmacytic colitis were incidental findings in a woodchuck with central nervous system signs. IBD was accompanied by necrotizing enteritis and/or bowel infarction in 10 other woodchucks, only 1 of which had diarrhea. One of these woodchucks had a colonicmesenteric volvulus, and death in the remaining woodchucks was attributed to extraintestinal lesions. Necrotizing enteritis and/or infarction were the dominant lesions in 11 woodchucks, many of which exhibited no signs of illness prior to death. Infectious etiology was suspected in 3 animals with necrotizing enteritis. Of these, Clostridium difficile and its endotoxin were isolated from the intestine in 1 woodchuck, and intranuclear inclusion bodies were observed in crypt epithelium in a second. In other species, intranuclear inclusion bodies have been associated with parvovirus enteritis. Additional tests (immunoflourescent antibody test [IFA] or examination of tissue by electron microscopy) were not done on this case. Two woodchucks had suppurative gastroenteritis, but no organism was cultured. Rectal prolapse, sometimes associated with colonic intussusception, is a rare condition seen in young woodchucks, pregnant dams, or dams that have recently whelped. Rectal prolapse and intussusception may result from straining associated with diarrhea or labor. If diagnosed and treated early, a prolapse can be manually reduced and kept in place with a purse-string suture. Intussusceptions require surgery. The ascending colon of the woodchuck consists of a short straight segment that is firmly attached to the body wall by the mesocolon, and two loops that lie free in the abdomen. The two loops occasionally twist upon each other forming an overhand knot that leads to strangulation of the colon. Woodchucks are typically found dead, having shown no prior clinical signs.

Intestinal obstruction can result from entrapment in diaphragmatic hernias or from stricture formation by omental adhesions. A partial obstruction may produce clinical signs such as anorexia, weight loss, and depression, whereas a total obstruction may result in sudden, unexpected death. 6.

Dental Problems

Dental problems, including broken incisors, abscessed/infected teeth, and long incisors caused by malocclusion are occasionally seen. Neoplasia involving the mouth (squamous cell carcinoma, ameloblastoma, fibrosarcoma) is extremely rare. Dental problems may cause systemic illness or difficulty eating, resulting in poor condition.

REFERENCES

Anderson, W. I., deLahunta, A., Hornbuckle, W. E., King, J. M., and Tennant, B. C. (1990a). Two cases of renal encephalopathy in the woodchuck (Marmota monax). Lab. Anim. Sci. 40, 86-88. Anderson, W. I., Scott, D. W., Hornbuckle, W. E., King, J. M., and Tennant, B. C. (1990b). Taenia crassiceps infection in the woodchuck: A retrospective study of 13 cases. Vet. Derm. 1, 85-92. Bailey, E. D. (1965). Seasonal changes in metabolic activity of non-hibernating woodchucks. Can. J. Zool. 43, 905-909. Baldwin, B. H., Tennant, B. C., Reimers, T. J., Cowan, R. G., and Concannon, P. W. (1985). Circannual changes in serum testosterone concentrations of adult and yearling woodchucks (Marmota monax). Biol. Reprod. 32, 804812. Beaudoin, R. L., Davis, D. E., and Murrell, K. D. (1969). Antibodies to larval Taenia crassiceps in hibernating woodchucks, Marmota monax. Exp. Parasitol. 24, 42-46. Childs, J. E., Colby, L., Krebs, J. W., Strine, T., Feller, M., Noah, D., Drenzek, C., Smith, J. S., and Rupprecht, C. E. (1997). Surveillance and spatiotemporal associations of rabies in rodents and lagomorphs in the United States, 1985-1994. J. Wildl. Dis. 33, 20-27. Christian, J., Steinberger, E., and McKinney, T. (1972). Annual cycle of spermatogenesis and testis morphology in woodchucks. J. Mammal. 53, 708716. Cohn, D. L., Erb, H. N., Georgi, J. R., and Tennant, B. C. (1986). Parasites of the laboratory woodchuck (Marmota monax). Lab. Anim. Sci. 36, 298-302. Concannon, P., Baldwin, B., and Tennant, B. (1984). Serum progesterone profiles and corpora lutea of pregnant, postpartum, barren, and isolated females in a laboratory colony of woodchucks (Marmota monax). Biol. Reprod. 30, 945-951. Concannon, P., Baldwin, B., Roberts, P., and Tennant, B. (1990). Endocrine correlates of hibernation-independent gonadal recrudescence and the limited late-winter breeding season in woodchucks, Marmota monax. J. Exp. Zool. Suppl. 4, 203-206. Concannon, P. W., Parks, J. E., Roberts, P. J., and Tennant, B. C. (1992). Persistent free-running circannual reproductive cycles during prolonged exposure to a constant 12L: 12D photoperiod in laboratory woodchucks (Marmota monax). Lab. Anim. Sci. 42, 382-391. Concannon, P., Roberts, P., Baldwin, B., Erb, H., and Tennant, B. (1993). Alteration of growth, advancement of puberty, and season-appropriate circannual breeding during 28 months of photoperiod reversal in woodchucks (Marmota monax). Biol. Reprod. 48, 1057-1070.

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Concannon, E, Roberts, E, Parks, J., Bellezza, C., and Tennant, B. (1996). Collection of seasonally spermatozoa-rich semen by electroejaculation of laboratory woodchucks (Marmota monax), with and without removal of bulbourethral glands. Lab. Anim. Sci. 46, 667-675. Concannon, P., Roberts, P., Baldwin, B., and Tennant, B. (1997a). Long-term entrainment of circannual reproductive and metabolic cycles by northern and southern hemisphere photoperiods in woodchucks (Marmota monax). Biol. Reprod. 57, 1008-1015. Concannon, P., Roberts, P., Ball, B., Schlafer, D., Yang, X., Baldwin, B., and Tennant, B. (1997b). Estrus, fertility, early embryo development, and autologous embryo transfer in laboratory woodchucks (Marmota monax). Lab. Anim. Sci. 47, 63-74. Concannon, P. W., Castracane, V. D., Rawson, R., and Tennant, B. C. (1999). Circannual changes in free-thyroxine, prolactin, testis function, and energy balance in woodchucks (Marmota monax). Am. J. Physiol. 277 (Regul. Integr. Comp. Physiol. 46), R1401-R1409. Cote, P. J., Pohl, C., Boyd, J., Tennant, B. C., and Gerin, J. L. (1990). a-Fetoprotein in the woodchuck model of hepadnavirus infection and disease: Immunochemical analysis of woodchuck a-fetoprotein and measurement in serum by quantitative monoclonal radioimmunoassay. Hepatology 11, 824-832. Davis, D. E. (1977). Role of ambient temperature in emergence of woodchucks (Marmota monax) from hibernation. Am. Midl. Nat. 97, 224-229. Fall, M. W. (1971). Seasonal variations in the food consumption of woodchucks (Marmota monax). J. Mammal. 52, 370-375. Fleming, W. J. (1978). Parasites and diseases of the woodchuck (Marmota monax) in Tompkins County, New York. Ph.D. thesis, Cornell University. Foley, G. L., Anderson, W. I., Schlafer, D. H., Hornbuckle, W. E., Baldwin, B. H., and Tennant, B. C. (1993). Neoplastic and non-neoplastic lesions of the reproductive tract of the woodchuck (Marmota monax). J. Zoo Wildl. Med. 24, 475-481. Georgi, J. R. (1985). "Parasitology for Veterinarians," 4th ed., p. 90. Saunders, Philadelphia. Graham, E. S. (1985). Clinicopathological investigation of woodchuck hepatitis virus infection. Ph.D. thesis, Cornell University. Hamilton, W. J., Jr. (1934). The life history of the rufescent woodchuck, Marmota monax rufescens Howell. Ann. Carnegie Mus. 23, 85-178. Magnarelli, L. A., and Swihart, R. K. (1991). Spotted fever group rickettsiae or Borrelia burgdorferi in Ixodes cookei (Ixodidae) in Connecticut. J. Clin. Microbiol. 29, 1520-1522. Mapes, C. R. (1950). The lancet fluke, a new parasite of the woodchuck. Cornell Vet. 40, 346-349. Mrozek, M., Lehr, B., Zillman, U., and Bannasch, P. (1994). A technique for serial liver biopsies in the woodchuck (Marmota monax). J. Exp. Anim. Sci. 37, 34-41. Panic, R., Scott, D., Tennant, B. C., Anderson, W. I., and Johnson, M. (1992). Skin disorders of the laboratory woodchuck (Marmota monax): A retrospective study of 113 cases (1980-1990). Cornell Vet. 82, 405-421. Peters, D. N., Steinberg, H., Anderson, W. I., Hornbuckle, W. E., Cote, P. J., Gerin, J. L., Lewis, R. M., and Tennant, B. C. (1992). Immunopathology of glomerulonephritis associated with chronic woodchuck hepatitis virus infection in woodchucks (Marmota monax). Am. J. Pathol. 141, 143-152. Podell, M., Pokras, M., Gerlach, P., and Jakowski, R. (1988). Meningioma in a woodchuck exhibiting central vestibular deficits. J. Wildl. Dis. 24, 695-699. Popper, H., Roth, L., Purcell, R. H., Tennant, B. C., and Gerin, J. L. (1987). Hepatocarcinogenicity of the woodchuck hepatitis virus. Proc. Natl. Acad. Sci. U.S.A. 84, 866-870. Rawson, R., Concannon, P. W., Roberts, P. J., and Tennant, B. C. (1998). Seasonal differences in resting oxygen consumption, respiratory quotient, and free thyroxine in woodchucks. Am. J. Physiol. 274, R963-R969. Roth, L. (1984). Hepatopathology of the woodchuck (Marmota monax). Ph.D. diss., Cornell University. Roth, L., Georgi, J. R., King, J. M., and Tennant, B. C. (1982). Parasitic en-

cephalitis due to Baylisascaris sp. in wild and captive woodchucks (Marmota monax). Vet. Pathol. 19, 658-662. Roth, L., and King, J. M. (1986). Congestive cardiomyopathy in the woodchuck, Marmota monax. J. Wildl. Dis. 22, 533-537. Roth, L., King, J. M., and Tennant, B. C. (1984). Primary hepatoma in a woodchuck (Marmota monax) without serologic evidence of woodchuck hepatitis infection. Vet. Pathol. 21, 607-608. Schecter, E. M., Summers, J., and Ogston, C. W. (1988). Characterization of a herpesvirus isolated from woodchuck hepatocytes. J. Gen. Virol. 69, 15911599. Schoen, E J. (1994). Blood vessels, inflammatory diseasemthe vasculitides. In "Pathologic Basis of Disease" (R. C. Cotran, V. Kumar, and S. L. Robbins, eds.), 5th ed., p. 490. Saunders, Philadelphia. Sinha Hikim, A. P., Sinha Hikim, I., Amador, A. G., Bartke, A., Woolf, A., and Russell, L. D. (1991a). Reinitiation of spermatogenesis by exogenous gonadotropins in a seasonal breeder, the woodchuck (Marmota monax), during gonadal inactivity. Am. J. Anat. 192, 194-213. Sinha Hikim, A. P., Woolf, A., Bartke, A., and Amador, A. G. (1991b). The estrous cycle of captive woodchucks (Marmota monax). Biol. Reprod. 44, 733-738. Sinha Hikim, A. P., Woolf, A., Bartke, A., and Amador, A. G. (1992). Further observations on estrus and ovulation in woodchucks (Marmota monax) in captivity. Biol. Reprod. 46, 10-16. Snyder, R. L. (1977). Longevity and disease patterns in captive and wild woodchucks. Proc. Am. Assoc. Zool. Parks and Aquariums, 535-552. Snyder, R. L. (1985). The laboratory woodchuck. Lab. Anim. 14, 20-32. Snyder, R. L., and Christian, J. J. (1960). Reproductive cycle and litter size of the woodchuck. Ecology 41, 647-655. Snyder, R. L., and Ratcliffe, H. L. (1969). Marmota monax: A model for studies of cardiovascular, cerebrovascular, and neoplastic disease. Acta Zool. Pathol. Antverp. 48, 265-273. Spurrier, W. A., Oeltgen, P. R., and Myers, R. D. (1987). Hibernation trigger from hibernating woodchucks Marmota monax induces physiological alterations and opiate-like responses in the primate Macaca mulatta. J. Thermal Biol. 12, 139-142. Tennant, B. C., and Gerin, J. L. (1994). The woodchuck model of hepatitis B virus infection. In "The Liver: Biology and Pathobiology" (I. M. Arias, J. Boyer, N. Fausto, W. B. Jakoby, D. Schachter, and D. A. Shafritz, eds.), pp. 1455-1466. Raven Press, New York. Tennant, B. C., Baldwin, B. H., Graham, L. A., Ascenzi, M. A., Hornbuckle, W. E., Rowland, P. H., Tokuyama, K., Yeager, A. E., Erb, H. N., Colacino, J. M., Lopez, J. M., Engelhardt, J. A., Bowsher, R. R., Richardson, R. D., Lewis, W., Cote, P. J., Korba, B. E., and Gerin, J. L. (1998). Antiviral activity and toxicity of fialuridine in the woodchuck model of hepatitis B virus infection. Hepatology 28, 179-191. Tyler, G. V., Summers, J. W., and Snyder, R. L. (1981). Woodchuck hepatitis virus in natural woodchuck populations. J. Wildl. Dis. 17, 297-301. Van Schoick, A., Baldwin, B., and Erb, H. (1996). Risk factors associated with the incidence of aortic rupture in the woodchuck, Marmota monax. [Unpublished]. Whitaker, J. O., Jr., and Schmeltz, L. L. (1973). External parasites of the woodchuck (Marmota monax) in Indiana. Ent. News 84, 69-72. Wong, D. C., Shih, J. W. K., Purcell, R. H., Gerin, J. L., and London, W. T. (1982). Natural and experimental infection of woodchucks with woodchuck hepatitis virus, as measured by new, specific assays for woodchuck surface antigen and antibody. J. Clin. Microbiol. 15, 484-490. Woolf, A., Cuff, J., and Gremillion-Smith, C. (1989). The use of subcutaneous ports in the woodchuck (Marmota monax). Lab. Anim. Sci. 39, 620-622. Young, R. A. (1984). Interrelationships between body weight, food consumption, and plasma thyroid hormone concentration cycles in the woodchuck, Marmota monax. Comp. Biochem. Physiol. 77A, 533-536. Young, R. A., and Sims, E. A. H. (1979). The woodchuck, Marmota monax, as a laboratory animal. Lab. Anim. Sci. 29, 770-780.

Chapter 9 Biology and Diseases of Rabbits Mark A. Suckow, David W. Brammer, Howard G. Rush, and Clarence E. Chrisp

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Use in Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. ComparativeAnatomy and Physiology . . . . . . . . . . . . . . . . . . . . . . . . B. Normative Physiological Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Management and Husbandry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Bacterial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Viral Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Protozoal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Arthropod and Helminth Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Mycotic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Management-Related Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Heritable Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Neoplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Miscellaneous Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

INTRODUCTION

Rabbits have been used extensively in a variety of biomedical research disciplines. The need for consistent research subjects has led to understanding of the basic biology and special needs of rabbits. This chapter will provide a s u m m a r y of care, management, and diseases of the laboratory rabbit. It is ironic that while effort is given to p r o m o t e the health of domestic rabbits, feral populations have the ability to explode to LABORATORY ANIMAL MEDICINE, 2nd edition

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plague proportions in areas of the world where natural predators and diseases are limited. In 1890, the rabbit population of Australia was estimated at 20 million. All of these individuals originated with one pair of rabbits introduced to the continent 31 years previously (Fox, 1994). It is further ironic that while effort is given to control infectious pathogens of domestic rabbits, in other circumstances such agents have been used to control feral populations. For example, m y x o m a virus has been used to control overpopulation of wild rabbits ( D i G i a c o m o and Mar6, 1994). Finally, although not intentionally released, Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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Class: Mammalia Order: Lagomorpha Family: Ochotonidae (pikas) Genus: Ochotona Species: 19 species

the calicivirus agent for rabbit hemorrhagic disease may have killed up to 30 million rabbits during a 1-month epidemic at Flinders Ranges National Park in Australia (Mutze et al., 1998). This outbreak is responsible for the death of 2.6 times as many rabbits as those used in biomedical research in the United States from 1973 through 1997.

A.

Taxonomy

The terms "rabbit" and "hare" are often misused when referring to common names or breeds of rabbits (Fox, 1994; Nowak and Paradiso, 1983). Animals classified in the genus Lepus are the only true hares. There are several genera that contain rabbits. Oryctolagus cuniculus is the only domesticated rabbit, and consequently the only species from which unique breeds are derived. Many breeds have been developed simply by selective breeding of O. cuniculus for different physical characteristics. Currently, 42 breeds are recognized by the American Rabbit Breeders Association. A list of these breeds is found in Table I. In addition to those described in Table I, over 100 different gene mutations have been described, and these phenotypes are used for the study of human disease. The inheritance properties of these mutations are described in detail elsewhere (Fox, 1994). The following list shows the complete taxonomic position of animals in the order Lagomorpha.

Table I

Breeds of Rabbits Recognizedby the American Rabbit Breeders Associationa American Blue & White American Checkered Giant American Chinchilla American Dutch American Sable Angora Belgian Hare Beverens Britannia Petite Californian Cavy Champagne d'Argent Cinnamon Creme d'Argent Dwarf Hotot English Spot Flemish Giant Florida White Fuzzy Lop Giant Chinchilla Harlequin

Family: Leporidae (rabbits and hares) Subfamily: Leporinae Genus/Species: Bunolagus monticularis (Bushman rabbit) Brachylagus idahoensis (Idaho pygmy rabbit) Caprolagus hispidus (hispid hare) Lepus, 22 species ("true" hares, jackrabbits) Nesolagus netscheri (Sumatra short-eared rabbit) Oryctolagus cuniculus (European rabbit, Old World rabbit) Pentalagusfurnessi (Amami rabbit) Poelagus marjorita (Bunyoro rabbit) Pronolagus, 3 species (rock hare) Romerolagus diazzi (volcano rabbit) Sylvilagus, 14 species (cottontail rabbits)

Havana Himalayan Holland Lop Hotot Jersey Wooly Lilac Lop Mini Lop Mini Rex Netherland Dwarf New Zealand Palomino Polish Rex Rhinelander Satin Silver Fox Silver Marten Silver Standard Chinchilla Tan

B.

Since 1973, the U.S. Department of Agriculture has reported the total number of certain species of animals used by registered research facilities (Animal and Plant Health Inspection Service, 1997). Table II indicates the total number of rabbits used in research as reported to the USDA for the period 1973-1997. Despite the overall drop in the number used in research, the rabbit is still a valuable model and tool for many disciplines. It is not a goal of this chapter to discuss in detail the different research uses of the rabbit. Rather, a few broad comments and examples of rabbit use will be offered. Table II

Numbers of Rabbits Used in Biomedical Research in the United States, 1973-1997a 1973 1974 1975 1976 1977 1978 1979

447,570 425,585 448,530 527,551 439,003 475,162 539,594

1980 1981 1982 1983 1984 1985 1986 1987 1988 1989

471,297 473,922 453,506 466,810 529,101 544,621 521,773 554,385 459,254 471,037

1990 1991 1992 1993 1994 1995 1996 1997

399,264 396,046 431,432 426,501 393,751 354,076 338,574 309,322

Total number of rabbits used in research as reported to the U.S. Department of Agriculture. Notice the trend toward reduced use of rabbits over the course of 25 years. a

Despite the different breed names and the use of the word hare for some breeds, all are derived from Oryctolaguscuniculus. a

Use in R e s e a r c h

9. BIOLOGY AND DISEASES OF RABBITS

One of the most common research uses of rabbits is in the production of polyclonal antibodies. The relatively large body size and blood volume, easy access to the vascular system, and an existent large body of information on the purification of rabbit immunoglobulins are a few reasons the rabbit is preferred over other common laboratory animal species for polyclonal antibody production (Stills, 1994). The Armed Forces Institute of Pathology (AFIP) has recognized at least 22 different spontaneous or induced diseases of the rabbit that are models of human diseases. Half of these models can be grouped into two categories: cancer and infectious agent models. Other recognized rabbit models of human disease include hydrocephalus induced by vitamin A deficiency (Newberne, 1974); hypervitaminosis A (Shenefelt, 1972); acute respiratory distress syndrome induced by phorbol myristate acetate (Salzer and McCall, 1991); diabetes mellitus (Roth and Conaway, 1983); inflammatory bowel disease (Rabin, 1980); methylmercury poisoning (Koller, 1979); and the Pelger-HuEt anomaly (Tvedten, 1983). There are six cancer models listed by the AFIP. The VX-2 tumor, spontaneous endometrial adenocarcinoma, monoclonal gammopathies, nephroblastoma, lymphoblastic leukemia, and malignant fibroma are all considered animal models of human neoplastic disease. The VX-2 carcinoma results from the malignant transformation of the viral-induced Shope papilloma. The tumor induces fulminating hypercalcemia, within 4 weeks of implantation (Young et al., 1978). Endometrial adenocarcinoma is most common in aged rabbits, with an incidence of 79% being reported in a colony of 5-year-old rabbits (Baba and von Haam, 1972). The other rabbit models of neoplasia described above are induced models. Monoclonal gammopathies can be induced in the rabbit in response to specific bacterial components (Hurvitz, 1975). Nephroblastoma is induced by administration of ethylnitrosourea to pregnant does (Haenichen and Stavrou, 1980). Finally, transgenic technology has been utilized to create a transgenic rabbit that develops acute [3lymphoblastic leukemia as a weanling (Sethupathi et al., 1993). The rabbit has been used extensively for infectious disease research, such as studies on Campylobacter enteritis (Caldwell and Walker, 1986), Chagas' disease (Texeira, 1986), cryptococcal meningitis (Perfect, 1985), Herpes simplex encephalitis (Schlitt and Bucher, 1989), and staphylococcal blepharitis (Mondino and Phinney, 1989). Another area in which the rabbit has been frequently employed as a model is in work related to cardiovascular disease. Numerous dietary modifications will induce or exacerbate cholesterol-induced atherosclerosis in the rabbit. A brief overview of some of these dietary modifications can be found elsewhere (Jayo et al., 1994). Research efforts into cholesterol metabolism have used the Watanabe heritable hyperlipidemic (WHHL) (Atkinson et al., 1992; Kita et al., 1981) and the St. Thomas Hospital strain rab-

331

bits (LaVille et al., 1987). The WHHL rabbit has a marked deficiency of low-density lipoprotein (LDL) receptors in the liver and other tissues. Selective breeding of the WHHL rabbit will increase the incidence of coronary artery atherosclerosis without increasing the incidence of aortic atherosclerosis (Watanabe et al., 1985). In contrast, the St. Thomas Hospital strain has a normal functioning LDL receptor but still maintains a hypercholesterolemic state (LaVille et al., 1987).

II.

A.

BIOLOGY

Comparative Anatomy and Physiology

1. Digestive System

The mouth of the rabbit is relatively small, and the oral cavity and pharynx are long and narrow. The dental formula is i2/1,c0/0,pm3/2,m2-3/3 • 2 = 26 or 28 teeth. A small pair of incisors is present directly caudal to the primary maxillary incisors and is referred to as the "peg" teeth. The peg teeth are used along with the primary incisors to bite and shear food. The absence of second incisors has been noted in some rabbit herds as a dominant trait (12/12or I2/i2). The teeth of rabbits erupt continuously throughout life and therefore will continue to grow and lengthen unless normal occlusion and use are sufficient to wear teeth to a normal length. Molars do not have roots and are characterized by deep enamel folds. Rabbits normally masticate food with a chewing motion that facilitates grinding of food by movement of the premolars and molars from side to side and front to back. The rabbit has four pairs of salivary glands, including the parotid, submaxillary, sublingual, and zygomatic. The parotid is the largest and lies laterally just below the base of the ear. The zygomatic salivary gland does not have a counterpart in humans. The esophagus of the rabbit has three layers of striated muscle that extend the length of the esophagus down to, and including, the cardia of the stomach. This is in contrast to humans and many other species of animals, who have separate portions of striated and smooth muscle along the length of the esophagus. There are no mucous glands in the esophagus of the rabbit. Although the stomach of the rabbit holds approximately 15% of the volume of the gastrointestinal tract, it is never entirely empty in the healthy rabbit. The gastric contents often include a large amount of hair ingested as the result of normal grooming activity. The stomach is divided into the cardia, fundus, and pylorus. The liver has four lobes. The gallbladder is found located on the right. From the liver, the common bile duct empties into the

332

MARK A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

duodenum posterior to the pylorus. Rabbits produce relatively large amounts of bile compared to other common species. The pancreas is diffuse within the mesentery of the small intestine and enters the duodenum 30 to 40 cm distal to the common bile duct. The small intestine of the rabbit is short relative to that of other species and comprises approximately 12% of the total length of the gastrointestinal (GI) tract. Because the GI tract of the rabbit is relatively impermeable to large molecules, kits receive most of their passive immunity via the yolk sac prior to birth rather than by the colostrum. Pale foci of lymphoid tissue referred to as Peyer's patches are found along the ileum, particularly near the cecal junction. The sacculus rotundus is a large bulb of lymphoid tissue located at this junction. The large intestine includes the cecum, the ascending colon, the transverse colon, and the descending colon. The ileocecal valve regulates flow of chyme into the cecum and retards reverse flow back into the ileum. The cecum is very large with a capacity approximately 10 times that of the stomach. The cecum ends in a blind sac, the appendix. The colon is divided into proximal and distal portions by the fusus coli, which serves to regulate the elimination of hard versus soft fecal pellets. Hard pellets comprise about two-thirds of the fecal output. Soft pellets, or "cecotrophs," have a high moisture content and are rich in nitrogen-containing compounds (Ferrando et al., 1970) and the B vitamins niacin, riboflavin, pantothenate, and cyanocobalamin. Rabbits consume cecotrophs directly from the anus to obtain significant nutritional benefit. Soft pellets are sometimes termed "night feces," since they are generally produced at night in domestic rabbits (Fig. 1). In contrast, the circadian rhythm of cecotrophy is reversed in

wild rabbits, occurring during the day when the animals are in their burrows (Hornicke, 1977).

2.

Nostrils of rabbits are well equipped with touch cells, and they have a well-developed sense of smell. Nasal breathing in rabbits is characterized by twitching of the nostrils at rates varying from 20 to 120 times per minute, although twitching may be absent in the relaxed rabbit. It has been speculated that inspiration occurs as the nostril moves up and that this serves to direct the flow of air over the turbinate bones where the olfactory cells are most concentrated. The musculature of the thoracic wall contributes little to respiratory efforts. Instead, rabbits rely mostly on the activity of the diaphragm. Because of this, artificial respiration is easily performed by alternating the head of the rabbit between the up position and the down position, 3 0 - 4 5 times per minute, while holding the animal. Compression and release of the chest wall is an ineffective means of artificial respiration in the rabbit. The pharynx of the rabbit is long and narrow, and the tongue is relatively large. These features make endotracheal intubation difficult to perform in the rabbit. The procedure is further complicated by the propensity of the rabbit to laryngospasm during attempts to intubate the trachea. The rabbit lungs consist of six lobes. Both right and left sides have cranial, middle, and caudal lobes, with the right caudal being further subdivided into lateral and medial portions. Flow volume of air to the left lung is higher than to the right due to the lower resistance of the proximal airways per unit volume (Yokoyama, 1979). In rabbits, lung volume increases with age, in contrast to that of humans and dogs, in which it decreases. Bronchial-associated lymphoid tissue (BALT) is present as distinct tissue.

3.

Fig. 1. Normalstomachcontents from a rabbit. Note the smooth,round mucoid night feces along with the amorphous food mass. Night feces are thought to originate from the cecum and are usually passed during the night and consumed by the rabbit. The night feces are easily distinguishedfrom the discrete oval fecal pellets produced during the day.

Respiratory System

Cardiovascular System

A unique feature of the cardiovascular system of the rabbit is that the tricuspid valve of the heart has only two cusps, rather than three as in many other mammals. A small group of pacemaker cells generates the impulse of the sinoatrial (SA) node in the rabbit, a feature that facilitates precise determination of the location of the pacemaker (Bleeker et al., 1980; Hoffmann, 1965; West, 1955). The SA and atrioventricular (AV) nodes are slender and elongated, and the AV node is separated from the annulus fibrosus by a layer of fat (Truex and Smythe, 1965). Additional unique anatomic features of the cardiovascular system of the rabbit have been utilized to advantage. The aortic nerve subserves no known chemoreceptors (Kardon et al., 1974; Stinnett and Sepe, 1979) and responds to baroreceptors only. Because the aortic nerve, which becomes the depressor nerve, runs alongside but separate from the vagosympathetic

9. BIOLOGYAND DISEASESOF RABBITS

333

trunk, it lends itself readily to implantation of electrodes (Karemaker et al., 1980). The blood supply to the brain is restricted mainly to the internal carotid artery. Blood supplied via the vertebral arteries is limited. The aorta of the rabbit demonstrates rhythmic contractions that arise from neurogenic stimulation in a pattern related to the pulse wave (Mangel et al., 1981). 4.

Urogenital System

The kidney of the rabbit is unipapillate in contrast to that of most other mammals, which is multipapillate. This feature increases the ease with which cannulization is performed. The right kidney lies more cranial than the left. Glomeruli increase in number after birth, whereas all of the glomeruli are present at birth in humans (Smith, 1951). Ectopic glomeruli are normal in the rabbit (Steinhausen et al., 1990). Blood vessels that perfuse the medulla remain open during many conditions under which vasoconstriction of the cortical tissue occurs; thus, the medullary tissue may be perfused while the cortex is ischemic (Trueta et al., 1947). In the rabbit, the clearance of creatinine is identical with the clearance of insulin, thus creatinine clearance can be used to accurately measure the glomerular filtration rate. This is not true for primates, rats, or guinea pigs, among others. The urine of adult rabbits is typically cloudy due to a relatively high concentration of ammonium magnesium phosphate and calcium carbonate monohydrate precipitates (Flatt and Carpenter, 1971). The urine may also take on hues ranging from yellow or reddish to brown. In contrast, the urine of young rabbits is typically clear, although healthy young rabbits may have albuminuria. The urine is normally yellow but can also take on reddish or brown hues once they begin to eat green feed and cereal grains. Normal rabbits have few cells, bacteria, or casts in their urine. The pH of the urine is typically alkaline at about 8.2 (Williams, 1976). A normal adult rabbit produces approximately 50-75 ml/kg of urine daily (Gillett, 1994), with does urinating more copiously than bucks. The urethral orifice of the buck is rounded, whereas that of the doe is slitlike. This feature is useful for distinguishing the sexes. The testes of the adult male usually lie within the scrotum; however, the inguinal canals that connect the abdominal cavity to the inguinal pouches do not close in the rabbit. For this reason, the testes can easily pass between the scrotum and the abdominal cavity. In particular, this feature necessitates closure of the superficial inguinal ring following orchiectomy by open technique, to prevent herniation. The reproductive tract of the doe is characterized by two uterine horns that are connected to the vagina by separate cervices (bicornuate uterus) (Fig. 2). A common tube, the urogenital sinus or vestibulum, is present where the urethra enters the vagina. The placenta is hemochorial, and maternal blood flows into sinuslike spaces where the transfer of nutrients and

Fig. 2. Rabbit uterus. Note two uterine horns each with its own cervix

(arrows).

other substances to the fetal circulation occurs (Jones and Hunt, 1983). Inguinal pouches are located lateral to the genitalia in both sexes. The pouches are blind and contain scent glands that produce white to brown secretions that may accumulate in the pouch. 5.

Metabolism

The metabolic rate of endotherms is generally related to the body surface area. Including the ears, the rabbit has a relatively low metabolic rate (MR); however, if the surface area of the ears is discounted, the MR of the rabbit is similar to that of other endotherms. Neonatal rabbits have an amount of body fat comparable to that of the human infant (16% of body weight) (Cornblath and Schwartz, 1976). The neonatal rabbit is essentially an ectotherm until about day 7 (Gelineo, 1964). The glucose reserves of the neonatal rabbit are quickly depleted, usually within about 6 hr after birth (Shelley, 1961). The fasting neonatal rabbit quickly becomes hypoglycemic and ketotic (Callikan and Girard, 1979). The normal rectal temperature of the adult New Zealand White rabbit at rest is approximately 38.5 ~ to 39.5~ (Ruckebusch et al., 1991). The ears serve an important thermoregulatory function. Because they have a large surface area and are highly vascular with an extensive arteriovenous anastomotic system, the ears help the rabbit sense and respond to cold versus warm temperatures (Kluger et al., 1972). In addition, the ears serve as a countercurrent heat-exchange system to help adjust body temperature. Early studies found that the body of the adult rabbit (3 kg body weight) consists of greater than 50% water (58%), with a half-time turnover of about 3.9 days and a loss of about 340 ml daily (Richmond et al., 1962). The amount of water ingested

334

MARK A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

varies with the amount and type of feed consumed and the environmental temperature. In general, rabbits will drink more water when consuming dry, pelleted feed than when consuming foodstuffs high in moisture, such as fresh greens. Conversely, rabbits deprived of water will decrease food consumption. After 3 days of complete water deprivation, the food intake falls to less than 2% of normal (Cizek, 1961).

B.

N o r m a t i v e Physiological Values

Normal values for various systems and parameters are provided as a general indication for these values in the rabbit. It is important to recognize, however, that most of these values have been obtained through the study of adult New Zealand White rabbits. Values can vary significantly between breeds, laboratories, methods of sampling and measurement, and individual rabbits due to age, sex, breed, health, handling, and husbandry (Hewitt et al., 1989; Lidena and Trautschold, 1986; Mitruka and Rawnsley, 1981; Woolford et al., 1986; Yu et al., 1979). For this reason, individual laboratories should strive to establish their own normal values, whenever possible. 1.

Hematologic Values

Values for hematologic parameters are shown in Table III. These values represent those typical of adult New Zealand White rabbits. In general, males have slightly greater hematocrit

Table III

Hematologic Valuesfor the Adult Rabbita Hematologic parameter Blood volume Plasma volume Hemoglobin Packed cell volume Erythrocytes Reticulocytes Mean corpuscularvolume (MCV) Mean corpuscularhemoglobin(MCH) MCH concentration(MCHC) Sedimentation rate White blood cells Neutrophils (heterophils) Lymphocytes Eosinophils Basophils Monocytes Platelets

Typical value 55- 65 ml/kg 28-50 ml/kg 9.8-14.0 gm/dl 34-43% 5.3-6.8 cells (106/lxl) 1.9-3.8% 60-69 fl 20-23 pg 31-35% 0.92-3.00 mm/hr 5.1-9.7 cells (103/ixl) 25- 46% 39- 68% 0.1-2.0% 2.0-5.0% 1.0-9.0% 158-650 (103/~1)

aValues obtained from the following sources: Burns and DeLannoy(1966), Gillett (1994),Kabataet al. (1991),Mitrukaand Rawnsley(1981),and Woolford et al. (1986).

and hemoglobin values than females (Mitruka and Rawnsley, 1981). Red blood cell (RBC) diameter reaches normal adult values of 6.7-7.9 mm (Jain, 1986). Anisocytosis is normal and accounts for variation in reported values for RBC diameter (Sanderson and Phillips, 1981). The life span of the rabbit RBC averages 57 days although some could survive up to 67 days (Vacha, 1983). Reticulocyte values are usually between 2% and 4% in healthy rabbits (Corash et al., 1988). Red blood cell sedimentation is minimal, with values of 1-3 mm/hr being typical (Schermer, 1967). Platelets have a pale blue cytoplasm and azurophilic granules when stained by standard methods (Jain, 1986; Sanderson and Phillips, 1981). The neutrophil of the rabbit is sometimes referred to as a "pseudoeosinophil" or "heterophil," due to the presence of red-staining granules in the cytoplasm. The heterophil (10-15 mm in diameter) is, however, smaller than the eosinophil (12-16 mm in diameter) (Sanderson and Phillips, 1981). In addition, the red granules of the heterophil are smaller than the red granules of the eosinophil. The nucleus of the eosinophil may be either bilobed or horseshoeshaped. Some rabbits demonstrate the Pelger-HuEt anomaly in which the heterophil nucleus is hyposegmented due to incomplete differentiation of the granulocytes (Jain, 1986). Although the typical presentation is that of a few Pelger cells in the circulation, one report describes a line of rabbits with uniform presence of Pelger cells in the circulation accompanied by high mortality (Schermer, 1967). The morphology ~of lymphocytes and monocytes is similar to that seen in other mammals. Both small (7-10 ~tm in diameter) and large (10-15 ~tm in diameter) lymphocytes are typically present (Jain, 1986; Sanderson and Phillips, 1981). The largest cell in the peripheral blood circulation of the rabbit is the monocyte, at 15-18 ~tm in diameter. Granules are not normally found in the cytoplasm of rabbit monocytes. 2.

Blood and Serum Chemistry and Enzyme Values

As mentioned earlier, chemistry values can vary because of a number of factors. For this reason, each laboratory should establish its own normal values. Aspartate aminotransferase (AST), formerly serum glutamate oxalate transaminase (SGOT), is present in the liver, heart, skeletal muscle, kidney, and pancreas. Collection of blood samples in rabbits by decapitation, cardiac puncture, or aortic incision, or the use of restraint that causes exertion will elevate AST levels due to muscle damage (Lidena and Trautschold, 1986). Similarly, levels of creatinine kinase are sensitive to muscle damage since that enzyme is present in skeletal muscle, brain, and heart (Lidena and Trautschold, 1986; Mitruka and Rawnsely, 1981). Although most mammals have two isoenzymes (intestinal and a liver/kidney/bone form) of alkaline phosphatase (AP), rabbits

335

9. BIOLOGY AND DISEASES OF RABBITS

are unique in having three forms of AP, including an intestinal form and two forms that are both present in the liver and the kidney (Noguchi and Yamashita, 1987). Values for blood and serum chemistry are shown in Table IV. Respiratory, Circulatory and Miscellaneous Biologic Parameters

Cardiovascular and respiratory function are often altered with experimental manipulation, anesthesia, or disease. Normal values for these parameters and other miscellaneous biologic characteristics of the rabbit are shown in Table V.

C.

Nutrition

Rabbits are strictly herbivorous with a preferred diet of herbage that is low in fiber and high in protein and soluble carbohydrate (Cheeke, 1987, 1994). Rabbits will generally accept Table IV Values of Serum Biochemical and Enzyme Parameters of the Adult Rabbita Biochemical parameter

Typical value

Total protein Globulin Albumin Glucose Sodium Chloride Potassium Phosphorus Calcium Magnesium Acid phosphatase Alkaline phosphatase Acid phosphatase Lactate dehydrogenase ~/-Glutamyltransferase Aspartate aminotransferase Creatine kinase Alanine aminotransferase (SGPT) Sorbitol dehydrogenase Urea nitrogen Creatinine Total bilirubin Uric acid Amylase Serum lipids Phospholipids Triglycerides Cholesterol Corticosterone

5.0-7.5 gm/dl 1.5-2.7 gm/dl 2.7-5.0 gm/dl 74-148 mg/dl 125 - 150 mEq/liter 92-120 mEq/liter 3.5-7.0 mEq/liter 4.0- 6.0 mg/dl 5.60-12.1 mg/dl 2.0-5.4 mg/dl 0.3-2.7 IU/liter 10- 86 IU/liter 0.30-2.70 IU/liter 33.5 - 129 IU/liter 10-98 IU/liter 20-120 IU/liter 25 - 120 IU/liter 25-65 IU/liter 170-177 U 5-25 mg/dl 0.5-2.6 mg/dl 0.2-0.5 mg/dl 1.0- 4.3 mg/dl 200-500 IU/liter 150- 400 mg/dl 40-140 mg/dl 50- 200 mg/dl 10-100 mg/dl 1.54 Ixg/dl

Values obtained from the following sources: Burns and DeLannoy (1966), Fox (1989), Gillett (1994), Kraus et al. (1984), and Loeb and Quimby (1989). a

Table V Respiratory, Circulatory, and Miscellaneous Biologic Parameters of the Rabbit a Parameter

Typical value

Life span Body weight GI transit time Number of mammary glands Diploid chromosome number Body temperature

5 - 7 years 2 -5 kg 4 -5 hr 8 or 10 44 38.5~176

Respiratory rate Lung weight (2.4 kg rabbit) Total lung capacity Minute volume Tidal volume Mean alveolar diameter

32-60 breaths/min 9.1 gm 111 +__14.7 ml 0.6 liter/min 4 - 6 ml/kg body weight 93.97 I~m

Heart rate pO2 pCO2 HCO3 Arterial oxygen Arterial systolic pressure Arterial diastolic pressure Arterial blood pH Interstitial fluid (IF) colloid osmotic pressure IF viscosity (water = 1) IF protein

200 -300 beats/min 85 - 102 mmHg 2 0 - 46 torr 12-24 mmol/liter 12.6-15.8% volume 90-130 mmHg 80-90 mmHg 7.2-7.5 13.6 mmHg 1.9 2.7

Cerebrospinal fluid (CSF) white blood cells CSF lymphocytes CSF monocytes

0 - 7 cells/mm3 40-79% 21- 60%

aValues obtained from the following sources: Barzago et al. (1992), Curiel et al. (1982), Gillett (1994), Kozma et al. (1974), Sanford and Colby (1980),

Suckow and Douglas (1997), and Zurovsky et al. (1995).

a pelleted feed more readily than one in meal form. When a meal diet is needed, a period of adjustment should be allowed for the rabbits to accommodate to the new diet. Examples of adequate diets are shown in Table VI. The exact nutrient requirements for individual rabbits vary with age, reproductive status, and health of the animal. Nutritional requirements for the domestic rabbit are shown in Table VII. On occasion, the need arises for use of highly purified diets. A suggested purified diet has been described elsewhere (Subcommittee on Rabbit Nutrition, 1977). It should be noted that overfeeding of laboratory rabbits resulting in obesity is common, but can be prevented by either reducing the amount of feed or by providing a low-energy, high-fiber maintenance diet. As mentioned earlier, rabbits engage in cecotrophy, and by doing so supplement their supply of protein and B vitamins. Rabbits fed a diet high in fiber ingest a greater quantity of cecotropes than those on a lower-fiber diet (Fekete and Bokori, 1985).

336

M A R K A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

Table VI

Table V I I

Examples of Adequate Diets for Commercial Production a

Nutrient Requirements of Rabbits Fed a d l i b i t u m (Percentage or Amount per Kilogram of Diet)a,b

Kind of animal Growth, 0.5- 4 kg

Maintenance, does and bucks, average 4.5 kg Pregnant does, average 4.5 kg

Lactating does, average 4.5 kg

Ingredients

Percentage of total diet b

Alfalfa hay Corn, grain Barley, grain Wheat bran Soybean meal Salt Clover hay Oats, grain Salt Alfalfa hay Oats, grain Soybean meal Salt Alfalfa hay Wheat, grain Sorghum grain Soybean meal Salt

50.00 23.50 11.00 5.00 10.00 0.50 70.00 29.50 0.50 50.00 45.50 4.00 0.50 40.00 25.00 22.50 12.00 0.50

a From

Subcommittee on Rabbit Nutrition (1977). Used with permission. bComposition given on an as-fed basis.

Prolonged feeding of diets high in calcium, such as those with a high level of alfalfa meal, can result in renal disease. Consumption of diets containing excessive vitamin D can result in calcification of soft tissues, including the liver, kidney, vasculature, and muscles (Fig. 3) (Besch-Williford et al., 1985). Diets that are either too high or too low in vitamin A can result in reproductive dysfunction and congenital hydrocephalus (Cheeke, 1987; DiGiacomo et al., 1992). The exact requirement for vitamin A in the rabbit has not been determined; however, a level of 10,000 IU/kg of diet is generally adequate. Vitamin E deficiency has been associated with infertility, muscular dystrophy, fetal death, neonatal death, and colobomatous microphthalmos in rabbits (Nielsen and Carlton, 1995; Ringler and Abrams, 1970, 1971). McDowell (1989) suggests that serum vitamin E levels of less than 0.5 ~tg/ml are indicative of hypovitaminosis E. Relative to other species, rabbits have a high water intake. In general, daily water intake is approximately 120 ml per kilogram of body weight. Consumption of water is influenced by environmental temperature, disease states, and feed composition and intake (Cizek, 1961). Consumption of diets high in fiber usually result in increased water intake. Water consumption also increases with food deprivation.

D.

Behavior

Rabbits are social animals and attempts at group housing often meet with success, although mature males will fight and can

Nutrients

Growth

Maintenance Gestation

Lactation

Energy and protein Digestible energy (kcal) Total digestible nutrients (%) Crude fiber (%) Fat (%)

2500.00 65.00

2100.00 55.00

2500.00 58.00

2500.00 70.00

10-12 c 2c

14 c 2c

10-12 c 2c

10-12 c 2~

16.00

12.00

15.00

17.00

0.4 0.22 300 - 400 0.6 0.2 c,e 0.3 c,e

__d __d 3 0 0 - 400 0.6 0.2 c,e

0.45 ~ 0.37 ~ 3 0 0 - 400 0.6 0.2 ~,e

0.75 c 0.5 3 0 0 - 400 0.6 0.2 c,e

0.3

0.3

0.3

Copper (mg) Iodine (mg)

3 0.2 ~

3 0.2 c

3 0.2 ~

3 0.2 c

Iron

~f

~f

~f

..__d

Manganese (mg) Zinc

8.5 f _d

2.5 f .__d

2.5 y d

2.5 f __d

580 0.83 c,d

___d g

> 1160 0.83 ~,d

d ~g

h 40 i -- j 180 39 1.2 c

h ~f mJ k

___h 40 h 0.2 ~ k

h 40 i ~J __k

k

k

k

k

~

k

0.65 0.6 0.6 0.3 c 1.1 c 0.6 c 1.1 c 0.6 c 0.2 r

h h h

h h h

___h h h

.__h

h

h

__h h h h

__h h ____h h

__h h h h

Crude protein (%) Inorganic nutrients Calcium (%) Phosphorus (%) Magnesium (mg) Potassium (%) Sodium (%) Chlorine (%)

c,e

c,e

c,e

Vitamins Vitamin A (IU) Vitamin A as carotene (mg) Vitamin D Vitamin E (mg) Vitamin K (mg) Niacin (mg) Pyri do xine (mg ) Choline (gm) Amino acids (%) Lysine Methionine + cystine Arginine Histidine Leucine Isoleucine Phenylalanine + tyrosine Threonine Tryptophan Valine

0.7 c

h ___h

h ___h

___h h

Glycine

__h

h

h

h

a From Subcommittee on Rabbit Nutrition (1977). Used with permission.

bNutrients not listed indicate dietary need unknown or not demonstrated. cMay not be minimum but known to be adequate. d Quantitative requirement not determined, but dietary need demonstrated. eMay be met with 5% NaC1. fConverted from amount per rabbit per day using an air-dry feed intake of 60 gm per day for a 1-kg rabbit. g Quantitative requirement not determined. hProbably required; amount unknown. i Estimated. J Intestinal synthesis probably adequate. kDietary need unknown.

9. BIOLOGY AND DISEASES OF RABBITS

Fig. 3. Calcifiedaorta resulting from excessivedietary Vitamin D.

inflict serious injury on one another (Love, 1994; Podberscek et al., 1991; Whary et al., 1993). Group-penned female rabbits allowed to choose between single or paired housing prefer being in the same cage with other rabbits (Huls et al., 1991). In general, rabbits are timid and nonaggressive. Some animals will display defensive behavior, typically characterized by thumping the cage floor with the rear feet, biting, and charging toward the front of the cage when opened. Laboratory-housed rabbits demonstrate diurnal behavior, in contrast to the nocturnal pattern exhibited by wild rabbits (Jilge, 1991).

E.

337

(Hafez, 1970). During periods of receptivity, the vulva of the doe usually becomes swollen, moist, and dark pink or red. Ovulation is induced and occurs approximately 10 to 13 hr after copulation. Interestingly, up to 25% of does fail to ovulate following copulation. Ovulation can also be induced by administration of luteinizing hormone (Kennelly and Foote, 1965), human chorionic gonadotropin (Williams et al., 1991), or gonadotropic releasing hormone (Foote and Simkin, 1993). Receptivity of the doe is usually signaled by vulvar changes as described above, restlessness, and rubbing of the chin on the hutch or cage. Vaginal cytology is generally not useful for determination of estrus or receptivity in the rabbit. Typically, the doe is brought to the buck's cage for breeding, since the doe can be very territorial and may attack the male in her own quarters. A period of 15 to 20 min is usually sufficient to determine compatibility of the doe and buck. If receptive, the doe will lie in the mating position and raise her hindquarters to allow copulation. If fighting or lack of breeding is observed, the doe may be tried with another buck. A single buck is usually sufficient to service 10 to 15 does. Does may be bred immediately after kindling; however, most breeders delay until after the kits have been weaned. Success at postpartum breeding varies, but one can produce a large number of kits in a relatively short time period by foster nursing the young and rebreeding the doe immediately. While conventional breeding, nursing, and weaning schedules allow for only 4 litters per year, early postpartum breeding allows for up to 11 litters per year.

Reproduction 3. Pregnancy and Gestation

1. Sexual Maturity

Puberty generally occurs between the ages of 5 - 7 months in the New Zealand White rabbit. Smaller breeds typically reach puberty earlier, and larger breeds a bit later. For example, Polish or Dutch rabbits are usually sexually mature by 4 months of age, while Flemish or Checkered Giant rabbits reach sexual maturity by 9 to 12 months. The breeding life of a doe typically lasts approximately 1 to 3 years, although some remain productive for up to 5 or 6 years. In later years, litter sizes usually diminish. In comparison, most bucks will remain reproductively useful for an average of 5 to 6 years. Because does often will engage in reproductive behavior before being able to ovulate, it is advisable not to breed does until they are fully grown.

Pregnancy can often be confirmed as early as day 14 of gestation by palpation of the fetuses within the uterus. Radiographic procedures permit pregnancy determination as early as day 11. Conception rates have been observed to have an inverse relationship with ambient temperature but not light cycle. Gestation in rabbits usually lasts for 30 to 33 days. Does beyond 2 to 3 weeks of gestation will usually refuse a buck. Does begin hair pulling and nest building during the last 3 to 4 days of gestation. A nesting box with shredded paper or other soft material such as straw should be provided to the doe several days prior to the expected kindling (parturition) date. The doe will usually line the box with her own hair. The nesting box should not be placed in the corner of the cage where the individual doe has been observed to urinate. 4. Pseudopregnancy

2. Reproductive Behavior

Does do not have a distinct estrous cycle, but rather demonstrate a rhythm with respect to receptivity to the buck. Receptivity is punctuated by periods (1-2 days every 4 - 1 7 days) of anestrus and seasonal variations in reproductive performance

Pseudopregnancy is common in rabbits and can follow a variety of stimuli, including mounting by other does, sterile matings by bucks, administration of luteinizing hormone, or the presence of bucks nearby. In such circumstances, ovulation is followed by a persistent corpus luteum that lasts 15 to 17 days.

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The corpus luteum or corpora lutea secrete progesterone during this time, causing the uterus and mammae to enlarge. The doe may have the appearance of a normally pregnant rabbit. Toward the end of pseudopregnancy, many does will begin to pull hair as part of ritual nest-building behavior. 5.

Parturition

The process of parturition is referred to as "kindling" as it relates to rabbits. Kindling normally occurs during the early morning hours and takes approximately 30 to 60 min. Impending kindling is often signaled by nest building and decreased food consumption during the preceding 2 to 3 days. Both anterior and breech presentations are normal in the rabbit. Fetuses retained beyond 35 days generally die and may harm future reproductive ability of the doe if not expelled. The average number of kits born is 7 to 9 per litter, although smaller litters and litters up to 10 kits are not uncommon. Litter size is influenced by breed, parity, nutritional status, and environmental factors. Polish rabbits usually have fewer than 4 kits per litter; Dutch or Flemish, 4 to 5; and New Zealand White, 8 to 10. After the young have been cleaned following parturition, the doe typically consumes the placenta. Cannibalism of the young by the doe sometimes occurs and may be related to environmental or hereditary factors or due to environmental stressors. 6.

Lactation

Does usually have either four or five pairs of nipples, while bucks have none. During the last week of pregnancy, marked development of the mammary gland occurs. The doe normally nurses the kits once daily for several minutes, usually in the early morning or in the evening, regardless of how many kits are present or how many times they attempt to suckle. Milk yield is normally between 160 and 220 gm/day. Maximum output occurs at 2 weeks following kindling and then declines during the fourth week. Rabbit milk contains approximately 12.5% protein, 13% fat, 2% lactose, and 2.5% minerals. Nursing may last 5 to 10 weeks. Kits may begin consuming solid food by 3 weeks of age, with weaning generally occurring by 5 to 8 weeks of age. F. 1.

M a n a g e m e n t and H u s b a n d r y

Housing

The facilities present in most modern research animal facilities would be suitable for housing rabbits. General construction should include adequate heating, ventilation, and air conditioning to house rabbits at appropriate temperature and humidity. In addition, lighting should be adequate to allow easy visualization of the rabbits. Surfaces, such as the floors, walls, and ceilings, should be easily sanitizable (Suckow and Douglas, 1997).

Rabbit cages should provide a safe environment with easy access to food and water. Adults can be caged individually or in compatible groups and should have sufficient floor space to lie down and stretch out. In the United States, minimum cage sizes are determined by the Animal Welfare Act (AWA) and the "Guide for the Care and Use of Laboratory Animals" (1996). In both cases, sizes vary with the weight of the animal. Currently, the AWA regulations and the "Guide" require 3.0 ft 2 of floor space and 14 inches of cage height for rabbits weighing 2 - 4 kg. Cages should be constructed of durable materials that will resist corrosion and harsh detergents and disinfectants used in cleaning. Consequently, in the research environment, rabbit cages are most often constructed of stainless steel or plastics. Rabbits are usually housed in cages with mesh or slatted floors to permit urine and feces to drop through into a catch pan. Mesh floors with catch pans do not prevent rabbits from engaging in the normal practice of coprophagy. Rabbits will play with objects placed in their cages. Huls et al. (1991) noted that rabbits would use wooden sticks, wooden rings, and brass wire balls as toys. Rabbits can also be housed in compatible pairs or groups, but one should take the possibility of aggression and pseudopregnancy into consideration before choosing this housing modality. 2.

Environment

Rabbits require cooler room temperatures than most other common species of laboratory animals. The "Guide" recommends that temperatures in rabbit rooms be maintained between 61 o and 72~ No specific illumination requirements for rabbits have been described. It is common practice to provide rabbits with 12-14 hr of light in the light-dark cycle. In breeding colonies, females should be provided with 14-16 hr of light. Ammonia production in rabbit rooms can be a signficant problem; therefore, rabbit rooms should be ventilated at 10-15 air changes per hr ("Guide," 1996). It is also important to change excreta pans often to prevent the buildup of ammonia. Rabbits are easily startled by sudden, loud noises. For this reason, they should not be housed near noisy species such as dogs or monkeys, nor should they be housed near noisegenerating operations such as the cage-wash area. 3.

Sanitation

Catch pans should be cleaned as often as necessary to prevent the formation of ammonia. Cages are generally sanitized on at least a weekly basis. Rabbit urine contains large amounts of protein and minerals, and often forms deposits on cages and catch pans. It is common practice to soak equipment having urine deposits in acid washes to remove the scale before washing.

9. BIOLOGY AND DISEASES OF RABBITS

III.

A. 1.

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DISEASES

Bacterial Diseases

Pasteurellosis

Pasteurellosis is a common disease of laboratory rabbits and is caused by Pasteurella multocida. It is a gram-negative, nonmotile, coccobacillus. Five capsular (A, B, D, E, and F) and 17 somatic serotypes are currently recognized. Rabbit isolates are most often capsular types A or D and somatic types 3 or 12 (DiGiacomo et al., 1991). Many isolates, however, are nontypable (Manning et al., 1989). Pasteurella multocida causes a variety of clinical syndromes in rabbits. The clinical presentation can include one or more of the following: rhinitis, sinusitis, pneumonia, otitis media, otitis interna, conjunctivitis, abscess formation, genital infection, and septicemia (DeLong and Manning, 1994). Rhinitis with or without sinusitis is the most common clinical manifestation of pasteurellosis in rabbits. It is commonly called "snuffles." In outdoor colonies, the incidence may be as high as 60%, and the disease is most common in the spring and fall (DiGiacomo et al., 1983). Rabbits present with a serous to mucopurulent nasal discharge, sneezing, coughing, and exudate on the fur of the forepaws. Infected rabbits may be clinically asymptomatic even though the organism is still present in the nasal passages. Rabbits with rhinitis often develop an associated conjunctivitis. Clinical signs include mucopurulent ocular exudate, chemosis, conjunctival reddening, swollen eyelids, epiphora, and hair loss around the eyes (DeLong and Manning, 1994). Pneumonia is also a common clinical condition in affected rabbits (DeLong and Manning, 1994). Both acute and chronic pneumonia may occur. Chronic pneumonia is often asymptomatic (DeLong and Manning, 1994; Flatt and Dungworth, 1971). In the research setting, animals often exhibit few clinical signs because the affected animal's respiratory demands in the cage are minimal. Affected animals may exhibit anorexia, depression, dyspnea, moist rales, and death. Pasteurella multocida can cause otitis media in rabbits, a clinically silent condition that may progress to otitis interna with torticollis (Fig. 4) (DeLong and Manning, 1994). Subcutaneous and visceral Pasteurella abscesses can also be clinically silent for long periods. Subcutaneous abscesses often rupture spontaneously to the outside. Rabbits that develop Pasteurella septicemia generally die acutely without any clinical signs. Female rabbits with acute genital tract infections may present with a serous, mucous, or mucopurulent vaginal discharge. Chronic genital tract infections in the female are often asymptomatic or may manifest as decreased fertility or abortion. Male rabbits may develop orchitis or epididymitis, exhibiting decreased fertility and enlarged, firm testicles.

Fig. 4. Head tilt (torticollis) in a rabbit with otitis interna related to infection with Pasteurella multocida. Torticollis is often accompanied by circling in the direction of the affected vestibular apparatus.

Rabbits become colonized with P. multocida early in life by the oral or respiratory routes. Direct contact is the most effective means of transmission, but aerosol transmission can also occur (DeLong and Manning, 1994). Veneral transmission occurs, but less commonly. Young rabbits generally become infected around the time of weaning. This probably corresponds with the decline in maternal antibody that occurs at this time (Glass and Beasley, 1989). The pharynx may be the first site colonized by P. multocida, with later spread to the nares and other organs (DeLong and Manning, 1994). Infection rates as high as 90% have been reported, and the rate of infection in colonies decreases with age (DeLong and Manning, 1994). Changes in temperature, experimental manipulation, pregnancy, and concurrent disease are frequently associated with the development of clinical signs. The specific pathologic findings will vary with the site of infection, but the underlying host response is characterized by acute or chronic suppurative inflammation with the infiltration of large numbers of neutrophils. Rhinitis and sinusitis are accompanied by a mucopurulent nasal exudate. Neutrophil infiltration of the tissues is extensive. The nasal passages are edematous, inflamed, and congested, and there may be mucosal ulcerations. The turbinate bones may atrophy (DiGiacomo et al., 1989; Chrisp and Foged, 1991). Purulent conjunctivitis may be present. Pneumonia is primarily cranioventral in distribution. The lungs can exhibit consolidation, atelectasis, and abscess formation. A purulent to fibrinopurulent exudate is evident, and there may be areas of hemorrhage and necrosis. In some rabbits, fibrinopurulent pleuritis and pericarditis are prominent features

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(Glavits and Magyar, 1990). This is probably due to elaboration of a heat-labile toxin in some strains of the bacteria (Chrisp and Foged, 1991). Acute hepatic necrosis and splenic lymphoid atrophy are also seen in association with the pleuritis and pneumonia induced by toxigenic strains. Otitis media is characterized by a suppurative exudate with goblet cell proliferation and lymphocytic and plasma cell infiltration. In female rabbits with genital tract infections, the uterus may be enlarged and dilated. In the early stages of infection, the exudate is watery; later it thickens and is cream-colored. The exudate contains numerous neutrophils. Focal endometrial ulceration can be found (Johnson and Wolf, 1993). In the male, the testes are enlarged and may contain abscesses. Systemic and visceral abscesses are characterized by a necrotic center, an infiltrate made up of polymorphonuclear neutrophils, and a fibrous capsule. Septicemia may only present as congestion and petechial hemorrhages in many organs. Early in the infectious process, P. multocida organisms likely colonize the nasopharyngeal mucosa of rabbits. Studies conducted in vitro have shown that P. multocida is an adhesive organism and has fimbriae (pili) (Glorioso et al., 1982; BonillaRuz and Garcia-Delgado, 1993). The factors that lead to subsequent spread to other organs are unknown. A variety of bacterial virulence factors likely play a role in protecting the organism from host defenses. These include resistance to phagocytosis by polymorphonuclear neutrophils, resistance to killing by serum and complement, toxin production, and endotoxin production. Culture of the organism is the most definitive means for diagnosis of pasteurellosis. Several serologic assays have also been described, including enzyme-linked immunosorbent assays (ELISA), indirect hemagglutination, and a gel diffusion precipitin test (Lukas et al., 1987; Zaoutis et al., 1991; Zimmerman et al., 1992; Kawamoto et al., 1994; Peterson, et al., 1997). A wide variety of bacterial extracts have been utilized to stimulate a humoral immune response in rabbits and other species. Presumably, the intent has been to stimulate opsonizing and/or bactericidal antibodies, but the mechanism(s) by which these vaccines stimulate protective immunity is (are) not well understood. In general, the host responds to a component vaccine effective against the homologous organism. In only a few preparations has protection against heterologous strains been demonstrated. Several antigens that provide partial protection have recently been described (Lu et al., 1988, 1991; Zimmerman et al., 1992; Ruffle and Alder, 1996). However, there is no commercial vaccine effective against P. multocida in rabbits. The control of pasteurellosis in research facilities is most easily accomplished through the use of Pasteurella-free rabbits from commercial vendors. Such animals can be maintained free of the disease by isolation away from infected animals. Personnel and management procedures should be established to pre-

vent the introduction of the organism into a Pasteurella-free colony. Minimally, rabbits suspected of harboring P. multocida should be isolated from other rabbits and species. Personnel practices such as frequent changing of lab coats and entering Pasteurella-free rabbit rooms only if individuals have had no other contact with other groups on that day should be undertaken. In addition, the use of laminar flow (outflow) housing and more rigorous personnel procedures (mask, gloves, gowns, etc.) can be used if the situation calls for a greater degree of containment. Development of Pasteurella-free rabbit colonies has been accomplished through culture and culling of animals shown to harbor the organism (Griffin, 1952). Once microbiologically negative animals have been selected for the colony, that group should be maintained in isolation from other animals. Suckow et al. (1996) were able to derive Pasteurella-free rabbits by treating pregnant does with enrofloxacin past kindling. Although the P. multocida could still be cultured from does, all kits from enrofloxacin-treated does were free of P. multocida. Clinical signs of rhinitis can often be eliminated by treatment with antibiotics. However, antibiotic therapy will generally not eliminate the organism from the nasal passages. More serious forms of the disease should be treated with antibiotics after culture and sensitivity testing of the organism. Procaine penicillin G (40,000 U/kg IM SID) is very effective against P. multocida but should be used with caution to prevent development of clostridial enterotoxemia. Enrofloxacin at a dosage of 5 mg/kg body weight administered in the drinking water has been reported as effective (Okerman et al., 1990). Parenteral enrofloxacin (5 mg/kg IM every 12 hr for 14 days) was also shown to be effective, and some rabbits became culture-negative for P. multocida by the end of the course of treatment (Broome and Brooks, 1991). Tilmicosin (25 mg/kg SC) was shown to be an effective treatment for pasteurellosis in New Zealand White rabbits (McKay et al., 1996). The most common research complication associated with pasteurellosis is infection of injection sites of rabbits immunized for production of polyclonal antisera. Death of rabbits from Pasteurella septicemia can also occur. Vascular cell adhesion molecule- 1 (VCAM- 1) is expressed by endothelial cells during inflammation. It has been shown that rabbits infected with P. multocida, compared with uninfected control animals, had increased VCAM-l-positive aortic endothelial cells (Richardson et al., 1997). 2.

Tyzzer's Disease

The etiologic agent of Tyzzer's disease is Clostridium piliforme, a gram-negative, bacillus-shaped, spore-forming bacterium. The disease occurs in many animal species in addition to rabbits. C. piliforme is an obligate intracellular pathogen. The

9. BIOLOGY AND DISEASES OF RABBITS

organism cannot be grown in artificial media and must be cultured in embryonated eggs or tissue culture (Fries, 1977). The disease occurs most often in young animals, particularly around the age of weaning. Affected rabbits exhibit profuse, watery diarrhea, anorexia, dehydration, lethargy, and staining of the hindquarters with feces. Rabbits often die 1-2 days after exhibiting clinical signs. In acute outbreaks, mortality may be as high as 90% (DeLong and Manning, 1994). Some animals may go on to develop a chronic infection characterized by weight loss and wasting. The organism is most likely transmitted via the fecal-oral route through ingestion of spores. Outbreaks occur most often in 6- to 12-week-old rabbits, but all age groups are susceptible (DeLong and Manning, 1994). In naive populations, the disease presents as an epizootic with high morbidity and mortality. In enzootically infected colonies, many animals may be subclinically infected, but only small numbers may demonstrate clinical signs (Fries, 1979). Stress may be important in precipitating disease in subclinically infected rabbits. Gross necropsy findings include hemorrhages on the serosal surface of the cecum; a thickened, edematous bowel wall; and foci of necrosis in the mucosa. The ileum and colon may also be affected. The liver typically has numerous pinpoint white foci throughout the parenchyma. Similar foci may be present in the myocardium. Histologically, the cecal lesions consist of subserosal hemorrhages, edema, and mucosal necrosis that may extend into deeper layers of the cecal wall. The hepatic foci noted at gross examination correspond to foci of necrosis surrounded by polymorphonuclear neutrophils. Multifocal necrotic myocarditis may be seen as well. Tangled

341

masses of rod-shaped bacteria can be found in the periphery of the lesions using special stains such as the Warthin-Starry silver stain or Giemsa stain (Fig. 5) (DeLong and Manning, 1994). Clostridium piliforme spores are shed in the feces of infected animals. The disease is transmitted to susceptible animals by the ingestion of spores contaminating the environment. Initially, the organism infects the intestinal tract; there is subsequent systemic spread to other organs, such as the liver and heart. Stress may play an important role in the development of clinical disease (DeLong and Manning, 1994). Diagnosis of Tyzzer's disease can be made by demonstrating characteristic intracellular bacteria in tissue sections stained with the Warthin-Starry silver stain or Giemsa stain (DeLong and Manning, 1994). Serologic assays are also utilized to diagnose Tyzzer's disease. Both indirect immunoflourescence and enzyme-linked immunosorbent assays (ELISA) can be used to detect antibodies to C. piliforme (Fries, 1977; Waggie et al., 1987). Proper husbandry managment plays an important role in the prevention of Tyzzer's disease. Sound husbandry practices that maintain high levels of sanitation and minimize stress should help to reduce the appearance of clinical disease. Within the animal research setting, only vendors whose animals are known to be free of the disease should be used. Methods used to screen for latent carriers, such as serum antibody titers or exposure of gerbils to rabbit feces, might be utilized. Commercial vaccines against Tyzzer's disease are not available. Thorough cleansing and disinfection are necessary to decontaminate facilities in which Tyzzer's disease has occurred. Surfaces should be cleaned with either 1% peracetic acid or 0.3% sodium hypochlorite (Ganaway, 1980). Spores can withstand

Fig. 5. Focalarea of necrosis in a rabbit liverdue to Tyzzer'sdisease. Notethe thin Clostridium piliforme organisms(arrows)within the lesion. Magnification: 400x, Warthin-Starry silver stain. Bar: 1500~tm.

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MARK A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

repeated freezing and thawing but are rendered noninfectious after 30 min at 80~ Clinical experience indicates that antibiotics, specifically tetracycline or oxytetracyline, may have some value in controlling epizootics of Tyzzer's disease. However, others believe that antibiotic treatment is ineffective (DeLong and Manning, 1994). The principal research complication associated with Tyzzer's disease is death of affected rabbits. Alterations in serum enzymes as a result of liver damage could also occur.

3.

Enterotoxemia

The primary causative agent of enterotoxemia in rabbits is Clostridium spiroforme (DeLong and Manning, 1994). Involvement of other Clostridium species has also been reported. Most recently, C. difficile was isolated from rabbits (Perkins et al., 1993, 1995). Clostridium perfringens type A, and C. welchii type A have also been reported as rabbit pathogens. Most often, affected animals die acutely without clinical signs. Watery brown diarrhea and staining of the perineal region may be seen. Affected animals may exhibit anorexia, dehydration, polydipsia, depression, pyrexia or hypothermia, bloat, and grinding of teeth. Enterotoxemia can affect rabbits of all ages but is seen primarily in weanling rabbits. Both isolated cases and epizootics can occur in colonies. In older rabbits, disruption of the normal gut flora can lead to the development of enterotoxemia. The cecum may be distended with excessive gas and dark brown fluid, and there may be serosal paintbrush hemorrhages. There may be mucosal hemorrhages and ulcers in the cecum. The colon may contain mucus or gas and dark brown fluid, and may feature an extension of the serosal hemorrhages. The ileum may also be involved. The acute inflammatory exudate and pseudomembrane formation characteristic of C. difficile infections in humans have not been reported in rabbits. Young rabbits likely develop the disease because of the change in gut flora associated with weaning. However, several reports of clostridial disease in adult rabbits have been recorded. These cases may have been associated with other factors that permitted proliferation of clostridial organisms, such as antibiotic administration, coinfections with other bacteria, or other stressors (DeLong and Manning, 1994). Cases of primary clostridial infection with no obvious predisposing factors have also been reported (Perkins et al., 1995). Isolation of the causative organism is required to definitively diagnose enterotoxemia. Procedures for culturing C. spiroforme have been described (Borriello and Carmen, 1983). Holmes et al. (1988) recommended centrifugation of the intestinal contents at 20,000 g for 15 min, followed by culture of the supernatant-pellet interface. Clostridium spiroforme has a characteristic helically coiled, semicircular appearance in fecal smears or after growth in vitro. For a definitive diagnosis, the supernatant from centrifuged

cecal contents can be analyzed for presence of the iota toxin (coupled with bacterial isolation). Both in vivo and in vitro assays have been described (Patton et al., 1978; Yonushonis et al., 1987; Perkins et al., 1993, 1995). Control of enterotoxemia in rabbits should focus on preventing disruption of gastrointestinal flora through utilization of proper husbandry and veterinary practices. There are no vaccines available to prevent enterotoxemia in rabbits. Around the time of weaning, rabbits should not be overfed and should be provided with sufficient dietary fiber. Abrupt changes in feed should be avoided. Copper sulfate has been advocated as a feed additive to reduce toxin production by Clostridium (DeLong and Manning, 1994). Antibiotics should be used judiciously in rabbits as they may precipitate enterotoxemia. Parenteral administration is preferred over oral administration. Treatment for enterotoxemia should include supportive fluid therapy. Although antibiotics are often recommended, there is little evidence that they are of value in enterotoxemia (Carman and Wilkins, 1991). Oral cholestyramine (an ion exchange resin) has been proposed for treatment because it binds bacterial toxins (Lipman et al., 1992). The principal research complication associated with enterotoxemia is the death of affected rabbits.

4.

Colibacillosis

In earlier literature, the role of Escherichia coli as a causative agent of diarrhea in rabbits was unclear because E. coli often proliferates when rabbits develop diarrhea for any reason. Other studies have demonstrated that certain strains of E. coli are capable of causing disease in rabbits. Escherichia coli strain RDEC-1 (rabbit diarrhea E. coli) has satisfied Koch's postulates (Cantey and Blake, 1977). RDEC-1 is now serotyped as O15:H, one of the more virulent strains that affects weanling rabbits. Strains (many of which are in serogroup O103) expressing the eae gene are most common and are particularly pathogenic in rabbits (Blanco et al., 1996). This gene encodes intimin, an outer membrane protein required for development of attaching and effacing lesions (Agin et al., 1996). Also of importance are serotypes O109:H2, O103:H2, O15:H, O128, and O132 (DeLong and Manning, 1994). Colibacillosis typically affects 4 to 6-week-old weanlings, but 1- to 2-week-old suckling rabbits can also be affected. There are three clinical syndromes associated with colibacillosis depending on the infecting strain of bacteria: neonatal diarrhea with high mortality; weanling diarrhea with high mortality; and weanling diarrhea with low mortality. Suckling rabbits typically present with severe yellow diarrhea and high mortality. Weanling rabbits are more likely to develop profuse, watery diarrhea with dehydration, anorexia, weight loss, stunted growth, and death if the infecting strain is highly virulent. Diarrhea can be mild in weanlings infected with strains of low pathogenicity. Neonatal diarrhea with high mortality is most often associated

9. BIOLOGYAND DISEASES OF RABBITS with serotype O 109:H2; weanling diarrhea with high mortality with serotype O 103:H2 or O 15 :H; and weanling diarrhea with low mortality with serotypes O123, O128, and O132 (DeLong and Manning, 1994). The ileal, cecal, and colonic walls may be thickened and edematous, and there may be mucosal ulcerations. The cecal contents are watery and brown, and there may be serosal hemorrhages. In neonates, the entire intestinal tract may be affected and contain yellow-brown feces. Mesenteric lymph nodes may also be enlarged. Histologically, there is villus atrophy and fusion in the ileum, cecum, and colon. The epithelium is flattened and disorganized, and there is focal necrosis of the mucosal epithelium. Neutrophils are present in the lamina propria, and the submucosa is edematous. Colonies of coliforms may be found on the intestinal surface. Neutrophils and enterocytes may be present in the intestinal lumen. Attachment of coliforms to the intestinal mucosal surface and effacement of the epithelial cells lead to a loss of the microvillus border and secretory diarrhea (Okerman, 1987). Escherichia coli can be readily cultured from the feces of rabbits with diarrhea. However, definitive diagnosis requires somatic and flagellar serotyping to correlate the strain with known enteropathogenic strains. The use of proper husbandry techniques is important in controlling colibacillosis in rabbit herds. Good sanitation practices are especially important in stopping the spread of organisms. Commercial vaccines for colibacillosis are not available for rabbits. Treatment for colibacillosis consists primarily of supportive care, such as fluid and electrolyte replacement. Antibiotics such as chloramphenicol and neomycin have been used successfully (DeLong and Manning, 1994). No specific research complications other than mortality have been reported. 5.

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Fig. 6. Treponematosis.Ulcerationwith exudation and crusting on the prepuce of a rabbit.

Histopathologic examination of treponemal lesions reveals epidermal hyperkeratosis, hyperplasia, and acanthosis with ulceration. There may be an exudative crust over the ulcer. Macrophage and plasma cell infiltration is present. Spirochetes may be found in the lesion with Warthin-Starry silver stains. The regional lymph nodes may be hyperplastic. The organism penetrates the mucous membrane in order to establish infection. Clinical signs may not appear for 3 - 6 weeks after exposure, and seroconversion may not occur until 8-12 weeks after exposure. Diagnosis of treponematosis can be made by demonstrating spirochetes in the lesions. The organism has a characteristic spiral morphology. In addition, in wet mounts of scrapings from lesions examined by dark-field microscopy, the organism demonstrates corkscrew motility. Serologic diagnosis can be made using the same assays as are used to diagnose T. pallidum infection in humans because the

Treponematosis

Treponematosis in rabbits is caused by Treponema paraluis cuniculi. It is a gram-negative, spiral-shaped rod and is closely related to T. pallidum, the causative agent of human syphilis. Typical treponemal lesions occur in vulvar or preputial areas (Fig. 6) and begin with swelling and erythema, often with vesicles or papules. Lesions at other mucocutaneous junctions can also occur (Fig. 7). These lesions progress to ulceration, followed by scaling and crusting over the ulcer. The regional lymph nodes may become enlarged. The lesions are chronic in nature but may resolve after many weeks. Other names for treponematosis include venereal spirochetosis and rabbit syphilis. All lagomorphs are susceptible. Clinically apparent disease is uncommon in rabbitries, while serologic evidence of infection is common. The organism is transmitted between rabbits during breeding. Rarely, the organism is found in nonbreeding rabbits.

Fig. 7. Treponematosis.Ulcerationwithexudationand crustingon the nares of a rabbit.

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two organisms share many antigens. Several tests are available that vary in sensitivity and specificity. The microhemagglutination T. pallidum test is used as a screening test because it is easy to use and sensitive. The venereal disease research laboratory slide test (VDRL) and the rapid plasma reagin card test (RPR) are widely available. New breeding animals should not be introduced into colonies known to be free of treponematosis. If animals must be introduced, they should be quarantined, tested serologically, and examined for lesions typical of treponematosis. There are no commercial vaccines to prevent the disease in rabbits. Penicillin is effective for treatment of treponematosis. The recommended treatment consists of three injections of benzathine procaine penicillin (42,000-84,000 IU/kg) given at 7-day intervals. Lesions should heal within 2 weeks, and RPR and VDRL titers will decline within several months. Fluorescent treponemal antibody-absorption test (FTA-ABS) titers may persist for up to a year. If all animals are treated simultaneously, this regimen can eradicate infection from a colony. Rabbits infected with T. cuniculi are unsuitable for use in research on human trepanematosis because of the close relationship between the rabbit and human pathogens. Specifically, the presence of shared antigens would complicate infection and immunologic studies. 6.

Proliferative Enteropathy

Proliferative enteropathy associated with Lawsonia intracellularis infection has been described in rabbits (Hotchkiss et al., 1996; Duhamel et al., 1998). Lawsonia intracellularis is a curved, gram-negative, obligate intracellular bacterium that plays a key role in the development of proliferative bowel disease in hamsters (Stills, 1991) and pigs (McOrist et al., 1993). The organism has been associated with a fatal outbreak of proliferative enteritis in rhesus monkeys (Klein et al., 1999). In rabbits, weanlings are most commonly affected. Clinical disease is characterized by diarrhea, depression, and dehydration, which resolves within 1 to 2 weeks. Disease rarely results in death. Rabbits can be infected with L. intracellularis in the absence of clinical signs (Duhamel et al., 1998). One outbreak with high mortality was associated with dual infection with enteropathogenic E. coli and L. intracellularis (Schauer et al., 1998). Grossly, the most striking finding is thickening and corrugation of the ileum. The jejunum, cecum, and proximal colon are variably affected as well. The mesenteric lymph nodes may be enlarged in some animals. In clinically affected rabbits, the cecal contents appear watery. Microscopically, the intestinal mucosa is thickened; and crowded, elongated, and sometime branching crypts can be observed (Hotchkiss et al., 1996). Inflammation is not present in all cases; however, infiltrates consisting of plasma cells and histiocytes can be observed in some sections. Small intestinal villi are often blunted.

Lawsonia intracellularis can be found most easily within the cytoplasm of immature crypt epithelial cells of the ileum. It has not been cultured in cell-free media, but isolates from other species can be grown in cultured enterocytes (Lawson et al., 1993; Stills, 1991). Diagnosis can be made by histologic identification of rod-shaped to curved to spiral, silver-staining bacteria within the apical cytoplasm of crypt enterocytes (Hotchkiss et al., 1996). Immunohistochemistry can also be used to identify L. intracellularis organisms in crypt and villous enterocytes (Schauer et al., 1998). Alternatively, identification of specific nucleotide sequences by the polymerase chain reaction can be used to identify the organism in jejunal, ileal, or colonic tissues of infected rabbits. Treatment of ill rabbits should be based on symptoms and isolation of sick animals is advised. Severely diarrheic rabbits should be administered parenteral fluids, and supplemental heat provided to those that become hypothermic.

B. 1.

Viral diseases

Poxvirus Infections

Myxomatosis, caused by myxoma virus, has a worldwide distribution and is endemic in the brush rabbit (Sylvilagus bachmani) in the United States. Rabbits of the genus Oryctolagus are particularly susceptible and often develop a fatal disease characterized by numerous mucinous skin lesions. Histopathology shows these "myxomas" to be composed of undifferentiated stellate mesenchymal cells embedded in a matrix of mucinous material and interspersed with capillaries and inflammatory cells (DiGiacomo and Mar6, 1994). Definitive diagnosis depends on culture of the virus from infected tissues. Since the disease is spread by fleas and mosquitoes as well as by direct contact, control measures should include prevention of contact with arthropods and quarantine of infected rabbits. Vaccines have been used in Europe with some success. Rabbits of the genus Sylvilagus develop fibroma-like lesions that may be indistinguishable from those caused by rabbit fibroma virus. The two diseases have been distinguished by inoculation of fibroma material into Oryctolagus rabbits. They develop a fatal disease if the myxoma virus is the etiologic agent, or fibromas if rabbit fibroma virus is responsible. Rabbit (Shope) fibroma virus is a poxvirus that is antigenically related to myxoma virus. Fibromatosis is endemic in wild rabbits; however, an outbreak in commercial rabbits caused extensive mortality (Joiner et al., 1971). Usually, less virulent strains cause skin tumors in domestic rabbits (Ratio et al., 1973). The disease is probably spread by arthropods, although definitive evidence is lacking (DiGiacomo and Mar6, 1994). Fibromas are fiat, subcutaneous, easily movable tumors; whereas papillomas arise from the skin, are heavily keratinized, and project outward.

345

9. BIOLOGYAND DISEASESOF RABBITS Rabbit pox is a rare disease induced by a poxvirus that has caused outbreaks of fatal disease in laboratory rabbits in the United States and Holland (DiGiacomo and Mar6, 1994). Rabbits with the disease may or may not present with "pox" lesions in the skin. The animals have a fever and nasal discharge 2 or 3 days after infection. Most rabbits have eye lesions including blepharitis, conjunctivitis, and keratitis with subsequent corneal ulcers. Skin lesions, when present, are widespread. They begin as a rash and progress to papules up to 1 cm in diameter by 5 days postinfection. The lymph nodes are enlarged, the face is often edematous, and there may be lesions in the oral cavity. At gross necropsy, there are extensive nodules in many organs, and there is widespread necrosis. Characteristic cytoplasmic inclusions seen in many poxvirus infections are rare in this disease. The virus is apparently spread by aerosols and is difficult to control. 2.

Herpesvirus Infections

Two herpesviruses have been isolated from rabbit kidney cultures. These are Leporid herpesvirus 1 (Herpesvirus sylvilagus), isolated from cottontail rabbits, and Leporid herpesvirus 2 (Herpesvirus cuniculi), isolated from domestic rabbits. Neither of these isolates has been shown to cause naturally occurring disease. Experimentally, Leporid herpesvirus 1 causes a lymphoproliferative disease in inoculated cottontail rabbits (DiGiacomo and Mar6, 1994). Acute mortality was associated with an unknown herpesvirus isolated from the kidneys of 4 adult rabbits from two commercial rabbitries (Onderka et al., 1992). Experimental inoculation of rabbits with the virus reproduced the disease syndrome. The virus has not been well documented. 3.

Papillomavirus Infections

The cottontail rabbit is the natural host of the cottontail (Shope) papillomavirus, which causes horny warts primarily on the neck, shoulders, and abdomen. The disease has a wide geographic distribution with the highest incidence occurring in rabbits in the Midwest (DiGiacomo and Mar6, 1994). However, natural outbreaks in domestic rabbits have been reported (Hagen, 1966). In these natural outbreaks, papillomas were more common on the eyelids and ears. A small percentage of papillomas are transformed into squamous cell carcinoma, indicating that this virus is oncogenic. The virus has been transmitted experimentally by arthropods. Therefore, arthropod control could be used as a means to prevent the disease from being transmitted to domestic rabbits. This virus is used extensively as a model for the study of oncogenic virus biology and as a model for the induction of protective immunity against papillomaviruses (Salmon et al., 1997; Sundaram et al., 1998). Rabbit oral papillomatosis is caused by a different virus than that causing cottontail rabbit papillomatosis. Naturally occurring lesions have been seen in laboratory rabbits and appear as

small, white, discrete growths on the ventral surface of the tongue. Microscopic examination shows them to be typical papillomas. Most lesions eventually regress spontaneously (DiGiacomo and Mar6, 1994). 4.

Rotavirus Infections

A number of studies have shown that rotavirus infections in rabbits are common (DiGiacomo and Mar6, 1994). Many colonies of rabbits are serologically positive, and rotavirus can be isolated readily from rabbit feces. However, attempts to experimentally produce clinical disease have had variable results. Mild diarrhea is usually seen, but in some studies there has been significant mortality. It is probable that rotavirus is only mildly pathogenic in rabbits and may require the presence of other organisms in order to produce clinical disease. In combined experimental infections with both rotavirus and Escherichia coli, the inoculation of both organisms led to more serious clinical signs than when given alone, indicating that rotavirus may have been a more significant determinant in the manifestation of this disease (Thouless et al., 1996). These investigators also showed that older rabbits were naturally more resistant to the combined infection with rotavirus and E. coli. Very young rabbits appear to be protected from rotavirus infection by passive immunity, when present, but are quite susceptible when there is none (Schoeb et al., 1986). Rabbits of weaning age seem to be the most susceptible. This is also the time when they are most likely to be subjected to diet changes that may contribute to a change in microbial flora. 5.

Coronavirus Infections

Pleural effusion disease/infectious cardiomyopathy was diagnosed in rabbits inoculated with Treponema pallidum-infected stocks of testicular tissue. Because these treponemes could not be grown in vitro, the organism was propagated by passage in rabbits. The stocks were contaminated with a coronavirus, although it is not known whether this virus originated from rabbits or was a virus of human origin that had adapted to rabbits. With continued passage, the virus became more virulent, and significant mortality ensued. Evidence indicated that it was not transmitted by direct contact. Rabbits died due to congestive heart failure, and microscopic examination showed there was widespread necrosis of the heart muscle. It has been suggested that infection with this virus might be a model for the study of virus-induced cardiomyopathy (DiGiacomo and Mar6, 1994). Rabbit enteric coronavirus has been isolated from tissue cultures from rabbits (LaPierre et al., 1980) and has been associated with one naturally occurring outbreak of diarrhea in a barrier-maintained breeding colony (Eaton, 1984). These rabbits developed severe diarrhea, and most died within 48 hr of onset of clinical signs. Attempts to reproduce the disease led to watery diarrhea, which lasted a short time; however, none of the

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rabbits died. It is quite probable that other microorganisms or unknown environmental factors contributed to the severity of this outbreak. 6.

Calicivirus Infections

Rabbit hemorrhagic disease (RHD), determined to be caused by a calicivirus, was first reported in China in 1984 and has since spread to other parts of Asia and Europe. More recently, the virus escaped from an island near Australia and has since caused widespread deaths on the mainland (Chasey, 1997). In addition, outbreaks have recently been reported in the United States and Mexico. The incubation period may be as short as 1 or 2 days, and sudden death with no previous signs is not uncommon. Clinical signs may included lethargy, anorexia, and fever. Periportal hepatic necrosis is the only consistent microscopic lesion, and the animals die due to disseminated intravascular coagulation with deep venous thromboses. The virus had not been successfully grown in vitro; however, diagnosis can be confirmed with negative-contrast electron microscopy of liver tissue. Specific antibodies can be detected by monoclonal antibody ELISA or by hemagglutination inhibition. The agent resists drying, can be carried on fomites, and may be transmitted via respiratory and intestinal secretions (Mitro and Krauss, 1993). Any rabbit colonies with this disease should be quarantined and depopulated, and the environment thoroughly cleansed and disinfected. Another calicivirus, European brown hare virus, has caused disease in hares in several countries in Europe (DiGiacomo and Mar6, 1994). Animals present with necrotic hepatitis, hemorrhages in the trachea and lungs, and pulmonary edema. Results of experimental inoculation of domestic rabbits are mixed, with some investigators reporting a disease similar to RHD, while others failed to induce disease. The virus is similar to that of RHD, but not identical. A monoclonal antibody ELISA is available, and control measures are similar to those for RHD. 7.

Other Viral Infections

Several other viruses have been isolated from rabbit tissues, but have not been shown to produce disease. These include paramyxoviruses and bunyaviruses. Serologic titers to togaviruses and flaviviruses have also been demonstrated in rabbit antisera (DiGiacomo and Mar6, 1994).

C. 1.

P r o t o z o a l Diseases

Hepatic Coccidiosis

Hepatic coccidiosis is caused by the parasite Eimeria stiedae, which has also been referred to as Monocystis stiedae, Coccidium oviforme, and Coccidium cuniculi (Hofing and Kraus,

1994). The age of the host strongly affects parasite development and oocyst production. Four-month-old, coccidia-free rabbits experimentally infected with E. stiedae produced fewer oocysts than similarly infected 2-month-old rabbits (Gomez-Bautista et al., 1987). The clinical disease has a wide range of manifestations. Mild infections often result in no apparent disease. Most clinical signs are the result of interruption of normal hepatic function and blockage of the bile ducts. Diarrhea can occur at the terminal stages of the disease (Hofing and Kraus, 1994). Enlargement of the liver (hepatomegaly) is common. The liver normally is approximately 3.7% of the body weight, but rabbits with severe hepatic coccidiosis may have livers that contribute to greater than 20% of the body weight (Lund, 1954a). Serum bilirubin levels can rise to 305 mg/dl, increasing as soon as day 6 of infection and increasing through days 20-24 before moderating (Rose, 1959). Decreased growth rates and weight loss are common. Joyner et al. (1987) demonstrated that infested rabbits begin to lose weight within 15 days. Eimeria stiedae is found worldwide, although rabbits bred for use in research are commonly free of the parasite. Transmission occurs by the fecal-oral route, as for other coccidia. The organism has also been experimentally transmitted by intravenous, intraperitoneal, and intramuscular administration of oocysts (Pell6rdy, 1969). Necropsy often shows the liver to be enlarged and discolored, with multifocal yellowish white lesions of varying size. Exudate in the biliary tree is common, along with dilatation of bile ducts. Microscopically, papillomatous hyperplasia of the ducts along with multiple life-cycle stages of the organism in the biliary epithelium can be seen. Smetana (1933) demonstrated that infection of the entire liver occurred following ligation of the right bile duct and inoculation of E. stiedae oocysts. The study also showed that infection occurred earliest within the small intrahepatic ducts, leading to the theory that infection occurred via blood or lymph. The precise life cycle is still undetermined, although a number of studies have examined it (Rose, 1959; Horton, 1967; Owen, 1970). Sporozoites have been demonstrated in the lymph nodes following experimental inoculation (Rose, 1959; Horton, 1967). Diagnosis can be made by examination of fecal material, by either flotation or concentration methods. Oocysts can also be detected within the gallbladder exudate (Hofing and Kraus, 1994). Alternatively, oocysts can sometimes be observed by microscopic examination of impression smears of the cut surface of the liver. Control of the infection until development of natural immunity is one strategy to minimize the severity of disease. Davies et al. (1963) demonstrated that immunity occurs following a light infection with E. stiedae. In the rabbit, immunity to Eimeria may be lifelong (Pell6rdy, 1965; Niilo, 1967). Prevention of hepatic coccidiosis with sulfaquinozaline in the feed (250 ppm) was shown to prevention infection in the face of experimental

9. BIOLOGY AND DISEASES OF RABBITS challenge with 100,000 sporulated oocysts (Joyner et al., 1987). Sulfonamides have been shown effective against Eimeria spp. (Jankiewicz, 1945; Horton-Smith, 1947; Lund, 1954b; Hagen, 1958; Tsunoda et al., 1968). Development of the organism was arrested by treatment with 0.02% sulfamerazine sodium administered continually in the drinking water (Peterson, 1950). Thorough sanitation of potentially contaminated surfaces is critical to control of coccidiosis. Potential research complications arising from hepatic coccidiosis are considerable. The resulting liver damage and decreased weight gains can complicate both the supply of rabbits for research as well as adversely affect the research protocol. 2.

Intestinal Coccidiosis

There are at least eight different pathogenic species of intestinal coccidia in rabbits, including E. intestinalis, E. flavescens, E. irresidua, E. magna, E. media, E. piriformis, E. neoleporis, and E. perforans (Varga, 1982). All of these coccidia are presented here as a group rather than as individual species of intestinal coccidia. Although intestinal coccidiosis may be subclinical, symptoms can range from mild to severe and can result in death of the animal. Postweanling rabbits are the most likely to experience mortality related to intestinal coccidiosis. Clinical signs also depend on the species of coccidia that are present. Severe diarrhea, weight loss, or mild reduction in growth rate are all possibilities. Death is usually associated with severe dehydration subsequent to diarrhea (Frenkel, 1971). Intestinal coccidiosis is a common rabbit disease worldwide (Varga, 1982). Transmission is by the fecal-oral route, through ingestion of sporocysts. Unsporulated oocysts are passed in the feces and are not infective. Such oocysts will, however, sporulate to an infective stage within 3 days after shedding; thus, it is important that sanitation be frequent enough to remove infective stages from the environment. The oocyst burden of feces can be enormous. Gallazzi (1977) demonstrated that an asymptomatic carrier of intestinal coccidia had 408,000 oocysts/gm of feces and that a rabbit with diarrhea could have in excess of 700,000 oocysts/gm of feces. Environmental contamination with oocysts can be a problem when large numbers of oocysts are being excreted. Lesions are apparent in the small and large intestines. Necrotic areas of the intestinal wall appear as white foci (Pakes, 1974; Pakes and Gerrity, 1994). The location and extent of the lesions depend on the species of coccidia. The life cycles of Eimeria spp. are similar to those of other coccidia. Schizogony, gametogony, and sporogony are the three phases of this life cycle. Other sources can be consulted for greater detail on the life cycle of this protozoan (Rutherford, 1943; Davies et al., 1963; Pell6rdy, 1965). Diagnosis of intestinal coccidiosis can be made through identification of the oocysts in the feces (Pakes, 1974; Pakes

347

and Gerrity, 1994). Using polymerase chain reaction (PCR) technology, a diagnostic test has been developed to detect Eimeria spp. in the feces. The test can be used to detect as few as 30 sporulated oocysts in rabbit feces (Cere et al., 1996). A 5S ribosomal RNA species-specific probe exists for E. tenella, a common parasite of poultry; however, the test is also useful for differentiating E. tenella from other Eimeria species (Stucki et al., 1993). Since intestinal coccidiosis is most common in postweanling rabbits, prevention of the disease should focus on the preweaning period. An oral vaccination has been developed and consists of a nonpathogenic strain of E. magna. This vaccine is sprayed into the nest box when rabbits are 25 days of age. The preweanling rabbits develop immunity subsequent to infection with the nonpathogenic strain and are then resistant to wild-type strains of E. magna at 35 days of age (Drouet-Viard et al., 1997). Prevention and control of infection can be accomplished by providing 0.02% sulfamerazine or 0.05% sulfaquinoxaline in the drinking water (Kraus et al., 1984). A combination of sulfaquinoxaline, strict sanitation, and elimination of infected animals has been shown to eliminate intestinal coccidiosis from a rabbit breeding colony (Pakes and Gerrity, 1994). As for hepatic coccidiosis, sulfaquinoxaline provided in the feed (250 ppm) is an effective treatment. 3.

Cryptosporidiosis

The protozoan organism Cryptosporidium cuniculus has been found in the intestinal tract of the rabbit (Inman and Takeuchi, 1979; Rehg et al., 1979). The name is based on the assumption of host specificity (Pakes and Gerrity, 1994), although C. parvum has been shown to have a wide host range across mammalian species, including humans (Current and Garcia, 1991). Transmission is likely be ingestion of thick-walled sporulated oocysts. Clinical signs related to cryptosporidiosis have not been well described in the rabbit, although one report describes small intestinal dilatation observed during surgery in a rabbit that did not have diarrhea (Inman and Takeuchi, 1979). Histopathology of the small intestine of the reported rabbit was characterized by shortened, blunted villi and mild edema of the lamina propria. The lacteals of the ileum were also dilated. Interestingly, inflammatory response was observed. 4.

Encephalitozoonosis

The etiologic agent responsible for encephalitozoonosis is Encephalitozoon cuniculi. This agent is historically known by the name Nosema cuniculi (Pakes and Gerrity, 1994). The disease was first described in 1922 as an infectious encephalomyelitis causing motor paralysis in young rabbits (Wright and Craighead, 1922). Although named for the motor paralysis in the young rabbit, the disease is usually latent in rabbits. Other clinical signs can

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include convulsions, tremors, torticollis, paresis, and coma (Pattison et al., 1971). Routes of transmission are not known. The organism has been found in the urine of infected rabbits (Yost, 1958). Transmission has occurred by oral administration of urine from infected rabbits (Cox et al., 1979). Evidence for vertical transmission in the rabbit has been reported (Hunt et al., 1972). This case report describes 2 litters of rabbits that were delivered by cesarean section, raised in a germfree environment, and fed sterile food. At 2 months of age, 2 were sacrificed due to poor weight gain. At necropsy, typical lesions of encephalitozoonosis were seen. The kidneys commonly demonstrate lesions. Typically, there are multiple white, pinpoint areas or gray, indented areas on the renal cortical surface (Kraus et al., 1984). Microscopically, these areas are characterized by granulomatous inflammation. Interstitial infiltration of lymphocytes and plasma cells and tubular degeneration may also be present (Flatt and Jackson, 1970). Granulomatous encephalitis is a characteristic lesion (Fig. 8) (Pakes and Gerrity, 1994). Lesions of the spinal cord can also occur (Koller, 1969). The organisms are often not observed in histologic sections of the lesions. Organisms may be seen floating free in the tubules of the kidney (Pakes and Gerrity, 1994). Although the pathologic changes associated with the organism have been well described, there is little known concerning the development of the disease. The organism can be found in the tissues without an inflammatory response (Pakes and Gerrity, 1994). It has been postulated that the rupture of cells containing the organism may induce the granulomatous reaction (Koller, 1969). Encephalitozoonosis is also a newly recognized disease in immunodeficient humans. It is recommended that

such individuals seek medical counsel prior to handling rabbits. Isolates from humans have been shown to be infectious for rabbits (Mathis et al., 1997). Definitive diagnosis can be made using several different methods. Histologic examination of tissues and observation of the organism is definitive. The E n c e p h a l i t o z o o n organism does not stain well with hematoxylin and eosin, and is better demonstrated using Giemsa stain, Gram stain, or Goodpasturecarbol fuchsin stain (Pakes, 1974). Many different serologic tests exist for the organism. The indirect fluorescence antibody technique has shown good results in screening large colonies of rabbits (Cox and Gallichio, 1978). Other tests include the complement fixation test, an immunoperoxidase test (Wosu et al., 1977; Gannon, 1978), a microagglutination test (Shadduck and Geroulo, 1979), and an enzyme immunoassay (Cox et al., 1981). An indirect fluorescence antibody test has been used to identify spores in the urine and tissues. Advances in diagnostic techniques have been made in human medicine due to the susceptibility of immunosuppressed patients to this particular infection. Several PCR tests for diagnosis and species differentiation of encephalitozoonosis have been developed (Croppo et al., 1998; Franzen et al., 1998; Weiss and Vossbrinck, 1998). Although these tests have generally not been used for diagnostic purposes in rabbits, they offer a wide range of diagnostic possibilities in humans. Amplification of the organism from urine, tissue biopsies, and feces has been described (Weiss and Vossbrinck, 1998). Prevention and control of the organism in the colony are done by elimination of the organism from the colony. Because this is a latent disease in rabbits, serologic methods must be used to

Fig. 8. Granulomatousencephalitis related to infection with Encephalitozoon cuniculi. The E. cuniculi organisms are rarely seen within such lesions. Magnification: 1000x. Bar: 3750 ~tm.

9. BIOLOGYAND DISEASESOF RABBITS

349

identify carriers of the organism. The indirect fluorescence antibody test has been used successfully to identify infected rabbits (Cox, 1977). The elimination of infected rabbits must be accompanied by disinfection of the environment. Several disinfectants have been effective against this organism. Encephalitozoon was killed by 2% (v/v) Lysol, 10% (v/v) Formalin, and 70% (v/v) ethanol (Shadduck and Polley, 1978). Successful treatment in the rabbit has not been reported (Pakes and Gerrity, 1994). Albendazole has been used successfully in human cases of E. intestinalis (Weber et al., 1994; Molina et al., 1998). This drug may show some promise for treatment in rabbits; however, the majority of infections in rabbits are asymptomatic. Encephalitozoonosis is most commonly an asymptomatic disease, which makes it difficult to determine the effects it may have on research. Granulomatous reactions would obviously complicate renalphysiology and neurologic research. Depression of the IgG response and an increase in the IgM response to Brucella abortus antigens has been demonstrated in rabbits infected with Encephalitozoon organisms (Cox, 1977). Natural killer cell activity is also increased in mice infected with the organism.

D.

Arthropod and Helminth Diseases

Fig. 9. Crusty exudate from the ear of a rabbit infested with ear mites (Psoroptes cuniculi).

1. Psoroptes cuniculi (Rabbit Ear Mite) Psoroptes cuniculi is a nonburrowing mite and the causative agent of psoroptic mange, also called ear mange, ear canker, or otoacariasis. The organism is distributed worldwide. Lesions occur primarily in the inner surfaces of the external ear. The lesions are pruritic and can result in scratching, head shaking, pain, and even self-mutilation (Hofing and Kraus, 1994). A tan, crusty exudate accumulates in the ears over the lesions and can become quite extensive and thick (Fig. 9). The skin under the crust is moist and reddened. The ears may become malodorous. All stages of the mite (egg, larva, protonymph, and adult) occur on the host. Early in the infestation, mites feed on sloughed skin cells and lipids. As local inflammation increases, they ingest serum, hemoglobin, and red blood cells (DeLoach and Wright, 1981; Hofing and Kraus, 1994). The entire life cycle is complete in 21 days. Mites are relatively resistant to drying and temperature and can survive off the host for 7-20 days in a temperature range of 5~176 and relative humidity of 20-75%. Lesions are characterized histologically by chronic inflammation, hypertrophy of the Malpighian layer, parakeratosis, and epithelial sloughing. An allergic response to the mites and mite feces and saliva is likely involved (Hofing and Kraus, 1994). Mites are large enough to be seen with the unaided eye or with an otoscope. Material scraped from the inner surface of the ear can also be examined using a dissecting microscope. Mites are

oval-shaped with well-developed legs that project beyond the body margin. Adult males measure 431-547 ~tm X 322-462 ~tm, and females measure 403-749 ~tm X 351-499 ~tm (Hofing and Kraus, 1994). Several successful treatments have been reported. Prior to local treatment, the ears should be cleaned gently to remove accumulated exudate. One treatment involves the application of 3% rotenone in mineral oil (1:3) every 5 days for 30 days. Ivermectin is an effective treatment at dosages of 4 0 0 - 4 4 0 ~tg/kg SC or IM (Wright and Riner, 1985; Curtis et al., 1990; McKellar et al., 1992). One or two doses were utilized for effective treatment. Bowman et al. (1992) reported an efficacy of 99.6% in rabbits with a single dose of 200 ~tg by the SC route. It is generally recommended that the entire group of rabbits be treated at the same time. Heat (40~ and desiccation (< 20% humidity) will kill parasites that are not on the host (Arlain et al., 1984).

2. Cheyletiella spp. (C. parasitovorax, C. takahasii, C. ochotonae, C. johnsoni) Cheyletiella mites are nonburrowing skin mites of rabbits. They are distributed worldwide. Several closely related species

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MARK A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

have been reported to occur on rabbits, namely, C. parasitovorax, C. takahasii, C. ochotonae, and C. johnsoni (Hofing and Kraus, 1994). The anatomic site most commonly infested is the area over the scapulae. There may be mild hair loss in the area, and the skin may have a gray-white scale (Fig. 10) (Cloyd and Moorhead, 1976). Affected rabbits do not scratch, and there is no evidence of pruritis (Hofing and Kraus, 1994). All stages (egg, larva, pupa, and adult) in the life cycle occur on the host. Mites remain in association with the keratin layer of the skin and feed on tissue fluid (Myktowyz, 1957). Transmission is probably by direct contact. Skin lesions are mild or nonexistent. When present, lesions are characterized by mild dermatitis, hyperkeratosis, and an inflammatory cell inflitrate (Hofing and Kraus, 1994). Mites can be isolated by scraping or brushing fur in the affected areas. Samples may be cleared with 5-10% potassium hydroxide to improve viewing. Mites can be identified under a dissecting microscope. The female measures 450 X 200 gm, and the male is 320 • 160 gm. Cheyletiella mites have a large, distinctive curved claw on the palpi (Pegg, 1970). Topical acaricides are often used and are effective at controlling infestation. Alternatively, ivermectin may be used as described for Psoroptes cuniculi. Cheyletid mites can cause a transient dermatitis in humans who are in regular contact with infested animals (Cohen, 1980; Lee, 1991). For this reason, these mites are considered a zoonotic pathogen. 3. Sarcoptes scabiei

Sarcoptes scabiei is a burrowing mite and the causative agent of sarcoptic mange. Mites of the genus Sarcoptes are generally considered to be one species, S. scabiei, but are often further

identified by a variety name corresponding to the host species (e.g., S. scabiei var. cuniculi). The organisms are commonly referred to as itch or scab mites. The disease has a worldwide distribution. Notoedric mites (Notoedres cati) are similar to sarcoptic mites in morphology, life cycle, and public health significance. Mites burrow and produce an intensely pruritic dermatitis. The lesions occur primarily on the face, neck, and ears of rabbits. Affected rabbits will exhibit intense pruritis. There is often hair loss and abrasions as a result of the scratching. Serous encrustations on the skin and secondary bacterial infections are common. Lesions are most common on the head (Hofing and Kraus, 1994). Anemia and leukopenia can also be observed in affected rabbits (Arlain et al., 1988). All stages of sarcoptic mange mites occur on the host. The females burrow into the skin to lay eggs. Young larvae can also be found in the skin while older larvae, nymphs, and males reside on the skin surface. Mites feed on lymph and epithelial cells (Hofing and Kraus, 1994). Amyloidosis of the liver and glomerulus has been reported in rabbits with severe infestation (Arlain et al., 1990). Because Sarcoptes is a burrowing mite, skin scrapings are necessary to diagnose infestation. Samples may be cleared with 5-10% potassium hydroxide. Female mites measure 3 0 3 - 4 5 0 ~tm X 2 5 0 - 3 5 0 ~tm. The body shape is round, and the legs are very short. Ivermectin is effective at eliminating infestation at 100 ~tg/kg administered subcutaneously. Sarcoptes can cause a self-limiting dermatitis in humans. Transmission is by direct contact. 4.

A wide variety of arthropod parasites have been reported in wild rabbits but are extremely rare in laboratory rabbits. For an extensive listing the reader is referred to other sources (Hofing and Kraus, 1994). 5.

Fig. 10. Hair loss and white scaling in a rabbit infested with skin mites (Cheyletiella spp.). A more typical location for this lesion is on the back in the scapular region.

Other Arthropod Parasites

Oxyuriasis (Pinworm Infestation)

Pinworms are occasionally found in the cecum and colon in laboratory rabbits. Historically, the rabbit pinworm was identified as Oxyuris ambigua, but this name is synonymous with the more contemporary name, Passalurus ambiguus (Hofing and Kraus, 1994). Even when rabbits have heavy oxyurid burdens, clinical signs are not usually apparent (Erikson, 1944; Soulsby, 1968). One case report describes unsatisfactory breeding performance and poor condition in a rabbit herd infested with the parasite. Passalurus ambiguus can readily be found in wild rabbits as well as in domestic and research rabbits (Hofing and Kraus, 1994). Transmission occurs easily, given that individual rabbits have been found with over 1000 adult parasites (Hofing and

9. BIOLOGY AND DISEASES OF RABBITS

Kraus, 1994) and that embryonated eggs pass out in the feces and are immediately infective (Taffs, 1976). Mature pinworms are found in the lumen of the cecum or colon of the rabbit. After ingestion, the eggs hatch in the small intestine, and the larvae molt. Development continues, and maturation occurs in the cecum. The prepatent period is between 56 and 64 days (Taffs, 1976). Several successful treatment strategies for rabbit oxyuriasis have been reported. Piperazine citrate at 100 mg/100 ml of drinking water for 1 day was successful in eliminating infestation (Hofing and Kraus, 1994). Fenbendazole mixed in the food for 5 days was effective at several dose levels. At 12.5 ppm, 99% of adult and most immature pinworms were eliminated. At 25 and 50 ppm, fenbendazole eliminated all immature and adult pinworms (Duwell and Brech, 1981). One gram of phenothiazine in 50 gm of feed has also been used. Subcutaneous doses of ivermectin (0.4 mg/kg) were reported to be ineffective in reducing the burden of Passalurus organisms in field populations of snowshoe hares (Lepus americanus) (Sovell and Holmes, 1996).

E.

Mycotic Diseases

Fungal forms are omnipresent in the environment. Evaluations of airborne fungi in an animal facility showed that counts of viable fungus particles were, in general, low. Penicillium was the most commonly recovered type, Aspergillus fumigatus was rarely recovered, and dermatophytes were not recovered. It appeared that bedding was the principal source of these fungi and that outdoor airborne fungi did not markedly contribute to the indoor air fungi identified (Burge et al., 1979.) 1.

Superficial Mycoses

Dermatophytosis is synonymous with the more colloquial descriptive term, "ringworm." The clinical disease is common among pet rabbits but is seen infrequently in laboratory-bred and -maintained animals. This is likely the result of the higher standard of husbandry, especially disinfection, followed by most research facilities. Marginal husbandry practices, poor nutrition, environmental stressors, overcrowding, excessive heat or humidity, extremes of age, and pregnancy are all factors that might precipitate clinical disease. Clinical dermatophytosis most commonly affects the occasional individual, although epizootic outbreaks have been described (Flatt et al., 1974). Endemic dermatophytosis that spread to employees and their families has also been described (Szili and Kohalmi, 1981). It should be noted that dermatophytosis is a zoonotic disease, and affected rabbits should be handled in a manner that will minimize the exposure of personnel to the pathogen. The causative agent most commonly identified with clinical dermatophytosis is Trichophyton mentagrophytes, with Mi-

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crosporum canis being identified on occasion. In rare instances, T. rubrum or M. gypseum is isolated (Flatt et al., 1974). Transmission of the agent occurs through direct contact with affected individuals or with macroconidia and arthrospores in the environment. Fomites can be a significant source of infection, particularly objects such as hairbrushes or other equipment that might be used without proper disinfection between animals. Asymptomatic carriers are not uncommon, with one study isolating T. mentagrophytes from 36% of clinically normal rabbits (Lopez-Martinez et al., 1984) Clinical disease is characterized by patchy alopecia with crusting, especially on the head and face. Lesions are often erythematous. The disease may spread to the paws, ears, and other sites. The lesions are typically pruritic, circular, and 1-2 cm in diameter, and have a peripheral raised rim of acute inflammation and broken hairs. Similar to dermatophytosis in other species, the lesion expands radially with central healing. Hyperkeratosis and acanthosis are characteristic histologic findings, with acute and chronic inflammatory cells diffusely infiltrating the underlying dermis. Focal abscesses of the hair follicles within the perimeter of the lesion commonly occur because of secondary bacterial invasion. Special stains such as periodic acid-Schiff, Gridley fungus stain or Gomori methenaminesilver stain are required to visualize mycelia and arthrospores. Although the lesions described above are characteristic of dermatophytosis, diagnostic procedures should be performed to definitively differentiate the condition from other skin diseases such as acaritic mange, fur pulling, moist dermatitis, malnutrition, spirochetosis, seasonal molting, behavioral vice, and bacterial dermatopathy. Following a physical examination and determination of the clinical history of the animal, skin scrapings with mineral oil and 10% potassium hydroxide (KOH) should be performed. The mineral oil scraping should be examined for ectoparasites. The KOH scraping should be placed in the KOH for 3 0 - 4 0 min and then gently heated for 10 min prior to examination for mycelia or arthrospores. In either case, scrapings should be taken from the periphery of the lesion. Dermatophytosis can also be confirmed by viewing the lesion under a Wood's lamp. Some isolates of Microsporum fluoresce under Wood's lamp illumination. However, Trichophyton and some Microsporum isolates do not fluoresce, thus a negative result with the Wood's lamp does not rule out dermatophytosis. Finally, samples of hair plucked from the edge of the lesion can be cultured for dermatophytes, using special media such as dermatophyte test media (DTM) or Sabouraud's agar. A positive culture should be followed by confirmation of fungal forms on a KOH skin scraping preparation or in the hair follicles by histopathology of a biopsy. Isolation of rabbits suspected of having an active dermatophyte infection is critical, since people and other rabbits and animals are at risk if exposed. Affected rabbits can be treated with griseofulvin (25 mg/kg) by gastric intubation once daily for 14 days. Affected rabbit colonies can be effectively treated with

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medicated diets containing 0.375 gm of griseofulvin per lb of diet for 14 days (Hagen, 1969). Alternatively, affected rabbits can be treated with 1% copper sulfate applied as a dip or with a dilution of a metastabilized chlorous acid-chlorine dioxide compound applied as either a dip or a spray (Franklin et al., 1991). 2. Deep and Systemic Mycoses

Systemic and deep mycoses are rare in rabbits. Aspergillosis associated specifically with Aspergillus flavus or A. fumigatus has been reported sporadically (Flatt et al., 1974). Pulmonary aspergillosis has been described in otherwise healthy young rabbits at a rabbitry in Japan (Matsui et al., 1985). Lesions contained hyphae surrounded by eosinophilic "asteroid bodies." Isolation ofA. fumigatus from the reproductive tract of an adult female rabbit that aborted at an advanced stage of pregnancy and the associated placenta has also been reported (Boro et al., 1978). Pneumocystis carinii is a microorganism present in the lungs of many mammal species. Although the exact taxonomic classification has been debated, recent studies strongly suggest that P. carinii is a fungus (Edman et al., 1988; Stringer et al., 1992; Kwon-Chung, 1994; Calliez et al., 1996; Stringer, 1996). Ultrastructural studies of organism morphology indicate that different Pneumocystis species or subspecies may exist between rabbits, rats, and mice (Nielsen et al., 1998). It is generally a harmless microorganism in immunocompetent individuals and has been identified in clinically normal rabbits (Mata, 1959; Sheldon, 1959a; Soulez et al., 1989; Cundiff et al., 1994). Animals with a less than fully functional immune system are susceptible to more severe infections. In rabbits, respiratory dis-

ease accompanied by pulmonary lesions has been reported in young or debilitated animals (Blazek, 1960; Blazek and Pokomy, 1963; Poelma and Broeckhuizen, 1972; Soulez et al., 1989). One report involving weanlings describes recovery of most clinically affected rabbits within 2 to 3 weeks (Sheldon, 1959b). Severely affected animals have histologic lesions characterized by extensive interstitial pneumonia with infiltration of mononuclear cells.

F.

Management-Related Diseases

1. Gastric Trichobezoar (Hair Ball)

The discovery of a hair ball in a rabbit is often an incidental finding during necropsy (Fig. 11). Indeed, up to 21% of rabbits have been found to have gastric trichobezoars during routine necropsy (Leary et al., 1984). If the trichobezoar causes partial or complete blockage, clinical signs of intestinal obstruction will result. Death can occur due to prolonged anorexia and metabolic imbalances (Gillett et al., 1983). It appears that obstruction of the pylorus, and not the volume of the gastric mass, is the critical factor in determining the clinical progress of the animal (Leary et al., 1984). Gastric rupture can also result from an obstructive trichobezoar (Gillett et al., 1983). Diagnosis is often difficult because the clinical signs are nonspecific and the disease often progresses gradually. Some cases involving acute pyloric obstruction result in sudden clinical disease and rapid clinical decline of the animal. Manual palpation may indicate presence of a firm mass in the cranial abdomen. Gastric radiographs using contrast media may aid in the diagnosis, but definitive diagnosis is often made during exploratory surgery (Gillett et al., 1983). Treatment of trichobezoar is often unsuccessful. Oral administration of mineral oil at 10 ml per day has been reported (Suckow and Douglas, 1997). Alternatively, oral administration of 5-10 ml of fresh pineapple juice daily has been reported as a possible treatment modality (Harkness and Wagner, 1995). If medical treatment does not resolve the condition, a gastrotomy should be performed. Early surgical intervention is important in such cases, as other, subsequent metabolic abnormalities may quickly increase the surgical risk to the rabbit (Bergdall and Dysko, 1994). 2. Traumatic Vertebral Fracture (Broken Back)

Fig. 11. Gastric trichobezoar from a rabbit. Note the large mass of hair entwined with ingesta occupying most of the lumenal space of the stomach.

Subluxation or compression fractures of lumbar vertebrae are often secondary to struggling during restraint, particularly when the hindquarters of the rabbit are not supported (Bergdall and Dysko, 1994). The seventh lumbar vertebra (L7) or its caudal articular processes are considered the most frequent sites of fractures, with fracture occurring more commonly than dislocation (Flatt et al., 1974). Clinical signs include posterior paresis

353

9. BIOLOGYAND DISEASESOF RABBITS or paralysis, loss of sensation in the hindlimbs, urinary and/or fecal incontinence, and perineal staining. Diagnosis is made based on clinical signs, history of recent restraint, struggling or other trauma, and palpation or radiographic analysis of the vertebral column. Euthanasia of affected animals is usually warranted. Moderate cases (subluxation with spinal edema) may resolve over time. The decision to euthanatize should be based on severity of clinical signs. Supportive care includes regular expression of the urinary bladder and prevention and treatment of decubital ulcers. Corticosteroid and diuretic therapy may be effective for cases of subluxation with spinal edema (Bergdall and Dysko, 1994). 3.

Ulcerative Pododermatitis

Although the condition is often referred to as "sore hocks," the correct name is ulcerative pododermatitis. Despite the name, the condition rarely affects the hocks, but rather occurs most frequently on the plantar surface of the metatarsal and, to a lesser extent, the metacarpal regions. The condition is believed to be initiated by wire-floor housing, foot stomping, or having thin plantar fur pads. Poor sanitation may worsen the condition. Larger rabbits are more commonly affected.

G.

these, such as the Watanabe heritable hyperlipidemic (WHHL) rabbit, have been used as disease research models. Spontaneous heritable conditions in the rabbit either can involve a single gene or can be polygenic. In addition, artificially created transgenic rabbits with disease conditions have been developed; however, that process is beyond the scope of the current discussion. Several of the more common heritable diseases are discussed in greater detail below. 1. Hydrocephalus

Hydrocephalus refers to dilatation of the cerebral ventricles and is usually accompanied by an accumulation of cerebrospinal fluid within the dilated spaces (Fig. 12). Some cases of hydrocephalus in rabbits have been presumed to be related to a single autosomal recessive gene (hy/hy); however, occurrence with other abnormalities suggests that inheritance may be more complicated (Lindsey and Fox, 1994). In some cases, the condition appears to be inherited along with various ocular anomalies as an autosomal gene with incomplete dominance. In addition, hydrocephalus can occur in rabbits as a congenital condition related to hypovitaminosis A in pregnant does (Lindsey and Fox, 1994). In contrast, the condition may also be the result of an inherited underlying defect in vitamin A metabolism.

Heritable Diseases

A number of heritable and congenital anomalies occur in rabbits. About one-third of them are related to pelage types and colors, one-third to blood groups and tissue antigen types, and the remainder to anatomic variants and heritable diseases. Some of

2.

Buphthalmia (Hydrophthalmia, Congenital or Infantile Glaucoma)

Buphthalmia is inherited as an autosomal recessive trait, although penetrance is presumably incomplete since severity and

Fig. 12. Dorsalview of a rabbit with hydrocephalus,with top of the calvariumremoved. The ventricles are enlarged secondaryto abnormal accumulationof cerebrospinal fluid.

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MARK A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

the age of onset vary greatly and some b u / b u individuals do not develop buphthalmia (Hanna et al., 1962). The condition is common in New Zealand White rabbits bred for research and other purposes. Clinical signs are usually seen in individuals older than 3 - 4 months of age, but rabbits that become buphthalmic demonstrate increased intraocular pressures as early as 3 months of age (Burrows et al., 1995). Animals may be affected either unior bilaterally. Typical changes include increased corneal diameter (Fig. 13), often with a cloudy or bluish tint, corneal edema, increased corneal vascularity, and flattening of the cornea. In some cases, the cornea ulcerates and ruptures. There is also a marked reduction in semen concentration in buphthalmics, with a decrease in libido and decreased spermatogenesis in affected males (Fox et al., 1969). The condition is associated with abnormal production and removal of aqueous humor from the anterior chamber. Impaired aqueous outflow may be due to incomplete cleavage of the drainage angle with abnormal insertion of uveal tissue into the cornea (Tesluk et al., 1982). Alternatively, it may be related to deposition of fibrin in the trabecular tissue, leading to obstruction of drainage. In affected individuals, there is an absence or underdevelopment of outflow channels of the ciliary body and sclera. By 3 months of age, decreased aqueous outflow and increased intraocular pressure can be detected. As the intraocular pressure increases, the globe enlarges since the scerla is still immature. Structural changes may include widening of the angle, thickening of Descmet's membrane, atrophy of the ciliary process, and excavation of the optic disk. Specific treatment of buphthalmia has not been described for rabbits; however, affected individuals should not be used for breeding purposes.

3.

Mandibular prognathism is the most common inherited disease of domestic rabbits. The condition is inherited as an autosomal recessive trait (mp/mp) with incomplete penetrance (Fox and Crary, 1971; Huang et al., 1981; Lindsey and Fox, 1994). Normally, the lower incisors occlude with the large upper incisors, as well as with a pair of small secondary incisors that are immediately caudal to the primary maxillary incisors. The lower set of incisors typically wear against the upper set during normal biting activity, along an arc formed by biting movements of the lower incisors, while the maxillary secondary incisors wear at right angles to the mandibular incisors. The incisors wear more quickly at the posterior aspect in rabbits, partly because the enamel layer is thinner on that side. Affected rabbits have a normal dental formula. The specific abnormality associated with mandibular prognathism is that the maxilla is short relative to a mandible of normal length. Thus, although the mandible appears abnormally long, the primary defect involves the maxilla. In rabbits, the teeth (including the molars and premolars) grow continuously throughout life. The incisors, for example, grow at the rate of 2.0-2.4 mm/week. When occlusion is normal, the teeth wear against one another and in this way remain a normal length. However, when occlusion is abnormal because of conditions that include mandibular prognathia, the teeth may become greatly elongated because typical attrition of the incisors does not occur. In affected animals the lower incisors often extend anterior to the upper incisors and protrude from the mouth, while the upper primary incisors grow past the lower incisors and curl within the mouth (Fig. 14). In some instances, the upper incisors curl around dorsally and lacerate the mucosa of the hard palate. Secondary infection and abscessation may occur in such cases. Malocclusion related to mandibular prognathia may be clinically apparent as early as 2 - 3 weeks of age, but is more typically seen in older rabbits. Clinical signs may include anorexia and weight loss. If severe enough and left untreated, affected animals will starve since they cannot properly prehend and masticate food. Overgrown teeth should be trimmed every 2 - 3 weeks or more frequently if needed. Trimming is preferably performed with a dental bur to avoid cracking the tooth, which may happen more frequently if a bone or wire cutter is used. Care should be taken to avoid exposing the pulp cavity as the result of excessive trimming. Because the condition is hereditary, use of affected animals as breeding stock should be avoided. 4.

Fig. 13. A New ZealandWhite rabbit with buphthalmos.Note the enlarged globe. Buphthalmosoften occurs secondaryto glaucomain the rabbit, in which it is an autosomalrecessivetrait.

Mandibular Prognathism (Malocclusion, Walrus Teeth, Buckteeth)

Splay Leg

A number of disorders characterized clinically by complete abduction of one or more legs and the inability to assume a normal standing position are described by the term "splay leg" (Fig. 15). Young kits 3 to 4 weeks of age are most commonly af-

355

9. BIOLOGY AND DISEASES OF RABBITS

exorotation of the limb at the hip, while rabbits that are unable to compensate are clinically affected. Although the precise pathogenesis of splay leg is not entirely understood, at least some cases are ascribed to inherited disorders. Typical clinical signs are secondary to femoral endotorsion, with a shallow acetabulum but without luxation of the femur at the hip. The semitendinosus muscle of affected animals is abnormal, with smaller fibers and abnormal mitochondria. Some reports suggest that the condition is associated with inherited achondroplasia of the hip and shoulder, while others indicate that a recessively inherited anteversion of the femoral head can be involved (Lindsey and Fox, 1974, 1994). In the Dutch breed, splay leg has been associated with either a single recessive gene with incomplete penetrance or as a polygenic condition with environmental modulation (Joosten et al., 1981). It has been further speculated that some cases of splay leg are the result of teratologic malformations (Flatt et al., 1974).

Fig. 14. Dentalmalocclusion involving the incisors in a rabbit. The upper incisors often curl back into the mouth and may lacerate the hard palate. The lowerincisors can curl outwardand be fractured off, leavingno evidenceof malocclusion other than staining of the chin with saliva.

fected. Affected rabbits cannot adduct limbs and have difficulty in making normal locomotory movements. Most commonly, animals are affected in the right rear limb, although the condition may be uni- or bilateral and may affect the anterior, posterior, or all four limbs. Rabbits with splay leg may have difficulty in accessing food and water; thus, attention to adequate nutrition is required as part of a proper clinical response. The clinical signs of splay leg may be due to an overall imbalance of development of the neural, muscular, and skeletal systems. Possibly, some animals compensate with torsion and

5. Inherited Self-Mutilating Behavior Self-mutilating behavior in a Checkered cross (cross between English Spot, German Checkered Giant, and Checkered of Rhineland rabbits) is reported to occur as an inherited trait (Ig!auer et al., 1995). Autotraumatization of the feet and pads was observed. The abnormal behavior could be interrupted by administration of haloperidol.

6. Atropine Esterase Activity Although not manifested as a disease, the presence of serum atropine esterase allows rabbits to inactivate atropine when administered for therapeutic purposes (Stormont and Suzuki, 1970; Liebenberg and Linn, 1979). The enzyme also permits rabbits to consume diets containing belladonna compounds. The enzyme is controlled by the semidominant gene E s t - 2 E Three phenotypes are recognized depending on the number of genes expressed. The enzyme first appears in the serum at 1 month of age, and enzyme levels are greater in females than in males (Lindsey and Fox, 1974, 1994). The Est-2F gene is linked to genes controlling the black pigments in the coat (Sawin and Crary, 1943; Fox and van Zutphen, 1977; Forster and Hannafin, 1979).

H.

Fig. 15. Splayleg in two New Zealand White rabbits. The conditionis characterized by inabilityto adduct the limbs. The rabbits shownare affected in the hindlimbs only (left) and all limbs (right).

Neoplasia

Historically, spontaneous neoplasia in the laboratory rabbit has not been widely reported. This is probably because neoplasia in the rabbit is very uncommon before 2 years of age, and many laboratory rabbits are not maintained beyond this age. Instead, neoplasia is more common with increasing age in rabbits, as it is in most mammals (Weisbroth, 1994). However, because

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of increasing use of specific pathogen-free rabbits, the use of better feeding and husbandry practices, and the increasing tendency to maintain antibody-producing rabbits for many years, neoplasia may actually be one of the more common spontaneous diseases of laboratory rabbits. Table VIII shows the rabbit tumors observed at the University of Michigan Unit for Laboratory Animal Medicine over a period of approximately 30 years. Almost all rabbits with neoplasia were over 2 years of age. Tumors induced by viruses are discussed under viral diseases. 1.

Neoplasia of Genitourinary System and Mammary Gland

Uterine adenocarcinoma is by far the most common tumor in rabbits. Typically, the disease is present as multiple tumors and is malignant, often metastasizing to the liver, lungs, and other organs. There is evidence that inheritance plays a role in susceptibility, but parity does not. Uterine leiomyomas and leiomyosarcomas (Table VIII) (Weisbroth, 1994) are much less common. There are a few reports of vaginal squamous cell carcinomas (Weisbroth, 1994) and an ovarian hemangioma has been described (Greene and Strauss, 1949). Mammary adenocarcinomas are fairly common in older female rabbits and may occur in animals with uterine adenocarcinoma (Table VIII) (Weisbroth, 1994). Papillomas have been described, but mammary adenocarcinomas are much more important. These malignant tumors may metastasize, but the cause of death in affected rabbits is often due to uterine adenocarcinoma. Serial biopsy studies indicate that these tumors are preceded by cystic mastopathy and changes in the adrenal and pi-

tuitary glands (Greene, 1965). Another paper has described the presence of small prolactin-secreting pituitary adenomas in rabbits with mammary dysplasia (Lipman et al., 1994). Testicular tumors in the rabbit appear to be relatively uncommon. Interstitial tumors are the most common testicular tumor in the rabbit (Fig. 16). Seminomas and teratomas have also been reported (Weisbroth, 1994). Embryonal nephromas are one of the most common tumors in laboratory rabbits. These tumors are often found incidentally, occur in younger animals, and seldom cause clinical signs (Weisbroth, 1994). There has been only one report of a renal carcinoma in the rabbit (Kaufman and Quist, 1970) and one report of a leiomyoma arising in the urinary bladder (Weisbroth, 1974). 2.

Neoplasia of Hematopoietic System

Malignant lymphomas (lymphosarcomas) are relatively common in rabbits. They may occur in rabbits that are less than 2 years of age (Weisbroth, 1994), but older rabbits may also be affected (Table VIII). According to Weisbroth (1994), a tetrad of lesions is often seen. These lesions include enlarged kidneys, splenomegaly, hepatomegaly, and lymphadenopathy. Older rabbits have presented with skin nodules and eye lesions (Table VIII); however, malignant lymphomas in the rabbit are seldom leukemic. Most cases of malignant lymphoma appear to resemble the lymphoblastic subtype as seen in humans and mice. Malignant lymphoma is more prevalent in some strains of rabbits than others, and there is some evidence for a retroviral cause of lymphomas in rabbits (Weisbroth, 1994). True thymo-

Table VIII

Neoplasia in Laboratory Rabbits at the Universityof Michigan Tumor type

Number

Femalea

Male

Uterine adenocarcinoma Mammary adenocarcinoma Malignant lymphoma Basal cell tumor Uterine leiomyosarcoma Embryonal nephroma Thyroid adenoma Fibrosarcoma (foot) Neurofibrosarcoma (foot) Osteosarcoma Retroperitoneal lipoma Rhabdomyosarcoma (leg) Squamous cell carcinoma (gingiva) Testicular teratoma Interstitial cell tumor Total

23 9 3 3 2 1 1 1 1 1 1 1 1 1 1 50

23 9 2 3 2 0 1 1 1 1 1 1 0 0 0 45

N/A N/A 1 0 N/A 1 0 0 0 0 0 0 1 1 1 5

aThe population evaluated consisted primarily of females, many of which were aged adults.

Fig. 16. Interstitialcell tumor in the testis of a rabbit (top). Note the atrophy of the contralateral testis (bottom).

9. BIOLOGYAND DISEASESOF RABBITS

357

mas (containing both lymphoid and epithelial components) (Vernau et al., 1995) and plasma cell myelomas (Pascal, 1961) are rare in rabbits. One case of myeloid leukemia has been reported (Meier et al., 1972).

a thyroid adenocarcinoma (Dinges and Kovac, 1972), and one thyroid adenoma has been seen (Table VIII).

3.

A number of other case reports of single tumors are found in the literature. These include a peritoneal mesothelioma (Lichtensteiger and Leathers, 1987), an intracranial teratoma (Bishop, 1978), an ependymoma (Kinkier and Jepsen, 1979), a neurofibrosarcoma (Table VIII), two hemangiosarcomas (Pletcher and Murphy, 1984), and a malignant fibrous histiocytoma (Yamamoto and Fujishiro, 1989). There are a few very old reports of lung tumors dating to the first part of the twentieth century and cited by Weisbroth (1994).

Neoplasia of Intestinal Tract

Bile duct adenomas and carcinomas are said to be rather common tumors in rabbits. Weisbroth (1994) has speculated that they may be preceded by and associated with bile duct hyperplasia induced by infection with Eimeria stiedae. Other tumors of the intestinal tract appear to be very uncommon. These include a squamous cell carcinoma of the gingiva (Table VIII), mucoepidermoid carcinoma (thought to be derived from salivary gland tissue) (Gillett and Gunther, 1990), gastric adenocarcinomas (Weisbroth, 1994), and a pancreatic adenocarcinoma (Roudebush, 1977), as well as gastric and intestinal leiomyosarcomas (Weisbroth, 1994). 4.

Neoplasia of Skin and Subcutaneous Tissue

Basal cell tumors are reported to be rare (Weisbroth, 1994), but they may be underreported (Table VIII) (Li and Schlafer, 1992). Squamous cell carcinomas are also uncommon, and there is no apparent predilection for any particular area of the body (Weisbroth, 1994). Other cited skin-associated tumors include a trichoepithelioma (Altman et al., 1978), a sebaceous gland carcinoma (Port and Sidor, 1978), and two malignant melanomas (Hotchkiss et al., 1994). 5.

Neoplasia of Bone, Muscle, and Connective Tissue

Osteosarcomas are extremely rare in rabbits, and most have arisen in the mandible or maxilla, with only one found in a long bone (Weisbroth, 1994) (Table VIII). No primary tumors arising in cartilage have been described, although some of the reported osteosarcomas have had cartilaginous elements. One tumor of skeletal muscle, a rhabdomyosarcoma, has been seen (Table VIII). A few fibrosarcomas are cited by Weisbroth (1994) and one fibrosarcoma involving the foot has been seen (Table VIII). 6.

7.

8.

Miscellaneous Neoplasia

Neoplasia Models Derived from Rabbits

There are several tumor models in which the cells used for inoculation were originally derived from rabbit tumors. These include the v x - 2 carcinoma (Kidd and Rous, 1940), the Brown Pearce carcinoma (Brown and Pearce, 1923), and the Greene melanoma (Greene, 1958). The v x - 2 carcinoma originated in a squamous cell carcinoma in a rabbit carrying a Shope papilloma. The most common modern use of this transplantable tumor is as a model for the study of various cancer treatment modalities for metastatic tumors (Stetson et al., 1991). The Brown Pearce carcinoma arose from a tumor in a rabbit testis, but the exact tissue of origin of the tumor was never determined. The tumor was readily transplantable and caused stable metastases. Because some tumors regress, even after widespread metastases, this tumor has been used as a model for the study of tumor immunology (Weisbroth, 1994). The Greene melanoma arose in the flank organ of a hamster and could be readily transplanted to homologous hosts and some heterologous hosts, but not to the rabbit (Greene, 1958). However, lines eventually developed that could be transplanted to the rabbit. This transplantable tumor is commonly used as a model for the study of the physiology of human ocular melanomas and treatment modalities for ocular melanomas (Weisbroth, 1994).

Endocrine Gland Neoplasia

As with tumors of many other organs in the rabbit, reports of endocrine gland tumors are also uncommon. Previously, only 2 pituitary gland tumors had been described (Weisbroth, 1994); however, Lipman et al. (1994), have reported a series of 9 cases in rabbits with mammary dysplasia. Some of these tumors were microscopic. Adrenal tumors of rabbits are rarely reported (Weisbroth, 1994), and no cases of islet cell tumors were found in the literature. Chen (1986) has discussed the ectopic adrenal cortical nodules found in rabbits. There has been one report of

I. 1.

Miscellaneous Diseases

Hydrometra

Hydrometra has been described as a clinical condition of rabbits. All cases were in unmated rabbits that were used experimentally for the production of serum antibodies (Morrell, 1989; Hobbs and Parker, 1990; Bray et al., 1991). Clinical signs include abdominal distension and tachypnea. Cases are characterized by distension of the uterine horns with a transudative fluid. One case was associated with uterine torsion (Hobbs and

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MARK A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

Parker, 1990), and one case had apparently resolved with diuretic therapy, only to return later (Bray et al., 1991). 2.

Liver Lobe Torsion

Most cases of liver lobe torsion in rabbits involve the caudate lobe (Bergdall and Dysko, 1994), although one case report described torsion of the left hepatic lobe (Wilson et al., 1987). Most reported cases have been incidental findings at necropsy. In one report, a rabbit was observed to be jaundiced, anemic, and anorexic, with elevated alanine aminotransferase. Torsion of the caudate liver lobe was seen at necropsy (Fitzgerald and Fitzgerald, 1992). 3.

Urolithiasis

Calcium carbonate and triple phosphate crystals are present in the urine of normal rabbits. These crystals contribute to the cloudy consistency of the urine (Williams, 1976). Uroliths may form from these crystals under certain conditions. Urolithiasis resulting in hematuria has been described in New Zealand White rabbits (Garibaldi et al., 1987). Predisposing factors include genetics, metabolic disturbances, nutritional imbalance, decreased water consumption, and concurrent infections. Labranche and Renegar (1996) reported a case of urolithiasis with hydronephrosis in a New Zealand White rabbit. This condition must be distinguished from hematuria caused by endometrial venous aneurysm in female rabbits (Bray et al., 1992). 4.

Lumbar Hernia

Herniation of the kidney along with perinephric fat has been reported (Suckow and Grigdesby, 1993). The affected rabbit was clinically normal except for a subcutaneous mass that had passed through the body wall. The precise etiology is not known, although it was speculated that herniation might have occurred as the result of unreported trauma. 5.

Anomalous Nasolacrimal Duct Apparatus

Occlusion of the nasolacrimal duct, presumably due to accumulation of fat droplets, has been described as a putative cause of epiphora in some rabbits (Marini et al., 1996). Although the obstruction occurred at the dorsal flexure, it is not clear if this was due to congenital rather than acquired stenosis.

REFERENCES

Agin, T. S., Cantey, J. R., Boedeker, E. C., and Wolf, M. K. (1996). Characterization of eaeA gene from rabbit enteropathogenic Escherichia coli strain

RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions. FEMS Microbiol. Lett. 144, 249-258. Altman, N. H., Damaray, S. Y., and Lamborn, P. B. (1978). Trichoepithelioma in a rabbit. Vet. Pathol. 15, 671-672. Animal and Plant Health Inspection Service 41-35-054: Animal Welfare Report Fiscal Year 1997 (1997). Report of the Secretary of Agriculture to the President of the Senate and the Speaker of the House of Representatives. Arlain, L. G., Runyan, B. S., Sorlie, B. S., and Estes, S. A. (1984). Hostseeking behavior of Sarcoptes scabei. Am. Acad. Dermatol. 11, 594-598. Arlain, L. G., Ahmed, M., and Vyszenski-Moher, D. L. (1988). Effects of Sarcoptes scabei var. canis (Acari: Sarcoptidae) on blood indexes of parasitized rabbits. J. Med. Entomol. 25, 360-369. Arlain, L. G., Bruner, R. H., Stuhlman, R. A., Ahmed, M., and VyszenskiMoher, D. L. (1990). Histopathology in hosts parasitized by Sarcoptes scabei. J. Parasitol. 76, 889-894. Atkinson, J. B., Swift, L. L., and Virmani, R. (1992). Watanabe heritable hyperlipidemic rabbits. Familial hypercholesterolemia. Am. J. Pathol. 140, 749-753. Baba, N., and von Haam, E. (1972). Animal model: Spontaneous adenocarcinoma in aged rabbits. Am. J. Pathol. 68, 653-656. Barzago, M. M., Botolotti, A., Omarini, D., Aramayona, J. J., and Bonati, M. (1992). Monitoring of blood gas parameters and acid-base balance of pregnant and non-pregnant rabbits (Oryctolagus cuniculus) in routine experimental conditions. Lab. Anim. 26, 73-78. Bergdall, V. K., and Dysko, R. C. (1994). Metabolic, traumatic, mycotic, and miscellaneous diseases of rabbits. In "The Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), 2nd ed., pp. 335-353. Academic Press, Orlando, Florida. Besch-Williford, C., Matherne, C., and Wagner, J. (1985). Vitamin D toxicosis in commercially reared rabbits. Lab. Anim. Sci. 35, 528. Bishop, L. (1978). Intracranial teratoma in a domestic rabbit. Vet. Pathol. 15, 525-530. Blanco, J. E., Blanco, M., Blanco, J., Mora, A., Balaguer, L., Mourino, M., Juarez, A., and Jansen, W. H. (1996). O serogroups, biotypes, and eae genes in Escherichia coli strains isolated from diarrheic and healthy rabbits. J. Clin. Microbiol. 34, 3101-3107. Blazek, K. (1960). Die pneumocystis-pneumonie beim feldhasen. (Lepus europaeus Pallas 1778). Zentralbl. Allergy. Pathol. 101, 484-489. Blazek, K., and Pokorny, B. (1963). Pneumocystic pneumonia in the field hare (Lepus europaeus Pallas 1778). In "Progress in Protozoology" (J. Ludvik, J. Lorn, and J. Vara, eds.), pp. 485-486. Academic Press, New York. Bleeker, W. K., Mackay, A. J., Masson-PeVet, M., Bouman, L. N., and Becker, A. E. (1980). Functional and morphological organization of the rabbit sinus node. Circ. Res. 46, 11-22. Bonilla-Ruz, L. E, and Garcia-Delgado, G. A. (1993). Adherence of Pasteurella multocida to rabbit respiratory epithelial cells in vitro. Rev. Latinoam. Microbiol. 35, 361-369. Boro, B. R., Sarmah, A. K., Goswami, J. N., and Chakrabarty, A. K. (1978). A case of abortion in a rabbit due to Aspergillusfumigatus infection. Vet. Rec. 103, 287-288. Borriello, S. P., and Carmen, R. J. (1983). Association of iota-like toxin and Clostridium spiroforme with both spontaneous and antibiotic-associated diarrhea and colitis in rabbits. J. Clin. Microbiol. 17, 414-418. Bowman, D. D., Fogelson, M. L., and Carbone, L. G. (1992). Effect of ivermectin on the control of ear mites (Psoroptes cuniculi) in naturally infected rabbits. Am. J. Vet. Res. 53, 105-109. Bray, M. V., Gaertner, D. J., Brownstein, D. G., and Moody, K. D. (1991). Hydrometra in a New Zealand White rabbit. Lab. Anim. Sci. 41, 628-629. Bray, M. V., Weir, E. C., Brownstein, D. G., and Delano, M. L. (1992). Endometrial venous aneurysm in three New Zealand white rabbits. Lab. Anim. Sci. 42, 360-362. Broome, R. L., and Brooks, D. L. (1991). Efficacy of enrofloxacin in the treatment of respiratory pasteurellosis in rabbits. Lab. Anim. Sci. 41, 572-576.

9. BIOLOGY AND DISEASES OF RABBITS

Brown, W. H., and Pearce, L. (1923). Studies based on a malignant tumor of the rabbit. I. The spontaneous tumor and associated abnormalities. J. Exp. Med. 37, 601-630. Burge, H. A., Solomon, W. R., and Williams, P. (1979). Aerometric study of viable fungus spores in an animal care facility. Lab. Anim. 13, 333-338. Burns, K. E, and DeLannoy, C. W. Jr. (1966). Compendium of normal blood values of laboratory animals, with indications of variations. Toxicol. Appl. Pharmacol. 8, 429-438. Burrows, A. M., Smith, T. D., Atkinson, C. S., Mooney, M. P., Hiles, D. A., and Losken, H. W. (1995). Development of ocular hypertension in congenitally buphthalmic rabbits. Lab. Anim. Sci. 45, 443-444. Caldwell, M. B., and Walker, R. I. (1986). Campylobacter enteritis, model no. 329. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.). Fascicle 15, Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Calliez, J. C., S6guy, N., Denis, C. M., Aliouat, E. M., Mazars, E., Polonelli, L., Camus, D., and DeiCas, E. (1996). Pneumocystis carinii: An atypical fungal microorganism. J. Med. Vet. Mycol. 34, 227-239. Callikan, S., and Girard, J. (1979). Perinatal development of glucogenic enzymes in rabbit liver. Biol. Neonate 36(1-2), 78-84. Cantey, J. R., and Blake, R. K. (1977). Diarrhea due to Escherichia coli in the rabbit: A novel mechanism. J. Infect. Dis. 135, 454-462. Carman, R. J., and Wilkins, T. D. (1991). In vitro susceptibility of rabbit strains of Clostridium spiroforme to antimicrobial agents. Vet. Microbiol. 28, 391397. Cere, N., Humbert, J. E, Licois, D., Corvione, M., Afanassieff, M., and Chanteloup, N. (1996). A new approach for the identification and the diagnosis of Eimeria media parasite of the rabbit. Exp. Parasitol. 82, 132138. Chasey, D. (1997). Rabbit hemorrhagic disease: The new scourge of Oryctolagus cuniculus. Lab. Anim. 31, 33-44. Cheeke, P. R. (1987). "Rabbit Feeding and Nutrition." Academic Press, New York. Cheeke, P. R. (1994). Nutrition and nutritional diseases. In "The Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.) 2nd ed. pp. 321-333. Academic Press, Orlando, Florida. Chen, H. H. C. (1986). Ectopic adrenal cortical nodules in the mesorchium of New Zealand White rabbits. Lab. Anim. Sci. 36, 537-538. Chrisp, C. E., and Foged, N. T. ( 1991). Induction of pneumonia in rabbits by use of a purified protein toxin from Pasteurella multocida. Am. J. Vet. Res. 52, 56-61. Cizek, L. J. (1961). Relationship between food and water ingestion in the rabbit. Am. J. Physiol. 201, 557-566. Cloyd, G. C., and Moorhead, D. P. (1976). Facial alopecia in the rabbit associated with Cheyletiella parasitovorax. Lab. Anim. Sci. 26, 801-803. Code of Federal Regulations (CFR), Title 9; Parts 1, 2, and 3 (Docket 89-130), Federal Register, Vol. 54, No. 168 (August 31, 1989); and 9 CFR Part 3 (Docket 90-218), Federal Register, Vol. 56, No. 32 (February 15, 1991). Cohen, S. R. (1980). Cheyletiella dermatitis: A mite infestation of rabbit, cat, dog, and man. Arch. Dermatol. 116, 435-437. Corash, L., Rheinschmidt, M., Lieu, S., Meers, P., and Brew, E. (1988). Fluorescence-activated flow cytometry in the hematology clinical laboratory. Cytometry Suppl. 3, 60-64. Cornblath, M., and Schwartz, R. (1976). "Disorders of Carbohydrate Metabolism in Infancy," 2nd ed. Saunders, Philadelphia. Cox, J. C. (1977). Altered immune responsiveness associated with Encephalitozoon cuniculi infection in rabbits. Infect. Immun. 15, 392-395. Cox, J. C., and Gallichio, H. A. (1978). Serological and histological studies on adult rabbits with recent, naturally acquired encephalitozoonosis. Res. Vet. Sci. 24, 260-261. Cox, J. C., Hamilton, R. C., and Attwood, H. D. (1979). An investigation of the route and progression of Encephalitozoon cuniculi infection in adult rabbits. J. Protozool. 26, 260-265.

359 Cox, J. C., Horsburgh, R., and Pye, D. (1981). Simple diagnostic test for antibodies to Encephalitozoon cuniculi based on enzyme immunoassay. Lab. Anim. 15, 41-43. Croppo, G. P., Moura, H., DaSilva, A. J., Leitch, G. J., Moss, D. M., Wallace, S., Slemenda, S. B., Pieniazek, N. J., and Visvesvara, G. S. (1998). Ultrastructure, immunoflourescence, western blot, and PCR analysis of eight isolates of Encephalitozoon (Septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with AIDS. J. Clin. Microbiol. 36, 1201-1208. Cundiff, D. D., Besch-Williford, C. L., Hook, R. R., Jr., Franklin, C. L., and Riley, L. K. (1994). Characterization of cilia-associated respiratory bacillus isolates from rats and rabbits. Lab. Anim. Sci. 44, 305-312. Curiel, T. J., Perfect, J. R., and Durack, D. T.(1982). Leukocyte subpopulations in cerebrospinal fluid of normal rabbits. Lab. Anim. Sci. 32, 622-624. Current, W. L., and Garcia, L. S. (1991). Cryptosporidiosis. Clin. Microbiol. Rev. 4, 325-358. Curtis, S. K., Housley, R., and Brooks, D. L. (1990). Use of ivermectin for treatment of ear mite infestation in rabbits. J. Am. Vet. Med. Assoc. 196, 11391140. Davies, S. E M., Joyner, L. P., and Kendall, S. B. (1963). "Coccidiosis." Oliver & Boyd, Edinburgh. DeLoach, J. R., and Wright, P. C. (1981). Ingestion of rabbit erythrocytes containing 51Cr-labeled hemoglobin by Psoroptes spp. that originated on cattle, mountain sheep, or rabbits. J. Med. Entomol. 18, 345-348. DeLong, D., and Manning, P. J. (1994). Bacterial diseases. In "Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), pp. 129-170. Academic Press, Orlando, Florida. DiGiacomo, R. E, and Mar6, C. J. (1994). Viral diseases. In "Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), 2nd ed., pp. 171-204. Academic Press, Orlando, Florida. DiGiacomo, R. E, Garlinghouse, L. E., and VanHoosier, G. L. (1983). Natural history of infection with Pasteurella muItocida in rabbits. J. Am. Vet. Med. Assoc. 183, 1172-1175. DiGiacomo, R. E, Deeb, B. J., Giddens, W. E., Bernard, B. L., and Chengappa, M. M. (1989). Atrophic rhinitis in New Zealand White rabbits infected with Pasteurella multocida. Am. J. Vet. Res. 50, 1460-1465. DiGiacomo, R. E, Allen, V., and Hinton, M. H. (1991). Naturally acquired Pasteurella multocida subsp, multocida infection in a closed colony of rabbits: Characteristics of isolates. Lab. Anim. 25, 236-241. DiGiacomo, R. E, Deeb, B. J., and Anderson, R. J. (1992). Hypervitaminosis A and reproductive disorders in rabbits. Lab. Anim. Sci. 42, 250-254. Dinges, H. P., and Kovac, W. (1972). Ein metastasierendes schildrusen carcinom beim kaninchen. Z Versuchstierkunde. 14, 197-204. Drouet-Viard, E, Coudert, P., Licois, D., and Boivin, M. (1997). Vaccination against Eimeria magna coccidiosis using spray dispersion of precocious line oocysts in the nest box. Vet. Parasitol. 70, 61-66. Duhamel, G. E., Klein, E. C., Elder, R. O., and Gebhart, C. J. (1998). Subclinical proliferative enteropathy in sentinel rabbits associated with Lawsonia intracellularis. Vet. Pathol. 35, 300-303. Duwell, D., and Brech, K. (1981). Control of oxyuriasis in rabbits by fenbendazole. Lab. Anim. 15, 101-105. Eaton, P. (1984). Preliminary observations on enteritis associated with a coronavirus-like agent in rabbits. Lab. Anim. 18, 71-74. Edman, J. C., Kovacs, J. A., Masur, H., Santi, D. V., Elwood, H. J., and Sogin, M. L. (1988). Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi. Nature 334, 519-522. Erikson, A. B. (1944). Helminth infection in relation to population fluctuations in snowshoe hare. J. Wildl. Manag. 8, 134-153. Fekete, S., and Bokori, J. (1985). The effect of the fiber and protein level of the ration upon the cecotrophy of rabbits. J. Appl. Rabbit Res. 8, 68-71. Ferrando, R., Wolter, R., Vilat, J. C., and Megard, J. P. (1970). Teneur en acides amines des deux cat6gories de feces du lapin: Caecotrophes et feces dur6s. C. R. Hebd. Seances Acad. Sci., 20, 2202-2205.

360

MARK A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

Fitzgerald, A. L., and Fitzgerald, S. D. (1992). Hepatic lobe torsion in a New Zealand White rabbit. Canine Pract. 17, 16-19. Flatt, R. E., and Carpenter, A. B. (1971). Identification of crystalline material in urine of rabbits. Am. J. Vet. Res. 32, 655-658. Flatt, R. E., and Dungworth, D. L. (1971). Enzootic pneumonia in rabbits: Naturally occurring lesions in the lungs of apparently healthy young rabbits. Am. J. Vet. Res. 32, 621-626. Flatt, R. E., and Jackson, S. J. (1970). Renal nosematosis in young rabbits. Vet. Pathol. 7, 492-497. Flatt, R. E., Weisbroth, S. H., and Kraus, A. L. (1974). Metabolic, traumatic, mycotic, and miscellaneous disease of rabbits. In "The Biology of the Laboratory Rabbit" (S. H. Weisbroth, R. E. Flatt, and A. L. Kraus, eds.), 1st ed., pp. 435-453. Academic Press, Orlando, Florida. Foote, R. H., and Simkin, M. E. (1993). Use of gonadotropic releasing hormone for ovulating the rabbit model. Lab. Anim. Sci. 43, 383-385. Forster, R. P., and Hannafin, J. A. (1979). Influence of a genetically determined atropinesterase on atropine inhibition of the "smoke (dive) reflex" in rabbits. Gen. Pharmacol. 10, 41-46. Fox, R. R. (1989). The rabbit. In "The Clinical Chemistry of Laboratory Animals" (W. E Loeb and F. W. Quimby, eds.), pp. 41-46. Pergamon, New York. Fox, R. R. (1994). Taxonomy and genetics. In "Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), 2nd ed., pp. 1-26. Academic Press, Orlando, Florida. Fox, R. R., and Crary, D. D. (1971). Mandibular prognathism in the rabbit. Genetic studies. J. Hered. 62, 23-27. Fox, R. R., and van Zutphen, L. F. (1977). Strain differences in the prealbumin serum esterases of JAX rabbits. J. Hered. 68, 227-230. Fox, R. R., Crary, D. D., Babino, E. J. Jr., and Sheppard, L. B. (1969). Buphthalmia in the rabbit. Pleiotropic effects of the (bu) gene and a possible explanation of mode of gene action. J. Hered. 60, 206-212. Franklin, C. L., Gibson, S. V., Caffrey, C. J., Wagner, J. E., and Steffen, E. K. (1991). Treatment of Trichophyton mentagrophytes infection in rabbits. J. Am. Vet. Med. Assoc. 198, 1625-1627. Franzen, C., Muller, A., Hartmann, P., Hegener, P., Schrappe, M., Diehl, V., Fatkenheuer, G., and Salzberger, B. (1998). Polymerase chain reaction for diagnosis and species differentiation of microsporidia. Folia Parasitol. (Praha) 45, 140-148. Frenkel, J. K. (1971). Protozoal diseases of laboratory animals. In "Pathology of Protozoal and Helminthic Diseases" (R. A. Marcial-Rojas, ed.), pp. 318369. Williams & Wilkins, Baltimore. Fries, A. S. (1977). Studies on Tyzzer's disease: Isolation and propagation of Bacillus piliformis. Lab. Anim. Sci. 11, 75-78. Fries, A. S. (1979). Studies on Tyzzer's disease: A long term study on the humoral antibody response in mice, rats, and rabbits. Lab. Anim. 13, 37-41. Gallazzi, D. (1977). Cyclical variations in the excretion of intestinal coccidial oocysts in the rabbit. Folia Vet. Lat. 7, 371-380. Ganaway, J. R. (1980). Effect of heat and selected chemical disinfectants upon the infectivity of spores of Bacillus piliformis (Tyzzer's disease). Lab. Anim. Sci. 30, 192-196. Gannon, J. (1978). The immunoperoxidase test diagnosis of Encephalitozoon cuniculi in rabbits. Lab. Anim. 12, 125-127. Garibaldi, B. A., Fox, J. G., Otto, G., Murphy, J. C., and Pecquet-Goad, M. E. (1987). Hematuria in rabbits. Lab. Anim. Sci. 37, 769-772. Gelineo, S. (1964). Organ systems in adaptation: The temperature regulating system. In "Handbook of Physiology" (D. B. Dill, E. F. Adolph, and C. G. Wilber, eds.), Sect. 4, pp. 259-282. Am. Physiol. Soc., Washington, D.C. Gillett, C. S. (1994). Selected drug dosages and clinical reference data. In "The Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and D. E. Newcomer, eds.), 2nd ed., pp. 467-472. Academic Press, Orlando, Florida. Gillett, C., and Gunther, R. (1990). Mandibular mucoepidermoid carcinoma in a rabbit. Lab. Anim. Sci. 40, 422-423. Gillett, N. A., Brooks, D. L., and Tillman, P. C. (1983). Medical and surgical

management of gastric obstruction from a hairball in the rabbit. J. Am. Vet. Med. Assoc. 183, 1176-1178. Glass, L. S., and Beasley, J. N. (1989). Infection with and antibody response to Pasteurella multocida and Bordetella bronchiseptica in immature rabbits. Lab. Anita. Sci. 39, 406-410. Glavits, R., and Magyar, T. (1990). The pathology of experimental respiratory infection with Pasteurella multocida and Bordetella bronchiseptica in rabbits. Acta Vet. Hung. 38, 211-215. Glorioso, J. C., Jones, G. W., Rush, H. G., Pentler, L. J., DaRif, C. A., and Coward, J. E. (1982). Adhesion of type A Pasteurella multocida to rabbit pharyngeal cells and its possible role in rabbit respiratory tract infections. Infect. Immun. 35, 1103-1109. Gomez-Bautista, M., Rojo-Vazquez, F. A., and Alunda, J. M. (1987). The effect of host's age on the pathology of Eimeria stiedae infection in rabbits. Vet. Parasitol. 24, 47-57. Greene, H. S. N., and Strauss, J. S. (1949). Multiple primary tumors in the rabbit. Cancer Res. 2, 673-691. Greene, H. S. N. (1958). A spontaneous melanoma in the hamster with a propensity for amelanotic alteration and sarcomatous transformation during transplantation. Cancer Res. 18, 422-425. Greene, H. S. N. (1965). Diseases of the rabbit. In "The Pathology of Laboratory Animals" (W. W. Ribelin and J. R. McCoy, eds.), pp. 340-348. Thomas, Springfield, Illinois. Griffin, C. A. (1952). Respiratory infection among rabbits. Proc. Anita. Care Panel 3, 3-13. "Guide for the Care and Use of Laboratory Animals" (1996). Committee to Revise the Guide for the Care and Use of Laboratory Animals. National Research Council, National Academy Press, Washington, D.C. Haenichen, T., and Stavrou, D. (1980). Nephroblastoma, model no. 193. In "Handbook: Animal Models of Human Disease" (C. C. Capen, D. B. Hackel, T. C. Jones, and G. Migaki, eds.), Fascicle 9. Registry of Comparative Pathology, Armed Forces Institute of Pathology. Washington, D.C. Ha.fez, E. S. E. (1970). Rabbits. In "Reproduction and Breeding Techniques for Laboratory Animals" (E. S. E. Hafez, ed.), Chap. 16. Lea & Febiger, Philadelphia. Hagen, K. W., (1958). The effects of continuous sulfaquinoxaline feeding on rabbit mortality. Amer. J. Vet. Res. 19, 494-496. Hagen, K. W. (1966). Spontaneous papillomatosis in domestic rabbits. Bull. Wildl. Dis. Assoc. 2, 108-110. Hagen, K. W. (1969). Ringworm in domestic rabbits: Oral treatment with griseofulvin. Lab. Anim. Care 19, 635-638. Hanna, B. L., Sawin, P. B., and Sheppard, L. B. (1962). Recessive buphthalmos in the rabbit. Genetics 47, 519-529. Harkness, J. E., and Wagner, J. E. (1995). "The Biology and Medicine of Rabbits and Rodents." Williams & Wilkins, Baltimore. Hewitt, C. D., Innes, D. J., Savory, J., and Willis, M. R. (1989). Normal biochemical and hematological values in New Zealand White rabbits. Clin. Chem. 35, 1777-1779. Hobbs, B. A., and Parker, R. F. (1990). Uterine torsion associated with either hydrometra or endometritis in two rabbits. Lab. Anim. Sci. 40, 535-536. Hoffman, B. E (1965). Atrioventricular conduction in mammalian hearts. Ann. N.Y. Acad. Sci. 127, 105-112. Hofing, G. L., and Kraus, A. L. (1994). Arthropod and helminth parasites. In "Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), 2nd ed., pp. 335-353. Academic Press, Orlando, Florida. Holmes, H. T., Sonn, R. J., and Patton, N. M. (1988). Isolation of Clostridium spiroforme from rabbits. Lab. Anim. Sci. 38, 167-168. Hornicke, H. (1977). Coecotrophy in rabbits--A circadian function. J. Mammal. 58, 240-242. Horton, R. J. (1967). The route of migration of Eimeria stiedae (Lindemann, 1865). Sporozoites between the duodenum and bile duct of the rabbit. Parasitology 57, 9-17.

9. BIOLOGY AND DISEASES OF RABBITS

Horton-Smith, C. (1947). The treatment of hepatic coccidiosis in rabbits. Vet. J. 103, 207-213. Hotchkiss, C. E., Norden, H., Collins, B. R., and Ginn, P. E. (1994). Malignant melanomas in two rabbits. Lab. Anim. Sci. 44, 377-379. Hotchkiss, C. E., Shames, B., Perkins, S. E., and Fox, J. G. (1996). Proliferative enteropathy of rabbits: The intracellular Campylobacter-like organism is closely related to Lawsonia intracellularis. Lab. Anim. Sci. 46, 623-627. Huang, C. M., Mi, M. P., and Vogt, D. W. (1981). Mandibular prognathism in the rabbit: Discrimination between single-locus and multifactorial models of inheritance. J. Hered. 72, 296-298. Huls, W. L., Brooks, D. L., and Bean-Knudsen, D. (1991). Response of adult New Zealand White rabbits to enrichment objects and paired housing. Lab. Anim. Sci. 41, 609-612. Hunt, R. D., King, N. W., and Foster, H. L. (1972). Encephalitozoonosis: Evidence for vertical transmission. J. Infect. Dis. 126, 212-214. Hurvitz, A. I. (1975). Monoclonal gammopathies, model no. 57. In "Handbook: Animal Models of Human Disease" (C. C. Capen, D. B. Hackel, T. C. Jones, and G. Migaki, eds.), Fascicle 4. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Iglauer, E, Beig, C., Dimigen, J., Gerold, S., Gocht, A., Seeburg, A., Steier, S., and Willmann, E (1995). Hereditary compulsive self-mutilating behaviour in laboratory rabbits. Lab. Anim. 29, 385-393. Inman, L. R., and Takeuchi, A. (1979). Spontaneous cryptosporidiosis in an adult female rabbit. Vet. Pathol. 16, 89-95. Jain, N. C. (1986). "Schalm's Veterinary Hematology," 4th ed. Lea & Febiger, Philadelphia. Jankiewicz, H. A. (1945). Liver coccidiosis prevented by sulfasuxidine. J. Parasitol. Suppl. 13, 15. Jayo, J. M., Schwenke, D. C., and Clarkson, T. B. (1994). Atherosclerotic research. In "The Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), 2nd ed., pp. 367-380. Academic Press, Orlando, Florida. Jilge, B. (1991). The rabbit: A diurnal or nocturnal animal? J. Exp. Anim. Sci. 34, 170-175. Johnson, J. A., and Wolf, A. M. (1993). Ovarian abscesses and pyometra in a domestic rabbit. J. Am. Vet. Med. Assoc. 203, 667-669. Joiner, G. N., Jardine, J. H., and Gleiser, C. A. (1971). An epizootic of Shope fibromatosis in a commercial rabbitry. J. Am. Vet. Med. Assoc. 159, 15831587. Jones, T. C., and Hunt, R. D. (1983). "Veterinary Pathology," 5th ed. Lea & Febiger, Philadelphia. Joosten, H. E P., Wartz, P., and Verbeek, H. O. E (1981). Splayleg: A spontaneous limb defect in rabbits. Genetics, gross anatomy, and microscopy. Teratology 24, 87-104. Joyner, L. P., Catchpole, J., and Berrett, S. (1987). Eimeria stiedae in rabbits: The demonstration of responses to chemotherapy. Res. Vet. Sci. 34, 64-67. Kabata, J., Gratwohl, A., Tichelli, A., John, L., and Speck, B. (1991). Hematologic values of New Zealand White rabbits determined by automated flow cytometry. Lab. Anim. Sci. 41, 613-617. Kardon, M. B., Peterson, D. E, and Bishop, V. S. (1974). Beat-to-beat regulation of heart rate by afferent stimulation of the aortic nerve. Am. J. Physiol. 227, 598-600. Karemaker, J. M., Borst, C., and Schreurs, A. W. (1980). Implantable stimulating electrode for baroreceptor afferent nerves in rabbits. Am. J. Physiol. 239, H706-H709. Kaufman, A. E, and Quist, K. D. (1970). Spontaneous renal carcinoma in a New Zealand White rabbit. Lab. Anim. Care 20, 530-532. Kawamoto, E., Sawada, T., Sato, T., Suzuki, K., and Maruyama, T. (1994). Comparison of indirect haemagglutination test, gel-diffusion precipitin test, and enzyme-linked immunosorbent assay for detection of serum antibodies to Pasteurella multocida in naturally and experimentally infected rabbits. Lab. Anim. Sci. 28, 19-25. Kennelly, J. J., and Foote, R. H. (1965). Superovulatory response of pre- and

361 post-pubertal rabbits to commercially available gonadotropins. J. Reprod. Fertil. 9, 177-183. Kidd, J. G., and Rous, P. (1940). A transplantable rabbit carcinoma originating in a virus-induced papilloma and containing the virus in masked or altered form. J. Exp. Med. 71, 813-838. Kinkier, R. J., and Jepsen, P. L. (1979). Ependymoma in a rabbit. Lab. Anim. Sci. 29, 255-256. Kita, T., Brown, M. S., Watane, Y., and Goldstein, J. L. (1981). Deficiency of low density lipoprotein receptors in liver and adrenal gland of the WHHL rabbit, an animal model of familial hypercholesterolemia. Proc. Natl. Acad. Sci. U.S.A. 78, 2268-2272. Klein, E. C., Gebhart, C. J., and Duhamel, G. E. (1999). Fatal outbreaks of proliferative enteritis caused by Lawsonia intracellularis in young colonyraised rhesus macaques. J. Med. Primatol. 28, 11-18. Kluger, M. J., Gonzalez, R. R., and Hardy, J. D. (1972). Peripheral thermal sensitivity in the rabbit. Am. J. Physiol. 222, 1031-1034. Koller, L. D. (1969). Spontaneous Nosema cuniculi infection in laboratory rabbits. J. Am. Vet. Med. Assoc. 155, 1108-1114. Koller, L. D. (1979). Methylmercury toxicity, model no. 158. In "Handbook: Animal Models of Human Disease" (T. C. Jones, D. B. Hackel, and G. Migaki, eds.), Fascicle 8. Registl:y of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Kozma, C., Macklin, W., Cummins, L. M., abd Nauer, R. (1974). The anatomy, physiology, and biochemistry of the rabbit. In "The Biology of the Laboratory Rabbit" (S. H. Weisbroth, R. E. Flatt, and A. L. Kraus, eds.), pp. 5 0 72. Academic Press, New York. Kraus, A. L., Weisbroth, S. H., Flatt, R. E., and Brewer, N. (1984). Biology and diseases of rabbits. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and F. M. Loew, eds.), pp. 207-240. Academic Press, Orlando, Florida. Kwon-Chung, K. J. (1994). Phylogenetic spectrum of fungi that are pathogenic to humans. Clin. Infect. Dis. 19(Suppl.), S 1-$7. Labranche, G. S., and Renegar, K. B. (1996). Urinary calculus and hydronephrosis in a New Zealand White rabbit. Contemp. Topics Lab. Anim. Sci. 35, 71-73. LaPierre, J., Marsolais, G., Pilon, P., and Descoteaux, J. P. (1980). Preliminary report on the observation of a coronavirus in the intestine of the laboratory rabbit. Can. J. Microbiol. 26, 1204-1208. LaVille, A., Turner, P. R., Pittilo, R. M., Martini, S., Marenah, C. B., Rowles, P. M., Morris, G., Thomson, G. A., Woolf, N., and Lewis, B. (1987). Hereditary hyperlipidemia in the rabbit due to overproduction of lipoproteins, I. Biochemical studies. Arteriosclerosis 7, 105-112. Lawson, G. H. K., McOrist, S., Jasni, S., and Mackie, R. A. (1993). Intracellular bacteria of porcine proliferative enteropathy: Cultivation and maintenance in vitro. J. Clin. Microbiol. 31, 1136-1142. Leary, S. J., Manning, P. J., and Anderson, L. C. (1984). Experimental and naturally-occuring gastric foreign bodies in laboratory rabbits. Lab. Anim. Sci. 34, 58-61. Lee, B. W. (1991). Cheyletiella dermatitis: A report of fourteen cases. Cutis 47, 111-114. Li, X., and Schlafer, D. H. (1992). A spontaneous skin basal cell tumor in a black French minilop rabbit. Lab. Anim. Sci. 42, 94-95. Lichtensteiger, C. A., and Leathers, C. W. (1987). Peritoneal mesothelioma in the rabbit. Vet. Pathol. 24, 464-466. Lidena, J., and Trautschold, I. (1986). Catalytic enzyme activity concentration in plasma of man, sheep, dog, cat, rabbit, guinea pig, rat, and mouse. J. Clin. Chem. Clin. Biochem. 24, 11-18. Liebenberg, S. P., and Linn, J. M. (1980). Seasonal and sexual influence on rabbit atropinesterase. Lab. Anim. 14, 297-300. Lindsey, J. R., and Fox, R. R. (1974). Inherited diseases and variations. In "Biology of the Laboratory Rabbit" (S. H. Weisbroth, R. E. Flatt, and A. L. Kraus, eds.), 1st ed., pp. 377-401. Academic Press, Orlando, Florida. Lindsey, J. R., and Fox, R. R. (1994). Inherited diseases and variations. In

362

MARK A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

"Biology of the Laboratory Rabbit" (E J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), 1st ed., pp. 293-319. Academic Press, Orlando, Florida. Linn, J. M., and Liebenberg, S. P. (1979). In vivo detection of rabbit atropinesterase. Lab. Anim. Sci. 29, 335-337. Lipman, N. S., Weischedel, A. K., Connors, M. J., Olsen, D. A., and Taylor, N. S. (1992). Utilization of cholestyramine resin as a preventative treatment for antibiotic (clindamycin) induced enterotoxemia in the rabbit. Lab. Anim. 26, 1-8. Lipman, N. S., Zhao, Z. B., Andrutis, K. A., Hurley, R. J., Fox, J. G., and White, H. J. (1994). Prolactin-secreting pituitary adenomas with mammary dysplasia in New Zealand White rabbits. Lab. Anim. Sci. 44, 114-120. Loeb, W. E, and Quimby, E W. (1989). Appendices. In "The Clinical Chemistry of Laboratory Animals" (W. E Loeb and E W. Quimby, eds.), pp. 419-509. Pergamon, New York. Lopez-Martinez, R., Mier, T., and Quirarte, M. (1984). Dermatophytes isolated from laboratory animals. Mycopathologia 88, 111-113. Love, J. A. (1994). Group housing: Meeting the physical and social needs of the laboratory rabbit. Lab. Anim. Sci. 44, 5-11. Lu, Y. S., Afendis, S. J., and Pakes, S. P. (1988). Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits. Infect. Immun. 56, 1532-1537. Lu, Y. S., Aguila, H. N., Lai, W. C., and Pakes, S. P. (1991). Antibodies to outer membrane proteins but not to lipopolysaccharide inhibit pulmonary proliferation of Pasteurella multocida in mice. Infect. Immun. 59, 1470-1475. Lukas, V. S., Ringler, D. H., Chrisp, C. E., and Rush, H. G. (1987). An enzymelinked immunosorbent assay to detect serum IgG to Pasteurella multocida in naturally and experimentally infected rabbits. Lab. Anim. Sci. 37, 60-64. Lund, E. E. (1954a). Estimating relative pollution of the environment with oocysts of Eimeria stiedae. J. Parasitol. 40, 663-667. Lund, E. E. (1954b). The effect of sulfaquinoxaline on the course of Eimeria stiedae infection in the domestic rabbit. Exp. Parasitol. 3, 497-503. Mangel, A., Fahim, M., and van Breemen, C. (1981). Rhythmic contractile activity of the in vivo rabbit aorta. Nature 289, 892-894. Manning, P. J., DiGiacomo, R. E, and DeLong, D. (1989). Pasteurellosis in laboratory animals. In "Pasteurella and Pasteurellosis" (C. Adlam and J. M. Rutter, eds.), pp. 263-302. Academic Press, Orlando, Florida. Marini, R. P., Foltz, C. J., Kersten, D., Batchelder, M., Kaser, W., and Li, X. (1996). Microbiologic, radiographic, and anatomic study of the nasolacrimal duct apparatus in the rabbit (Oryctolagus cuniculus). Lab. Anim. Sci. 46, 656-662. Mata, A. D. (1959). Latent infection by Pneumocystis carinii in rabbits in Mexico City. Rev. Inst. Salubr. Enferm. Trop. Mexico City 19, 357-360. Mathis, A., Michel, M., Kuster, H., Muller, C., Weber, R., and Deplazes, P. (1997). Two Encephalitozoon cuniculi strains of human origin are infectious to rabbits. Parasitology 114, 29-35. Matsui, T., Taguchi-Ochi, S., Takano, M., Kuroda, S., Taniyama, H., and Ono, T. (1985). Pulmonary aspergillosis in apparently healthy young rabbits. Vet. Pathol. 22, 200-205. McDowell, L. R. (1989). "Vitamins in Animal Nutrition." Academic Press, San Diego. McKay, S. G., Morck, D. W., Merrill, J. K., Olson, M. E., Chan, S. C., and Pap, K. M. (1996). Use of tilmicosin for treatment of pasteurellosis in rabbits. Am. J. Vet. Res. 57, 1180-1184. McKellar, Q. A. (1992). Clinical and pharmacological properties of ivermectin in rabbits and guinea pigs. Vet. Rec. 25, 71-73. McOrist, S., Jasni, S., Mackie, R. A., Maclntyre, N., Neef, N., and Lawson, G. H. K. (1993). Reproduction of porcine proliferative enteropathy with pure cultures of ileal symbiont intracellularis. Infect. Immun. 61, 4286-4292. Meier, H., Fox, R. R., and Cary, D. D. (1972). Myeloid leukemia in the rabbit. Cancer Res. 32, 1785-1787. Mitro, S., and Krauss, H. (1993). Rabbit hemorrhagic disease: A review with special reference to its epizootiology. Eur. J. Epidemiol. 9, 70-78. Mitruka, B. M., and Rawnsley, H. M. (1981). "Clinical Biochemical and Hema-

tological Reference Values in Normal Experimental Animals and Normal Humans," 2nd ed. Masson, New York. Molina, J. M., Chastang, C., Goguel, J., Michiels, J. E, Sarfati, C., DesportesLivage, I., Horton, J., Derouin, E, and Modai, J. (1998). Albendazole for treatment and prophylaxis of microsporidiosis due to Encephalitozoon intestinalis in patients with AIDS: A randomized double-blind controlled trial. J. Infect. Dis. 177, 1373-1377. Mondino, B. J., and Phinney, R. (1989). Staphylococcal blepharitis, model no. 361. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.), Fascicle 17. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D. C. Morrell, J. M. (1989). Hydrometra in the rabbit. Vet. Rec. J. Br. Vet. Assoc. London: The Association. 125, 325. Mutze, G., Cooke, B., and Alexander, P. (1998). The initial impact of rabbit hemorrhagic disease on European rabbit populations in South Australia. J. Wildl. Dis. 34, 221-227. Myktowycz, R. (1957). Ectoparasites of the wild rabbit, Oryctolagus cuniculus (L.) in Australia. CSIRO Wildl. Res. 2, 1-4. Newberne, P. M. (1974). Vitamin A deficiency hydrocephaly, model no. 38. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.), Fascicle 3. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Nielsen, J. N., and Carlton, W. W. (1995). Colobomatous microphthalmos in a New Zealand White rabbit, arising from a colony with suspected vitamin E deficiency. Lab. Anim. Sci. 45, 320-322. Nielsen, M. H., Settnes, O. P., Aliouat, E. M., Cailliez, J. C., and Dei-Cas, E. (1998). Different ultrastructural morphology of Pneumocystis carinii derived from mice, rats, and rabbits. APMIS 106, 771-779. Niilo, L. (1967). Acquired resistance to reinfection of rabbits with Eimeria magna. Can. Vet. J. 8, 201-208. Noguchi, T., and Yamashita, Y. (1987). The rabbit differs from other mammalian species in the tissue distribution of alkaline phosphatase isoenzymes. Biochem. Biophys. Res. Commun. 143, 15-19. Nowak, R. M., and Paradiso, J. L. (1983). "Walker's Mammals of the World," 4th ed., pp. 473-492. Johns Hopkins Univ. Press, Baltimore. Okerman, L. (1987). Enteric infections caused by non-enterotoxigenic Escherichia coli in animals: Occurrence and pathogenicity mechanisms. A review. Vet. MicrobioI. 14, 33-46. Okerman, L., Devriese, L. A., Gevaert, D., Uyttebroek, E., and Haesebrouck, E (1990). In vivo activity of orally administered antibiotics and chemotherapeutics against acute septicaemic pasteurellosis in rabbits. Lab. Anim. 24, 341-344. Onderka, D. K., Papp-Vid, G., and Perry, A. W. (1992). Fatal herpesvirus infection in commercial rabbits. Can. Vet. J. 33, 539-543. Owen, D. (1970). Life cycle of Eimeria stiedae. Nature 227, 304. Pakes, S. P. (1974). Protozoal diseases. In "The Biology of the Laboratory Rabbit" (S. H. Weisbroth, R. E. Flatt, and A. L. Kraus, eds.), 1st ed., pp. 263286. Academic Press, Orlando, Florida. Pakes, S. P., and Gerrity, L. W. (1994). Protozoal diseases. In "Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), 2nd ed., pp. 205-230. Academic Press, Orlando, Florida. Pascal, R. R. (1961). Plasma cell myeloma in the brain of a rabbit. Cornell Vet. 51,528-535. Pattison, M., Clegg, E G., and Duncan, A. L. (1971). An outbreak of encephalomyelitis in broiler rabbits caused by Nosema cuniculi. Vet. Rec. 88, 404-405. Patton, N. M., Holmes, H. T., Riggs, R. J., and Cheeke, P. R. (1978). Enterotoxemia in rabbits. Lab. Anim. Sci. 28, 536-540. Pegg, E. J. (1970). Three ectoparasites of veterinary interest communicable to man. Med. Biol. Illus. 20, 106-110. Pell6rdy, L. (1965). "Coccidia and Coccidiosis." Hungarian Academy of Science, Budapest. Pell6rdy, L. (1969). Parenteral infection experiments with Eimeria stiedae. Acta Vet. Acad. Sci. Hung. 19, 171-182.

9. BIOLOGY AND DISEASES OF RABBITS

Perfect, J. R. (1985). Cryptococcal meningitis, model no. 318. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.), Fascicle 14. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Perkins, S. E., Fox, J. G., Taylor, N. S., Green, D. L., and Lipman, N. S. (1993). Clostridium dificile enterotoxemia in specific pathogen-free rabbits. Contemp. Topics Lab. Anim. Sci. 32, 20 (abstract). Perkins, S. E., Fox, J. G., Taylor, N. S., Green, D. L., and Lipman, N. S. (1995). Detection of Clostridium difficile toxins from the small intestine and cecum from rabbits with naturally acquired enterotoxemia. Lab. Anim. Sci. 45, 379-384. Peterson, E. H. (1950). The prophylaxis and therapy of hepatic coccidiosis in the rabbit by the administration of sulfonamides. Vet. Med. 45, 170-172. Peterson, R. R., Deeb, B. J., and DiGiacomo, R. E (1997). Detection of antibodies to Pasteurella multocida by capture enzyme immunoassay using a monoclonal antibody against P37 antigen. J. Clin. Microbiol. 35, 208-212. Pletcher, J. M., and Murphy, J. C. (1984). Spontaneous malignant hemangiosarcomas in two rabbits. Vet. Pathol. 21, 542-544. Podberscek, A. L., B lackshaw, J. K., and Beattie, A. W. (1991). The behaviour of group penned and individually caged laboratory rabbits. Appl. Anim. Behav. Sci. 28, 353-359. Poelma, E G., and Broeckhuizen, S. (1972). Pneumocystis carinii in hares, Lepus europaeus Pallas, in the Netherlands. J. Parasitenkd. 40, 195-202. Port, C. D., and Sidor, M. A. (1978). A sebaceous gland carcinoma in a rabbit. Lab. Anim. Sci. 28, 215-216. Rabin, B. S. (1980). Animal model: Immunologic model of inflammatory bowel disease. Am. J. Pathol. 99, 253-256. Raflo, C. P., Olsen, R. G., Pakes, S. P., and Webster, W. S. (1973). Characterization of a fibroma virus isolated from naturally-occurring skin tumors in domestic rabbits. Lab. Anim. Sci. 23, 525-532. Rehg, J. E., Lawton, G. W., and Pakes, S. P. (1979). Cryptosporidium cuniculus in the rabbit (Oryctolagus cuniculus). Lab. Anim. Sci. 29, 656-660. Richardson, M., Fletch, A., Delaney, K., DeReske, M., Wilcox, L. H., and Kinlough-Rathbone, R. L. (1997). Increased expression of vascular cell adhesion molecule-1 by the aortic endothelium of rabbits with Pasteurella multocida pneumonia. Lab. Anim. Sci. 47, 27-35. Richmond, C. R., Langham, W. H., and Trujillo, T. T. (1962). Comparative metabolism of tritiated water by mammals. J. Cell. Comp. Physiol. 59, 45-53. Ringler, D. H., and Abrams, G. D. (1970). Nutritional muscular dystrophy and neonatal mortality in a rabbit breeding colony. J. Am. Vet. Med. Assoc. 157, 1928-1934. Ringler, D. H., and Abrams, G. D. (1971). Laboratory diagnosis of vitamin E deficiency in rabbits fed a faulty commercial ration. Lab. Anim. Sci. 21, 383-388. Rose, M. E. (1959). A study of the life cycle of Eimeria stiedae (Lindemann, 1865) and the immunological response of the host. Ph.D. thesis, Cambridge Univ. Roth, S. I., and Conaway, H. H. (1983). Diabetes mellitus, model no. 266. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.), Fascicle 12. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Roudebush, P. (1977). Pancreatic adenocarcinoma in a domestic rabbit. Vet. Med. Small Anim. Clin. 72, 467-468. Ruckebusch, Y., Phaneuf, L. P., and Dunlop, R. (1991). "Physiology of Small and Large Animals." Dekker, Philadelphia. Ruffle, C. G., and Alder, B. (1996). Cloning, sequencing, expression, and protective capacity of the oma87 gene encoding the Pasteurella multocida 87kilodalton outer membrane antigen. Infect. lmmun. 64, 3161-3167. Rutherford, R. L. (1943). The life cycle of four intestinal coccidia of the domestic rabbit. J. Parasitol. 29, 10-32. Salmon, J., Ramoz, N., Cassonnet, P., Orth, G., and Breitburd, E (1997). A cottontail rabbit papillomavirus strain (CRPVb) with striking divergent E6 and E7 oncoproteins: An insight in the evolution of papillomaviruses. Virol. 235, 228-234.

363 Salzer, W. L., and McCall, C. E. (1991). Acute respiratory distress syndrome, model no. 275. Supplemental update. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.), Fascicle 18. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Sanderson, J. H., and Phillips, C. E. (1981). "An Atlas of Laboratory Animal Haematology." Oxford Univ. Press (Clarendon), Oxford. Sanford, T. D., and Colby, E. D. (1980). Effect of xylazine and ketamine on blood pressure, heart rate, and respiratory rate in rabbits. Lab. Anim. Sci. 30, 519-523. Sawin, P. B., and Crary, D. D. (1943). Atropinesterase, a genetically determined enzyme in the rabbit. Proc. Natl. Acad. Sci. U.S.A. 29, 55-59. Schauer, D. B., McCathey S. N., Daft, B. M., Jha, S. S., Tatterson, L. E., Taylor, N. S., and Fox, J. G. (1998). Proliferative enterocolitis associated with dual infection with enteropathogenic Escherichia coli and Lawsonia intracellularis in rabbits. J. Clin. Microbiol. 36, 1700-1703. Schermer, S. (1967). "The Blood Morphology of Laboratory Animals," 3rd ed. Davis, Philadelphia. Schlitt, M., and Bucher, A. P. (1989). Herpes simplex encephalitis, model no. 352. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.), Fascicle 17. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Schoeb, T. R., Casebolt, D. B., Walker, V. E., Potgieter, L. N. D., Thouless, M. E., and DiGiacomo, R. E (1986). Rotavirus-associated diarrhea in a commercial rabbitry. Lab. Anim. Sci. 36, 149-152. Sethupathi, P., Spieker-Polet, H., Picken, M. M., Zaryczny, J., and Knight, K. L. (1993). Acute B-lymphoblastic leukemia, model no. 275. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.), Fascicle 19. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Shadduck, J. A., and Geroulo, M. J. (1979). A simple method for the detection of antibodies to Encephalitozoon cuniculi in rabbits. Lab. Anim. Sci. 29, 330-334. Shadduck, J. A., and Polley, M. B. (1978). Some factors in the in vitro infectivity and replication of Encephalitozoon cuniculi. J. Protozool. 25, 491-496. Sheldon, W. H. (1959a). Subclinical pneumocystis pneumonitis. Am. J. Dis. Child. 97, 287-297. Sheldon, W. H. (1959b). Experimental pulmonary Pneumocystis carinii infection in rabbits. J. Exp. Med. 110, 147-160. Shelley, H. J. (1961). Glycogen reserves and their changes at birth. Br. Med. Bull, 17, 137-143. Shenefelt, R. E. (1972). Gross congenital malformations. Animal model: Treatment of various species with a large dose of vitamin A at known stages in pregnancy. Am. J. Pathol. 66, 589-592. Smetana, H. (1933). Coccidiosis of the live rabbit. II. Experimental study on the mode of infection of the liver by sporozoites of Eimeria stiedae. Arch. Pathol. 15, 330-339. Smith, H. W. (1951). "The Kidney," p. 535. Oxford Univ. Press, New York. Soulez, B., Dei-Cas, E., Charet, P., Mongeot, G., Caillaux, M., and Camus, D. (1989). The young rabbit: A nonimmunosuppressed model for Pneumocystis carinii pneumonia. J. Infect. Dis. 160, 355 - 356. Soulsby, E. J. L. (1968). "Helminth, Arthropods, and Protozoa of Domestic Animals." Williams & Wilkins, Baltimore. Sovell, J. R., and Holmes, J. C. (1996). Efficacy of ivermectin against nematodes infecting field populations of snowshoe hares (Lepus americanus) in Yukon, Canada. J. Wildl. Dis. 32, 23-30. Steinhausen, M., Endlich, K., and Wiegman, D. L. (1990). Glomerular blood flow. Kidney Int. 38, 769-784. Stetson, P. L., Normolle, D. P., Knol, J. A., Johnson, N. J., Yang, Z. M., Sakmar, E., Prieskorn, D., Terrio, P., Knutsen, C. A., and Ensminger, C. D. (1991). Biochemical modulation of 5-bromo-2'-deoxyuridine and 5-iodo-2'deoxyuridine incorporation into DNA in VX2 tumor-bearing rabbits. J. Natl. Cancer Inst. 83, 1659-1667. Stills, H. E, Jr. (1991). Isolation of an intracellular bacteria from hamsters

364

MARK A. SUCKOW, DAVID W. BRAMMER, HOWARD G. RUSH, AND CLARENCE E. CHRISP

(Mesocricetus auratus) with proliferative enteritis and reproduction of the disease with a pure culture. Infect. Immun. 59, 3227-3236. Stills, H. E, Jr. (1994). Polyclonal antibody production. In "The Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), 2nd ed., pp. 435-448. Academic Press, Orlando, Florida. Stinnett, H. O., and Sepe, E J. (1979). Rabbit cardiovascular responses during PEEP before and after vagotomy. Proc. Soc. Exp. Biol. Med. 162, 485-494. Stormont, C., and Suzuki, Y. (1970). Atropinesterase and cocainesterase of rabbit serum: Localization of the enzyme activity in isozymes. Science 167, 200-202. Stringer, J. R. (1996). Pneumocystis carinii: What is it exactly? Clin. Microbiol. Rev. 9, 489-498. Stringer, J. R., Edman, J. C., Cushion, M. T., Richards, F. E, and Watanabe, J. (1992). The fungal nature of Pneumocystis. J. Med. Vet. Mycol. 30(Suppl. 1), 271-278. Stucki, U., Braun, R., and Roditi, I. (1993). Eimeria tenella: Characterization of a 5S ribosomal RNA repeat unit and its use as a species specific probe. Exp. Parasitol. 76, 68-75. Subcommittee on Rabbit Nutrition, Committee on Animal Nutrition, Board on Agriculture and Renewable Resources, National Research Council (1977). "Nutrient Requirements of Rabbits," 2nd ed. National Academy of Sciences, Washington, D.C. Suckow, M. A., and Douglas, E A. (1997). "The Laboratory Rabbit." CRC Press, Boca Raton, Florida. Suckow, M. A., and Grigdesby, C. F. (1993). Spontaneous lateral abdominal (lumbar) hernia in a New Zealand White rabbit. Lab. Anim. Sci. 43, 106107. Suckow, M. A., Martin, B. J., Bowersock, T. L., and Douglas, E A. (1996). Derivation of Pasteurella multocida-free rabbit litters by enrofloxacin treatment. Vet. Microbiol. 51, 161-168. Sundarum, P., Tigelaar, R. E., and Brandsma, J. L. (1998). Intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (CRPV) L1 gene protects against virus challenge. Vaccine 16, 613-623. Szili, M., and Kohalmi, I. (1981). Endemic Trichophyton mentagrophytes infection of rabbit origin. Mykosen 24, 412-420. Taffs, L. E (1976). Pinworm infection in laboratory rodents: A review. Lab. Anim. 10, 1-13. Texeira, A. R. L. (1986). Chagas' disease, model no. 334. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.), Fascicle 15. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Tesluk, G. C., Peiffer, R. L., and Brown, D. (1982). A clinical and pathological study of inherited glaucoma in New Zealand White rabbits. Lab. Anim. 16, 234-239. Thouless, M. E., DiGiacomo, R. E, and Deeb, B. J. (1996). The effect of combined rotavirus and Escherichia coli infection in rabbits. Lab. Anim. Sci. 46, 381-385. Trueta, J., Barclay, A. E., Daniel, P. M., Franklin, K. J., and Prichard, M. M. L. (1947). "Studies on the Renal Circulation." Blackwell, Oxford. Truex, R. C., and Smythe, M. Q. (1965). Comparative morphology of the cardiac conduction tissue in animals. Ann. N.Y. Acad. Sci. 127, 19-33. Tsunoda, K., Imai, S., Tsutsumi, Y., and Inouye, S. (1968). Intermittent medication of sulfadimethoxine and sulfamonomethoxine for the treatment of coccidiosis in domestic rabbits. Nat. Inst. Anim. Health Q. Tokyo. 8, 74-80. Tvedten, H. W. (1983). Pelger-HuEt anomaly, model no. 278. In "Handbook: Animal Models of Human Disease" (C. C. Capen, T. C. Jones, and G. Migaki, eds.), Fascicle 12. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Vacha, J. (1983). Red cell life-span. In "Red Blood Cells of Domestic Animals" (N. S. Agar and P. G. Board, eds.), pp. 67-132. Elsevier, Amsterdaml Varga, I. (1982). Large-scale management systems and parasite populations: Coccidia in rabbits. Vet. Parasitol. 11, 69-84. Vernau, K. M., Grahn, B. H., Clarke-Scott, H. A., and Sullivan, N. (1995). Thymoma in a rabbit with hypercalcemia and periodic exophthalmus. J. Am. Vet. Med. Assoc. 206, 820-822.

Waggie, K. S., Spencer, T. H., and Ganaway, J. R. (1987). An enzyme-linked immunosorbent assay for detection of anti-Bacillus piliformis serum antibody in rabbits. Lab. Anim. Sci. 37, 176-179. Watanabe, Y., Ito, T., and Shiomi, M. (1985). The effect of selective breeding on the development of coronary atherosclerosis in WHHL rabbits. An animal model for familial hypercholesterolemia. Atherosclerosis (Shannon, Ireland) 56, 71-79. Weber, R., Bryan, R. T., Schwartz, D. A., and Owen, R. L. (1994). Human microsporidial infections. Clin. Microbiol. Rev. 7, 426-461. Weisbroth, S. H. (1974). Neoplastic diseases. In "The Biology of the Laboratory Rabbit" (S. H. Weisbroth, R. E. Flatt, and A. L. Kraus, eds.), 1st ed., pp. 331-375. Academic Press, San Diego. Weisbroth, S. H. (1994). Neoplastic diseases. In "The Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.) 2nd ed., pp. 259-289. Academic Press, Orlando, Florida. Weiss, L. M., and Vossbrinck, C. R. (1998). Microsporidiosis: Molecular and diagnostic aspects. Adv. Parasitol. 40, 351-395. West, T. C. (1955). Ultramicroelectric recording from cardiac pacemaker. J. Pharmacol. Exp. Ther. 115, 283-290. Whary, M., Peper, R., Borkowski, G., Lawrence, W., and Ferguson, F. (1993). The effects of group housing on the research use of the laboratory rabbit. Lab. Anim. 27, 330-336. Williams, C. S. E (1976). "Practical Guide to Laboratory Animals." Mosby, St. Louis. Williams, J., Gladen, B. C., and Turner, T. W. (1991). The effects of ethylene dibromide on semen quality and fertility in the rabbit: Evaluation of a model for human seminal characteristics. Fundam. Appl. Toxicol. 16, 687-691. Wilson, R. B., Holscher, M. A., and Sly, D. L. (1987). Liver lobe torsion in a rabbit. Lab. Anim. Sci. 37, 506-507. Woolford, S. T., Schroer, R. A., Gohs, E X., Gallo, P. P., Brodeck, M., Falk, H. B., and Ruhren, R. (1986). Reference range database for serum chemistry and hematology values in laboratory animals. J. ToxicoL Environ. Health 18, 161-188. Wosu, N. J., Olsen, R., Shadduck, J. A., Koestner, A., and Pakes, S. P. (1977). Diagnosis of experimental encephalitozoonosis in rabbits by complement fixation. J. Infect. Dis. 135, 944-948. Wright, J. H., and Craighead, E. M. (1922). Infectious motor paralysis in young rabbits. J. Exp. Med. 36, 135-140. Wright, P., and Riner, J. (1985). Comparative efficacy of injection routes and doses of ivermectin against Psoroptes in rabbits. Am. J. Vet. Res. 46, 752754. Yamamoto, H., and Fujishiro, K. (1989). Pathology of spontaneous malignant fibrous histiocytoma in a Japanese White rabbit (Oryctolagus cuniculus). Lab. Anim. Sci. 38, 165-169. Yokoyama, E. (1979). Flow-volume curves of excised right and left rabbit lungs. J. Appl. Physiol. 46, 463-468. Yonushonis, W. P., Roy, M. J., Carman, R. J., and Sims, R. E. (1987). Diagnosis of spontaneous Clostridium spiroforme iota enterotoxemia in a barrier rabbit breeding colony. Lab. Anim. Sci. 37, 69-71. Yost, D. H. (1958). Encephalitozoon infection in laboratory animals. J. Natl. Cancer Inst. 20, 957-960. Young, D. M., Ward, J. M., and Prieur, D. J. (1978). Hypercalcemia of pregnancy. Animal model: VX-2 carcinoma of rabbits. Am. J. Pathol. 93, 619622. Yu, L., Pragay, D. A., Chang, D., and Wicher, K. (1979). Biochemical parameters of normal rabbit serum. Clin. Biochem. 12, 83-87. Zaoutis, T. E., Reinhard, G. R., Cioffe, C. J., Moore, P. B., and Stark, D. M. (1991). Screening rabbit colonies for antibodies to Pasteurella multocida by an ELISA. Lab. Anim. Sci. 41, 419-422. Zimmerman, T. E., Deeb, B. J., and DiGiacomo, R. F. (1992). Polypeptides associated with Pasteurella multocida infection in rabbits. Am. J. Vet. Res. 53, 1108-1112. Zurovsky, Y., Mitchell, G., and Hattingh, J. (1995). Composition and viscosity of interstitial fluid of rabbits. Exp. Physiol. 80, 203-210.

Chapter 10 Microbiological Quality Control for Laboratory Rodents and Lagomorphs William R. Shek and Diane J. Gaertner

I. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Biosecurity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chainof Adventitious Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Containmentand Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Microbiological (Health) Surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. DiagnosticMethodologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Designand Implementation of a Surveillance Program . . . . . . . . . . . C. Interpretation of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

OVERVIEW

Microbiological quality control for laboratory animals, composed of biosecurity and health surveillance, is essential to guard against the research complications and public health dangers that have been associated with adventitious infections. Laboratory animal biosecurity consists of all measures taken to prevent, contain, and eradicate adventitious infections. To institute an effective biosecurity program, one must understand the chain of infection, including the environmental and animal reservoirs, the sources of i n f e c t i o n m such as wild rodents, supplies, people, and biological m a t e r i a l s - - a n d the modes of transmission. Based on the sources of infection, risk factors are defined and controlled. A pest control program is put in place; supplies are disinfected by physical or chemical processes; air and water are filtered; personnel don gowns; and biological maLABORATORY ANIMAL MEDICINE, 2nd edition

365 366 366 366 367 371 372 372 382 385 387

terials are screened for viral contamination by rodent antibody production tests, in vitro virus isolation, or PCR (polymerase chain reaction). Should an adventitious infection occur, control and eradication are most reliably achieved by depopulation and disinfection, followed by repopulation with SPF replacements or rederived descendants of the infected colony. When this approach is not feasible, however, other control measures such as a breeding moratorium, chemotherapy, or vaccination may be attempted, although these have limited applicability and are risky. In all cases, steps should be taken to ensure that the likely sources of infection and associated risk factors are controlled or eliminated. Because even the most rigorous biosecurity cannot guarantee that adventitious infections won't occur, health surveillance, including laboratory methods to detect inapparent infections and identify specific etiologic agents, should be performed routinely on both breeding and research colonies. To develop a Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

WILLIAM R. SHEK AND DIANEJ. GAERTNER

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microbiological monitoring program that is both effective and practical, choices need to be made regarding the agents for which to screen, the type and number of animals to be sampled, and the sampling frequency. Program implementation is accomplished by systematically recording these choices and incorporating them into testing schedules. Although the primary methodologies for detection of parasites, bacteria, and viruses are direct gross and microscopic examination, cultural isolation, and serology, respectively, a combination of methodologies is frequently employed to make a definitive diagnosis. The newest of these methodologies, molecular testing by PCR (see p. 378), has made direct detection of viruses and other fastidious microorganisms in clinical specimens practical. Because no laboratory assay is completely accurate, it cannot be emphasized enough that all unexpected positive findings must be confirmed by testing additional samples and by using alternative assays and diagnostic methodologies to corroborate primary test results.

II.

INTRODUCTION

It has been amply documented that adventitious infections of laboratory animals with certain microorganisms can interfere with research. Infections may result in clinical disease and pathological changes, especially in perinatal and immunodeficient animals (Barthold et al., 1985; Gaertner et al., 1989; Jacoby et al., 1987; Schoeb et al., 1986; Waggie et al., 1981; Walzer et al., 1989; Weir et al., 1988). Although infections of postweaning, immunocompetent animals are often subclinical, they can lead to contamination of biological materials and abnormal responses to experimental treatments (Bonnard et al., 1976; McKisic et al., 1993; Peck et al., 1983; Riley et al., 1960; Rowe et al., 1962). Furthermore, some microorganisms indigenous to laboratory animal species are zoonotic agents that have caused disease in people (Anderson et al., 1983; Deibel et al., 1975; Hjelle et al., 1994; Lee and Johnson, 1982; Lewis et al., 1965). It is therefore essential for laboratory animal breeders and users alike to implement and maintain a microbiological quality control program that includes strict biosecurity and comprehensive microbiological, or health, surveillance (Fox and Loew, 1983; Jacoby and Lindsey, 1997; Small, 1984; Wagner et al., 1991; Weisbroth et al., 1998). Both aspects of quality assurance will be reviewed in this chapter. Although microbiological quality control for rodents will be emphasized, the concepts considered are applicable to laboratory animals in general.

III.

BIOSECURITY

Laboratory animal biosecurity consists of all measures taken to prevent, contain, and eradicate adventitious infections. In the

case of gnotobiotic animals that are axenic or have a defined microflora consisting of a few nonpathogenic bacteria, biosecurity measures must entirely exclude exogenous microorganisms. This is accomplished by housing gnotobiotic animals in isolators supplied with sterile food, bedding, and water (Trexler, 1983). Most research animals are not gnotobiotic but instead are classified as specific pathogen-free (SPF) or conventional (Jacoby and Lindsey, 1998). SPF animals are those that have tested negative for a limited list of exogenous viruses, bacteria, and parasites that may cause disease or otherwise interfere with research. The level of biosecurity appropriate to SPF animals depends largely on their immune status and the ease with which they can be replaced (White et al., 1998). Immunocompetent animals housed in open cages in barrier rooms will develop a complex microflora that includes opportunistic pathogens such as P n e u m o c y s t i s carinii and P s e u d o m o n a s aeruginosa and will still be suitable for most research. On the other hand, opportunistic pathogens are likely to cause disease in immunodeficient or immunosuppressed animals (Flynn, 1963; Rosen and Berk, 1977; Waggie et al., 1988; Walzer et al., 1989; Weir et al., 1986). Because opportunists are difficult to exclude from barrier rooms, immunocompromised and valuable genetically modified strains are frequently housed under stricter conditions, in isolators or filter-top microisolation cages (Sedlacek and Mason, 1977). Conventional animals are maintained with minimal biosecurity and health surveillance and thus have a nominally defined microflora that often includes pathogens (Foster, 1980; Trexler, 1983).

A.

Chain of Adventitious Infection

Effective biosecurity requires an understanding of the chain of adventitious infection, including reservoirs, sources, and modes of transmission (Fig. 1). The reservoir, or ecological niche, of a microorganism can be an animal species or the environment (Brachman, 1996). For example, the reservoir for lymphocytic choriomeningitis virus (LCMV) in the wild is the mouse (Lehmann-Grube, 1982), whereas Listeria m o n o c y t o g e n e s is found in various avian and mammalian species as well as throughout the environment (Broome et al., 1998). The source of an organism for transmission to a susceptible host is not necessarily the same as its reservoir. The source of L. m o n o c y togenes for an SPF colony might be food or bedding that was contaminated by carrier animals or the environment. The distinction between reservoir and source is important in the case of laboratory animal biosecurity because, in general, it is more practical to eliminate or control a pathogen's source than its reservoir. The modes by which an infection can be transmitted to a susceptible host are direct animal-to-animal contact and indirect transfer via an inanimate vehicle, also termed a fomite, or an animate vector. Contact transmission is vertical when it takes place in utero or at birth, or horizontal if it occurs postpartum

10. MICROBIOLOGICALQUALITYCONTROLFOR LABORATORYRODENTS AND LAGOMORPHS

Fig. 1. Chainof adventitious infection for laboratoryrodents.

through the transfer of droplets or by intimate contact, as exemplified by venereal diseases. Fomite transmission can be airborne or by way of common vehicles such as food, water, and bedding. Airborne transmission refers to the spread of contaminated droplet nuclei (i.e., the residue of dried droplets) or dust for a distance of more than several feet (Brachman, 1996). A vector is an animal, typically an arthropod, involved in the spread of infection. Vectors can be biological, that is, essential to the life cycle of the pathogenic organism, or mechanical (Brachman, 1996; Cohen, 1998; Prince et al., 1991; Waggie et al., 1994). Given that most pathogens are obligate parasites with a limited host range, it stands to reason that wild and domestic rodents are the principal reservoir of adventitious infection for laboratory rodents. Most rodent pathogens are transmitted efficiently by direct animal-to-animal contact (Parker and Reynolds, 1968; Shek et al., 1998; Thigpen et al., 1989; Yang et al., 1995). An exception is lactate dehydrogenase-elevating virus (LDV), which is not readily transmitted from mouse to mouse by natural means, even though it causes a persistent viremia and is excreted in large amounts (Brinton, 1982). In the laboratory, LDV appears to be transmitted mainly by parenteral injection of mice with contaminated biological materials. In this regard, it is noteworthy that LDV is among the most common contaminants of tumors maintained by passage in mice (Collins and Parker, 1972; Nicklas et al., 1993; Riley, 1974). Because contact transmission is usually horizontal, the majority of rodent pathogens can be eliminated through derivation by cesarean section or embryo transfer. Vertical transmission, however, is common for a few agents, such as LCMV in mice (Lehmann-Grube, 1982) and cytomegalovirus in guinea pigs (Choi and Hsiung, 1978).

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Fomite transmission with soiled bedding as the common vehicle has been demonstrated for various rodent pathogens. On the other hand, soiled bedding does not transmit cilia-associated respiratory (CAR) bacillus (Cundiff et al., 1995) and appears to be an inefficient mode of transmission for Sendai virus (Artwohl et al., 1994; Dillehay et al., 1990). Airborne transmission is, in general, of little consequence when the number of infected animals is small. A recent study, however, provides evidence that it is important when the reservoir of infection is a large breeding colony (Henderson et al., 1998). Arthropod vectors play a minor role in the transmission of rodent pathogens. Lice are known biological vectors for the erythrocyte parasites E p e r y t h r o z o o n coccoides and H e m o b a r tonella muris of mice and rats, respectively (Hildebrandt, 1982), but neither the louse vectors nor the rickettsial parasites have been encountered recently (Jacoby and Lindsey, 1998). Both insects and people have been incriminated as mechanical vectors for adventitious viral infections (Ishii et al., 1974; Tietjen, 1992). To summarize, adventitious infection occurs when an etiologic agent is accidentally transmitted from its reservoir, most often animals of the same species, into an SPF animal colony by direct animal-to-animal contact or indirectly through a fomite or vector.

B.

Prevention

A biosecurity program should emphasize prevention, which is undoubtedly preferable to containment and eradication. In analytical epidemiology, risk factors are the characteristics of affected individuals, which correlate with illness, as smoking is a risk factor for lung cancer (Cohen, 1998). As noted, wild rodents are an important source (and reservoir) of pathogens. A risk factor associated with this source is an inadequate pest control program. Biosecurity should minimize the risk factors associated with potential sources of infection and modes of transmission (Table I). 1.

Contact Transmission

Contact transmission can occur when wild, or escaped, rodents enter an SPF colony or when infected laboratory animals are transferred from one colony to another. Wild rodents have been shown to carry a variety of pathogens (Behnke, 1975; Bhatt et al., 1986a; Childs et al., 1989; Skinner et al., 1977; Smith et al., 1993a). The risk of their contaminating an SPF colony is expected to increase when a rodent control program is not in place or the structural barriers to entry are inadequate (Lussier et al., 1988). Pest control services are best provided by a reputable and licensed commercial vendor. Animal facilities should be constructed and maintained so that potential nest areas and routes of ingress or egress are not present. All holes and cracks in the facility should be sealed. Trapping devices should be used to detect and eliminate loose rodents. Those that are

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Table I

Risk Factors for Adventitious Infection Transmission Contact

Fomite

Vector

Source Wild or escaped rodents Transferred rodents Personnel Food, bedding, supplies Water Biologics Airborne Insect (mechanical and biological) Personnel (mechanical)

Risk factors Pest control program inadequate, structural defects Source colony is conventional, health surveillance not recent or routine Manipulating animals without wearing gown, mask, and disinfected gloves Disinfection inadequate or not done Not treated (e.g., not filtered or chlorinated) Inoculated into animals without mouse antibody production (MAP) testing Contaminated colony on site Pest control program inadequate, structural defects Contact with reservoir, access to multiple colonies, unprotected contact with laboratory animal

captured alive should be identified as to species, handled as if they were infected, anesthetized, and bled for serology prior to euthanasia. Food, bedding, and garbage attract loose rodents and therefore should be stored off the floor in a secure area in sealed containers (Hoddenbach et al., 1997; Small, 1983). The risk of introducing pathogens through an animal transfer depends, in part, on the degree of certainty that the source colony is SPE The chance that an adventitious infection will go undiagnosed increases when the sample size is small (Dubin and Zietz, 1991) or surveillance is done infrequently (Selwyn and Shek, 1994). The accuracy of test results also depends on samples being appropriate for the diagnostic methodology. Animals sampled for serology need to be immunocompetent and given sufficient time to seroconvert (Parker and Reynolds, 1968; Peters and Collins, 1983; Smith, 1983a). In the case of pathology, bacteriology, and parasitology, it is helpful to sample animals of multiple ages, because the prevalence of infection with some bacteria and parasites is age-dependent. For instance, enteric protozoa are readily observed in weanlings but not in older rodents. Conversely, because of a long life cycle, patent infections with the mouse pinworm Aspicularis tetraptera are most often found in adolescent rather than weanling mice (Wescott, 1982). It has become standard practice to ship rodents and rabbits in filtered containers to prevent contamination during transit. Animals in containers with damaged filters are undoubtedly at increased risk for adventitious infections and therefore should not be brought into an SPF facility. The risk of contamination is reduced by direct shipment in vehicles dedicated to SPF animals as opposed to air shipment, because animals shipped by air are more likely to be exposed to vermin or infected animals from other vendors in holding areas (Rehg and Toth, 1998). Unless laboratory animals are obtained from a regular supplier that practices rigorous biosecurity and performs routine and comprehensive microbiological surveillance, it is strongly recommended that the animals be quarantined upon receipt. Quarantined animals need to be maintained in a manner that not only protects them from adventitious infection but also contains any infectious agents that they may be carrying. Containment is

particularly important when quarantining animals with an undefined microflora or from a conventional colony. Air pressure in quarantine rooms or isolation units should be negative relative to common corridors, and materials for disposal should be disinfected or placed in sealed containers before being removed from the quarantine area. Finally, personnel access should be kept to a minimum (Rehg and Toth, 1998). Quarantine programs have been classified as passive when animals are observed only for clinical disease, or active if their microbiological status is also assessed by laboratory testing (Small, 1984). An active quarantine is considered preferable because of the ample evidence that subclinical infections can have adverse effects on research (Bhatt et al., 1986b). The quarantine period, which starts when the most recently received animals are placed into a quarantine, should not be less than several weeks, to allow time for seroconversion to infectious agents acquired in transit. 2.

Fomite Transmission: Supplies

The risk of fomite transmission may be reduced by using physical and chemical processes to sterilize or disinfect equipment and supplies. Sterilization is the elimination or inactivation of all microorganisms, whereas disinfection is less complete. For example, a disinfection process might destroy vegetative bacteria but not bacterial spores (Block, 1991). Supplies for gnotobiotic colonies must be sterilized, whereas disinfection, or pasteurization, generally suffices for supplies being transferred into an SPF colony (Foster et al., 1964; Foster, 1980; Trexler, 1983). Rational selection of a disinfection or sterilization process is aided by knowledge of the process's mechanism of action and the physiochemical characteristics of the microorganisms to be eliminated. In general, bacterial spores, freeliving stages of parasites (e.g., pinworm eggs and protozoan cysts), and nonenveloped viruses are resistant to inactivation (Ganaway, 1980; Hoover et al., 1985; Leland, 1991; Prince et al., 1991; Russell, 1992; van der Gulden and van Erp, 1972). The best method for disinfection is also determined by the process's applicability to a particular medium (e.g., air, food,

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10. M I C R O B I O L O G I C A L QUALITY C O N T R O L F O R LABORATORY RODENTS AND L A G O M O R P H S

water, surfaces), hazards and toxicity of treatment, ease of application, and cost (Russell, 1991). a.

Physical Processes of Disinfection

Physical processes of disinfection, such as autoclaving and electromagnetic irradiation, are the treatments of choice for food and bedding. In contrast to chemical disinfection, these methods do not leave a residue or by-products that may be toxic for or cause physiologic changes in animals (Hermann et al., 1982). Raw materials used in the preparation of animal feed and bedding frequently have a high bacterial count. The heating of food to 7 5 - 8 0 ~ during pelleting substantially reduces the bacterial count but is not sufficient to inactivate thermostable pathogens. In addition, food and bedding may become recontaminated after processing (Clarke et al., 1977). Therefore, they should be sterilized or pasteurized for gnotobiotic or SPF rodent colonies, respectively. As mentioned, this has traditionally been accomplished by autoclaving (i.e., saturated steam heat) or gamma irradiation. In comparison with gamma irradiation, autoclaving is less expensive but causes a greater reduction in the nutritional value of food (Ferrando et al., 1981). Another drawback of autoclaving is the difficulty in achieving uniform steam penetration and temperature throughout a load (Small, 1983). Presterilization vacuum cycles help preserve the nutritional value of food by promoting rapid and uniform steam penetration, which allows autoclave times to be kept short (Foster et al., 1964; Maerki et aL, 1989). Gamma radiation, usually emitted from a 6~ source, is a type of ionizing radiation. Although ionizing irradiation has a variety of physical and biochemical effects, it mainly renders microorganisms nonviable by causing breakage in their nucleic acid (Silverman, 1991). Ultraviolet (UV) radiation (210328 nm), which does not possess sufficient energy to cause ionization, also inactivates microorganisms by damaging their DNA but does not cause DNA breakage. Instead, UV irradiation produces thymine and other pyrimidine dimers. As one might expect, the bactericidal activity of UV irradiation is max -j imal near the peak of DNA absorption, which is 260 nm (Russell, 1991). Gamma radiation passes through solid objects; by contrast, UV radiation does not and therefore is effective only for disinfection of surfaces and drinking water. UV inactivation of microbes in drinking water is reduced as the UV-light source loses intensity or becomes dirty and by the presence of particles and dissolved organics in the water (Sobsey, 1989). Nonetheless, UV irradiation is an attractive option for water disinfection because it is virucidal and, in contrast to chlorination, does not convert organic precursors into potentially carcinogenic trihalomethanes (Flood, 1995). The radiosensitivity of organisms has been shown to correlate with genome volume and the ability of the organism to repair DNA damage (Silverman, 1991). This is the reason why comparatively small viruses, such as parvoviruses, are highly re-

sistant to UV and gamma irradiation (Hanson and Wilkinson, 1993), as are bacterial spores, protozoan cysts, and vegetative bacteria with highly efficient DNA repair capabilities (Russell, 1991). Accordingly, irradiation should not be relied on as the sole treatment for sterilization of supplies intended for gnotobiotic rodents. Filtration is the process most often employed to remove microbes from air and water (Denyer, 1992; Levy and Leahy, 1991). Depth filters entrap and adsorb, whereas membrane filters exclude particles according to pore size. Depth filters have high "dirt-handling" capacity, and therefore they are used for high-efficiency particulate air (HEPA) filtration and for clarification of particle-laden liquids. Because depth filters have no meaningful pore size, they are given nominal ratings to indicate the efficiency with which they retain particles of a particular size. The 99.97% rating given HEPA filters is based on the efficiency with which they retain 0.3 ~tm particles (Avery, 1996). A filtration process can be classified according to the minimum size of particles retained as microfiltration (range 0.110.0 ~tm), ultrafiltration (range 1000-1,000,000 molecular weight), or reverse osmosis (low-molecular-weight molecules, including salts). Microfiltration of water retains bacteria, fungi, and their spores, but it cannot be relied upon to exclude viruses (Block, 1991). Removal of virus from water can be achieved, however, by ultrafiltration or reverse osmosis. Although there are no reports implicating water as source of adventitious viral infections for laboratory rodents, the possibility should be taken seriously because rodents are susceptible to infection with viruses that are taxonomically related to waterborne human viruses (Table II). Characteristically, waterborne viruses are of small to medium size, nonenveloped (and hence stable), and shed in the feces (Block and Schwartzbrod, 1989). b.

Chemical Disinfectants

Chemical disinfectants are commonly utilized to decontaminate a room or an isolator before the introduction of SPF animals and to treat the surfaces of materials and containers being brought into an SPF colony or removed from a quarantined colony (Small and New, 1981). Water is often disinfected through Table II Waterborne Human and Related Rodent Viruses Family

Waterborne human viruses

Related rodent viruses

Picornaviridae Reoviridae

Poliomyelitic virus 1, 2, 3 Reovirus 1, 2, 3 Rotavirus 1, 2, 3, 4 Human coronavirus Human adenoviruses 1-33

TMEV a Reovirus 1, 2, 3 Mouse rotavirus MHV, SDAV b Mouse adenovirus 1, 2

Coronaviridae Adenoviridae

Theiler's murine encephalomyelitis virus. bMouse hepatitis virus and sialodacryoadenitis virus. a

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WILLIAM R. SHEK AND DIANE J. GAERTNER

chemical processes such as chlorination (Hermann et al., 1982; Homberger et al., 1993) or ozonation (Flood, 1995; Shek et al., 1991). Chemical disinfectants inactivate microorganisms by acting as denaturants that disrupt protein or lipid structures, reactants that form or break covalent bonds, or oxidants (Table III) (Prince et al., 1991). Various schemes have been developed to link the physiochemical characteristics of microorganisms with susceptibility to chemical inactivation. For example, the Klein-DeForest scheme for viruses associates sensitivity to disinfectants with viral solubility (Table IV). Phenolics and quaternary ammonium compounds, which disrupt lipid membranes, are more potent against lipophilic, enveloped viruses than against hydrophilic, nonenveloped viruses. Oxidants attack all organic compounds and thus inactivate hydrophilic as well as lipophilic viruses (Klein and DeForest, 1983; Prince et al., 1991). A disinfection scale for all microbial taxons likely to be encountered in laboratory animals, derived from one proposed by Prince et al. (1991) is presented in Table V. In brief, this scale restates the generalization made at the beginning of this section that enveloped viruses and vegetative bacteria are considerably easier to inactivate than are nonenveloped viruses, bacterial endospores, and free-living parasite stages. For the most part, a disinfectant that has been shown to inactivate microorganisms of a particular susceptibility group will inactivate infectious agents in more susceptible groups. Thus, a disinfectant that inactivates parvoviruses will certainly kill Staphylococcus aureus. The potency of a disinfectant can be enhanced through chemical modification or the addition of synergistic ingredients to the formulation. Conversely, physical factors, including temperature, pH, and the chemical "demand" of the medium being treated, can diminish potency by reducing the concentration or stability of the active form of the disinfectant. Using chlorine as a case in point, increasing the pH or temperature of water reduces the concentration of hypochlorous acid (HOC1) in favor of the hypochlorite (OC1-) ion, which is less biocidal. Chlorine is a strong oxidant that reacts not only with living microorganisms but also with inorganic reducing substances such as ferrous iron and organic impurities, including dissolved proteins.

Table III Chemical Disinfectant Categories Category

Examples

Denaturants

Quaternary ammonium compounds (benzalkonium chloride) Phenolics Alcohols Aldehydes (formaldehyde, glutaraldehyde) Ethylene oxide Halogens (chlorine bleach, chlorine dioxide, povidone-iodine) Peroxygens (H202, peracetic acid) Ozone

Reactants Oxidants

These reactions exert a chemical demand that reduces the concentration of free chlorine available for disinfection (Dychadala, 1991; Flood, 1995; Russell, 1991; Wickramanayake and Sproul, 1991). Association with dirt and organic matter has been shown to protect microorganisms from disinfectants (Grossgebauer et al., 1975; Russell, 1992; Small and New, 1981; Wickramanayake and Sproul, 1991). Upon colonizing surfaces, bacteria such as P s e u d o m o n a s aeruginosa are notorious for forming biofilms, i.e., large clumps of bacteria surrounded in slime that resist chemical disinfectants (Potera, 1996). It is therefore crucial that soiled surfaces be sanitized before being disinfected in order to reduce chemical demand and to ensure that microorganisms are adequately exposed to disinfectant. Biofilms, which are likely to accumulate in water systems, can reportedly be removed by treatment with H202 or alkaline peroxide (Klein and DeForest, 1983; Kramer, 1992). 3.

Fomite Transmission: Biological Materials

A substantial risk of adventitious infection is posed by inoculation of rodents with biological materials that have not been screened for extraneous viruses. Recent ectromelia virus outbreaks have been linked to contaminated serum (Dick et al., 1996; Lipman et al., 1999). The viral contamination rate is reportedly highest for transplantable tumors passaged in vivo, whereas that for cells grown in culture is comparatively low (Collins and Parker, 1972; Nicklas et al., 1993). Failing to screen biological materials for rodent viruses can also have public health consequences, because LCMV has been a relatively prevalent contaminant of cell lines (Bhatt et al., 1986a; Lewis et al., 1965; Simon et al., 1982). Hence, biological materials should be tested for rodent viruses and, additionally, for extraneous bacteria, fungi, and mycoplasma before being inoculated into SPF rodents. Surveillance for rodent viral contaminants has traditionally been carried out by the mouse and rat antibody production (MAP and RAP) tests and by other in vivo and cultural isolation techniques (Lussier, 1991; Smith, 1986a; Waggie et al., 1994; Weisbroth et al., 1998). Investigators, though, may unwisely avoid rodent antibody production testing because of the time and expense involved. Polymerase chain reaction (PCR) assays for viruses provide an accurate, rapid, and less costly alternative to MAP testing (Riley et aL, 1999). 4.

Vector Transmission

Previously in this chapter, it was noted that although biological vectors are rarely involved in the transmission of rodent pathogens, both insects and people have been incriminated as mechanical vectors. People are also carriers of opportunistic bacteria such [3-hemolytic streptococci and Staphylococcus aureus (Foster, 1996; Patterson, 1996). The keys to controlling insects--mostly flies and cockroaches--are deterrence to entry,

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10. MICROBIOLOGICAL QUALITY CONTROL FOR LABORATORY RODENTS AND LAGOMORPHS

Table IV Klein-DeForest Scheme for Viral Sensitivity to Disinfectants Category

Solubility

Structure

Sensitivity

A

Lipophilic

Lipid envelope + capsid

Marked

B

Hydrophilic

Naked capsid

Slight

C

Intermediate

Partially lipophilic capsid

Moderate

Examples a Paramyxovirus (Sendai, PVM) Coronavirus (MHV, SDAV) Arenavirus (LCMV) Picornavirus (TMEV) Parvovirus (MVM, MPV, KRV, RPV) Adenovirus (MAdV-1, -2) Reovirus (Reo-3) Rotavirus (EDIM virus, IDIR virus)

a PVM, pneumonia virus of mice; MHV, mouse hepatitis virus; SDAV, sialodacryoadenitis virus; LCMV, lymphocytic choriomeningitis virus; TMEV, Theiler's murine encephalomyelitis virus; MVM, minute virus of mice; MPV, mouse parvovirus; KRV, Kilham's rat virus; RPV, rat parvovirus; MAdV, mouse adenovirus; EDIM, epizootic diarrhea of infant mice; IDIR, infectious diarrhea of infant rats.

sanitation, and the application of control methods, resorting last to the use of insecticides that might alter rodent physiology (Small, 1983). Entomologists with a detailed understanding of insect life cycles can often minimize or obviate chemical use. Risk factors for personnel becoming vehicles of infection include (1) exposure to a reservoir, such as an infected colony; (2) access to multiple colonies, especially going from conventional to SPF; and (3) unprotected human-animal contact, as exemplified by a technician handling animals without wearing disinfected gloves. To state the obvious, because people who care for and use research animals do not themselves live in isolators or barrier rooms, contact between people and reservoirs of infection can never be completely avoided. However, practices can be instituted that reduce this risk. Animal care technicians should be prohibited from having pet rodents. In many institutions, visitors are permitted to enter animal facilities only if they have not had recent contact with laboratory animals. Breeders with large production rooms may have a dedicated staff for each room. Access to smaller colonies, for which a dedicated staff is not practical, should still be limited, and the flow of people and supplies should always be from "clean" to "dirty." Personnel entering a barrier room should gown in a manner that keeps areas of

Table V Approximate Scale for Susceptibility of Laboratory Rodent Pathogens to Disinfectants Susceptibility category a

Type of microorganism Enveloped viruses, non-spore-forming bacteria Partially lipophilic, nonenveloped viruses Hydrophilic, nonenveloped viruses Bacterial endospores and parasite ova and cysts

a

Susceptibility decreases from A to D.

exposed skin to a minimum in order to reduce the potential for transmitting infectious agents. Alternatively, it has become common practice to limit animal-human contact by housing rodents in microisolation cages (Sedlacek and Mason, 1977) or isolators (Trexler, 1983). Contact is limited further by manipulating rodents in a laminar flow hood and by handling them with disinfected forceps.

C.

Containment and Eradication

A variety of options is available for dealing with an adventitiously infected laboratory animal colony. When SPF replacement animals can be obtained, it is standard practice to depopulate and disinfect. Certainly, animals infected with a zoonotic agent should be euthanized, decontaminated, and then safely discarded. Because pathogens often cause immunological perturbations, and because these disturbances can persist even in recovered animals (Compton et al., 1993), the use of infected animals in immunological research should be avoided. It is clearly contraindicated to do research involving tissues or organs that are the targets of an infectious agent. Laboratory animals that have undergone an adventitious viral infection should not be used for passaging cell lines or as a source of tissues and fluids for subsequent experiments. A virus might contaminate these materials, especially if it causes a persistent infection (Riley et al., 1960), has a broad host range (Bhatt et al., 1986a), or has a predilection for replicating in rapidly dividing cells (Bonnard et aL, 1976; McKisic et al., 1993). With the advent of transgenic technology, the use of genetically modified strains in biomedical research has grown dramatically. These and other valuable mutant strains are often difficult to replace. In such instances, derivation by cesarean section or by embryo transfer is considered the most dependable process for eliminating pathogens that are not vertically transmitted. Another option applied to nonpersistent infections

WILLIAM R. SHEK AND DIANE J. GAERTNER

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of immuncompetent hosts with enveloped viruses (e.g., Sendai virus and SDAV) is to break the cycle of infection by instituting a 6 to 8 week moratorium on breeding and on the introduction of susceptible animals (Bhatt and Jacoby, 1985). During this period, it is expected that all animals in the colony will recover from infection and stop shedding virus and that the excreted virus will quickly become noninfectious. A time-efficient alternative to a breeding moratorium is to start a new colony with seropositive, noncontagious breeders (Brammer et al., 1993). One should exercise caution when attempting to break the cycle of infection in a transgenic colony, because of the possibility that genetic modification has made the transgenic strain immunodeficient. Determining whether the viral infection has been eradicated is best accomplished by serologic surveillance of sentinels instead of the colony offspring that may have maternal antibodies. Chemotherapy has been used for infections with bacteria and parasites, often with the principal goal of preventing disease rather than eradicating the infection (Bhatt et al., 1981; Bhatt and Jacoby, 1987; Ganaway et al., 1965; Nikkels and Mullink, 1999). It is difficult to achieve eradication when the etiologic agent is stable outside of the host, because infection is likely to recur once chemotherapy has ended. On the other hand, endoparasitic infections of rodents have been eradicated by utilizing microisolation cages to prevent reinfection in combination with the highly potent anthelminthics ivermectin or fenbendazole (Flynn et al., 1999; Wescott et al., 1976). Antibiotic treatments have been shown to eliminate infections with bacteria that do not survive for long ex vivo, including Pasteurella pneumotropica (Goelz et al., 1996) and Helicobacter hepaticus (Foltz et al., 1996; Russell et al., 1995). Even when effective, however, antibiotic treatments may be too expensive or laborious to be practical for continuous or large-scale use. It is probably more practical to treat pregnant females prior to derivation to reduce the likelihood of vertical transmission. Although vaccination of laboratory rodents is not a common practice, there are notable examples where it has been employed to prevent disease and curtail the spread of infection. Mice have been vaccinated with vaccinia virus to control ectromelia (i.e., mousepox) virus outbreaks (Bhatt et al., 1981; Bhatt and Jacoby, 1987). Sendai virus and Bordetella bronchiseptica vaccines have been administered to mice (Eaton et al., 1982; Kimura et al., 1979) and guinea pigs (Ganaway et al., 1965; Nikkels and Mullink, 1999), respectively, to prevent the pneumonia caused by active infections. The principal drawbacks of vaccination are similar to those of chemotherapy in that vaccination must be continued until the sources of infection have been controlled, and routine vaccination of large production colonies is impractical. Vaccination may not be effective for every individual animal. In addition, vaccination may lessen morbidity but still not prevent infection and the resultant detrimental effects on host physiology. Serologic surveillance after vaccination can be problematic if it is not possible to distinguish

antibodies to the vaccine from those formed in response to infection with the pathogen. In summary, control and eradication are most reliably achieved by depopulation, disinfection, and repopulation with SPF replacements or derived descendants of the infected colony. When this approach is not feasible, other control measures such as a breeding moratorium, chemotherapy, or vaccination may be attempted, although these have limited applicability and are risky. In all cases, steps should be taken to ensure that the likely sources of infection are adequately disinfected or eliminated.

IV. M I C R O B I O L O G I C A L (HEALTH) SURVEILLANCE

Microbiological surveillance of both breeding and research colonies should be performed routinely because even the most rigorous biosecurity cannot be guaranteed to exclude all adventitious infections. Surveillance must include microbiologic laboratory methods to detect inapparent infections and to identify specific etiologic agents, because infections are usually subclinical or disease signs are not diagnostic. These methodologies include (1) gross and microscopic examination of animal specimens; (2) cultural and in vivo isolation of microorganisms; (3) infectious agent detection and identification by microscopic, biochemical, serologic, and genetic (or "molecular") techniques; and (4) serology for detection of microbial antibodies formed in response to infection (Isenberg, 1998; Washington, 1996).

A.

Diagnostic Methodologies

1. Gross and Microscopic Examination of Animal Specimens

Despite the increasing availability of rapid and specific in vitro assays, gross and microscopic examination of animal specimens continues to be an essential component of laboratory animal health surveillance. Examination of animal specimens may reveal disease during the active phase of an infection, prior to seroconversion (Allen et al., 1981; Bhatt et al., 1981). It is sometimes the most reliable diagnostic methodology when a specific in vitro test is unavailable or unsatisfactory (Cundiff et al., 1992; Gibson et al., 1987). Examination has uncovered the existence of hitherto unrecognized (i.e., "emerging") etiologic agents, as was the case for Helicobacter hepaticus, which was discovered to be the cause of hepatitis and hepatocellular carcinoma in mice in a long-term toxicology study (Ward et al., 1994a). Finally, gross and microscopic examinations of animal specimens are fundamental to laboratory animal pathology and

10. MICROBIOLOGICALQUALITYCONTROLFOR LABORATORYRODENTSAND LAGOMORPHS parasitology. As discussed below, examination may be combined with other techniques to arrive at specific diagnoses. a.

Pathology

Tissues and organs are inspected for gross abnormalities during routine health monitoring. Selected specimens may then be examined microscopically for histopathological changes after tissue sections are stained with hematoxylin and eosin (Weisbroth et al., 1998). Special stains can be applied to tissue sections to enhance the visibility of certain pathogens (Clifford et al., 1995; Gibson et al., 1987; Hoover et al., 1985; Thompson et al., 1982; Waggie et al., 1983; Ward et al., 1994b). Microbial antigens or nucleic acid in tissue sections can be specifically stained by immunohistochemistry (Allen et al., 1981; Brownstein and Barthold, 1982; Cera et al., 1994; Hall and Ward, 1984; Jacoby et al., 1975; Sundberg et al., 1989) or in situ hybridization (Gaertner et al., 1993; Jacoby et al., 1995), respectively. b.

Parasitology

Low-power dissecting microscopy is used to inspect the pelage and skin of laboratory animal carcasses for mites and lice, and the macerated gastrointestinal tract for adult helminths (Flynn, 1973; Weisbroth, 1982). Microscopic examination of skin scrapings may be necessary to detect mites, such as Demodex and Notoedres, which burrow into the epidermis (Weisbroth, 1979a; Wescott, 1982). It has been reported that fur mites can be found in a higher percentage of mice by microscopic examination of adhesive tape applied to the dorsal fur than by checking the skin or skin scrapings (West et al., 1992). The use of a pelt digestion method is also effective (Owen, 1972). Infections with enteric protozoa are diagnosed by examining wet mounts of mucosal scrapings of the small and large intestines. This is typically done with a phase-contrast microscope, which makes it possible to see unstained microorganisms (Brock, 1970; Weisbroth et al., 1996). Fecal flotation has been shown to be superior to intestinal wet mounts and histology for demonstrating coccidia in rabbits (Weisbroth et al., 1996). Helminth ova are also detected by fecal flotation and by the perianal tape test in the case of pinworms belonging to genus Syphacia, but direct examination of the gastrointestinal tract for adult helminths is most reliable (Huerkamp, 1993; West et al., 1992). 2.

Microbial Isolation in Culture or Animals

Microbial isolation is a traditional methodology that is essential for fulfilling Koch's postulates to prove that a particular microorganism is the cause of a specific disease. Because it is both definitive and sensitive, isolation is often the standard with which other assays are compared (Brownstein et al., 1985; Chang et al., 1997; Davidson et al., 1981; Lukas et al., 1987a;

373

Manning et al., 1987; Shames et al., 1995). Isolation techniques are used in routine health surveillance to monitor animals (and their supplies and environment) for bacteria and to screen biological materials for viral contaminants. Virus isolation is not practical for routine animal monitoring, because immunocompetent animals usually clear viral infections rapidly; thus, the period during which virus can be isolated is short (Jacoby, 1986; Parker and Reynolds, 1968). In addition, different viral species and strains have diverse host ranges in culture and tissue tropisms in vivo, making host and specimen selection problematic. Some fastidious viruses, such as mouse thymic virus (MTLV), will not grow in any cell-culture system (Morse, 1988). Although virus isolation is not practical for routine animal monitoring, it has been important for disease diagnosis and for corroborating the results of other tests (Allen et al., 1981; Dick et al., 1996). a.

Bacteriology

Bacterial monitoring of laboratory animals generally begins by inoculating artificial, cell-free agar and broth media with animal or environmental specimens. The specimens, media, and culture conditions are chosen to favor the isolation and cultivation of potentially pathogenic bacteria while limiting the growth of commensal and autochthonous microorganisms (Ganaway, 1976; Orcutt, 1980; Weisbroth, 1979b). The animal sites most often sampled--the upper respiratory tract and the large intestinemmay possess a complex microflora that can overgrow cultures and obscure colonies of interest. To mitigate this problem, specimens are cultured with selective media that contain additives, such as dyes or antibiotics, to inhibit the growth of certain microorganisms. MacConkey's agar, for example, contains crystal violet and bile salts that selectively inhibit the growth of gram-positive bacteria, while allowing most gram-negative bacteria to grow (Forbes et al., 1998). Media for the isolation of Helicobacter from fecal or intestinal specimens contain a mixture of antibiotics to selectively inhibit the growth of the intestinal microflora (Fox et al., 1999). Overgrowth can be further reduced by culturing sites that normally do not possess a microflora to obscure invasive bacteria. Tracheal cultures from Bordetella bronchiseptica-infected animals contain few extraneous bacteria, making it easier to view B. bronchiseptica colonies. Corynebacterium kutscheri is most reliably isolated from the submaxillary lymph nodes of infected rats (Brownstein et al., 1985). Enrichment media are used to encourage the growth of particular bacteria, which are at low concentration in a specimen containing many microorganisms. Selenite broth is an enrichment medium that is used to recover salmonellas from feces or the intestinal tract (Orcutt, 1980). Media are categorized as differential when they allow colonies to be morphologically differentiated based on metabolic characteristics. On MacConkey's agar, lactose-fermenting bacteria produce pink to red colonies whereas colonies of non-lactose fermenters remain

WILLIAM R. SHEK AND DIANE J. GAERTNER

374

colorless (Forbes et al., 1998). Cultures are usually incubated aerobically at 35~176 because the majority of clinically important bacteria are facultative anaerobes that will grow under these conditions, whereas the strict anaerobes that constitute the autochthonous microflora will not. A few fastidious bacteria require special growth conditions. Mycoplasmas require media supplemented with serum as a source of cholesterol and an atmosphere with additional CO2 because they are capnophilic (Davidson et al., 1981; Freundt, 1983; Orcutt, 1980). Campylobacter and Helicobacter species must be cultivated in a microaerophilic environment (Fox et al., 1999; Meanger and Marshall, 1989). Clostridium piliforme can be grown in embryonated chicken eggs and mammalian cell culture, but to date not with artificial, cell-free media (Riley et al., 1994). After incubation, cultures are examined to assess colonial morphology, and suspicious colonies are selected for further characterization. Cellular morphology, size, and motility are evaluated by examining a wet mount of an isolate with a phasecontrast microscope or a slide of Gram-stained cells with a bright-field microscope. If still suspect, an isolate is speciated, often using biochemical methods that include individual assays (e.g., catalase) and multitest systems (MacFaddin, 1980). Serotyping may also be necessary or helpful for isolate identification (Washington, 1996) and, doubtless, strain typing with molecular methods will soon become commonplace (Tenover, 1998). b.

Virology

Viruses are obligate intracellular parasites that are incapable of replicating on their own, outside of a susceptible host cell. Most viruses have a limited host range; i.e., they infect certain animal species but not others. In an animal host, viruses infect discrete populations of cells, tissues, and organs; this is known as the viral tropism. Sendai virus is referred to as pneumotropic, to indicate that it principally replicates in the lung (Brownstein, 1986). The reoviruses are described as pantropic because they replicate in many host tissues and organs (Tyler and Fields, 1986). The host systems used to isolate viruses in diagnostic laboratories include cell culture, embryonated eggs, and laboratory animals, particularly neonatal mice. Most cell culture is done with continuous cell lines that have the potential to divide indefinitely (Hawkes, 1979; Landry and Hsiung, 1992). However, primary cell cultures enzymatically dispersed directly from animal tissues (Greenlee et al., 1982) and explant cultures consisting of tissue fragments (Paturzo et al., 1987; Smith and Paturzo, 1988) are still used to isolate viruses that cannot otherwise be grown in vitro. To isolate a range of viruses, cell cultures of several types, often including primary cells as well as continuous cell lines, need to be inoculated to accommodate the variety of viral host ranges and tissue tropisms (Landry and Hsiung, 1992). By contrast, one or two host cell types might suffice when the goal is to isolate a single virus. Virus infection

of cultured cells may induce degenerative changes, such as syncytial cell formation or cell lysis, that are referred to as cytopathic effects, or CPE. These changes may be characteristic and aid in identification of the infecting agent. Other viruses may produce effects that are not distinctive, or they may be noncytopathic (Hawkes, 1979; Landry and Hsiung, 1992). In these instances, evidence of virus replication can be obtained by alternative methods such as hemadsorption, for viruses like Sendai that agglutinate red blood cells (Chanock, 1979), or immunofluorescence with virus-specific antibodies (Smith, 1986a). When available, electron microscopy can be a rapid way of observing the morphology of a virus isolate (Allen et aL, 1981; Jonas et al., 1969; Vonderfecht et al., 1988; Wallace et al., 1981). Serologic methods can be combined with electron microscopy for virus identification (Doane, 1992). Although cell culture is the predominant host system for virus isolation and cultivation, laboratory animals, and to a lesser extent embryonated chicken eggs, are still utilized, especially when monitoring for a panel of viruses or for one that is particularly fastidious. Use of a natural animal host can expedite virus isolation by avoiding the time that a field strain may require to adapt to growth in culture. Animal hosts are less susceptible than cell culture to nonspecific specimen toxicity and bacterial or fungal contamination (Hawkes, 1979; Rowe et al., 1959; Rowe et al., 1962). The sensitivity of virus isolation in vivo is enhanced by using multiple routes of inoculation to accommodate the variety of viral tropisms (Parker and Reynolds, 1968). Following inoculation with a biological specimen, animals are observed daily for disease and mortality. Morbidity can usually be increased by inoculating immunologically immature neonatal animals (Barthold et al., 1982; Jacoby et al., 1987), immunodeficient hosts such as nude mice (Barthold et al., 1985; Gaertner et aL, 1989; Weir et al., 1988), or laboratory animal strains for which a particular viral infection is more pathogenic (Brownstein et al., 1981; Parker et al., 1978). A notable exception occurs when the viral disease is immune-mediated, as exemplified by LCMV infection of mice. Following intracranial inoculation of LCMV, immunocompetent adult mice develop lymphocytic choriomeningitis, whereas T lymphocyte-deficient mice do not (Cole and Nathanson, 1974). Morbidity is not a reliable indicator, because viral infections of immunocompetent, postweaning animals are often asymptomatic. Moreover, certain laboratory animal viruses are nonpathogenic, even in neonatal and immunodeficient hosts (Jacoby et al., 1996). A more dependable method for determining whether an animal has been infected with a virus is serology for virus-specific antibodies. This is the basis of the MAP and RAP tests, alluded to above, for detection of murine viruses in biological specimens (Collins and Parker, 1972; Nicklas et al., 1993). Rodent antibody production tests are accurate and comprehensive because mice and rats, as natural hosts, are highly susceptible to infection with field as well as laboratory strains of murine viruses and because serologic

10. MICROBIOLOGICALQUALITYCONTROLFOR LABORATORYRODENTSAND LAGOMORPHS assays are sensitive and specific. The MAP test is recommended by regulatory agencies worldwide for detecting murine viruses in rodent-based biological products for use in humans. In the MAP test, immunocompetent postweaning mice, free of exogenous viruses, are inoculated with a specimen (i.e., test article) by multiple routes. The MAP mice are then housed in strict isolation to prevent adventitious infection. After at least 4 weeks, blood is collected from study mice, and sera are assayed for virus-specific antibodies by serologic methods described below. Detection of specific antibodies is tantamount to identifying infectious virus in the test article (Collins and Parker, 1972; Nicklas et al., 1993; Rowe et al., 1962). MAP test mice may also be tested for immunity to LCMV by intracranial challenge with a lethal dose of LCMV administered no sooner than 2 weeks after test article inoculation. Should the test article contain LCMV, the study mice would be preimmunized and thus survive the challenge. Otherwise, the MAP mice would be nonimmune and would succumb to the challenge within 6 - 9 days (Lehmann-Grube, 1982). Redundant testing of study mice for exposure to LCMV by serology and lethal challenge is justified because of the public health significance of this virus. Serology is not used to demonstrate LDV because this virus does not elicit an easily measured antibody response. Instead, the level of serum or plasma LDH activity is measured; a 10- to 20-fold increase above normal is consistent with, but not specific for, LDV infection (Brinton, 1982). To confirm that elevated LDH activity is due to an infectious virus, sera from test article-inoculated mice are passaged into additional SPF mice. Detection of significantly elevated LDH activity in serum or plasma from the passage mice corroborates the diagnosis of LDV. Because the LDH assay is not specific, it is being replaced in most laboratories with LDV-specific PCR assays (Chen and Plagemann, 1997; Goto et al., 1998). The sensitivity of the MAP test has been reported to be similar to that of other in vivo and cultural infectivity assays, although it has been shown to be more or less sensitive for particular viruses (DeSousa and Smith, 1989; Lewis and Clayton, 1971; Morse, 1989; Rowe et al., 1959). The range of viruses detected by rodent antibody production tests is of course limited by the available serologic assays. Other viruses that have yet to be discovered, or for which serologic assays do not exist, might be revealed by CPE in cell culture or morbidity in an animal host (Hartley and Rowe, 1960; Rowe and Capps, 1961). 3.

Infectious Agent Detection

For reasons just discussed, cultural isolation is not suitable for routine surveillance of laboratory animals for viruses and certain fastidious bacteria. The rodent antibody production tests for detecting viruses in biological specimens, although sensitive and specific, are time-consuming, taking at least 5 weeks to complete. An alternative to isolation is to analyze the specimen

375

directly for the presence of potential pathogens. Microscopic examination of specimens can provide the most rapid means of detecting microorganisms, but the organism concentration must be high, and further characterization is often needed. Advances in immunodiagnostics and the advent of molecular methods, notably the PCR, have made possible the development of highly sensitive, rapid assays for detection and identification of microorganisms directly in clinical specimens and after cultivation as well. a.

Serology

Diagnostic serology can be divided into two broad categories: (1) antibody assays in which known antigen is employed to determine whether a specimen, usually a serum sample, contains antibodies to a particular infectious agent (this category, which is particularly important in viral monitoring, will be reviewed separately in the next section; and) (2) antigen assays in which specific antibodies are used to detect or identify microorganisms according to their antigenic makeup. It should be kept in mind when interpreting the results of an antigen assay that a given antigen might be represented on a number of different microorganisms. Therefore, although an antigen-antibody reaction is itself highly specific, the results of serological identification of a microorganism may be ambiguous (Rose, 1999). A common usage of antigen assays in laboratory animal health surveillance is to serotype isolates of bacteria for which a linkage between clinical significance and serotype has been established. More than 1000 antigenic types of Salmonella have been delineated by agglutination with antisera to somatic O and flagellar H antigens (Ganaway, 1982; Giannella, 1996). [3-Hemolytic streptococci usually have group-specific, cell-wall carbohydrate (C) antigens, which are the basis of the popular Lancefield classification system. To determine the Lancefield group of an isolate, soluble C antigen is extracted from the organisms and reacted with the typing sera. Homologous reactions, indicating that an immune serum contains antibodies to the C antigen, can be demonstrated by precipitation or by the agglutination of antibody-coated latex particles (Fig. 2) (Corning et al., 1991; Patterson, 1996; Washington, 1996). Neutralization, complement fixation, and hemagglutination inhibition (HAI) tests are traditional serologic methods that discriminate among related viral strains (Beards et al., 1980; Chanock, 1979; Lee et al., 1985; Lussier et al., 1987; Schmaljohn et al., 1985), such as those that constitute the rodent parvoviruses (Table VI) (Siegl, 1976). With regard to routine virus monitoring of laboratory animals, however, these methods are generally performed as antibody assays with known antigen to delineate the strain specificity and thus the etiology of the viral antibody response (Parker et al., 1965; Parker et al., 1979; Smith et al., 1993b; Takahashi et al., 1986). Among the antigen assays, labeled antibody methods have been preferred for direct identification of microorganisms in

WILLIAM R. SHEK AND DIANEJ. GAERTNER

376

Specific antibody bound to latex bead + ' 0 ' ~I) 4 ) ,0,

Test Specimen . (Bacterial Antigen)

Agglutination

r

Fig. 2. Serotypingof bacteria by latex agglutination. (Adaptedfrom Washington, 1996,Fig. 10-2,p. 158.)

animal specimens because they combine the virtues of simplicity and sensitivity. Moreover, they can be made highly specific through the incorporation of monoclonal antibodies (Greenberg et al., 1983; Kovacs et al., 1989; Kristensson and Orvell, 1983). Standard labels include fluorescent dyes, with fluorescein isothiocyanate (FITC) being the most popular; enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP); and radioisotopes (Rose, 1999). Because isotopes are infrequently used in laboratory animal diagnostics, this discussion will focus on immunofluorescence and enzyme immunoassays. Most methods that utilize labeled antibodies or antigens are heterogeneous, solid-phase immunoassays. The term solid phase refers to the surface, frequently a glass microscope slide or wells in a plastic microtiter plate, to which an antigen or capture antibody is attached. Heterogeneous indicates that each incubation period is followed by a wash step to separate antigenantibody complexes bound to the solid phase from unbound antigen or antibody. The wash step also removes interfering

Table VI

Specificity of RodentParvovirusHAI

a

HAI titerc Antiserumb

KRV

H-1

MVM

RV H-1 virus MVM

160 ---

-20,480 --

10,240

Adapted from Siegl (1976). bKRV,Kilham'srat virus; MVM,minute virus of mice. c - titer less than 20. a

substances in a specimen that would compromise the sensitivity or specificity of a corresponding homogeneous assay. Immunofluorescence assay results are read with a fluorescence microscope or fluorometer directly following the final labeledantibody incubation and wash steps, whereas an additional incubation with substrate is required to develop the results of an enzyme immunoassay. Most substrates are chromogenic, producing a colored product, at a rate proportional to the quantity of enzyme-labeled antigen-antibody complexes that have attached to the solid phase. Color development can be read visually in a qualitative or semiquantitative fashion or with a spectrophotometer to obtain quantitative optical density readings (Chan, 1987; Mahoney and Chernesky, 1999; Rose, 1999; Voller et al., 1982). In diagnostic and experimental laboratory animal microbiology, the antigen assay methodology to which labeled antibodies are most frequently applied is immunocytochemistry for the identification of microbial antigens in cell cultures or animal tissues (Allen et al., 1981; Brownstein et al., 1981; Cera et al., 1994; Dick et al., 1996; Jacoby et al., 1975; Sundberg et al., 1989; Tanishita et al., 1984; Weir et al., 1986, 1988). Standard specimens for immunocytochemistry are cells or cryostat-cut tissue sections that have been fixed in cold acetone to preserve microbial antigens and make cell membranes permeable to antibodies. When diagnosing a disease retrospectively, however, it may be that only formalin-fixed, paraffin-embedded tissues are available. Immunochemical staining of such tissues can be performed, provided that tissue sections are first digested with trypsin to unmask microbial antigens (Brownstein and Barthold, 1982; Elias et al., 1987; Swoveland and Johnson, 1979). Immunocytochemistry methods are classified as direct or indirect (Fig. 3). Antibodies to the target microorganism are labeled in the direct method, which therefore has just one antibody incubation step. In the indirect method, the binding of unlabeled antigen-specific antibodies to a specimen is detected by labeled secondary antispecies IgG antibodies, also referred to as antiimmunoglobulins. Protein A and protein G, derived from the cell wall of Staphylococcus aureus and certain streptococci, respectively, bind certain IgG subclasses from various species and thus can sometimes be substituted for species-specific secondary antibodies (DeLellis, 1981; Hrapchak, 1980; Mahoney and Chernesky, 1999). Other popular modifications of the indirect method that amplify signal by increasing the concentration of enzyme bound to the solid phase are the peroxidaseantiperoxidase (PAP) and avidin-biotin-enzyme complex (ABC) techniques (Hsu et al., 1980; Milios and Leong, 1988; Nerurkar et al., 1982). The ABC system makes use of the strong interaction between avidin, an egg-white protein, or streptavidin from Streptomyces avidinii, and the low-molecular-weight vitamin biotin coupled to antibodies and enzymes (Wilchek and Bayer, 1984). The main advantages of the indirect method of immunochemical staining, in comparison with the direct method, are better sensitivity and the ability to perform a variety of

10. MICROBIOLOGICALQUALITYCONTROL FOR LABORATORYRODENTSAND LAGOMORPHS A. Direct

377

B. Indirect

~

Fluorescein-Labeled IgGAnti-SpeciesIgG

~ Agent-Specifc

Fluorescein-Labeled Agent-Specific Antibody

Antibody

Jf

J

Specimen

Fig. 3. Direct(A) and indirect (B) immunofluorescence.(Adaptedfrom Mahonyand Chernesky, 1999,Fig. 2, p. 206.)

tests without having to prepare labeled antigen-specific antibodies for each one. On the other hand, the increased sensitivity of an indirect method may be associated with more background, especially if the labeled secondary antibodies react with immunoglobulins in the tissue section. Background in enzyme immunoassays can also result from endogenous tissue enzymes, such as peroxidases, that nonspecifically catalyze the conversion of substrate to product (DeLellis, 1981). During the 1980s, enzyme immunoassays to detect microbial antigens in body fluids achieved widespread use in diagnostic microbiology as a whole, but not in laboratory animal health surveillance. However, there have been reports in which a commercial human rotavirus enzyme immunoassay was applied to the diagnosis of mouse rotavirus infections (Jure et al., 1988; Newsome and Coney, 1985). This was possible because the assay targeted a common, inner-capsid antigen shared by all human and animal group A rotaviruses, including those of mice (Greenberg et al., 1983). The assay employed a double antibody sandwich method (Fig. 4) in which rotavirus-specific anti-

4. Enzyme Substrate 3.

[~

Color

Enzyme-Labeled Detector Antibody

2. TestSpecimenwith MicrobialAntigen 1. Capture

Antibody Solid-PhaseSurface

Fig. 4. Doubleantibodysandwichenzymeimmunoassayfor microbialantigens. (AdaptedfromMahonyand Chernesky, 1999,Fig. 1A, p. 206.)

bodies, coated onto polystyrene beads, captured rotavirus antigens present in a fecal or intestinal specimen. The test sample was followed by HRP-conjugated rotavirus antibodies and substrate to demonstrate antigen binding to the beads. Interestingly, the authors of one study attributed a high prevalence of falsepositive results with this assay to a substance (probably a protein) in nonautoclaved feed that nonspecifically bound to the beads and activated the substrate (Jure et al., 1988). An inhibition enzyme immunoassay for the infectious diarrhea of infant rats (IDIR) group B rotavirus (formerly rotavirus-like virus) was developed to evaluate the relevance of such a method for diagnosing group B and other non-group A rotavirus infections in people (Vonderfecht et al., 1985, 1988). The limited sensitivity of antigen-detection solid-phase immunoassays because of background noise explains, in part, why few such assays have been developed for laboratory animal health surveillance. b.

M o l e c u l a r Diagnostics

Dramatic advances in molecular biology during recent years have coincided with a shift from antigen immunoassays to molecular assays for microbial genomic sequences. This shift has been most prohounced for tests performed directly on clinical specimens, because molecular methods, particularly the PCR, have proven to be substantially more sensitive than their immunoassay counterparts (Wilde et al., 1990). Molecular techniques are better able to differentiate among strains or isolates of microbial species than the traditional strain typing procedures, such as serotyping (Gentsch et al., 1992; Tenover, 1998; Tenover et al., 1994; Ushijima et al., 1992). However, it should be kept in mind that detection of a microbial genomic sequence does not necessarily indicate that infectious microorganisms are present. Just as the specificity of immunoassays is a characteristic of antigen-antibody reactions, so too is the specificity of molecular methods a consequence of the unique pairing that occurs between nucleotide bases on complementary strands of DNA or

378

WILLIAM R. SHEK AND DIANE J. GAERTNER

RNA. Double-stranded DNA will separate into single strandsm i.e., denature--at high temperature (e.g., 90~176 and renature according to complementary base pairing when incubated at a lower temperature (e.g., 65 ~C). This process, termed nucleic acid hybridization, can also occur between a strand of DNA and a strand of RNA (Cooper, 1997). Hybridization underlies the principal strategies for demonstrating target sequences of microbial DNA or RNA in clinical specimens. These strategies are (1) direct detection with a complementary DNA (cDNA) or cRNA probe and (2) biochemical amplification of a target (or probe) nucleic acid sequence (Tang and Persing, 1999; Tenover, 1998). The common formats for probe hybridization assays correspond to those employed for immunoassays. They include liquid phase, solid phase, and, in in situ hybridization, the molecular equivalent of immunocytochemistry. Liquid-phase hybridization assays have the advantages of simplicity and speed, but solid-phase assays are also popular in research and clinical laboratories. In a solid-phase assay, a nucleic acid specimen may be bound directly to a nitrocellulose or nylon membrane, or may be separated first by electrophoresis into fragments of different sizes and then blotted onto a membrane. The latter technique is named Southern blotting (after the developer E. M. Southern) if the reporter probe is used to detect DNA or Northern blotting (a play on words) when the probe hybridizes to RNA (Cundiff et al., 1994a; Hsu and Choppin, 1984). Alternatively, target sequences in the specimen can be captured with an unlabeled cDNA probe attached to a microtiter plate well (Goto and Itoh, 1996). Irrespective of the format, a probe hybridization assay involves (1) denaturing the sample nucleic acid; (2) incubating the probe together with the sample under conditions that permit stable probe-target hybrids to form; and (3) detecting the hybrids, usually by measuring the signal emitted by a label (Fig. 5). Reporter probes, like antibodies, can be labeled directly or indirectly with radioisotopes, enzymes that act on chromogenic or chemiluminescent substrates, or fluorophores. The specificity of a hybridization assay is a function of

the probe sequence and the stringency of the reaction conditions. Probes can be complementary to genomic sequences that identify the group, species, or strain of a microorganism. The usefulness of labeled antibody probes and nucleic acid probes for direct detection of microorganisms in clinical specimens is limited by fixed target quantities in specimens and background due to nonspecific binding of the labeled probes (Mahoney and Chernesky, 1999; Tang and Persing, 1999). Both the difficulties of isolating fastidious organisms and the sensitivity limitations of labeled probe assays have been bypassed by the recent development of practical and robust technologies for rapid biochemical amplification of target (or probe) nucleic acid sequences entirely in vitro. T h e best developed and most widely used of these, the PCR (polymerase chain reaction), was the invention for which Kary Mullis was awarded the Nobel Prize for Medicine in 1993 (Mullis, 1990). PCR has become the predominant methodology for demonstrating infectious agents in clinical specimens, including those from laboratory animals, because of its simplicity, speed, sensitivity, and specificity. The emphasis in laboratory animal diagnostics has been to develop PCR assays for viruses (Besselsen et al., 1995a, b; Eiden et al., 1991; Hjelle et aL, 1994; Kunita et al., 1992) and for other microorganisms that are difficult to cultivate or are noncultivable (Battles et aL, 1995; Beckwith et al., 1997; Cundiffet al., 1994b; Feldman et aL, 1999; Goto and Itoh, 1996). Viral PCR assays are replacing cultural isolation and the rodent antibody production tests to screen biological specimens for viral contamination (Chang et al., 1997; Chen and Plagemann, 1997; Riley et al., 1999; Yagami et al., 1995). They are also being used to diagnose laboratory animal infections (Casebolt et al., 1997; Matthaei et al., 1998; Shames et al., 1995; Weisbroth et al., 1999) and to test the environment for sources of adventitious infection (Henderson et al., 1998). It is unlikely, however, that the PCR will replace serology in routine viral surveillance, because many viral infections are short-lived and convalescence of the host is complete, making attempts at virus detection futile regardless of the assay sensitivity.

,•••E

Add labeled probe DNA nzyme Enzym~-~l, / Substrate ......Jy~'~~~ ~ " Enzyme ~ Color/Chemi~ : ~ uU U L ~ ~ = ~ ~ luminescence

~/ 7

Native DNA (target)

Heat ,.

~

Denatured (single stranded) DNA

Probe DNA hybridized to target DNA

Fig. 5. Hybridizationwith an enzyme-labeledDNAprobe. (AdaptedfromTenover, 1998,Fig. 14-1,p. 153.)

10.

MICROBIOLOGICALQUALITYCONTROL FOR LABORATORYRODENTS AND LAGOMORPHS

The specificity of amplification in the PCR is provided by synthetic oligonucleotide primers (15-20 bases long) that hybridize, or anneal, to complementary sequences in the target nucleic acid. The primers determine the sequences that are replicated, because the DNA polymerase used in the PCR can initiate synthesis of a complementary DNA strand only by extending a hybridized primer. The primers chosen for screening assays generally target conserved regions of the microbial genome, such as the parvovirus NS-1 gene (Irving et al., 1993), to minimize the occurrence of false-negative results. For diagnostic PCR assays, on the other hand, specificity is emphasized by selecting primers (and probes) that bind to species- or strain-specific genomic sequences (Battles et al., 1995; Beckwith et al., 1997; Besselsen et al., 1995b; Lu et al., 1995; Shames et al., 1995). Primers for bacterial PCR assays are generally designed from ribosomal RNA (rRNA) gene sequences because the rRNA genes have been extensively analyzed, and they contain both conserved and differential sequences (Greisen et al., 1994). In a standard PCR assay, two primers are designed to bind in

379

opposite directions to complementary strands of the target DNA. The sequence between the two primer-binding sites (usually 100-200 base pairs) is amplified exponentially with each PCR cycle, which consists of the three steps illustrated in Fig. 6. A PCR assay consists of 30-50 cycles, each lasting little more than a minute, that are performed automatically by a programmable heating block called a thermocycler. The automatic, rapid cycling that is the essence of the PCR is possible because the Taq bacterial DNA polymerase used is stable even at the high temperatures used to denature DNA (Cooper, 1997; Tang and Persing, 1999; Tenover, 1998). The PCR can be used to amplify RNA as well as DNA targets, but RNA targets must first be transcribed into cDNA templates by reverse transcriptase (RT). Heat-stable RT allows RT-PCR to be done in a single step. RT-PCR is particularly useful for detection of RNA viruses, e.g., MHV (Casebolt et al., 1997; Homberger et al., 1991; Matthaei et al., 1998; Taylor and Copley, 1993). Detection and analysis of PCR products are facilitated by the substantial quantity of target DNA that can be amplified from a small number of initial template copies. It is common by PCR

Fig. 6. Stepsof polymerase chain reaction (PCR). First, nucleic acid isolated from a clinical specimen is denatured at high temperature (e.g., 95~ Next, primers are allowedto anneal to their complementaryamplificationtarget sequences at a lower temperature (e.g., 55~ In the final step, the DNA polymerase synthesizes copies of the target sequencesby extending the primers. (Adaptedfrom Cooper, 1997,Fig. 3.27 p. 114.)

380

to obtain readily detectable quantities of DNA from just a single template copy. In contrast, approximately 100,000 copies of a target nucleic acid sequence are required for detection by blot hybridization (Cooper, 1997). Typically, the products of PCR are separated according to size by electrophoresis on eithidium bromide-stained agarose gels. When a gel is exposed to ultraviolet light, stained DNA fragments appear as fluorescent bands. The sizes of bands are compared with the expected product size to corroborate the specificity of the reaction. A PCR product can be further analyzed by digestion with restriction endonucleases that cleave double-stranded DNA at sites containing specific short nucleotide sequences (Xiao et al., 1992). Following digestion, the number and size of the bands in an electrophoretogram can help verify the identity of a PCR product or characterize the microorganism from which the product was amplified. For example, restriction enzyme analysis has been used to determine the species of Helicobacter detected by PCR with genus-specific primers (Riley et al., 1996a). Other more specific methods for analyzing the PCR product are probe hybridization and DNA sequencing. Microtiter plate-based hybridization assays that utilize colorometric or chemiluminescent detection systems are more practical than conventional blotting methods and can be 10- to 100-fold more sensitive than ethidium bromide-agarose gel electrophoresis (Tang and Persing, 1999). Amplification and hybridization occur concurrently in the fluorogenic 5'-nuclease assay in which an oligonucleotide probe, labeled with both a fluorescent reporter dye and a quencher dye, is included in the PCR reaction mixture. During the extension step of the PCR cycle, the probe, which anneals to the DNA template between the forward and reverse primers, is digested by the exonuclease activity of the Taq polymerase. Once separated from the quencher, the sequencespecific reporter dye signal can be read with a fluorometer (Gibson et al., 1996). The exquisite sensitivity of the PCR, which is its main advantage, is also its principal drawback. As was mentioned, it is not uncommon for a PCR assay to be capable of detecting a single copy of target nucleic acid, nor is it unusual for a single copy of template to be amplified 1 million-fold. Therefore, contamination of negative specimens with target DNA from previously amplified templates, positive controls, or positive samples represents a major challenge to use of the PCR for high-throughput testing of clinical specimens. Various measures are taken to prevent cross-contamination, including physical separation of preand postamplification procedures, decontamination of work surfaces with chemicals or UV irradiation, and enzymatic digestion or chemical inactivation of amplified template. Conversely, PCR sensitivity can be diminished by specimens such as feces or whole blood that alter the reaction environment or otherwise inhibit target amplification by the Taq polymerase (Wilde et al., 1990). This inhibition can be detected by including an internal assay control or by spiking a duplicate reaction with control template; inhibition is prevented by purifying DNA

WILLIAM R. SHEK AND DIANE J. GAERTNER

or RNA from the specimen. Because of the cost of reagents, patent royalties, equipment, and the space needed to separate preamplification from postamplification procedures, PCR assays will probably not be used in the immediate future for the diagnosis of infections that are easily demonstrated by conventional methods (Tang and Persing, 1999). 4.

Serology for Detection of Antibodies to Infectious Agents

Antibody immunoassays are the mainstay of viral surveillance in laboratory animals, because viral infections of immunocompetent animals are mostly transient, whereas viral antibody responses are easily detected for prolonged periods (Jacoby, 1986; Parker and Reynolds, 1968). In addition, a single specimen of serum can be tested for antibodies to a panel of viruses by assays that are inexpensive, rapid, sensitive, and specific (Smith, 1986b). Finally, viruses are very contagious and spread rapidly through a colony of animals kept in open cages. Under such conditions, the percentage of seropositives is high, and therefore the sample size for routine viral surveillance can be small. An important caveat is that seroconversion rates appear to be decreasing as the use of microisolation and ventilated-rack caging systems has become more common. Although serology is a sensitive and specific methodology for viral and mycoplasmal (Davidson et al., 1981) monitoring, its value for bacterial surveillance has been less clear. Because bacteria are genetically and antigenically complex and many antigens are shared across species, bacterial serology with whole-cell antigen is likely to detect cross-reacting antibodies to clinically irrelevant bacterial species or strains. Poor sensitivity can occur when antigen purified from one bacterial strain does not cross-react with antibodies to others (Manning et al., 1994; Motzel and Riley, 1991). Bacterial serology can also yield false-negative results when noninvasive bacteria that colonize the skin or mucous membranes do not stimulate detectable antibody production. Nonetheless, serology has been employed, along with other diagnostic methodologies, to monitor laboratory animals for infections with fastidious bacteria such as Clostridium piliforme (Riley et al., 1994; Waggie et al., 1987), Helicobacter hepaticus (Fox et al., 1996), and CAR bacillus (Lukas et al., 1987b; Matsushita et aL, 1987). Serologic assays have also been developed for gram-negative bacteria such as Pasteurella pneumotropica (Boot et al., 1995), P. multocida (Lukas et al., 1987a), and Bordetella bronchiseptica from which specific lipopolysaccharide antigen can be purified (Manning et al., 1987). Antibody assay methods include conventional, or traditional, tests such as complement fixation (CF), hemagglutination inhibition (HAI), and neutralization, as well as nonradioisotopic solid-phase immunoassays, notably the enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence assay (IFA) (Mahoney and Chernesky, 1999; Rose, 1999; Smith, 1986b). Most serologic tests are performed in a 96-well

381

10. MICROBIOLOGICAL QUALITY CONTROL FOR LABORATORY RODENTS AND LAGOMORPHS

microtiter plate format to facilitate automation and minimize reagent usage (Parker et al., 1965; Voller et al., 1982). An exception is the IFA, which is generally performed using Tefloncoated multiwell glass microscope slides (Lyerla and Forrester, 1979). When performed correctly, serologic tests include controis to distinguish between specific and nonspecific reactions and standard positive and negative control sera to verify assay sensitivity and specificity, respectively. a.

Traditional Serologic Methods

Although the CF method can be applied to test for antibodies to most infectious agents, it is no longer in routine use because it is time-consuming and not very sensitive (Schmidt, 1969). For viruses that agglutinate red blood cells, the HAI test method (Fig. 7), which is predicated on the ability of specific antibodies to inhibit virus-mediated hemagglutination, is still commonly employed as a confirmatory test, because it is simple to perform and can distinguish among antibodies to different species or strains of viruses (Table VI) (Chanock, 1979; Siegl, 1976; Smith et al., 1993b). In the standard HAI test method, each serum sample is titrated in duplicate. One dilution is incubated with virus; the other, without. When hemagglutination is detected in the virus wells but not in the control wells, the sample is considered HAI antibody-negative. If hemagglutination is inhibited in the virus wells, and the control wells show no evidence of hemagglutination, the result is recorded as a titer, which is the reciprocal of the highest serum dilution to inhibit virus-mediated hemagglutination. Significant titers (-> 10 or 20, depending on the virus) can also be caused by nonspecific inhibitors of hemagglutination, which can be removed by heat inactivation, enzymatic digestion, and other treatments (Hier-

Fig. 7. Viralhemagglutination inhibition (HAI) test. Serial dilutions of a serum specimen are incubated with viral antigen in V-bottom microtiter plate wells. A suspension of red blood cells is then added. The species of blood cells and the incubation temperature vary according to the virus. If the serum specimen contains antibodiesto the viralhemagglutinin, these will coat the virus and prevent it from agglutinating the red blood cells. Nonagglutinated red blood cells appearin the well bottom as a button that streams when the plate is tilted. Conversely, if the sample is HAI antibody-negative, red blood cells do not stream but instead blanket the well bottom, indicating that hemagglutination has occurred.

Table VII

HAI Interpretation Hemagglutination Antigen

Control m

+

+

Result Negative Positivea Agglutinationb

Positive if antibody titer 1>10 or 20, depending on viral antigen. bResult considered nonspecific.

a

holzer et al., 1969). Hemagglutination in the control wells indicates that the serum contains agglutinins that will mask the presence of specific antibodies (Table VII). b.

Solid-Phase Immunoassays

The indirect ELISA is the method most often used to screen serum samples for antibodies to infectious agents, because it is highly sensitive (Davidson et al., 1981; Ferner et al., 1987; Parker et al., 1979; Peters and Collins, 1981) and amenable to automation (Cerra et al., 1990). In addition, assay results can be read with a spectrophotometer and sent to a computer to be compiled into reports. The steps of the indirect ELISA are depicted in Fig. 8 (Mahoney and Chernesky, 1999; Voller et al., 1982). To detect nonspecific antibody binding, each sample is simultaneously incubated in an antigen-coated test well and a separate tissue-control well (Fig. 9). A sample that gives positive or nonspecific results by ELISA (or another primary assay) should always be retested by an alternative method. A modification of the indirect ELISA method that is particularly useful for evaluating the specificity of a preliminary positive result is Western blotting (Fig. 10). It is comparable to the DNA blotting methods alluded to above, except that electrophoresis is used to separate proteins instead of DNA fragments, and the blot is

Fig. 8. Indirectenzyme-linkedimmunosorbentassay (ELISA)for microbial antibodies. (Adaptedfrom Mahony and Chernesky, 1999,Fig. 4A, p. 208.)

382

Fig. 9. Interpretation of ELISA results by comparison of color in antigen and tissue control wells. For a viral antibody assay, the tissue-control (TC) well is coated with an extract of uninfected cells of the type used to propagate the virus. For a microorganism that is not grown in cell culture, the tissue-control well can be coated with a related but antigenically distinguishable microorganism. For example, the tissue-control well for Mycoplasma pulmonis might be coated with M. arthritidis. Nonspecific binding is discouraged by using special protein solutions for "blocking" wells after the antigen-coating step and as diluents for serum and conjugate. The sample is considered antibody-negative when color development in the antigen (AG) well is minimal. The sample is evaluated as antibody-positive when the intensity of color in the antigen well is moderate to strong, but little or no color develops in the tissue control well. A reaction is nonspecific when moderate to strong color develops in the tissue-control well in addition to the antigen well. Results may be read visually or with a spectrophotometer.

probed with labeled antibody instead of complementary nucleic acid (Mahoney and Chernesky, 1999; Minion et al., 1984; Motzel and Riley, 1991). The IFA is rarely used as a primary screening assay, although it is generally as sensitive as the corresponding ELISA (Kraft et al., 1982; Parker et al., 1979; Smith, 1983a,b; Smith et al., 1984). The reason for its rare use for this purpose is that the IFA does not lend itself to automation, and the results must be read manually. The steps of the IFA are similar to those of the indirect ELISA. Briefly, virus-infected cells and uninfected cells are fixed to wells on a glass slide, using cold acetone. The binding of primary antibodies to the solid phase in the IFA is demonstrated with an FITC-labeled antispecies immunoglobulin. After being washed to remove unbound conjugate, slides are covered with buffered mounting medium and examined with a fluorescence microscope. Bright, granular fluorescence is typical of an antibody-virus reaction, whereas diffuse fluorescence is characteristic of nonspecific reactions. The location of fluorescence is also an important factor. In the case of certain DNA viruses, such as the rodent parvoviruses (MVM, KRV, and H-1), strong nuclear fluorescence is characteristic (Cross and Parker, 1972). The ability of IFA to include evaluation of fluorescence morphology and location in the interpretation of reactions is its

W I L L I A M R. SHEK AND DIANE J. G A E R T N E R

Fig. 10. Western blot analysis of antibody specificity. In confirmatory Western blot analysis, antigen proteins are denatured and separated according to their molecular weight by polyacrylamide gel electrophoresis with the detergent sodium dodecyl sulfate (SDS-PAGE). The electrophoresis gel is blotted onto a nitrocellulose membrane, and strips cut from the membrane are incubated with primary sera, including an immune control (C +), a nonimmune control ( C - ) , and the test samples (S 1-$6). The assay is developed according to the steps of the indirect ELISA. The enzyme-substrate reaction produces bands at sites in the blot where primary antibody bound. The specificity of a test serum reaction is evaluated by comparison with the C + reaction. When the test serum band pattern matches that of the C + or is consistent with a known pattern for the agent, the test serum result is interpreted as positive (+). If, on the other hand, the test serum pattern does not match that of the C + or other known pattern, the test serum reaction is interpreted as nonspecific (NS). The absence of bands is a negative result ( - ) . (Adapted from Mahony and Chernesky, 1999, Fig. 6B, p. 209.)

major advantage over other serologic methods. For the rodent parvoviruses, the IFA detects cross-reacting antibodies better than the standard ELISA because the virus-infected cells that constitute the IFA antigen contain nonstructural viral proteins not found in conventional ELISA antigen consisting of purified viral particles (Smith et al., 1993b). Using recombinant technology, however, it is now possible to produce large quantities of nonstructural viral antigen for ELISA (Riley et al., 1996b). Because recombinant antigens are noninfectious, they are especially appropriate for detecting antibodies to zoonotic viruses such as LCMV (Homberger et al., 1995).

B.

Design and Implementation of a Surveillance Program

To develop a microbiological monitoring program that is both effective and practical, choices need to be made regarding the

10. MICROBIOLOGICALQUALITY CONTROL FOR LABORATORYRODENTS AND LAGOMORPHS agents for which to screen, the type and number of animals to be sampled, and the sampling frequency. Program implementation is accomplished by systematically recording these choices and incorporating them into testing schedules. 1. Selection of Infectious Agents

In addition to being based on laboratory animal health and research effects, the selection of infectious agents to be excluded from rodent colonies is determined by the colony microbiological status. Gnotobiotic animal colonies must be monitored for any exogenous microorganism. SPF rodent colonies are expected to be free of ectoparasites, metazoan endoparasites, and pathogenic enteric protozoa. They are also expected to test negative for antibodies to most exogenous viruses, regardless of pathogenicity. This is because viruses are obligate intracellular parasites that alter the metabolism of the host cells they infect (Oldstone et al., 1982). The bacteria that need to be excluded from an SPF colony depend on the immune status of the animals in it. As mentioned, immunocompetent animals remain healthy and suitable for most research provided they are kept free from infection with a small number of "primary" pathogens. The list of bacteria for immunodeficient animals is expanded to include opportunists that are likely to cause disease in these strains. The lists of etiologic agents for which SPF rodents and rabbits are monitored are largely the same throughout the world, with some differences between those used in the United States and those used in Europe. The agent list and reporting formats in Europe are approved by the Federation of European Laboratory Animal Science Associations (FELASA) (Rehbinder et al., 1996). The microorganisms selected for monitoring in mice and rats have been compiled, and the basis for their selection has been categorized, in the "Manual of Microbiologic Monitoring of Laboratory Animals," authored by Japanese and American scientists and published by the U.S. National Institutes of Health (Waggie et al., 1994). These lists of microbes can be expected to expand, although not dramatically, as husbandry practices evolve, as additional studies are published on the clinical and research effects of particular infectious agents, and as new pathogens are discovered. 2.

Sampling

Accurate, meaningful results require that an adequate number of the appropriate animals be sampled on a sufficiently frequent basis. The animals selected for testing should be representative of the microbiological condition of the colony as a whole. This is best accomplished by selecting animals of different ages, sexes, and strains, because infections and positive assay results may have an age-, sex-, or strain-dependent distribution. Alternatively, sentinel animals, typically but not always of the same species as that being monitored, can be tested. To be used successfully, sentinels should be housed in a manner that maximizes their exposure to the microflora of the

383

principal animals being monitored. In general, infections are transmitted most efficiently through animal contact. Fomite transmission, commonly via soiled bedding, is usually effective, whereas airborne spread can be unreliable even for highly infectious viruses (Artwohl et al., 1994; Cundiff et al., 1995; Dillehay et al., 1990; Parker and Reynolds, 1968; Thigpen et al., 1989; Yang et al., 1995). Airborne spread is further slowed when microisolation or ventilated caging is used. There are occasions when it is helpful to use sentinels of one species to monitor principals of a second. One such occasion is when little is known about the viruses that infect a species, which is the case for gerbils (Clark, 1984). It is arguably more meaningful to do serology on sentinel mice or rats to determine whether gerbils are shedding murine viruses than to test the gerbils themselves. A different species might be chosen as a sentinel because it is more likely to become ill following infection than is the principal species. Because gerbils are uniquely susceptible to Tyzzer's disease, they have been used as sentinels to detect latent Clostridium piliforme infections in other rodent species (Gibson et al., 1987). Animal selection is influenced by the diagnostic methodology. For serology, the animals sampled should be immunocompetent and able to mount a strong serum antibody response to infection. Such a response is typical of disease-resistant inbred strains (Brownstein et al., 1981) and outbred stocks (Parker et al., 1978). Because serum antibodies take, on average, 2 - 3 weeks to develop (Parker and Reynolds, 1968; Peters and Collins, 1983; Smith, 1983a), sentinels should be kept in a colony for at least 1 month. Sick animals should be allowed to convalesce and seroconvert before they are tested. In the case of production colonies, retired breeders are recommended because they have had ample time to become infected and seroconvert. For pathology, bacteriology, and parasitology, it is especially important to sample animals of multiple ages, because the prevalence of infection with some bacteria and parasites is agedependent. Along with, or as an alternative to, sampling multiple age groups, the diagnosis of certain latent infections may be facilitated by testing immunodeficient or immunosuppressed animals. Immunosuppression to provoke Tyzzer's disease is used in the diagnosis of C. piliforme infections (Riley et al., 1994; Waggie et al., 1981). Guidelines regarding sample sizes for detection of adventitious infections have been developed by using various statistical formulas. In essence, these formulas demonstrate that the sample size required for detecting infection with a certain degree of confidence increases as the prevalence of infection, or positive reactors, decreases. Sample size is also related to the number of animals in a colony in a way that most nonstatisticians find paradoxical. That is, the number of animals that must be sampled to achieve a certain level of confidence increases as the colony size decreases (Dubin and Zietz, 1991). It is important to distinguish between prevalence and incidence. Prevalence is the percentage of positive animals at a point in time in

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WILLIAM R. SHEK AND DIANE J. GAERTNER

a designated area. Incidence is the percentage of new positives over a designated period. The binomial distribution formula for determining sample size is often cited in discussions of laboratory animal health surveillance (Small, 1984). According to this formula, if the prevalence of infection or positive reactors is 30%, 8-10 animals must be sampled to realize a 95% probability of detecting at least 1 infected or assay-positive animal. To achieve the same level of confidence for a presumed prevalence of 10% requires a sample size of 25-30 animals. The correctness of sample sizes calculated with the binomial formula depends on certain assumptions being met--including random spread of infection and a colony size of at least 100 animals m and on the accuracy of the prevalence estimate. Estimates of the prevalence of infection that are conservatively low for animals kept in open cages may be too high for those housed in microisolators. The trend away from open cages toward filter-top microisolation cages may result in smaller effective population sizes and adventitious infections that spread more slowly and have a lower prevalence. As just shown, the calculated sample size for a low prevalence of infection, such as 10%, can be utterly unrealistic. In addition, the relevance of the sampling formulas to sentinel animals kept on pooled, soiled bedding has not been addressed. Thus, the use of statistical formulas to determine sample size is of uncertain value. In practice, the number of animals monitored ends up being a compromise between the desire to achieve a high degree of certainty versus the availability of animals for monitoring and the cost of testing. The frequency of testing should be adjusted based on historical contamination rates (Selwyn and Shek, 1994). As mentioned, gnotobiotic and immunodeficient colonies are usually maintained in isolators or microisolators to achieve the high level of biosecurity necessary to sustain a defined or limited microflora. Contamination of gnotobiotic colonies with extraneous bacteria and fungi through physical defects in an isolator or inadequately disinfected supplies is more common than are adventitious viral and parasitological infections. Therefore, bacteriology should be performed more frequently than serology and parasitology on gnotobiotic colonies. Bacteriology should also be performed often on immunodeficient rodents for which

many opportunistic bacteria are pathogenic, but it can be done less regularly on immunocompetent SPF colonies because most bacterial contaminants of isolator-, microisolator-, and barrierreared rodents are not primary pathogens. In barrier rooms where adventitious infections are most frequently caused by viruses, serology should be performed more often than bacteriology and parasitology. 3.

Implementation

Implementation of health surveillance requires a systematic approach for translating the decisions on agent selection and sampling into a program of consistent and routine testing. The first step in this process is to record the viruses, bacteria, fungi, and parasites for which each species is to be monitored. Then assays for these agents are combined into serology, bacteriology, parasitology, and pathology panels. Serology panels consist of antibody assays identified by method and agent. Bacteriology panels are composed of sampling sites and lists of the primary pathogens and opportunists to be found at these sites. Pathology panels specify the tissues and organs to be examined. Several panels may be defined for a species in order to reflect the frequency with which certain infectious agents have been found. In the case of serology, basic profiles that include commonly found viruses are performed more often than are comprehensive profiles to which rarely detected agents have been added. Next, test protocols are constructed by combining assay panels with the appropriate samples. For example, retired breeders might be selected for serology, whereas parasitology would be performed on weanlings and young adults (Table VIII). Finally, testing frequencies are combined with test protocols to form schedule templates (Table IX), which are assigned to colonies to create schedules (Table X). This may be done manually or by computer. On the dates indicated in the schedule, the number and type of samples designated in the protocol are collected and submitted to a diagnostic laboratory. A submission form that contains the protocol information should be sent with the samples. Result reports should be analyzed and filed in an organized fashion (e.g., according to facility, room, and species).

Table VIII

Rat HealthMonitoringProtocol Bacteriology Age (number) Retired Breeder (4) 8-12 wks (4) 4--5 wks (4)

Serology

Pathology

Nasal~Cecum

+

+

+

+

+ +

+

Parasitologyo Lymph

Ecto

+

+

Endo

+ a

_~_ +

For lymphnode culture for Corynebacterium kutscheri. bMicroscopicexaminationsof skin and pelage for ectoparasites (Ecto) and of gastrointestinaltract for helminths (Endo) and protozoa(Proto).

a

Proto

385

10. MICROBIOLOGICAL QUALITY CONTROL FOR LABORATORY RODENTS AND LAGOMORPHS

Table IX Schedule Template

Step

Protocol

1 2 3 4

Comprehensive health monitoring b Serology only Serology only Comprehensive health monitoring

a

NUMBER OF ANIMALS

Offset a (weeks)

IDEAL TEST

l N

KNOWN

Weeks to next step. bComprehensive health monitoring includes serology, bacteriology, pathology, and parasitology.

C.

~

KNOWN

4 4 4

REACTIONINTENSITY/TITER IV DIVIDING LINE

Interpretation of Results

When gnotobiotic or SPF laboratory animals have been used from the start, the interpretation of diagnostic test results is, for the most part, qualitative. The goal is to determine whether the animals tested have been exposed to a particular infectious agent. Accurate quantification of antibody levels or numbers of bacteria, for example, is only important insofar as clearly negative and positive results are easier to interpret than are equivocal results near the dividing line between positive and negative. The ideal test is one that in all cases clearly distinguishes between exposed and unaffected animals. With a typical test, however, a certain percentage of results are inaccurate, in that

NUMBER OF ANIMALS

TYPICAL TEST

l

FALSE 7 NEGATIVES

DIVIDING~I~ FALSE LINE POSITIVES

Fig. 11. Comparison of ideal and typical serology tests. (From Weisbroth et al., 1998, Fig. 6, p. 283.)

Table X Colony Schedule Colony: Species: Start Date:

X Rat 1 Jan 98 Test date

Protocol

1 Jan 98 29 Jan 98 26 Feb 98 26 Mar 98

Comprehensive testing Serology only Serology only Comprehensive testing

samples from unaffected animals may give false-positive reactions and those from exposed animals may yield false-negative results (Fig. 11) (Tyler and Cullor, 1989). To this point in the chapter, the terms sensitivity and specificity have been used in their analytical sense. In that context, sensitivity is the ability of an assay to detect small amounts of analyte (e.g., antigen, antibody, or target DNA); specificity describes the selectivity of an assay reaction. These terms are defined somewhat differently in a diagnostic or statistical context. A sensitive assay is one that produces a low percentage of false-negative results or, conversely, a high percentage of truepositive results in tests performed on exposed animals. A spe-

cific assay is one that gives a low percentage of false-positive resuits or, conversely, a high percentage of true-negative results in tests performed on unaffected animals (Fig. 12) (Zweig and Robertson, 1987).

Assay result

',

Positive

~

Microbial status Exposed Unaffected .... TP

Negative i

i

i

FP

FN .

.

.

Sensitivity =

I

.

TN ,

TP TP + FN

TN Specificity = . . . . . . . . . . . . TN +FP

,,,,,

x 100

x 100

Fig. 12. Definition of assay sensitivity and specificity. TP, True positive; FP, false positive; TN, true negative; FN, false negative. (From Weisbroth et al., 1998, Fig. 7, p. 284.)

386

W I L L I A M R. SHEK AND DIANE J. G A E R T N E R

Table Xl Examples of Sample Selection Errors Result False negative

Methodology Serology Bacteriology/parasitology All

False positive

Serology

All

Error Acutely ill; serum antibodies not yet detectable Immunodeficient or immunosuppressed; weak or no antibody response Older and recovered from infection Site where organism is not resident Small sample size Sentinels not adequately exposed via soiled bedding or contact to infectious agents carried by principals Rodent strain with autoimmune disease a Immunized or inoculated with biological material (e.g., tumor cells) a Maternal antibodies b Sentinels housed under less strict conditions than principals (e.g., principals kept in microisolation cages, but sentinels are in open cages)

Sera from animals with autoimmune disease or from those inoculated with biological materials may contain antibodies that react with microbial or nonmicrobial constituents in the antigen preparation. Antibodies to nonmicrobial constituents may not be detected in the control, leading to a false-positive result. bFalse positive in that maternal antibodies are not a response by the animal sampled to an infection. a

Besides being a consequence of the limits of test sensitivity and specificity, false-positive and false-negative results can be due to sample selection and laboratory errors. Examples of sample selection errors are shown in Table XI. Myriad laboratory errors can cause inaccurate results, including improper sample preparation and storage, sample mix-ups, deviation from accepted procedures, and result transcription mistakes. False-positive results should be suspected when reactions are borderline-positive or the prevalence of positive specimens is low. As demonstrated in Fig. 13, the predictive value of positive results for a highly specific assay becomes negligible when the percentage of positive samples is low, e.g., less than 15% (LaRegina and Lonigro, 1988; Zweig and Robertson, 1987). First-time positive findings should always be confirmed before acting. Confirmation is accomplished by repeat testing of the positive samples, by testing additional samples, and by using alternative assays and diagnostic methodologies to corroborate primary test results. For example, sera that are Mycoplasma pulmonis ELISA-positive might be repeat-tested for specific antibodies by IFA. Additional animals from the suspect colony could be cultured for mycoplasma and examined grossly and microscopically for lung lesions. Finally, mycoplasma isolates could be identified as M. pulmonis with species-specific antisera or by PCR with a species-specific primer set. Once results are confirmed, the options for eliminating or containing an infection discussed in Section III should be followed. It is worth reemphasizing that it is counterproductive to start a new SPF colony without first investigating the sources of the infection and making the necessary procedural and facility modifications to prevent a recurrence. In summary, no diagnostic test always gives accurate results. False-positive and false-negative results occur because of the incomplete specificity or sensitivity of tests and because of

TP

=

PV (+)

TP + FP

x 100

10% Prevalence i

iii

ii

ii

i

i

i

i

i

Microbial status Exposed Unaffec!ed

Assay result Positive ,

9800

Negative .

.

4500

i,

,

.

.

'

200

'1

85,500

.

9800 PV (+)=

i

i

iiii

ii

i

9800 + 4500

x 100 = 68.5%

1% Prevalence

iiii |11

.

.

.

.

.

.

.

.

.

.

.

.

Microbial status Assay result .......... Exposed . . . . . . . Unaffected ,

,

,,

,

Positive

. . . . . . . . . . .

980

Negative ,,

.

.

.

.

.

.

.

.

.

.

.

.

.

.

J

,,

4950

20 ,,

,

94,050 .

980 PV (+) = . . . . . . . . 980 + 4950

llll

x 100 = 16.5%

Fig, 13. Effect of prevalence on the predictive value of positive results (PV [ + ]) for an assay with a specificity of 95%. TP, true positive; FP, false positive. (From Weisbroth et al., 1998, Fig. 8, p. 284.)

10. MICROBIOLOGICAL QUALITY CONTROL FOR LABORATORY RODENTS AND LAGOMORPHS

sample selection and laboratory errors. Consequently, it is prudent to always confirm unexpected positive findings before deciding on a course of action. This is accomplished by repeat testing of the same and additional samples, using a variety of diagnostic methodologies.

REFERENCES

Allen, M. A., Clarke, G. L., Ganaway, J. R., Lock, A., and Werner, R. M. (1981). Pathology and diagnosis of mousepox. Lab. Anim. Sci. 31, 599-608. Anderson, L. M., Leary, S. L., and Manning, P. J. (1983). Rat-bite fever in animal research laboratory personnel. Lab. Anim. Sci. 33, 292-294. Artwohl, J. E., Cera, L. M., Wright, M., Medina, L., and Kim, L. J. (1994). The efficacy of a dirty bedding sentinel system for detecting Sendai virus infection in mice: comparison of clinical signs and seroconversion. Lab. Anim. Sci. 44, 73-75. Avery, B. (1996). HEPA and ULPA filter testing. In "NAFA Guide to Air Filtration." (K. Sufka, R. Harden, and S. M. McKenna, eds.), pp. 8.1-8.7. National Air Filtration Association, Washington, D.C. Barthold, S. W., Smith, A. L., Lord, P. E S., Bhatt, P. N., Jacoby, R. O., and Main, A. J. (1982). Epizootic typhlocolitis in suckling mice. Lab. Anim. Sci. 32, 376-383. Barthold, S. W., Smith, A. L., and Povar, M. L. (1985). Enterotropic mouse hepatitis virus infection in nude mice. Lab. Anim. Sci. 35, 613-618. Battles, J. K., Williamson, J. C., Pike, K. M., Gorelick, P. L., Ward, J. M., and Gonda, M. A. (1995). Diagnostic assay for Helicobacter hepaticus based on nucleotide sequence of its 16S rRNA gene. J. Clin. Microbiol. 33, 1344-1347. Beards, G. M. P. J. N., Thouless, M. E., and Flewett, T. H. (1980). Rotavirus serotypes by serum neutralisation. J. Med. Virol. 5, 231-237. Beckwith, C. S., Franklin, C. L., Hook, R. R., Besch-Williford, C., and Riley, L. K. (1997). Fecal PCR assay for diagnosis of Helicobacter infection in laboratory rodents. J. Clin. Microbiol. 35, 1620-1623. Behnke, J. M. (1975). Aspiculuris tetraptera in wild Mus musculus. The prevalence of infection in male and female mice. J. Helminthol. 49, 85-90. Besselsen, D. G., Besch-Williford, C., Pintel, D. J., Franklin, C. L., Hook, R. R., and Riley, L. K. (1995a). Detection of newly recognized rodent parvoviruses by PCR. J. Clin. Microbiol. 33, 2859-2863. Besselsen, D. G., Besch-Williford, C., Pintel, D. J., Franklin, C. L., Hook, R. R., and Riley, L. K. (1995b). Detection of H- 1 parvovirus and Kilham rat virus by PCR. J. Clin. Microbiol. 33, 1699-1703. Bhatt, P. N., and Jacoby, R. O. (1985). Epizootiological observations of natural and experimental infection with sialodacryoadenitis virus. Lab. Anim. Sci. 35, 129-134. Bhatt, P. N., and Jacoby, R. O. (1987). Effect of vaccination on the clinical response, pathogenesis, and transmission of mousepox. Lab. Anim. Sci. 37, 610-614. Bhatt, P. N., Downs, W. G., Buckley, S. M., Casals, J., Shope, R. E., and Jonas, A. M. (1981). Mousepox epizootic in an experimental and a barrier mouse colony at Yale University. Lab. Anim. Sci. 31, 560-564. Bhatt, P. N., Jacoby, R. O., and Barthold, S. W. (1986a). Contamination of transplantable murine tumors with lymphocytic choriomeningitis virus. Lab. Anim. Sci. 36, 136-139. Bhatt, P. N., Jacoby, R. O., Morse, H. C., and New, A. E. (1986b). "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research." Academic Press, Orlando, Florida. Block, J. C., and Schwartzbrod, L. (1989). Waterborne viruses. In "Detection

387

and Identification of Viruses in Water Systems," pp. 1-22. VCH Publishers, New York. Block, S. S. (1991). Definition of terms. In "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.), pp. 18-25. Lea and Febiger, Gainesville, Florida. Bonnard, G. D., Manders, E. K., Campbell, D. A., Herbermann, R. B., and Collins, M. J. (1976). Immunosuppressive activity of a subline of mouse EL-4 lymphoma: evidence for minute virus of mice causing the inhibition. J. Exp. Med. 143, 187-205. Boot, R., Thuis, H. C. W., Veenema, J. L., and Bakker, R. H. G. (1995). An enzyme-linked immunosorbent assay (ELISA) for monitoring rodent colonies for Pasteurella pneumotropica antibodies. Lab. Anim. 29, 307-313. Brachman, P. S. (1996). Epidemiology, In "Medical Microbiology" (S. Baron, ed.), pp. 145-152. Univ. of Texas Medical Branch at Galveston. Brammer, D. W., Dysko, R. C., Spilman, S. C., and Oskar, P. A. (1993). Elimination of sialodacryoadenitis virus from the rat production colony by using seropositive breeding animals. Lab. Anim. Sci. 43, 633-634. Brinton, M. A. (1982). Lactate dehydrogenase elevating virus. In "The Mouse in Biomedical Research: Diseases" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), pp. 194-208. Academic Press, New York. Brock, T. D. (1970). The procaryotic cell: Microscopical methods. In "Biology of Microorganisms." pp. 19-29. Prentice-Hall, Englewood Cliffs, New Jersey. Broome, C., Pinner, R., and Schuchat, A. (1998). Listeria monocytogenes. In "Infectious Diseases" (S. L. Gorbach, J. G. Bartlett, and N. R. Blacklow, eds.), pp. 1750-1755. Saunders, Philadelphia. Brownstein, D. G. (1986). Sendai virus. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 37-61. Academic Press, Orlando, Florida. Brownstein, D. G., and Barthold, S. W. (1982). Mouse hepatitis virus immunofluorescence in formalin or Bouin's-fixed tissues using trypsin digestion. Lab. Anim. Sci. 32, 37-39. Brownstein, D. G., Smith, A. L., and Johnson, E. A. (1981). Sendai virus infection in genetically resistant and susceptible mice. Am. J. Pathol. 105, 156-163. Brownstein, D. G., Barthold, S. W., Adams, R. L., Terwilliger, G. A., and Aftosmis, J. G. (1985). Experimental Corynebacterium kutscheri infection in rats: bacteriology and serology. Lab. Anim. Sci. 35, 135-138. Casebolt, D. B., Qian, B., and Stephensen, C. B. (1997). Detection of enterotropic mouse hepatitis virus fecal excretion by polymerase chain reaction. Lab. Anim. Sci. 47, 6-10. Cera, L. M., Artwohl, J. E., Wright, M., and Kim, L. J. (1994). Immunohistochemical detection of localized Sendai virus antigen in preserved mouse tissue. Lab. Anim. Sci. 44, 88-90. Cerra, M. A., Oskar, P. A., and Shek, W. R. (1990). Robotic enzyme-linked immunosorbent assay (ELISA) system for detection of rodent viral antibodies. Lab. Robotics Autom. 2, 119-131. Chan, D. W. (1987). General principle of immunoassay. In "Immunoassay: A Practical Guide" (D. W. Chan and M. T. Perlstein, eds.), pp. 1-23. Academic Press, Orlando, Florida. Chang, A., Havas, S., Borellini, E, Ostrove, J. M., and Bird, R. E. (1997). A rapid and simple procedure to detect the presence of MVM in conditioned cell fluids or culture media. Biologicals 25, 415-419. Chanock, R. M. (1979). Parainfluenza viruses. In "Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections" (E. H. Lennette and N. J. Schmidt, eds.), pp. 611-632. American Public Health Association, Washington, D.C. Chen, Z., and Plagemann, P. G. (1997). Detection of lactate dehydrogenaseelevating virus in transplantable mouse tumors by biological and RT-PCR assays and its removal from the tumor cell. J. Virol. Methods 65, 227-236. Childs, J. E., Glass, G. E., Korch, G. W., and LeDuc, J. W. (1989). Effects of hantaviral infection on survival, growth, and fertility in wild rat (Rat-

388 tus norvegicus) populations of Baltimore, Maryland. J. Wildl. Dis. 25, 469-476. Choi, Y. C., and Hsiung, G. D. (1978). Cytomegalovrius infection in guinea pigs. 2. Transplacental and horizontal transmission. J. Infect. Dis. 138, 197-202. Clark, J. D. (1984). Biology and diseases of other rodents. In "Laboratory Animal Medicine" (J. Fox, B. Cohen, and E Loew, eds.), pp. 183-206. Academic Press, Orlando, Florida. Clarke, G. L., Coates, M. E., Eva, J. K., Ford, D., Milner, C. K., O'Donoghue, P. N., Scott, P. P., and Ward, R. J. (1977). Dietary standards for laboratory animals. Lab. Anim. 11, 1-28. Clifford, C. B., Walton, B. J., Reed, T., Coyle, M. B., White, W. J., and Amyx, H. L. (1995). Hyperkeratosis in athymic nude mice caused by a coryneform bacterium: Microbiology, transmission, clinical signs, and pathology. Lab. Anim. Sci. 45, 131-139. Cohen, M. L. (1998). Epidemiology of community-acquired infections. In "Infectious Diseases" (S. L. Gorbach, J. G. Bartlett, and N. R. Blacklow, eds.), pp. 101-122. Saunders, Philadelphia. Cole, G. A., and Nathanson, N. (1974). Lymphocytic choriomeningitis virus. Prog. Med. Virol. 18, 94-119. Collins, M. J., and Parker, J. C. (1972). Murine virus contaminants of leukemia viruses and transplantable tumors. J. Natl. Cancer Inst. 49, 1139-1143. Compton, S. R., Barthold, S. W., and Smith, A. L. (1993). The cellular and molecular pathogenesis of coronaviruses. Lab. Anim. Sci. 43, 15. Cooper, G. M. (1997). Fundamentals of molecular biology. In "The Cell," pp. 87-132. ASM Press, Washington, D.C. Corning, B. E, Murphy, J. C., and Fox, J. G. (1991). Group G streptococcal lymphadenitis in rats. J. Clin. Microbiol. 29, 2720-2723. Cross, S. S., and Parker, J. C. (1972). Some antigenic relationships of the murine parvoviruses: Minute virus of mice, rat virus, and H-1 virus. Proc. Soc. Exp. Biol. Med. 139, 105-108. Cundiff, D. C., Besch-Williford, C., and Riley, L. K. (1992). A review of the ciliaassociated respiratory bacillus. Charles River Lab. Ref Pap. (Fall), 1-4. Cundiff, D. C., Besch-Williford, C. L., Hook, R. R., Franklin, C. L., and Riley, L. K. (1994a). Characterization of cilia-associated respiratory bacillus isolates from rats and rabbits. Lab. Anim. Sci. 44, 305-312. Cundiff, D. C., Besch-Williford, C., Hook, R. R., Franklin, C. L., and Riley, L. K. (1994b). Detection of cilia-associated respiratory bacillus by PCR. J. Clin. Microbiol. 32, 1930-1934. Cundiff, D. C., Riley, L. K., Franklin, C. L., Hook, R. R., and Besch-Williford, C. (1995). Failure of a soiled bedding sentinel system to detect cilia-associated respiratory bacillus infection in rats. Lab. Anim. Sci. 45, 219-221. Davidson, M. K., Lindsey, J. R., Brown, M. B., Schoeb, T. R., and Cassell, G. H. (1981). Comparison of methods for detection of Mycoplasma pulmonis in experimentally and naturally infected rats. J. Clin. Microbiol. 14, 646-655. Deibel, R., Woodall, J. P., Decher, W., and Schryver, G. D. (1975). Lymphocytic choriomeningitis virus in man. J. Am. Med. Assoc. 232, 501-504. DeLellis, R. A. (1981). Basic techniques of immunohistochemistry. In "Diagnostic Immunohistochemistry" (R. DeLellis, ed.), pp. 7-15. Masson Publ., New York. Denyer, S. P. (1992). Filtration sterilization. In "Principles and Practice of Disinfection, Preservation, and Sterilization" (A. D. Russell, W. B. Hugo, and G. A. J. Ayliffe, eds.), pp. 557-572. Blackwell Science, Oxford. deSousa, M., and Smith, A. (1989). Comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses. J. Clin. Microbiol. 27, 185-187. Dick, E. J., Kittell, C., Meyer, H., Farrar, P. L., Ropp, S. L., Esposito, J. J., Buller, R. M. L., Neubauer, H., Kang, Y. H., and McKee, A. E. (1996). Mousepox outbreak in a laboratory mouse colony. Lab. Anim. Sci. 46, 602-611. Dillehay, D. L., Lehner, N. D. M., and Huerkamp, M. J. (1990). The effectiveness of a microisolator cage system and sentinel mice for controlling and detecting MHV and Sendai virus infections. Lab. Anim. Sci. 40, 367-370.

WILLIAM R. SHEK AND DIANE J. GAERTNER

Doane, E W. (1992). Electron microscopy and immunoelectron microscopy. In "Clinical Virology Manual" (S. Specter, and G. Lancz, eds.), pp. 89-109. Elsevier, New York. Dubin, S., and Zietz, S. (1991). Sample size for animal health surveillance. Lab. Anim. 20, 29-33. Dychadala, G. R. (1991). Chlorine and chlorine compounds. In "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.), pp. 131-151. Lea and Febiger, Gainesville, Florida. Eaton, G. J., Lerro, A., Custer, R. P., and Crane, A. R. (1982). Eradication of Sendai pneumonitis from a conventional mouse colony. Lab. Anim. Sci. 32, 384-386. Eiden, J. J., Wilde, J., Fir0ozmand, E, and Yolken, R. (1991). Detection of animal and human group B rotaviruses in fecal specimens by polymerase chain reaction. J. Clin. Microbiol. 29, 539-543. Elias, J. M., LaNeve, D., Gabore, D., and Margiota, M. (1987). Unmasking capacity of stored trypsin solution: rescue of immunoglobulins in formaldehyde-paraffin solution. J. HistotechnoL 10, 229-231. Feldman, S. H., Weisbroth, S. P., and Weisbroth, S. H. (1999). Detection of Pneumocystis carinii in rats by polymerase chain reaction: Comparison of lung tissue and bronchoalveolar lavage specimens. Lab. Anim. Sci. 46, 628-634. Ferner, W. T., Miskuff, R. L., Yolken, R. H., and Vonderfecht, S. L. (1987). Comparison of methods for detection of serum antibody to murine rotavirus. J. Clin. Microbiol. 25, 1364-1369. Ferrando, R., Vallette, J. P., Sain-Lebe, L., Bonnod, J., and Huard, M. (1981). Influence of autoclaving on gamma irradiation on the main constituents of a complete composed diet for laboratory rats and mice. Sci. Tech. Anim. Lab. 6, 5-13. Flood, K. M. (1995). "Efficacy of Alternative Disinfectants in the Inactivation of Kilham Rat Virus." Thesis, Univ. of Connecticut, Storrs. Flynn, B. M., Brown, P. A., Eckstein, J., and Strong, D. (1999). Treatment of Syphacia obvelata in mice using ivermectin. Lab. Anim. Sci. 39, 461-463. Flynn, R. J. (1963). Pseudomonas aeruginosa infection and its effects on biological and medical research. Lab. Anim. Care 13, 1-6. Flynn, R. J. (1973). "Parasites of Laboratory Animals." Iowa State Univ. Press, Ames. Foltz, C. J., Fox, J. G., Yan, L., and Shames, B. (1996). Evaluation of various oral antimicrobial formulations for eradication of Helicobacter hepaticus. Lab. Anim. Sci. 46, 193-197. Forbes, B. A., Sahm, D. E, and Weissfeld, A. S. (1998). Laboratory cultivation and isolation of bacteria. In "Bailey and Scott's Diagnostic Microbiology" (B. A. Forbes, D. E Sahm, and A. S. Weissfeld, eds.), pp. 150-166. Mosby, St. Louis. Foster, H. L. (1980). Gnotobiology. In "The Laboratory Rat: Biology and Disease" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), Vol. 1, pp. 4 3 58. Academic Press, San Diego. Foster, H. L., Black, C., and Pfau, E. (1964). A pasteurization process for pelleted diets. Lab. Anim. Sci. 14, 373-381. Foster, T. (1996). Staphylococcus. In "Medical Microbiology" (S. Baron, ed.), pp. 188-i97. Univ. of Texas Medical Branch at Galveston. Fox, J. G., and Loew, E M. (1983). Animal health surveillance and delivery systems. In "The Mouse in Biomedical Research: Normative Biology, Immunology, and Husbandry" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 69-82. Academic Press, San Diego. Fox, J. G., Li, X., Yan, L., Cahill, R. J., Hurley, R., Lewis, R., and Murphy, J. C. (1996). Chronic proliferative hepatitis in A/JCr mice associated with persistent Helicobacter hepaticus infection: A model of helicobacter-induced carcinogenesis. Infect. Immun. 64, 1548-1558. Fox, J. G., Dewhirst, E E., Tully, J. G., Paster, B. J., Yan, L., Taylor, K., Collins, M., Gorelick, P. L., and Ward, J. M. (1999). Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J. Clin. Microbiol. 32, 1238-1245. Freundt, E. A. (1983). Culture media for classic mycoplasmas. In "Methods

10. MICROBIOLOGICAL QUALITY CONTROL FOR LABORATORY RODENTS AND LAGOMORPHS in Mycoplasmology: Mycoplasma Characterization" (S. Razin, and J. G. Tully, eds.), Vol. 1, pp. 127-136. Academic Press, New York. Gaertner, D. J., Jacoby, R. O., Smith, A. L., Ardito, R. B., and Paturzo, E X. (1989). Persistence of rat parvovirus in athymic rats. Arch. Virol. 105, 259-268. Gaertner, D. J., Jacoby, R. O., Johnson, E. A., Paturzo, F. X., and Smith, A. L. (1993). Characterization of acute rat parvovirus infection by in situ hybridization. Virus Res. 28, 1-18. Ganaway, J. R. (1976). Bacteria, mycoplasma, and rickettsial diseases. In "The Biology of the Guinea Pig" (J. E. Wagner and E J. Manning, eds.), pp. 121135. Academic Press, New York. Ganaway, J. R. (1980). Effect of heat and selected chemical disinfectants upon infectivity of spores of Bacillus piliformis (Tyzzer's disease). Lab. Anita. Sci. 30, 192-196. Ganaway, J. R. (1982). Bacterial and mycotic diseases of the digestive system. In "The Mouse in Biomedical Research: Diseases" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 1-20. Academic Press, New York. Ganaway, J. R., Allen, A. M., and McPherson, C. (1965). Prevention of acute Bordetella bronchiseptica pneumonia in a guinea pig colony. Lab. Anita. Sci. 15, 156-162. Gentsch, J. R., Glass, R. I., Woods, E, Gouvea, V., Gorziglia, M., Flores, J., Das, B. K., and Bhan, M. K. (1992). Identification of group A rotavirus gene 4 types by polymerase chain reaction. J. Clin. Microbiol. 30, 1365-1373. Giannella, R. A. (1996). Salmonella. In "Medical Microbiology" (S. Baron, ed.), pp. 295-302. Univ. of Texas Medical Branch at Galveston. Gibson, S. V., Waggie, K. S., Wagner, J. E., and Ganaway, J. R. (1987). Diagnosis of subclinical Bacillus piliformis infection in a barrier-maintained mouse production colony. Lab. Anim. Sci. 37, 786-788. Gibson, U. E. M., Heid, C. A., and Willams, P. M. (1996). A novel method for real time quantitative RT-PCR. Genome Res. 6, 995-1001. Goelz, M. E, Thigpen, J. E., Mahler, J., Rogers, W. P., Locklear, J., Weigler, B. J., and Forsythe, D. B. (1996). Efficacy of various therapeutic regimens in eliminating Pasteurella pneumotropica from the mouse. Lab. Anim. Sci. 46, 280 -284. Goto, K., and Itoh, T. (1996). Detection of Clostridium piliforme by enzymatic assay of amplified cDNA segment in microtitration plates. Lab. Anim. Sci. 46, 493-496. Goto, K., Takakura, A., Yoshimura, M., Ohnishi, Y., and Itoh, T. (1998). Detection and typing of lactate dehydrogenase-elevating virus RNA from transplantable tumors, mouse liver tissues, and cell lines, using polymerase chain reaction. Lab. Anim. Sci. 48, 99-102. Greenberg, H., McAuliffe, V., Valdesuso, J., Wyatt, R., Flores, J., Kalica, A., Hoshino, Y., and Singh, N. (1983). Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies. Infect. Immun. 39, 91-99. Greenlee, J. E., Dodd, W. K., and Oster-Granite, M. L. (1982). An in vitro assay for K papovavirus of mice. J. Virol. Methods 4, 139-146. Greisen, K., Loeffelholz, M., Purohit, A., and Leong, D. (1994). PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in the cerebrospinal fluid. J. Clin. Microbiol. 32, 335-351. Grossgebauer, K., Spicker, G., and Peter, J. (1975). Experiments on terminal disinfection by formaldehyde vapor in the case of smallpox. J. Clin. Microbiol. 2, 519. Hall, W. C., and Ward, J. M. (1984). A comparison of the avidin-biotinperoxidase complex (ABC) and peroxidase-anti-peroxidase (PAP) immunocytochemical techniques for demonstrating Sendai virus infection in fixed tissue specimens. Lab. Anim. Sci. 34, 261-263. Hanson, G., and Wilkinson, R. (1993). Gamma radiation and virus inactivation. Art. Sci. 12, 1-5. Hartley, J. W., and Rowe, W. P. (1960). A new mouse virus apparently related to the adenovirus group. Virology 11, 645-649.

389

Hawkes, R. A. (1979). General principles underlying laboratory diagnosis of viral infections. In "Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections" (E. H. Lennette and N. J. Schmidt, eds.). American Public Health Association, Washington, D.C. Henderson, K. S., White, W. J., Cail, S. P., and Perkins, C. L. (1998). Environmental monitoring for the presence of rodent parvoviruses on barrier room air intake filters via the polymerase chain reaction (PCR). Lab. Anim. Sci. 48, 407. (Abstract). Hermann, L. M., White, W. J., and Lang, C. M. (1982). Prolonged exposure to acid, chlorine, or tetracycline in the drinking water: effects on delayed-type hypersensitivity, hemagglutination titers, and reticuloendothelial clearance rates in mice. Lab. Anim. Sci. 32, 603-608. Hierholzer, J. C., Suggs, M. T., and Hall, E. C. (1969). Standardized viral hemagglutination and hemagglutination-inhibition tests: 2. Description and statistical evaluation. Appl. Microbiol. 18, 824-833. Hildebrandt, P. K. (1982). Rickettsial and chlamydial diseases. In "The Mouse in Biomedical Research: Diseases" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 99-109. Academic Press, New York. Hjelle, B., Jenison, S., Torrez-Martine, N., Yamada, T., Nolte, K., Zumwalt, R., Maclnnes, K., and Myers, G. (1994). A novel hantavirus associated with an outbreak of fatal respiratory disease in the southwestern United States: Evolutionary relationships to known hantaviruses. J. Virol. 68, 592-596. Hoddenbach, G., Johnson, J., and Disalvo, C. (1997). "Mechanical Rodent Proofing Techniques: A Training Guide for National Park Service Employees." U.S. Dept. of the Interior, National Park Serv., Washington, D.C. Homberger, E R., Smith, A. L., and Barthold, S. W. (1991). Detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction. J. Clin. Microbiol. 29, 2789-2793. Homberger, E R., Zsuzsanna, E, and Thomann, E E. (1993). Control of Pseudomonas aeruginosa infection in mice by chlorine treatment of drinking water. Lab. Anita. Sci. 43, 635-637. Homberger, E R., Romano, T. E, Seiler, E, Hansen, G. M., and Smith, A. L. (1995). Enzyme-linked immunosorbent assay for detection of antibody to lymphocytic choriomeningitis virus in mouse sera, with recombinant nucleoprotein as antigen. Lab. Anita. Sci. 45, 493-496. Hoover, D., Bendele, S. A., Wightman, S. R., Thompson, C. Z., and Hoyt, J. A. (1985). Streptococcal enteropathy in infant rats. Lab. Anita. Sci. 35, 635-641. Hrapchak, B. B. (1980). Immunohistochemistry. In "Theory and Practice of Histotechnology" (D. C. Sheehan and B. B. Hrapchak, eds.), pp. 310-326. Battelle Press, Columbus, Ohio. Hsu, M., and Choppin, E W. (1984). Analysis of Sendai virus mRNAs with cDNA clones of viral genes and sequences of biologically important regions of the fusion protein. Proc. Natl. Acad. Sci. 81, 7732-7736. Hsu, S. M., Raine, L., and Fanger, H. (1980). A comparative study of the peroxidase-anti-peroxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am. J. Clin. Pathol. 75, 734-738. Huerkamp, M. J. (1993). Ivermectin eradication of pinworms from rats kept in ventilated cages. Lab. Anita. Sci. 43, 86-90. Irving, J. M., Chang, L. W. S., and Castillo, E J. (1993). A reverse transcriptasepolymerase chain reaction assay for the detection and quantitation of murine retroviruses. Bio/Technology 11, 1042-1046. Isenberg, H. D. (1998). Clinical microbiology. In "Infectious Diseases" (S. L. Gorbach, J. G. Bartlett, and N. R. Blacklow, eds.), pp. 123-145. Saunders, Philadelphia. Ishii, A., Yago, A., Nariuchi, H., Shirasaka, A., Wada, Y., and Matubashi, T. (1974). Some aspects on the transmission of hepatitis B antigen: model experiments by mosquitoes with murine hepatitis virus. Jpn. J. Exp. Med. 44, 495-501. Jacoby, R. O. (1986). Rat coronavirus. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (E N. Bhatt,

390 R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 625-638. Academic Press, Orlando, Florida. Jacoby, R. O., and Lindsey, J. R. (1997). Health care for research animals is essential and 'affordable. FASEB J. 11, 609-614. Jacoby, R. O., and Lindsey, J. R. (1998). Risks of infection among laboratory rats and mice at major biomedical research institutions. ILAR J. 39, 266-271. Jacoby, R. O., Bhatt, E N., and Jonas, A. M. (1975). Pathogenesis of sialodacryoadenitis in gnotobiotic rats. Vet. Pathol. 12, 196-209. Jacoby, R. O., Bhatt, E N., Gaertner, D. J., Smith, A. L., and Johnson, E. A. (1987). The pathogenesis of rat virus infection in infant and juvenile rats after oronasal inoculation. Arch. Virol. 95, 251-270. Jacoby, R. O., Johnson, E. A., Ball-Goodrich, L., Smith, A. L., and McKisic, M. D. (1995). Characterization of mouse parvovirus infection by in situ hybridization. J. Virol. 69, 3915-3919. Jacoby, R. O., Ball-Goodrich, L., Besselsen, D. G., McKisic, M. D., Riley, L. K., and Smith, A. L. (1996). Rodent parvovirus infections. Lab. Anita. Sci. 46, 370-380. Jonas, A. M., Craft, J., Black, C. L., Bhatt, E N., and Hilding, D. (1969). Sialodacryoadenitis in the rat. Arch. Pathol. 88, 613-622. Jure, M. N., Morse, S. S., and Stark, D. M. (1988). Identification of nonspecific reactions in laboratory rodent specimens tested by Rotazyme rotavirus ELISA. Lab. Anita. Sci. 38, 273-278. Kimura, Y., Aoki, H., Shimokata, K., Ito, Y., Takano, M., Hirabayashi, N., and Norrby, E. (1979). Protection of mice against virulent virus infection by a temperature-sensitive mutant derived from HVJ (Sendai virus) carrier culture. Arch. Virol. 61, 297-304. Klein, M., and DeForest, A. (1983). Principals of virus inactivation. In "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.), pp. 422-434. Lea and Febiger, Philadelphia. Kovacs, J. A., Halpern, J. L., Lundgren, B., Swan, J., Parrillo, J. E., and Masur, H. (1989). Monoclonal antibodies to Pneumocystis carinii: Identification of specific antigens and characterization of antigenic differences between rat and human isolates. J. Infect. Dis. 159, 60-70. Kraft, V., Meyer, B., Thunert, A., Deerberg, E, and Rehm, S. (1982). Diagnosis of Mycoplasma pulmonis infection of rats by an indirect immunofluorescence test compared with four other diagnostic methods. Lab. Anim. 16, 369-373. Kramer, D. N. (1992). Myths: Cleaning, sanitation, and disinfection. Dairy Food Environ. Sanit. 12, 507-509. Kristensson, K., and Orvell, C. (1983). Cellular localization of five structural proteins of Sendai virus studied with peroxidase-labelled Fab fragments of monoclonal antibodies. J. Gen. Virol. 64, 1673-1678. Kunita, S., Terada, E., Goto, K., and Kagiyama, N. (1992). Sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction. Lab. Anim. Sci. 42, 593-598. Landry, M. L., and Hsiung, G. D. (1992). Primary isolation of viruses. In "Clinical Virology Manual" (S. Specter and G. Lancz, eds.), pp. 43-65. Elsevier, New York. LaRegina, M. C., and Lonigro, J. (1988). Serologic screening for murine pathogens: Basic concepts and guidelines. Lab. Animal 17(6), 40-47. Lee, H. W., and Johnson, K. M. (1982). Laboratory-acquired infections with Hantaan virus, the etiologic agent of Korean hemorrhagic fever. J. Infect. Dis. 146, 645-651. Lee, E W., Gibbs, C. J., Jr., Gajdusek, D. C., and Yanagihara, R. (1985). Serotypic classification of hantaviruses by indirect immunofluorescent antibody and plaque reduction neutralization tests. J. Clin. Microbiol. 22, 940-944. Lehmann-Grube, E (1982). Lymphocytic choriomeningitis virus. In "The Mouse in Biomedical Research: Diseases." (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 231-266. Academic Press, New York. Leland, S. E. (1991). Antiprotozoan, antihelmintic, and other pest management

WILLIAM R. SHEK AND DIANE J. GAERTNER

compounds. In "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.). Lea and Febiger, Gainesville, Florida. Levy, R. V., and Leahy, T. J. (1991). Sterilization filtration. In "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.), pp. 527-552. Lea and Febiger, Gainesville, Florida. Lewis, A. M., Rowe, W. P., Turner, H. C., and Huebner, R. J. (1965). Lymphocytic-choriomeningitis virus in hamster tumor: Spread to hamster and humans. Science 150, 363-364. Lewis, V. J., and Clayton, D. M. (1971). An evaluation of the mouse antibody production test for detecting three murine viruses. Lab. Anim. Sci. 21, 203 -205. Lipman, N. S., Nguyen, H., and Perkins, S. (1999). Threat to U.S. mouse colonies [letter]. Science 284, 1123. Lu, J. J., Bartlett, M. S., Smith, J. W., and Lee, C. H. (1995). Typing of Pneumocystis carinii strains with type-specific oligonucleotide probes derived from nucleotide sequences of internal transcribed spacers of rRNA genes. J. Clin. Microbiol. 33, 2973-2977. Lukas, V. S., Ringler, D. H., Chrisp, C. E., and Rush, H. G. (1987a). An enzymelinked immunosorbent assay to detect serum IgG to Pasteurella multocida in naturally and experimentally infected rabbits. Lab. Anim. Sci. 37, 60-64. Lukas, V. S., Ruehl, W. W., and Hamm, T. E. (1987b). An enzyme-linked immunosorbent assay to detect serum IgG in rabbits naturally exposed to cilia-associated respiratory bacillus. Lab. Anim. Sci. 37, 533 Lussier, G. (1991). Detection methods for the identification of rodent viral and mycoplasmal infections. Lab. Anim. Sci. 41, 199-225. Lussier, G., Smith, A. L., Guenette, D., and Descoteaux, J. P. (1987). Serological relationship between mouse adenovirus strains FL and K87. Lab. Anim. Sci. 37, 55-57. Lussier, G., Guenette, D., and Descoteaux, J. P. (1988). Detection of antibodies to mouse thymic virus by enzyme-linked immunosorbent assay. Can. J. Vet. Res. 52, 236-238. Lyerla, H. C., and Forrester, F. T. (1979). "Immunofluorescence Methods in Virology, Course No. 8231-C." Centers for Disease Control, Atlanta. MacFaddin, J. E (1980). "Biochemical Tests for Identification of Medical Bacteria." Williams and Wilkins, Baltimore. Maerki, U., Rossbach, W., and Leuenberger, J. (1989). Consistency of laboratory animal food following incubation prior to autoclaving. Lab. Anim. 23, 319-323. Mahoney, J. B., and Chernesky, M. A. (1999). Immunoassays for the diagnosis of infectious diseases. In "Manual of Clinical Microbiology" (P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken, eds.), pp. 202214. ASM Press, Washington, D.C. Manning, P. J., Brackee, G., Naasz, M. A., DeLong, D., and Leary, S. L. (1987). A dot-immunobinding assay for the serodiagnosis of Pasteurella multocida infection in laboratory rabbits. Lab. Anim. Sci. 37, 615-620. Manning, P. J., DeLong, D., Swanson, D., and Shek, W. R. (1994). Rodent isolates of Pasteurella pneumotropica: Demonstration of O polysaccharide chains and serologically specific lipopolysaccharide antigens. Lab. Anim. Sci. 44, 399 (Abstract). Matsushita, S., Kashima, M., and Joshima, H. (1987). Serodiagnosis of ciliaassociated respiratory bacillus infection by the indirect immunofluorescence assay technique. Lab. Anim. 21, 356-359. Matthaei, K. I., Berry, J. R., France, M. P., Yeo, C., Garcia-Aragon, J., and Russell, P. J. (1998). Use of polymerase chain reaction to diagnose a natural outbreak of mouse hepatitis virus infection in nude mice. Lab. Anim. Sci. 48, 137-144. McKisic, M. D., Lancki, D. W., Otto, G., Padrid, P., Snook, S., Cronin, D. C., Lohmer, P. D., Wong, T., and Fitch, F. (1993). Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells. J. Immunol. 1511,419-428. Meanger, J. D., and Marshall, R. B. (1989). Campylobacter jejuni infection within a laboratory animal production unit. Lab. Anim. 23, 126-132.

10. MICROBIOLOGICAL QUALITY CONTROL FOR LABORATORY RODENTS AND LAGOMORPHS Milios, J., and Leong, A. S.-Y. (1988). Avidin-biotin-peroxidase complex reagents for routine histological application: A comparison of four commercial kits. J. Histotechnol. 11, 81-83. Minion, E C., Brown, M. B., and Cassell, G. H. (1984). Identification of crossreactive antigens between Mycoplasma pulmonis and Mycoplasma arthritidis. Infect. Immun. 43, 115-121. Morse, S. S. (1988). Mouse thymic virus (MTLV; murid herpesvirus 3) infection in athymic nude mice: Evidence for a T lymphocyte requirement. Virology 163, 255-258. Morse, S. S. (1989). Comparative sensitivity of infectivity assay and mouse antibody production (MAP) test for detection of mouse thymic (MTLV). J. Virol. Methods 28, 15-24. Motzel, S. L., and Riley, L. K. (1991). Bacillus piliformis flagellar antigens for serodiagnosis of Tyzzer's disease. J. Clin. Microbiol. 29, 2566-2570. Mullis, K. B. (1990). The unusual origin of the polymerase chain reaction. Sci. Am. 262, 56-65. Nerurkar, L. S., Jacob, A. J., Madden, D. L., and Sever, J. L. (1982). Detection of genital Herpes simplex infections by a tissue culture-fluorescent-antibody technique with biotin-avidin. J. Clin. Microbiol. 17, 149-154. Newsome, P. M., and Coney, K. A. (1985). Synergistic rotavirus and Escherichia coli diarrheal infection of mice. Infect. Immun. 47, 573-574. Nicklas, W., Kraft, V., and Meyer, B. (1993). Contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses. Lab. Anim. Sci. 43, 296-300. Nikkels, R. J., and Mullink, J. W. M. A. (1999). Bordetella bronchiseptica pneumonia in guinea-pigs: Description of the disease and elimination by vaccination. Z. Versuchstierkd. 13, 105-111. Oldstone, M. B. A., Sinha, Y. N., Blount, P., Tishon, A., Rodriguez, M., Von Wedel, R., and Lampert, P. W. (1982). Virus-induced alterations in homeostasis: Alterations in differentiated functions of infected cells in vivo. Science 218, 1125-1127. Orcutt, R. P. (1980). Bacterial diseases: Agents, pathology, diagnosis, and effects on research. Lab. Anim. 9, 28-43. Owen, D. (1972). Preparation and identification of common parasites of laboratory rodents. In "Medical Research Council, Laboratory Animal Centre Handbook No. 1," pp. 9-16. Her Majesty's Stationery Office, London. Parker, J. C., and Reynolds, R. K. (1968). Natural history of Sendai virus infection in mice. Am. J. Pathol. 88, 112-125. Parker, J. C., Tennant, R. W., Ward, T. G., and Rowe, W. P. (1965). Virus studies with germfree mice: 1. Preparation of serologic diagnostic reagents and survey of germfree and monocontaminated mice for indigenous murine viruses. J. Natl. Cancer Inst. 34, 371-380. Parker, J. C., Whiteman, M. D., and Richter, C. B. (1978). Susceptibility of inbred and outbred mouse strains to Sendai virus and prevalance of infection in laboratory rodents. Infect. Immun. 19, 123-130. Parker, J. C., O'Beirne, A. J., and Collins, M. (1979). Sensitivity of enzymelinked immunosorbent assay, complement fixation, and hemagglutination inhibition serological tests for detection of Sendai virus antibody in laboratory mice. J. Clin. Microbiol. 9, 444-447. Patterson, M. J. (1996). Streptococcus. In "Medical Microbiology" (S. Baron, ed.), pp. 199-215. Univ. Texas Medical Branch at Galveston. Paturzo, F. X., Jacoby, R. O., Bhatt, P. N., Gaertner, D. J., and Ardito, R. B. (1987). Persistence of rat virus in seropositive rats as detected by explant culture. Arch. Virol. 95, 137-142. Peck, R. M., Eaton, G. J., Peck, E., and Litwin, S. (1983). Influence of Sendai virus on carcinogenesis in strain A mice. Lab. Anim. Sci. 33, 154-156. Peters, R. L., and Collins, M. (1983). The enzyme-linked immunosorbent assay for mouse hepatitis and rat coronaviruses. Lab. Anim. 12, 18-25. Peters, R. L., and Collins, M. J. (1981). Use of mouse hepatitis virus antigen in an enzyme-linked immunosorbent assay for rat coronaviruses. Lab. Anim. Sci. 31, 472-475. Potera, C. (1996). Biofilms invade microbiology. Science 273, 1795-1797.

391

Prince, H. N., Prince, D., and Prince, R. (1991). Principles of viral control and transmission. In "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.), pp. 411-444. Lea and Febiger, Gainesville, Florida. Rehbinder, C., Baneux, E, Forbes, D., Van Herck, H., Nicklas, W., Rugaya, Z., and Winkler, G. (1996). FELASA recommendations for the health monitoring of mouse, rat, hamster, gerbil, guineapig, and rabbit experimental units. Lab. Anim. 30, 193-208. Rehg, J. E., and Toth, L. A. (1998). Rodent quarantine program: Purpose, principles, and practice. Lab. Anim. Sci. 48, 438-447. Riley, L. K., Franklin, C. L., Hook, R. R., and Besch-Williford, C. (1994). Tyzzer's disease: An update of current information. Charles River Lab. Tech. Bull. (Fall), 1-6. Riley, L. K., Franklin, C. L., Hook, R. R., and Besch-Williford, C. (1996a). Identification of murine helicobacters by PCR and restriction enzyme analyses. J. Clin. Microbiol. 34, 942-946. Riley, L. K., Knowles, R., Purdy, G., Salome, N., Pintel, D., Hook, R. R., Franklin, C. L., and Besch-Williford, C. (1996b). Expression of recombinant parvovirus NS 1 protein by a baculovirus and application to serologic testing of rodents. J. Clin. Microbiol. 34, 440-444. Riley, L. K., Catty, A. J., and Besch-Williford, C. L. (1999). PCR-based testing as an alternative to MAP testing. Contemp. Top. Lab. Anim. Sci. 39, 41-42. Riley, V. (1974). Persistence and other characteristics of the lactate dehydrogenase-elevating virus (LDH-virus). Prog. Med. Virol. 18, 198-213. Riley, V., Lilly, F., Huerto, E., and Bardell, D. (1960). Transmissible agent associated with 26 types of experimental mouse neoplasms. Science 132, 545 -547. Rose, N. R. (1999). Immunodiagnosis. In "Infectious Diseases" (S. L. Gorbach, J. G. Bartlett, and N. R. Blacklow, eds.), pp. 145-152. Saunders, Philadelphia. Rosen, D. D., and Berk, R. S. (1977). Pseudomonas eye infections in cyclophosphamide-treated mice. Invest. Ophthalmol. 16, 649-652. Rowe, W. P., and Capps, W. I. (1961). A new mouse virus causing necrosis of the thymus in newborn mice. J. Exp. Med. 113, 831-834. Rowe, W. P., Hartley, J. W., Estes, J. D., and Huebner, R. J. (1959). Studies of mouse polyoma virus infection. 1. Procedures for quantitation and detection of virus. J. Exp. Med. 109, 379-391. Rowe, W. P., Hartley, J. W., and Huebner, R. J. (1962). Polyoma and other indigenous mouse viruses. In "The Problems of Laboratory Animal Disease" (R. J. C. Harris, ed.), pp. 131-142. Academic Press, New York. Russell, A. D. (1991). Principles of antimicrobial activity. In "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.), pp. 29-58. Lea and Febiger, Gainesville, Florida. Russell, A. D. (1992). Factors influencing the efficacy of antimicrobial agents. In "Principles and Practice of Disinfection, Preservation, and Sterilization" (A. D. Russell, W. B. Hugo, and G. A. J. Ayliffe, eds.), pp. 7-88. Blackwell Science, Oxford. Russell, R., Haines, D. C., Anver, M. R., Battles, J. K., Gorelick, P. L., Blumenauer, L. L., Gonda, M. A., and Ward, J. M. (1995). Use of antibiotics to prevent hepatitis and typhlitis in male SCID mice spontaneously infected with Helicobacter hepaticus. Lab. Anim. Sci. 45, 373-378. Schmaljohn, C. S., Hasty, S. E., Dalrymple, J. M., LeDuc, J. W., Lee, H. W., von Bonsdorff, C. H., Brummer-Korvenkontio, M., Vaheri, A., Tsai, T. E, Regnery, H. L., Goldgaber, D., and Lee, P. W. (1985). Antigenic and genetic properties of viruses linked to hemorrhagic fever with renal syndrome. Science 227, 1041-1044. Schmidt, N. J. (1969). Complement fixation technique for assay of viral antigens and antibodies. In "Fundamental Techniques in Virology" (K. Habel and N. P. Salzman, eds.), pp. 263-275. Academic Press, New York. Schoeb, T. R., Casebolt, D. B., Walker, V. E., Potgieter, L. N. D., Thouless, M. E., and DiGiacomo, R. E (1986). Rotavirus-associated diarrhea in a commercial rabbitry. Lab. Anim. Sci. 36, 149-152. Selwyn, M. R., and Shek, W. R. (1994). Sample sizes and frequency of testing

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for health monitoring in barrier rooms and isolators. Contemp. Top. Lab. Anim. Sci. 33, 56-60. Shames, B., Fox, J. G., Dewhirst, E, Yan, L., Shen, Z., and Taylor, N. S. (1995). Identification of widespread Helicobacter hepaticus infection in feces in commercial mouse colonies by culture and PCR assay. J. Clin. Microbiol. 33, 2968-2972. Shek, W. R., Flood, K. M., Green, D., and Jennings, S. M. (1991). Inactivation of Kilham's rat virus (KRV) by ozonation. Annual meeting, AALAS, Buffalo. (Abstract). Shek, W. R., Paturzo, E X., Johnson, E. A., Hansen, G. M., and Smith, A. L. (1998). Characterization of mouse parvovirus infection among BALB/c mice from an enzootically infected colony. Lab. Anim. Sci. 48, 294-297. Siegl, G. (1976). The parvoviruses. Virol. Monogr. 15, 1-109. Silverman, G. J. (1991). Sterilization and preservation by ionizing irradiation. In "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.), pp. 566-579. Lea and Febiger, Gainesville, Florida. Simon, M., Domok, I., and Pinter, A. (1982). Lymphocytic choriomeningitis (LCM) virus carrier cell cultures in Hungarian laboratories. Acta Microbiol. Acad. Sci. Hung. 29, 201-208. Skinner, H. H., Knight, E. H., and Grove, R. (1977). Murine lymphocytic choriomeningitis: The history of a natural cross-infections from wild to laboratory mice. Lab. Anim. 11, 219-222. Small, J. D. (1983). Environmental and equipment monitoring. In "The Mouse in Biomedical Research: Normative Biology, Immunology, and Husbandry" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 83-101. Academic Press, San Diego. Small, J. D. (1984). Rodent and lagomorph health surveillance quality assurance. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 709-723. Academic Press, Orlando, Florida. Small, J. D., and New, A. (1981). Prevention and control of mousepox. Lab. Anim. Sci. 31, 616-621. Smith, A. L. (1983a). An immunofluorescence test for detection of serum antibody to rodent coronaviruses. Lab. Anim. Sci. 33, 157-160. Smith, A. L. (1983b). Response of weanling random-bred mice to inoculation with minute virus of mice. Lab. Anim. Sci. 33, 37-39. Smith, A. L. (1986a). Methods for potential application to rodent virus isolation and identification. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 753-776. Academic Press, Orlando, Florida. Smith, A. L. (1986b). Serologic tests for detection of antibody to rodent viruses. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 731-749. Academic Press, Orlando, Florida. Smith, A. L., Carrano, V. A., and Brownstein, D. (1984). Response of weanling random-bred mice to infection with pneumonia virus of mice (PVM). Lab. Anim. Sci. 34, 35-37. Smith, A. L., and Paturzo, E X. (1988). Explant cultures for detection of minute virus of mice in infected mouse tissue. J. Tissue Cult. Methods 11, 45-47. Smith, A. L., Singleton, G. R., Hansen, G. M., and Shellam, G. (1993a). A serologic survey for viruses and Mycoplasma pulmonis among wild house mice (Mus domesticus) in southeastern Australia. J. Wildl. Dis. 29, 219-229. Smith, A. L., Jacoby, R. O., Johnson, E. A., Paturzo, E, and Bhatt, P. N. (1993b). In vivo studies with an "orphan" parvovirus of mice. Lab. Anim. Sci. 43, 175-182. Sobsey, M. D. (1989). Inactivation of health-related microorganisms in water by disinfection processes. Water Sci. Technol. 21, 179-195. Sundberg, J. P., Burnstein, T., Shultz, L. D., and Bedigian, H. (1989). Identification of Pneumocystis carinii in immunodeficient mice. Lab. Anim. Sci. 39,213-218. Swoveland, P. T., and Johnson, K. P. (1979). Enhancement of fluorescent antibody staining of viral antigens in formalin-fixed tissues by trypsin digestion. J. Infect. Dis. 140, 758-764.

WILLIAM R. SHEK AND DIANE J. GAERTNER Takahashi, Y., Okuno, Y., Yamanouchi, T., Takada, N., and Yamanishi, K. (1986). Comparison of immunofluorescence and hemagglutination inhibition tests and enzyme-linked immunosorbent assay for detection of serum antibody in rats infected with hemorrhagic fever with renal syndrome virus. J. Clin. Microbiol. 24, 712-715. Tang, Y. W., and Persing, D. H. (1999). Molecular detection and identification of microorganisms. In "Manual of Clinical Microbiology" (P. R. Murray, E. J. Baron, M. A. Pfaller, E C. Tenover, and R. H. Yolken, eds.), pp. 215248. ASM Press, Washington, D.C. Tanishita, O., Takahashi, Y., Okuno, Y., Yamanishi, K., and Takahashi, M. (1984). Evaluation of focus reduction neutralization test with peroxidaseanti-peroxidase staining technique for hemorrhagic fever with renal syndrome virus. J. Clin. Microbiol. 20, 1213-1215. Taylor, K., and Copley, C. G. (1993). Detection of rodent RNA viruses by polymerase chain reaction. Lab. Anim. 28, 31-34. Tenover, E C. (1998). Molecular techniques for the detection and identification of infectious agents. In "Infectious Diseases" (S. L. Gorbach, J. G. Bartlett, and N. R. Blacklow, eds.), pp. 152-156. Saunders, Philadelphia. Tenover, E C., Arbeit, R., Archer, G., Biddle, J., Byrne, S., Goering, R., Hancock, G., Herbert, G. A., Hill, B., Hollis, R., Jarvis, W. R., Kreiswirth, D., Eisner, W., Maslow, J., McDougal, L. K., Miller, J. M., Mulligan, M., and Pfaller, M. A. (1994). Comparison of traditional and molecular methods of typing of Staphylococcus aureus. J. Clin. Microbiol. 32, 407-415. Thigpen, J. E., Lebetkin, E., Dawes, M., Amyx, H. L., Caviness, G. E, Sawyer, B. A., and Blackmore, D. E. (1989). The use of dirty bedding for detection of murine pathogens in sentinel mice. Lab. Anim. Sci. 39, 324-327. Thompson, R. E., Smith, T. E, and Wilson, W. R. (1982). Comparison of two methods used to prepare smears of mouse lung tissue for detection of Pneumocystis carinii. J. Clin. Microbiol. 16, 303-306. Tietjen, R. M. (1992). Transmission of minute virus of mice into a rodent colony by a research technician. Lab. Anim. Sci. 42, 422. (Abstract). Trexler, P. C. (1983). Gnotobiotics. In "The Mouse in Biomedical Research: Normative Biology, Immunology, and Husbandry" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 1-17. Academic Press, San Diego. Tyler, J. W., and Cullor, J. S. (1989). Titers, test, and truisms: Rational interpretation of diagnostic serologic testing. J. Am. Vet. Med. Assoc. 194, 1550-1558. Tyler, K. L., and Fields, B. N. (1986). Reovirus infection in laboratory rodents. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse, and A. E. New, eds.), pp. 277-303. Academic Press, Orlando, Florida. Ushijima, H., Koike, H., Mukoyama, A., Hasegawa, A., Nishimura, S., and Gentsch, J. (1992). Detection and serotyping of rotaviruses in stool specimens by using reverse transcription and polymerase chain reaction amplification. J. Med. Virol. 38, 292-297. van der Gulden, W. J. I., and van Erp, A. J. M. (1972). The effect of peracetic acid as a disinfectant on worm eggs. Lab. Anim. Sci. 22, 225-226. Voller, A., Bidwell, D. E., and Bartlett, A. (1982). "ELISA Techniques in Virology" (C. R. Howard, ed.), Vol. 5, pp. 59-91. Alan R. Liss, New York. Vonderfecht, S. L., Miskuff, R. L., Eiden, J. J., and Yolken, R. H. (1985). Enzyme immunoassay inhibition assay for the detection of rat rotavirus-like agent in intestinal and fecal specimens obtained from diarrheic rats and humans. J. Clin. Microbiol. 22, 726-730. Vonderfecht, S. L., Eiden, J. J., Musjuff, R. L., and Yolken, R. H. (1988). Kinetics of intestinal replication of group B rotavirus and relevance to diagnostic methodologies. J. Clin. Microbiol. 26, 216-221. Waggie, K. S., Hansen, G., Ganaway, J., and Spencer, T. H. (1981). A study of mouse strain susceptibility to Bacillus piliformis (Tyzzer's disease): The association of B-cell function and resistance. Lab. Anim. Sci. 31, 139-142. Waggie, K. S., Wagner, J. E., and Lentsch, R. H. (1983). A naturally occurring outbreak of Mycobacterium avium-intracellulare infections in C57BL/6N mice. Lab. Anim. Sci. 33, 249-250. Waggie, K. S., Spencer, T. H., and Ganaway, J. (1987). An enzyme-linked

10. MICROBIOLOGICAL QUALITY CONTROL FOR LABORATORY RODENTS AND LAGOMORPHS immunosorbent assay for detection of anti-Bacillus piliformis serum antibody in rabbits. Lab. Anim. Sci. 37, 176-179. Waggie, K. S., Hansen, G., Moore, T. D., Bukowski, M. A., and Allen, A. M. (1988). Cecocolitis in immunodeficient mice associated with an enteroinvasive lactose-negative E. coli. Lab. Anim. Sci. 38, 389-393. Waggie, K., Kagiyama, N., Allen, A. M., and Nomura, T. (1994). "Manual of Microbiologic Monitoring of Laboratory Animals." National Institutes of Health Publication 94-2498, Bethesda, Maryland. Wagner, J. E., Besch-Williford, C. L., and Steffen, E. K. (1991). Health surveillance of laboratory rodents. Lab. Anim. 20, 40-45. Wallace, G. D., Werner, R. M., Golway, P. L., Hernandez, D., Ailing, D. W., and George, D. A. (1981). Epizootiology of an outbreak of mousepox at the National Institutes of Health. Lab. Anim. Sci. 31, 609-615. Walzer, P. D., Kim, C. K., Linke, M. J., Pogue, C. L., Heurkamp, M. J., Chrisp, C. E., Lerro, A. V., Wixson, S. K., Hall, E., and Shultz, L. D. (1989). Outbreaks of Pneumocysitis carinii pneumonia in colonies of immunodeficient mice. Infect. Immun. 57, 62-70. Ward, J. M., Fox, J. G., Anver, M. R., Haines, D. C., George, C. V., Collins, M. J., Gorelick, P. L., Nagashima, K., Gonda, M. A., Gilden, R. V., Tully, J. G., Russel, R. J., Benveniste, R. E., Paster, B. J., Dewhirst, E E., Donovan, J. C., Anderson, L. M., and Rice, J. M. (1994a). Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J. Natl. Cancer Inst. 86, 1222-1227. Ward, J. M., Anver, M. R., Haines, D. C., and Benveniste, R. E. (1994b). Chronic active hepatitis in mice caused by Helicobacter hepaticus. Amer. J. Pathol. 145, 959-968. Washington, J. A. (1996). Principles of diagnosis. In "Medical Microbiology" (S. Baron, ed.), pp. 153-162. Univ. of Texas Medical Branch at Galveston. Weir, E. C., Brownstein, D. G., and Barthold, S. W. (1986). Spontaneous wasting disease in nude mice associated with Pneumocystis carinii infection. Lab. Anim. Sci. 36, 140-1 44. Weir, E. C., Brownstein, D., Smith, A., and Johnson, E. A. (1988). Respiratory disease and wasting in athymic mice infected with pneumonia virus of mice. Lab. Anim. Sci. 38, 133-137. Weisbroth, S. H. (1979a). Parasitic diseases. In "The Laboratory Rat: Biology and Diseases" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), Vol. 1, pp. 307-331. Academic Press, Orlando, Florida. Weisbroth, S. H. (1979b). Bacterial and mycotic diseases. In "The Laboratory Rat: Biology and Diseases" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds), Vol. 1, pp. 193-241. Academic Press, Orlando, Florida. Weisbroth, S. H. (1982). Arthropods. In "The Mouse in Biomedical Research: Diseases" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 385402. Academic Press, New York.

393

Weisbroth, S. H., Brobst, R. C., and Smiley, D. E (1996). Diagnosis of low level coccidiosis (Eimeria) infection in laboratory rabbits: Evaluation of detection methods. Lab. Anim. Sci. 35, 87-89. Weisbroth, S. H., Peters, R. L., Riley, L. K., and Shek, W. S. (1998). Microbiological assessment of laboratory rats and mice. ILAR J. 39, 272-290. Weisbroth, S. H., Geistfeld, J., Weisbroth, S. P., Williams, B., Feldman, S. H., Linke, M. J., Orr, S., and Cushion, M. T. (1999). Latent Pneumocystis carinii infection in commerical rat colonies: Comparison of inductive immunosuppressants plus histopathology, PCR, and serology as detection methods. J. Clin. Microbiol. 37, 1441-1446. Wescott, R. B. (1982). Helminths. In "The Mouse in Biomedical Research: Diseases" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 2, pp. 373-384. Academic Press, New York. Wescott, R. B., Malczewski, A., and Van Hoosier, G. L. (1976). The influence of filter-top caging on the transmission of pinworm infections in mice. Lab. Anim. Sci. 26, 742-745. West, W. L., Schofield, J. C., and Bennett, B. T. (1992). Efficacy of the "microdot" technique for administering topical 1% ivermectin for the control of pinworms and fur mites in mice. Contemp. Top. Lab. Anim. Sci. 31, 7-10. White, W. J., Anderson, L. C., Geistfeld, J., and Martin, D. G. (1998). Current strategies for controlling/eliminating opportunistic microorganism. ILAR J. 39, 291-305. Wickramanayake, G. B., and Sproul, O. (1991). Kinetics of the inactivation of microorganisms. In "Disinfection, Sterilization, and Preservation" (S. S. Block, ed.), pp. 72-84. Lea and Febiger, Gainesville, Florida. Wilchek, M., and Bayer, E. A. (1984). The avidin-biotin complex in immunology. Immunol. Today 5, 39-43. Wilde, J., Eiden, J., and Yolken, R. (1990). Removal of inhibitory sustances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reaction. J. Clin. Microbiol. 28, 1300-1307. Xiao, S. Y., Chu, Y. K., Knauert, E, Lofts, R., Dalrymple, J. M., and LeDuc, J. W. (1992). Comparison of hantavirus isolates using a genus-reactive primer pair polymerase chain reaction. J. Gen. Virol. 73, 567-573. Yagami, K., Goto, K., Ishida, J., Ueno, Y., Kajiwara, N., and Sugiyama, E (1995). Polymerase chain reaction for detection of rodent parvoviral contamination in cell lines and transplantable tumors. Lab. Anim. Sci. 45, 326-328. Yang, F. C., Paturzo, E X., and Jacoby, R. O. (1995). Environmental stability and transmission of rat virus. Lab. Anim. Sci. 45, 140-144. Zweig, M. H., and Robertson, E. A. (1987). Clinical validation of immunoassays: A well-designed approach to a clinical study. In "Immunoassay: A Practical Guide" (D. W. Chan and M. T. Perlstein, eds.), pp. 97-127. Academic Press, Orlando, Florida.

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Chapter 11 Biology and Diseases of Dogs Robert C. Dysko, Jean A. Nemzek, Stephen L Levin, George J. DeMarco, and Maria R. Moalli

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Use in Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Availability and Sources

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L a b o r a t o r y M a n a g e m e n t and H u s b a n d r y

Biology

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N o r m a l Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Nutrition

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Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Infectious Diseases

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M e t a b o l i c and Nutritional Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Iatrogenic Diseases

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Neoplastic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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M i s c e l l a n e o u s Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

A.

INTRODUCTION

Taxonomic Considerations

Dogs are mammals in the order Carnivora, suborder Caniformia (or superfamily Arctoidea), and family Canidae. The domesticated dog has been designated as Canisfamiliaris. Other members of the genus Canis include four species of jackals, the coyote (C. latrans), the red wolf (C. rufus), and the gray wolf (C. lupus). Canis familiaris is subdivided into approximately 400 breeds, ranging in size and shape from the teacup chihuahua to the large Irish wolfhound. The domesticated dog is LABORATORYANIMALMEDICINE, 2nd edition

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thought to have descended from a Eurasian subspecies of the gray wolf, possibly either the Indian wolf (C. lupus pallipes) or the Chinese wolf (C. lupus chanco). Wolf-dog hybrids have been produced when the species are brought together in captivity (Nowak, 1999).

B.

Use in Research

1. Historical Use of Dogs in Research

The dog played an important role as a laboratory animal in the early history of biomedical research, primarily because of its Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

ROBERT C. DYSKO ET AL.

396

status as a cooperative companion animal of reasonable size. Dogs were used in the mid-1600s by William Harvey to study cardiac movement, by Marcello Malpighi to understand basic lung anatomy and function, and by Sir Christopher Wren to demonstrate the feasibility of intravenous delivery of medications (Gay, 1984). The use of dogs continued as biomedical research advanced, and they were featured in many noteworthy studies, including those by Pavlov to observe and document the conditioned reflex response and by Banting and Best to identify the role of insulin in diabetes mellitus. For a comprehensive but concise review of the use of the dog as a research subject, the readers are directed to the manuscript by Gay (1984).

models of Duchenne muscular dystrophy in human children. Duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an X-linked recessive manner. The dystrophy in golden retrievers is caused by absence of the same protein and is inherited in the same way. The clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (Kornegay et al., 1994). Bedlington terriers have been used to study copper storage diseases (such as Wilson's disease), and the development of spontaneous diabetes mellitus and hypothyroidism in a variety of dogs has also been studied for comparisons with the human conditions.

2.

3.

Current Use of Dogs in Research

The breed of dog most commonly bred for use in biomedical research is the beagle. Some commercial facilities also breed foxhounds or other larger-breed dogs for use in surgical research studies. Some specific breeds with congenital or spontaneous disorders are also maintained by research institutions (see specific examples below). Random-source dogs used in research are most frequently mongrels or larger-breed dogs (e.g., German shepherd, Doberman pinscher, Labrador and golden retrievers) that are used for surgical research and/or training. According to a computerized literature search for beagle for the years 1998-1999, approximately 40% of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. Especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. The next most common areas of research using beagles were dental and periodontal disease and surgery (12% of publications), orthopedic surgery and skeletal physiology (7%), and radiation oncology (4%). Other research areas that utilized beagles included canine infectious disease, surgery, imaging, prostatic urology, and ophthalmology. Most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. Anesthetic protocols and systems for dogs are well established, and the organs of larger-breed dogs are often an appropriate size for trials of potential pediatric surgical procedures. Surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. There are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. A colony of gray collies is maintained at the University of Washington (Seattle) for the study of cyclic hematopoiesis. This condition is manifested by periodic fluctuations of the cellular components of blood, most notably the neutrophil population. These dogs are used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (Brabb et al., 1995). Golden retrievers affected with muscular dystrophy have been used as

Decline in Numbers Used

Although historically the dog has been a common laboratory animal, the use of dogs in research has been waning over the past few years. According to the U.S. Department of Agriculture (1998), the number of dogs used in research has declined from a high use of 211,104 in 1979 to only 75,429 in 1997 (Fig. 1). This decrease was caused by a variety of factors, including (but not limited to) increased cost, decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), and shift in scientific interest from pathophysiology to molecular biology and genetics. C.

Availability and Sources

Dogs used for research are generally segregated into two classes: purpose-bred and random-source. Purpose-bred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. Usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. Purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. They are usually

Fig. 1. Numberof dogs used by research, 1973-1997, accordingto the U.S. Department of Agriculture. (Datafrom U.S. Departmentof Agriculture, 1998).

397

11. B I O L O G Y AND DISEASES OF DOGS

vaccinated against canine distemper virus, parvovirus, adenovirus type 2, parainfluenza virus, Leptospira serovars canicola and icterohemorrhagiae, and Bordetella bronchiseptica. Rabies virus vaccination may also be included. Purpose-bred dogs are also usually treated prophylactically for helminths and ectoparasites, intestinal coccidia, and bacterial ear infections (R. Scipioni and J. Ball, personal communication, 1999). Random-source dogs are not bred specifically for use in research. They may be dogs bred for another purpose (e.g., hunting), retired racing dogs, or stray dogs collected at pounds or shelters. The health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. Randomsource dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. These dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. Conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. Nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations. Options for procurement of dogs for biomedical research typically include purchase from a U.S. Department of Agriculturedesignated Class A or Class B licensed dealer or directly from a municipal pound. The requirements for USDA licensure are detailed in Code of Federal Regulations (CFR), Title 9, Chapter 1 (1-1-92 edition), Subchapter A, Animal Welfare, 1.1, Definitions, and 2.1, Requirements and Application. Briefly, Class A licensees are breeders who raise all animals on their premises from a closed colony (suppliers of purpose-bred dogs are typically Class A dealers). Class B licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and then resell them to research facilities. There are additional regulations that apply to Class B dealers (such as holding periods and recordkeeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. Regulations regarding the sale of pound dogs to research facilities or Class B dealers vary from state to state and include some bans on this practice. The best resource for identification of possible vendors is the "Buyer's Guide" issue of the periodical Lab Animal. Typically the last issue of each year, the "Buyer's Guide" lists sources for both purpose-bred and random-source dogs and denotes such features as pathogen-free status, documentation of health status, and availability of specific breeds and timed pregnant females. Some suppliers also have separate advertisements within that issue of the journal.

D.

Laboratory Management and Husbandry

Federal regulations promulgated by the Animal and Plant Health Inspection Service, USDA, in response to the Animal

Welfare Act (7 CFR 2.17, 2.51, and 371.2[g]) are described in 9 CFR Chapter 1 (1-1-92 edition), Subchapter A, Animal Welfare. Regulations pertaining specifically to the care of dogs used in research are found in Subpart A, Specifications for the Humane Handling, Care, Treatment, and Transportation of Dogs and Cats, of Part 3 (Standards) of Subchapter A. Particular attention should be paid to Section 3.6c (Primary Enclosures-Additional Requirements for Dogs), because the space required for housing dogs is calculated using the length of the dog rather than the body weight (which is used for other species and also for dogs, according to National Research Council (NRC) guidelines). Section 3.8 (Exercise for Dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. The Institute of Laboratory Animal Research (ILAR) has written the "Guide for the Care and Use of Laboratory Animals" (Seventh edition, 1996). The "Guide" is the primary document used by institutional animal research programs to develop and design their programs, as well as by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and other animal care evaluation groups to facilitate site visits and inspections. The ILAR committee on dogs has also written "Dogs: Laboratory Animal Management " (1994). This publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the Animal Welfare Regulations and the requirements of the Public Health Service Policy on Humane Care and Use of Laboratory Animals." The reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting.

II.

A.

BIOLOGY

Normal Values

Growth data for beagles from a purpose-bred dog breeding facility are provided in Table I. Table II features hematology data from beagles from the same commercial facility. Table III lists serum and urine chemical data for beagles. Normal physiologic data for dogs (no breed specified) are provided in Table IV. The information presented in the tables represents a range of normal values that can vary, depending on the analytical method and equipment used as well as the age, breed, gender, and reproductive status of the animal.

B.

Nutrition

Good nutrition and a sound, balanced diet are essential to the health, performance, and well-being of the animal. The basic

ROBERT C. DYSKO ET AL.

398 Table I

Beagle GrowthChart from 3 Monthsto 1 Yearof Agea Beagle weightdata (in kg) Age (months) Mean 3 4 5 6 7 8 9 10 11 12

4.8 6.2 7.1 8.1 8.8 8.9 8.9 9.6 9.3 9.7

Male

Female

S.D.b

Cases

Mean

S.D.

Cases

1.01 1.03 1.11 1.29 1.51 1.59 1.67 2.11 1.73 1.78

108 8018 3850 2412 2252 1746 618 455 391 394

4.7 5.3 6.0 6.7 7.3 7.8 8.2 8.3 8.5 8.5

0.78 0.86 0.97 1.06 1.26 1.46 1.47 1.55 1.64 1.61

155 7187 3182 1485 1365 1220 501 430 351 463

aData fromR. Scipioniand J. Ball of MarshallFarmsUSA,Inc., NorthRose, New York. bS.D., standarddeviation.

nutrient requirements for dogs have been compiled by the NRC and represent the average amounts of nutrients that a group of animals should consume over time to maintain growth and prevent deficiencies (National Research Council, 1985). The reader is referred to these guidelines for useful reference points for management of an animal's diet during various physiologic states (e.g., gestation, lactation, maturational age). Most commercially available balanced dog diets are "closedformula" diets, in which the labeled specific minimum requirements for protein and fat, and the maximum values for ash and fiber, are met. These diets do not necessarily provide the identical composition of ingredients from batch to batch. Ingredient composition varies, depending on the cost relationships of the various ingredients as the manufacturer attempts to achieve the label requirements at the lowest ingredient cost. An "openformula" (or "fixed-formula") diet provides more precise dietary control. In these diets the ingredients are specified, and the percentage of each ingredient is kept constant from batch to batch. "Semipurified" diets provide for the strictest control of ingredients and are formulated from the purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. The animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. As a general rule, diets are generally safe for consumption up to 6 months following the manufacture date when stored at room temperature. Refrigeration may prolong the shelf life, but the best strategy is to use each lot based on the date of manufacture in order to prevent food from expiring and to ensure that only fresh diets are fed. Specifications for feeding and watering of dogs are provided in the regulations of the Animal Welfare Act. Recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. The basal

metabolic rate, or basal energy requirement (BER), refers to the amount of energy expended following sleep, 12-18 hours after food consumption, and during thermoneutral conditions (Kleiber, 1975; Lewis et al., 1987). The maintenance energy requirement (MER) is the amount of energy used by a moderately active adult animal in a thermoneutral environment, which in the dog is approximately twice the BER (Lewis et al., 1987). For dogs weighing greater than 2 kg, the MER may be calculated using this simplified linear equation: MER (metabolizable kcal/day) = 2(30 weightkg + 70) (National Research Council, 1985; Lewis et al., 1987). The quantity of a correctly balanced diet to be fed to each dog can then be determined by dividing the MER by the energy density of the diet. Fat provides three major dietary functions, including absorption of fat-soluble vitamins (A, D, E, and K), enhancement of palatability, and provision of essential (unsaturated) fatty acids. Dietary fat is an excellent, highly digestible energy source, providing 2.25 times more energy on a per weight basis than either soluble carbohydrates or proteins (Lewis et al., 1987). However, fats are not needed for this purpose when adequate carbohydrate and protein are present. Consumption of fat in excess of an animal's ability to metabolize it results in steatorrhea and has been related to the development of acute pancreatitis, whereas lack of dietary fat may lead to a fatty acid/energy deficiency. Fatty acid deficiency is associated with poor growth, poor physical performance, reduced reproductive performance, and weight loss. Dogs are considered to be "easy keepers," because they do not have as many absolute nutritional requirements as their domestic counterpart, the cat. However, they do possess a unique requirement for certain polyunsaturated fatty acids, a deficiency of which may predispose them to decreased growth rates and dermatologic abnormalities, such as "hot spots." Dogs require linoleic (f2-6) acid, an essential fatty acid (National Research Council, 1985), and more recently it has been demonstrated that the f2-3 fatty acids may play a role in maintaining healthy skin (Logas and Kunkle, 1994). Supplementation with a balanced essential fatty acid product (e.g., Derm Caps) may alleviate allergy-related dermatoses such as flea-bite dermatitis and pyoderma (Logas and Kunkle, 1994; Miller, 1989). Essential fatty acid deficiency can occur in dogs receiving low-fat dry dog food that has been stored too long, particularly under warm, humid conditions (Lewis et al., 1987). There are 22 a-amino acids, 10 of which cannot be synthesized in sufficient quantity to meet a dog's normal metabolic demands for growth and maintenance. Hence, as their name implies, these essential amino acids are required by all dogs and must be provided in the diet. The essential amino acids and the minimal requirements for growth are listed elsewhere (Lewis et al., 1987). Chronic excessive protein intake may be detrimental to the kidney by contributing to accelerated renal aging and subsequent glomerulosclerosis (Lewis et al., 1987). Conversely, inadequate protein intake results in retardation of growth and

399

11. BIOLOGY AND DISEASES OF DOGS Table II Hematology Data from Purpose-Bred Beagles a Beagles tested for period 2/28/99 through 9/01/99 Male

Female

Test b

Units

Cases

Mean value

S.D. b

Cases

Mean value

S.D. b

WBC RBC HGB HCT MCV MCH MCHC RDW HDW PLT MPV NEUTI Absolute LYMP-Absolute MONO-Absolute EOS~ Absolute BASO~ Absolute LUCre Absolute NEUTI% LYMPH~% MONO m % EOS I % BASOu% LUCM% LI MPXI Prothrombin time Reticulocyte

Xl03p~l x 1031xl gm/dl % fL pgm gm/dl % gm/dl X 1031xl fL X1031~l

4746 4746 4746 4746 4746 4746 4746 4746 4746 4746 4746 4746

13.50 6.27 13.77 43.76 69.86 21.97 31.46 15.30 1.45 390.28 6.72 7.60

2.90 0.46 1.05 3.16 2.74 0.88 0.82 0.90 0.12 105.41 1.03 2.17

133 133 133 133 133 133 133 133 133 133 133 133

26.3 26.3 26.3 26.3 26.3 26.3 26.3 26.3 26.3 26.3 26.3 26.3

4744 4744 4744 4744 4744 4744 4744 4744 4744 4744 4744 4744

13.61 6.47 14.34 45.47 70.39 22.19 31.54 15.27 1.47 386.47 6.86 7.55

2.85 0.55 1.34 3.94 2.72 0.92 0.78 0.90 0.13 106.99 1.01 2.07

139 139 139 139 139 139 139 139 139 139 139 139

34.6 34.6 34.6 34.6 34.6 34.6 34.6 34.6 34.6 34.6 34.6 34.6

x103txl

4746

4.87

1.11

133

26.3

4744

4.98

1.17

139

34.6

Xl031xl

4746

0.58

0.35

133

26.3

4744

0.64

0.31

139

34.6

x 103~1

4746

0.30

0.13

133

26.3

4744

0.26

0.13

139

34.6

X 1031xl

4746

0.08

0.03

133

26.3

4744

0.09

0.03

139

34.6

X 1031xl

4746

0.07

0.04

133

26.3

4744

0.07

0.04

139

34.6

% % % % % % Index Index sec

4746 4746 4746 4746 4746 4746 4746 4746 4374

55.83 35.56 4.28 2.24 0.58 0.52 2.05 1.10 6.05

6.74 6.54 2.32 0.98 0.25 0.28 0.27 3.82 0.12

133 133 133 133 133 133 133 133 122

26.3 26.3 26.3 26.3 26.3 26.3 26.3 26.3 10.4

4744 4744 4744 4744 4744 4744 4744 4744 4302

55.14 37.01 4.71 1.95 0.65 0.55 2.03 2.00 6.07

6.73 6.59 1.93 0.92 0.28 0.31 0.27 3.71 0.13

139 139 139 139 139 139 139 139 122

34.6 34.6 34.6 34.6 34.6 34.6 34.6 34.6 11.6

262

1.03

0.37

176

38.4

845

1.04

0.37

201

28.0

%

Mean age S.D. b age (days) (days)

Mean age S.D. b age (days) (days)

aData graciously provided by R. Scipioni and J. Ball of Marshall Farms USA, Inc., North Rose, New York. Beagles tested for period 2/28/99-9/01/99. bS.D., standard deviation; WBC, white blood cells; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; HDW, hemoglobin distribution width; PLT, platelets; MPV, mean platelet volume; NEUT, neutrophils; LYMP, lymphocytes; MONO, monocytes; EOS, eosinophils; BASO, basophils; LUC, large unstained cells; LI, lobularity index; MPXI, mean peroxidase activity index

reduction in production and/or performance. Protein deficiency, a potential consequence of decreased food intake, results in decreased energy intake. As a compensatory mechanism for a lack of fat or carbohydrate, body protein catabolism ensues in order to meet energy demands, thus exacerbating the negative protein balance and contributing to the clinical signs of edema/ascites, unkempt appearance, lethargy, and weight loss. Thus, caloric needs must be met before protein needs (Lewis et al., 1987), an important concept to bear in mind in the event of research experiments that may predispose to anorexia. In general, providing a good quality commercial diet that supplies the required

amount of amino acids and caloric requirements of the animal, while avoiding excess protein, will ensure nutritional stability and promote longevity. Appropriate mineral balance in the diet is very important. The best approach in the laboratory setting is to feed a commercial diet that has been formulated with the proper amount and balance of minerals for normal growth. The recommended amount of dietary minerals and the major causes and clinical signs of deficiencies are published elsewhere (Lewis, 1987). Determining the specific mineral involved in an imbalance can be a diagnostic challenge, because the clinical signs for several excesses/

4

0

0

R

O

B

E

R

T

C. D Y S K O E T AL.

Table III

Clinical Chemistry Data for the Beagle a Male Analyte

Units

Senlm Glucose Urea nitrogen Creatinine Sodium Potassium Chloride Bicarbonate Calcium Phosphorus Magnesium Iron Total Iron Binding Capacity Haptoglobin Alanine aminotransferase Aspartate aminotransferase Alkaline phosphatase Lactate dehydrogenase Sorbitol dehydrogenase ~/-Glutamyl transpeptidase Creatinine kinase Protein, total Albumin Cholesterol Triglycerides Bilirubin, total Bile acids Uric acid Luteinizing hormone Prolactin Growth hormone Thyroid stimulating hormone Adrenocorticotropic hormone Vasopressin Oxytocin Thyroxine, total Triiodothyronine, total Thyroxine, free Parathyroid hormone

mg/dl mg/dl mg/dl mEq/1 mEq/1 mEq/1 mEq/1 mg/dl mg/dl mg/dl I~g/dl Ixg/dl mg/dl IU/liter IU/liter IU/liter IU/liter IU/liter IU/liter IU/liter gm/liter gm/liter mg/dl mg/dl mg/dl I~mol/1 mg/dl ng/ml ng/ml ng/ml ng/ml pg/ml pg/ml pg/ml lxg/dl ng/dl ng/dl pg/ml

< 1 year

98.0 22.0 0.80 150.0 4.90 113.0

• 10.3 b -----7.1 -----0.13 _--.3.40 -----0.38 • 2.00

11.2 ___0.62 5.60 • 0.85

125 23.0 19.0 63 52

___37.5 ___7.4 -----5.1 ___21.1 • 28.4

106 59 30 175 62 0.5

___71.1 ___3.5 • 2.5 • 38.0 • 22.7 • 0.49

Female > 1 year

< 1 year

> 1 year

95.0 • 9.2 18.0 • 4.0 0.80 • 0.14 149.0 ___4.20 4.70 • 0.35 113.0 • 4.00 21.0 • 1.5 10.7 • 0.71 3.70 • 0.79 2.10 • 0.30 105 __. 37.5 c,d 359.6 d 62.5 • 18.75 d 29.0 • 11.4 21.0 _--_4.6 41 • 18.7 53 • 23.9 4.5 • 1.9 c'd 3.5 _ 1.8 c'd

98.0 • 7.9 17.0 ___4.3 0.80 ___0.11 152.0 _ 2.60 4.80 • 0.33 114.0 • 2.80

95.0 • 10.1 19.0 ___5.3 0.80 • 0.15 150.0 __+3.40 4.70 __. 0.35 113.0 • 3.50

11.1 • 0.47 4.80 ___0.83

10.8 ___0.55 3.80 ___ 1.18 2.20 __+0.28

25.0 20.0 60 49

26.0 __+ 15.0 20.0 ___4.5 43__. 17.0 58 _ 38.0

78 ___36.7 62 _ 4.4 36 __- 4.5 151 _ 31.5 58 • 19.0 0.4 • 0.38 2.6 • 0.40 d 1.15 • 1.43 c 0.2--10 c 9a 2.0 c <0.5 c 20-100 c 2.5-10 ~,d 0-30 c 1.5--3.0 ~,d 100 -- 200 c,d 0.8--2.0 ~,d 18_122 c,d

118 • 60.5 58 • 2.9

81 ___45.3 61 ___3.7

183 _ 38.6

176 • 41.9 65 ___22.9 0.4 __. 0.35

• 9.4 • 4.4 • 21.7 __. 28.0

55 __. 19.6 0.4 ___0.36

1.06 • 1.45 c

2.0 (basal) 12.6 (anestrus) 2.0 c <0.5 c 20-100 c 4-73 r

(continues)

deficiencies are similar and nonspecific. A definitive diagnosis is often made only after the diet has undergone analysis of the mineral components. Once the imbalance has been identified, the safest resolution to the problem is to discard the entire lot of misformulated diet. Attempting to correct the imbalance through oral supplementation is likely to be more harmful than beneficial, and it risks intensifying the problem by creating additional mineral imbalances. Vitamins function as enzymes that regulate a wide variety of physiologic processes. They are divided into two groups based on their solubility. The fat-soluble vitamins include A, D, E, and

K, whereas the rest are water-soluble. A list of the vitamins, their requirements, and clinical signs associated with deficiencies and toxicities is published elsewhere (Lewis et al., 1987). Cases of dietary deficiency are rarely encountered in the research setting, because laboratory dog chows are fortified with vitamins. Additional vitamin supplementation may occasionally be required during prolonged clinical illnesses, such as polyuria or diarrhea, which predispose to loss of water-soluble vitamins (B complex and C) (Lewis et al., 1987). However, as with minerals, routine supplementation of vitamins may induce inadvertent toxicity and exacerbation of an imbalance.

401

11. BIOLOGY AND DISEASES OF DOGS Table III (Continued) Clinical Chemistry Data for the Beagle

a

Male

Female

Analyte

Units

< 1 year

> 1 year

1,25-Dihydroxy vitamin D Cortisol Aldosterone Epinephrine Norepinephrine Insulin Progesterone

pg/ml i~g/dl ng/dl pg/dl pg/dl pdU/ml ng/ml

88----- 13

35 -----7.0 c'd 1.8--4.0 ~,d 6--31 c 100 -- 400 c,d 68--526 c,d 5_25~,d <0.2 c

Estradiol

pg/ml

<20 ~

ng/ml

1-7 c

ml/22hr gm/liter

186 _ 159 1.036 + 0.014 7.5 ___0.14 1013 • 539 126 • 68 89 • 56 110_ 77 102 • 58 3.3 • 1.3 147 • 85 42.1 • 29.1 16.2 • 13.0d 16.2 • 2.0 c'd 6.9 • 2.9 c'd 1.02 _ 0.56 d 0.5 • 0.2 d 1.23 • 1.21d 3.7 • 0.77c'd 3.89 • 0.42 c'd 8.8 "+" 1.0 c'd 234 • 16.4c,d 3.5 • 7.9 c'd 43.8 • 11.1 c,d

Testosterone Urine Volume Specific gravity pH Osmolality Chloride Sodium Potassium Phosphorus Calcium Creatinine Protein Alkaline phosphatase Lactate dehydrogenase N-Acetyl-13-o-glucosaminidase Acid phosphatase Aspartate aminotransferase Alanine aminotransferase Creatinine clearance Insulin clearance PAH clearance Glomerular filtration rate Urine flow rate PSP clearance

mOsm/kg mmol/liter mmol/liter mmol/liter mg/dl mg/dl mg/dl mg/24 hr IU/24hr IU/24hr U/liter IU/24hr IU/24 hr IU/24hr ml/min/kg ml/min/kg ml/min/kg

rrd/kglhr

ml/kg/hr %

aSummarized from Loeb and Quimby (1999). bMean • S.D.

C.

Reproduction

M a n a g e m e n t of a b r e e d i n g c o l o n y requires b r o a d k n o w l e d g e of the dog's anatomy, reproductive physiology, and b e h a v i o r a l needs during breeding, gestation, and parturition. A l t h o u g h a c o m p r e h e n s i v e discussion of the b i o l o g y of canine r e p r o d u c t i o n is b e y o n d the scope of this chapter, essential features of the b r o a d topics n o t e d above are presented. This section is largely b a s e d on i n f o r m a t i o n assimilated f r o m texts such as "Miller's A n a t o m y of the D o g " (Evans and C h r i s t e n s e n , 1993), "Veterinary R e p r o d u c t i o n and Obstetrics" ( A r t h u r et al., 1989), and an

< 1 year

> 1 year

5-11

< 1c (proestrus, estrus) > 10 c (diestrus, pregnancy) 25-50 c (proestrus, estrus) <20 c (pregnancy) <0.2 191 +_ 156

1019 ___517 126 ___68 107 • 61 108 __. 68 109 __.46 3.0 +_. 1.3 143 ___87

CBreed not specified. dGender not specified.

issue of V e t e r i n a r y C l i n i c s o f N o r t h A m e r i c a : S m a l l A n i m a l P r a c t i c e d e v o t e d to pediatrics of puppies and kittens (Hoskins, 1999).

1.

Anatomy and Reproductive Physiology of the Bitch

T h e ovaries of the bitch are attached to the dorsolateral walls of the a b d o m i n a l cavity caudal to the kidneys by the b r o a d liga m e n t s and are not p a l p a b l e abdominally. T h e uterus consists of the cervix, uterine body, and uterine horns. T h e cervix is an a b d o m i n a l organ, located a p p r o x i m a t e l y halfway b e t w e e n the

402

ROBERT C. DYSKO E T AL.

Table IV Normal PhysiologicData for Dogsa Parameter Body temperature Heart rate (beats/min) Respiration rate

Awake 99.5~176 (37.5~176 70-180 20-40

(breaths/min) Capillary refill time (sec) Arterial pH Arterial Pco2(mm Hg) Arterial Po2(mm Hg)

7.30-7.43 30-49 91-97

Arterial HCO 3 (mEq/liter) Arterial base excess (mEq/liter)

18-22 - 3 to +3

Anesthetized (if different)

pregnant. Serum progesterone levels peak during diestrus. The duration of anestrus is approximately 4 months. Anestrus is the stage of reproductive quiescence, characterized by an absence of ovarian activity and serum progesterone levels of less than 1 ng/ml. 2.

60 - 180 8-20

<1.5

90 -500 (if >50% inspired 02)

a Breed not specified. Modified from Birchard and Sherding (1994).

ovaries and the vulva. When the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. Catheterization of the cervix is usually not possible in the normal bitch at any stage of the reproductive cycle, except during or immediately following parturition. Thus, semen is deposited at the external cervical os during natural or artificial insemination. The vagina is a long musculomembranous canal that extends from the uterus to the vulva. When the vagina is examined, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva so as to avoid the deep ventral clitoral fossa. Examination should proceed at an angle of approximately 60 ~ until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. The bitch has a monoestrous cycle, with clinical estrus occurring predominantly in January or February and again in July or August (although it can occur at any time of year). The estrous cycle consists of four stages: proestrus, estrus, diestrus, and anestrus. The average duration of proestrus is 9 days. During this stage the vulva is enlarged, turgid, and firm, and a sanguinous vaginal discharge is present. Endocrinologically, proestrus is the follicular stage of the cycle, and estrogen levels peak at this time. Estrus generally lasts 9 days, and the vulva is softer and smaller than in proestrus. A vaginal discharge persists during estrus and may remain serosanguinous or become straw-colored. The endocrine feature of estrus is the luteinizing hormone (LH) surge, followed by ovulation within 24-72 hours. Diestrus begins approximately 9 days after the onset of standing heat. The end of this stage is 60 days later, which would be coincident with whelping if the bitch had become

Anatomy and Reproductive Physiology of the Male Dog

Components of the canine spermatic cord include the ductus deferens, the testicular artery and vein, the lymphatics and nerves, and the cremaster muscle. The cremaster muscle and pampiniform plexus aid in thermoregulation of the testicles, which are maintained at 2~176 lower than basal body temperature. Sweat glands in the scrotum assist in lowering the scrotal temperature through evaporation. The penis is a continuation of the muscular pelvic urethra and is attached to the ischiatic arch by two fibrous crura. It is composed of fibrous tissue and three cavernous sinuses: corpus cavernosum, corpus spongiosum penis, and corpus spongiosum glandis. The accessory sex glands of the dog consist of only a well-encapsulated prostate gland that surrounds the pelvic urethra, and ampullary glands at the termination of the vas deferens in the urethra. The dog does not have seminal vesicles or bulbourethral glands. The onset of puberty ranges from 5 to 12 months of age and is affected by breed, season, and nutritional and disease status. Testicular growth is rapid at this time, and the seminiferous tubules begin to differentiate. The Sertoli cells form the bloodtestis barrier, the tubules become hollow, and spermatogenesis commences. This process is initiated by the secretion of LH from the anterior pituitary, which stimulates the production of testosterone by the interstitial, or Leydig's, cells. Secretion of follicle-stimulating hormone (FSH) by the anterior pituitary stimulates the production of other key hormones by the Sertoli cells, including inhibin, androgen binding protein, and estrogen. FSH stimulates spermatogenesis in the presence of testosterone, while inhibin and estrogen play a role in a feedback loop on the pituitary gland to decrease FSH production. Spermatogenesis in the dog is completed in 45 days, with subsequent maturation of sperm occurring in the epididymis for approximately 15 days. Thus, the entire process from initiation of spermatogonial mitosis to delivery of mature sperm to the ejaculate is 60 days. A breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. Factors affecting male fertility include libido, ability to copulate, testicular size, and quality and number of sperm produced. Problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities that suppress sexual behavior. Animals with poor hindlimb conformation or with trauma to the back or hindlimbs may be unable to properly mount the female. There is a positive correlation with the size of the testicles as measured by scrotal circumference and the

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11. BIOLOGY AND DISEASES OF DOGS number of sperm produced. Finally, parameters used to assess the quality of sperm include motility, morphology, volume, and concentration. An ejaculate (5 ml) that contains approximately 500 million progressively motile sperm without significant morphological abnormalities (such as a kinked tail) is a good indicator of normal male fertility. In general, erection, which involves muscular contractions and increased arterial blood flow to the penis, is controlled by the parasympathetic nervous system, whereas ejaculation is under sympathetic control. On mounting, the initial thrusting and ejaculation of semen last about 1 minute. The bulbus glandis becomes enlarged, which lodges the penis in the female reproductive tract. The male then dismounts and brings one hindleg over the female, and the two continue to be joined "rear to rear," a position classically termed "the tie." Ejaculation of the accessory gland fluid continues for 5 - 3 0 minutes. The continued expulsion of prostatic fluid during the "tie" may serve to propel the semen from the vagina through the cervix into the uterus. Fertilization occurs in the oviduct and may occur as late as 8 days after coitus, because of the long life span of sperm in the dog. However, once ovulated, oocytes generally remain viable for only 12-24 hours. Therefore, the bitch should be bred prior to ovulation to ensure the presence of sperm for fertilization of live oocytes. 3.

Detection of Estrus and Pregnancy

Cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. Because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. The smear will not confirm the presence of ovulation, nor is it of prognostic value in normal bitches during anestrus. The percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. As cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. The nuclear-cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). The cells appear "anuclear" and are classified as "cornified" or "anuclear squames." Cornification occurs approximately 2 days prior to the estrogen peak and 4 days prior to standing heat. The percentage of cornified cells (of the total number of epithelial cells) decreases gradually to zero after the onset of diestrus. The vaginal cytology smear of the bitch changes from predominantly cornified to noncornified 6 days after ovulation. The day of this change is the first day of diestrus. Other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-

stained nuclei, and a high nuclear-cytoplasmic ratio). Based on vaginal cytology, the estrous cycle is classified as follows:

Proestrus, early: intermediate and superficial cells, red blood cells, and neutrophils. Proestrus, late: superficial cells, anuclear squames, and red blood cells. Estrus: more than 50% anuclear squames, superficial cells, with or without red blood cells. Diestrus: more than 50% intermediate cells, superficial cells and squames early, but becoming completely noncornified with neutrophils present as diestrus proceeds. Anestrus: small numbers of parabasal cells and intermediate cells, with or without neutrophils. Although vaginal cytology is a useful tool, it is not a substitute for observation of behavioral estrus, which is the best criterion to use in breeding management. During proestrus the male is attracted to the bitch and will investigate her hindquarters, but she will not accept breeding. The behavioral hallmark of estrus is standing receptivity toward the male. During this stage the bitch will exhibit "flagging," or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. In order to maximize the conception rate, and the number of pups whelped per egg ovulated, it is recommended to breed the bitch on days 1, 3, and 5 of the standing heat. 4.

Pregnancy

Fertilization is completed in the mid- to distal oviduct. Implantation is evident by areas of local endometrial edema 17-18 days after breeding. There is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. The dog has endotheliochorial placentation. The endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. The canine placenta is also classified as zonary and deciduate, indicating that the placental villi are arranged in a belt and that maternal decidual cells are shed with fetal placentas at parturition. The length of gestation is 59-63 days. Luteal progesterone is responsible for maintaining pregnancy, and canine corpora lutea retain their structural development throughout gestation. Serum progesterone rises from less than 1 ng/ml in late proestrus to a peak of 3 0 - 6 0 ng/ml during gestation, then declines to 4 - 5 ng/ml just prior to parturition. Progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility. Pregnancy detection can be performed by abdominal palpation of the uterus 28 days after breeding. The embryos and chorioallantoic vesicles form a series of ovoid swellings in the

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early gravid uterus. They are approximately 2 inches in length at 28-30 days, the time at which pregnancy is most easily and accurately diagnosed. By day 35 the uterus begins to enlarge diffusely, so that the vesicles (and, therefore, pregnancy) are difficult to identify by palpation. Fetal skeletons become calcified and are radiographically evident by day 42. Bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. Real-time ultrasound can be utilized for pregnancy detection of vesicles as early as 25-28 days. 5.

Parturition and the Neonate

An abrupt drop in body temperature to less than 100~ indicates impending parturition within 18-24 hours. The process of parturition has been divided into three stages, Stage 1 of labor lasts 6 - 1 2 hours and is characterized by uterine contractions and cervical dilation. During this stage, the bitch may appear restless, nervous, and anorexic. Other common clinical signs include hard panting and increased pulse and respiration rates. Fetal expulsion occurs during stage 2, which lasts approximately 3 - 6 hours. As the fetus engages the cervix, the neuroendocrine system induces the release of oxytocin; this is referred to as the Ferguson reflex. Oxytocin strengthens the uterine contractions and may elicit voluntary abdominal contractions as well. The bitch is usually recumbent during stage 2 but is able to inhibit this stage if labor if disturbed. The chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. Interestingly, posterior presentation is common in dogs but does not predispose to dystocia. The time interval between delivery of each pup is irregular, but the average time lapse is less than 1 hour between pups until parturition is complete. Veterinary assistance is necessary if the bitch remains in stage 2 for more than 5 hours without delivering the first pup, or for more than 2 hours before delivering subsequent pups. The placentas are expelled during stage 3 of labor, immediately following delivery of a pup, or up to 15 minutes thereafter. If two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. The bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. She will also sever the umbilical cord. The bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. Thermal support should be provided prior to parturition. Dogs housed on grated flooring should be provided with mats, and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. Shavings are discouraged as they have the potential to coat the umbilical cord, which may predispose to ascending infections. Heat lamps may be placed 24 hours prior to parturition and remain until all neonates dem-

onstrate vigorous and successful suckling behavior. However, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. If possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. Thus, monitoring of parturition is important, but human intervention should be minimal in order to prevent stress-induced cannibalism. Weak or debilitated puppies may be cannibalized by the bitch before the research staff recognizes the need for veterinary attention. The postpartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. In these cases, 5 - 2 0 units of oxytocin may be administered intramuscularly. Uterine involution occurs during anestrus within 4 - 5 weeks of parturition. During this time a greenish to red-brown vaginal discharge, or lochia, may be noted. Although lochia is normal, the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. Desquamation of the endometrium begins by the sixth postpartum week, with complete repair by 3 months. Newborn puppies are easily sexed by examination of the anogenital distance. In female puppies the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. Eyes are open at approximately 12 days, and ears are patent at approximately 1220 days. Solid food can be introduced between 4.5 and 6 weeks of age, and puppies can be weaned at 6 - 8 weeks. 6.

Artificial Insemination

Artificial insemination (AI) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as strictures, or when the bitch refuses to stand for breeding. Semen for AI is collected using a plastic centrifuge tube and rubber latex artificial vagina. The male is introduced to the bitch's scent and manually stimulated. After collection of the first two fractions, a sufficient amount of the third fraction, which consists predominantly of prostatic fluid, is collected to bring the total semen volume to 4 - 6 ml. The semen is then drawn into a sterile 10 or 12 ml syringe attached to a sterile disposable insemination pipette. The bitch is inseminated either standing or with raised hindquarters. A gloved index finger is inserted into the dorsal commissure of the vulva and directed craniodorsally until it is over the ischial arch. The tip of the insemination pipette is introduced and guided by the gloved finger toward the external cervical os. The semen is injected, and 2 - 3 ml of air are then flushed through the syringe and pipette. The pipette is withdrawn, and the gloved finger is used to feather the ceiling of the vagina until contractions of the vaginal musculature are palpable. The bitch's hindquarters are subsequently elevated to promote pooling of semen around the external cervical os. As with natural breeding, AI should be performed on days 1,

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11. BIOLOGY AND DISEASES OF DOGS 3, and 5 of standing heat, or on the days of maximal vaginal cornification. The bitch should be palpated for pregnancy approximately 4 weeks after the first insemination. 7.

False Pregnancy

False pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. The condition occurs after the decline in serum progesterone toward the end of diestrus. There is no age or breed predisposition. Pseudopregnancy does not predispose the bitch to reproductive disease or infertility. However, in the event of extreme discomfort due to mammary gland enlargement, bitches may be treated with mibolerone (Cheque Drops) at an oral dose of 16 ~tg/kg q24 hr for 3 - 5 days (Brown, 1984). 8.

Reproductive Life Span

Reproductive performance in the bitch is optimal prior to 4 years of age. Although normal cycle lengths are reported to occur up to the ages of 5 - 7 years, the interestrous interval tends to increase by 4 years of age. Cycling does not completely cease; however, after 8 years of age, bitches demonstrate significant decreases in conception rate and number of live pups whelped. By 8 - 9 years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia are extremely common.

D.

Behavior

Beagles have been a popular animal model because of their docile nature. They are easily handled and for the most part respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiogram (ECG) recordings, oral gavage, and venipuncture. Dogs are sexually mature by 6 - 9 months of age, but they are not socially mature until 18-36 months of age. The socialization process should begin early during development, when puppies are receptive to conspecific and human contact. For example, from 3 - 8 weeks of age, puppies are most capable of learning about how to interact with other dogs. Between weeks 5 and 12, puppies are most capable of learning how to interact with people. By 10-12 weeks of age dogs voluntarily wander and explore new environments. Thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. The extent to which breed affects behavior has been the subject of popular speculation but is difficult to prove. In general, breed-specific patterns do tend to emerge. For example, it appears that beagle pups are very motivated by food reward (Overall, 1997). This is not surprising, because the breed was selected

to work with its nose, and this may be a useful attribute for laboratory investigations that are predicated on food restriction. Canid social systems use signals and displays that minimize the probability of outright aggression. These behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. An excellent, illustrated discussion of normal canine behavior patterns can be found in the third chapter of "Clinical Behavioral Medicine for Small Animals" (Overall, 1997).

III.

DISEASES

By virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of the disorders that can affect this species. The authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting. Especially noted in this chapter are infectious diseases associated with the use of random-source dogs that have unknown vaccination history and have had intensive contact with other similar animals at pounds and/or shelters, or conditions seen frequently in the beagle, the most common breed used in biomedical research. For more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks, such as the "Current Veterinary Therapy" series (J. D. Bonagura and R. W. Kirk, eds.), "Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), and "Infectious Diseases of the Dog and Cat" (C. E. Greene, ed.). Full citations of some chapters from these texts are listed in the references (W. B. Saunders Co. of Philadelphia publishes all three texts.)

A.

Infectious Diseases

1. Bacterial, Mycoplasmal, and Rickettsial Diseases

Canine Infectious Tracheobronchitis (Kennel Cough Complex) Etiology. Infectious tracheobronchitis (ITB) is a highly contagious illness of the canine respiratory tract that usually manifests as an acute but self-limiting disease. Several organisms have been incriminated as causative for this condition: Bordetella bronchiseptica; canine parainfluenza virus (CPIV); canine adenovirus types 1 and 2 (CAV-1, CAV-2); canine herpesvirus; canine reovirus types 1, 2, and 3; and mycoplasms and ureaplasms.

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Clinical signs. Clinical infectious tracheobronchitis can be subdivided into mild or severe forms. The mild form is the more common presentation and is characterized by an acute onset of a loud, dry, hacking cough. Increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. Cough is easily elicited by tracheal palpation and may be more frequent with excitement or exercise. Otherwise the dog is typically asymptomatic, with normal body temperature, attitude, and appetite. Mild tracheobronchitis usually lasts 7-14 days, even if left untreated. The severe form of tracheobronchitis generally results from mixed infections complicated by poor general health, immunosuppression, or lack of vaccination. Secondary bronchopneumonia can occur and may be the determinant of severity (Sherding, 1994). Animals are clinically ill and may be febrile, anorexic, and depressed. Productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. These cases require more aggressive treatment and may be fatal. Epizootiology and transmission. The natural reservoir for Bordetella bronchiseptica is considered to be the respiratory tract of infected animals (Bemis, 1992). This bacterium is very easily spread by aerosol and direct contact, and fomite transmission is also possible (Bemis, 1992). Transmission is favored by confined housing of multiple animals. In experimental studies, B. bronchiseptica transmission to susceptible individuals was 100% (Thompson et al., 1976; McCandlish et al., 1978). The incubation period is 3-10 days. CPIV and CAV-2 are also spread by aerosols. Of these two viruses, CAV-2 is the most persistent, lasting for up to several months in the environment, whereas CPIV is fairly labile (Hoskins, 2000a). Both viruses can be destroyed by quaternary ammonium disenfectants.

Pathogenesis. The most common clinical isolates are CPIV and Bordetella bronchiseptica. However, B. bronchiseptica may be a commensal organism, and it is often recovered from asymptomatic animals. In cases of clinical infection, B. bronchiseptica attaches to the cilia on the mucosal surface of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. Infections with CPIV or CAV-2 alone are usually subclinical; coinfections with B. bronchiseptica or other microbes may result in clinical ITB (Keil and Fenwick, 1998; Wagener et al., 1994). The characteristic lesion from CPIV or CAV-2 infection is necrotizing tracheobronchiolitis (Dungworth, 1985). Pathogenic infection of the upper airways typically results in inflammation and ciliary dysfunction.

Diagnosis and differential diagnosis.

Diagnosis of infectious tracheobronchitis is often based on clinical signs. Isolation of Bordetella bronchiseptica or mycoplasma by nasal swabs allows only a presumptive diagnosis. Viral isolation or paired serology can be done but is often impractical and expensive. If cough persists for more than 14 days, other disease conditions

ROBERT C. DYSKO ET AL.

should be considered. Canine distemper virus infection, pneumonia, heartworm disease, tracheal collapse, and mycotic infections are differential diagnoses for dogs with similar signs. Bronchial compression as a result of left atrial enlargement, hilar lymphadenopathy, or neoplasia may also elicit a nonproductive cough (Johnson, 2000) and should be considered as a differential for ITB.

Prevention. Prevention is best achieved by avoiding exposure to infected animals, but this is oftentimes not practical. Dogs should be vaccinated prior to, or at the time of, admission to the animal research facility. Intranasal vaccine combinations for Bordetella bronchiseptica and CPIV are preferred. Intranasal vaccines protect against both infection and disease, can be given to dogs as young as 2 weeks of age, and can produce immunity within 4 days.

Control. Sanitation and ventilation are critical for control. The animal care staff must practice proper hygiene to prevent fomite transmission. Symptomatic animals should be isolated, and animal-to-animal contact avoided. Kennels should be disinfected with agents such as bleach, chlorhexidine (Nolvasan) or quaternary ammonium chloride (Roccal-D). Proper ventilation and humidity are important in controlling spread of these infectious agents; 15-20 air changes per hour at 50% relative humidity are recommended (Sherding, 1994). Treatment: No specific treatment is available for viral infections. Bordetella bronchiseptica is typically sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. Use of antibiotics is indicated when severe or persistent clinical signs occur, and it should be continued for 14 days. Use of empirical antibiotic treatment in mild cases may hasten the resolution of clinical signs. For severe or unresponsive infection, treatment should be based on bacterial culture sensitivity patterns; nebulized gentamicin may be helpful. Cough suppressants (e.g., dextromethorphan) should be avoided if the cough is bringing up mucus (productive); however, their use is indicated if coughing is causing discomfort or interfering with sleep. Bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction and minimizing discomfort. Research complications.

Because infectious tracheobronchitis results in altered respiratory tract histology and impaired mucociliary clearance, infected animals should not be used for pulmonary studies. Animals with clinical disease would also be poor surgical candidates.

b.

Group C Streptococcus Infections

Etiology. [3-Hemolytic Lancefield's group C streptococcus (Streptococcus zooepidemicus) is a gram-positive non-spore-

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11. BIOLOGYAND DISEASES OF DOGS forming coccus and an etiologic agent for pneumonia and septicemia in dogs.

Clinical signs. Clinical signs vary based on the organ system affected. Pneumonic disease is typically associated with coughing, weakness, fever, dyspnea, and hematemesis. Peracute death without clinical signs has been reported in a previously healthy research dog (Bergdall et al., 1996), and conjunctivitis can also be caused by this organism (Murphy et al., 1978). Epizootiology and transmission. Lancefield's group C streptococci have been isolated as commensal flora in the upper respiratory tract and the vagina of clinically normal dogs (Olson et al., 1973). Epizootics have been reported in both racing greyhounds and research colonies (Sundberg et al., 1981; Garnett et al., 1982). In these epizootics, and in the reported case of peracute death (Bergdall et al., 1996), recent transportation (within 7 days) was associated with the disease. As such, Lancefield's group C streptococcus may be an opportunistic pathogen in dogs. Pathologic findings. In the peracute case reported (Bergdall et al., 1996), hemorrhage from the mouth and nose and within the pleural cavity was the most striking lesion. Ecchymotic and petechial hemorrhages were seen on other organ surfaces. The lungs were heavy and wet, and blood oozed from cut surfaces. "Bull's-eye" lesions were observed on the pleural surface of affected lung lobes, similar to ischemic lesions seen with fungal infections (Fig. 2). Histologically, the lungs were characterized by areas of hemorrhage surrounding foci of degenerative neutrophils, blood, and necrotic debris. Gram-positive cocci were seen in both the lung and the tonsils. Pathogenesis. The pathogenesis for disease caused by Lancefield's group C streptococcus is unclear. Strain variation with respect to virulence and host immune factors is probably significant. Diagnosis and differential diagnosis. Definitive diagnosis is made based on bacterial culture and identification. Any cause of pneumonia and/or peracute death in dogs needs to be considered as a differential diagnosis. Bacterial pneumonias or septicemias can be caused by other pathogenic Streptococcus spp., Staphylococcus spp., Escherichia coli, Pasteurella multocida, Pseudomonas spp., Klebsiella pneumoniae, and Bordetella bronchiseptica. Nonbacterial causes include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. Prevention and control. Too little is known about the pathogenesis of Lancefield's group C streptococcus to make any recommendations about prevention and control.

Fig. 2. Bull's-eyelesions seen with peracute Lancefield's group C streptococcalpneumoniain a random-sourceresearch dog. Lesionson the lung surface are characterizedby a ringed pale area and central ulceration that oozedblood.

Treatment. Antibiotic therapy should be provided, based on culture and sensitivity. Intravenous fluids are indicated for febrile or systemically ill patients. For dyspneic patients, oxygen therapy and strict activity restriction are required. Research complications. Clearly, dogs with severe hemorrhagic pneumonia or septicemia are not appropriate for any research study. The association between epizootics of this disease and transportation shipment supports the philosophy of providing acclimation periods to animals upon arrival at research facilities to evaluate health status and enable the animals to normalize physiologically. c.

Leptospirosis

Etiology. Serovars of the spirochete Leptospira interrogans sensu lato cause canine leptospirosis. Disease in dogs is primarily due to serovars canicola, icterohemorrhagiae, grippotyphosa, pomona, and bratislava. Clinical signs. Leptospirosis may present as either an acute or a chronic problem. Clinical signs are nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, and vomiting. Animals may be febrile and may be reluctant to move, because of muscle or renal pain or meningitis. Icterus, congested mucous membranes, or signs referable to

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disseminated intravascular coagulation (petechial/ecchymotic hemorrhages, melena, epistaxis, or hematemesis) are also possible. Animals with peracute leptospirosis are characterized by septicemia, shock, vascular collapse, andrapid death. Uveitis, abortions, and stillbirths have also been associated with leptospirosis.

it develops, is most common in the acute phase. The combination of azotemia and icterus should alert the clinician to the possibility of leptospirosis. Disseminated intravascular coagulation is often a secondary complication. The severity and course of leptospirosis depend on the causative serovar and the age and immune status of the patient.

Epizootiology and transmission. Vaccination and reduced exposure to reservoir hosts have markedly decreased the prevalence of leptospirosis over the past 30 years. Wild animals, cattle, and rodents are reservoirs for Leptospira. The epidemiology of the disease is not static, and recent changes have been observed. Serovars pomona, grippotyphosa, and bratislava are becoming more common causes of canine disease, with canicola and icterohemorrhagiae becoming less common. This may be due to vaccination practices and increased movement of wildlife reservoirs (raccoons, skunks, and opossums) into urban/suburban areas. Rats have been implicated as important in the transmission of serovars canicola and icterohemorrhagiae (Rentko et al., 1992; Brown et al., 1996; Kalin et al., 1999). Transmission occurs primarily by environmental contact, and not directly from animal to animal. Infected hosts shed leptospires in urine, thereby contaminating the environment; naive animals are infected when the organisms contact mucous membranes or abraded skin. Recovered animals may shed organisms in their urine for months to years. The organisms are actually labile in the environment; moisture, moderate temperatures, and alkaline soil favor survival and subsequent transmission. Close contact, bites, ingestion of infected meat, and transplacental and venereal transmission are also possible. Leptospirosis is a zoonotic disease.

Diagnosis and differential diagnosis. Zinc toxicity in dogs most closely mimics the clinical syndrome of leptospirosis. Other causes of acute and chronic renal failure, icterus, and acute hepatic failure must also be considered. Paired serology is the most reliable means of definitive diagnosis; however, seroconversion may not occur until after the first week of infection.

Pathologic findings. The kidneys consistently have gross and microscopic lesions. In the acute phase of the infection, kidneys are swollen and have subcapsular and cortical ecchymotic hemorrhages. Petechial or ecchymotic hemorrhages and swelling of the lungs and liver may also be noted. Hepatic lesions during the acute phase consist of diffuse hemorrhage and focal areas of necrosis (Searcy, 1995). In chronic stages of leptospirosis the kidneys become small and fibrotic. Endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. Pathogenesis. Infection occurs after the leptospires penetrate a mucous membrane or abraded skin. The organisms then invade the vascular space and multiply rapidly. Several days postinfection the renal tubular epithelium (and, to a variable extent, the liver) is colonized. The hematogenous phase lasts 4 - 1 4 days. Acute renal failure or progressive renal failure leading to oliguria or anuria may occur. The most common clinical syndrome is chronic or subclinical infections after recovery from the acute phase (Greene, 2000). The nephritis may or may not be accompanied by hepatitis, uveitis, and meningitis. Icterus, if

Prevention and control. Vaccination for leptospirosis is standard veterinary practice. Bivalent inactivated bacterins for serovars of L. interrogans canicola and serovars of L. interrogans icterohemorrhagiae are commercially available. However, immunization does not prevent development of the carrier state or protect against other serovars. For outdoor-housed dogs, an effective program to prevent contact with wildlife reservoirs is important. Control requires identification and either treatment or elimination of carrier animals. Treatment. Penicillins are the drugs of choice for treating leptospiremia, and prompt use reduces fatal complications. Aggressive fluid therapy and supportive care may also be needed. Elimination of renal colonization and the carrier state can be accomplished with dihydrostreptomycin or doxycycline administration. Research complications. Dogs with clinical leptospirosis should not be used in research studies because of the effects of the disease on renal and hepatic function. d.

Campylobacteriosis

Etiology. Campylobacteriosis in dogs is caused by Campylobacter jejuni, a thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rod. Clinical signs. Most adult animals infected with C. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than 6 months of age (Greene, 2000; Burnens et al., 1992). In cases of clinical illness, small volumes of mucoid or watery diarrhea, with or without frank blood, are most commonly noted. These signs are usually mild, may be intermittent, and typically last 5-21 days. Tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea. Epizootiology and transmission. The role of C. jejuni as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (Sherding and Johnson, 1994).

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BIOLOGYAND DISEASESOF DOGS

Prevention and control.

Clinical signs of disease most often occur in dogs less than 6 months of age, although any age may be affected. Stress or immunosuppression may make animals more susceptible to clinical disease. Pound and shelter populations have the highest rates of fecal excretion of C. jejuni (Sherding and Johnson, 1994). Transmission is via the fecal-oral route, mostly through fecally contaminated food or water. Unpasteurized milk, poultry, and meat are other sources of infection. Campylobacter jejuni can be zoonotic; children and immunocompromised individuals are at the greatest risk.

Treatment. Neomycin, enrofloxacin, chloramphenicol, and doxycycline are all effective antibiotics. Treatment should be for 10-14 days, and bacterial cultures should be repeated 1 and 4 weeks after treatment.

Pathologic findings.

Research complications.

The actual lesions observed depend upon the mechanism of the enteropathy (Van Kruiningen, 1995). Enterotoxin production results in dilated fluid-filled bowel loops, with little or no histopathologic alteration. In cytotoxin-mediated disease, hyperemia and a friable, hemorrhagic mucosal surface are noted. On histopathology the mucosal surface is irregular and ulcerated, and a lymphocytic-plasmacytic ileitis or colitis may be seen. When translocation occurs, the lamina propria becomes edematous and congested, with focal accumulation of granulocytes in the crypts and lamina propria. Focal areas of epithelial hyperplasia and decreased numbers of goblet cells are also noted. With Warthin-Starry silver staining, C. jejuni may be seen between enterocytes but only rarely inside them.

Pathogenesis.

Clinical disease may be produced by several different mechanisms after the Campylobacter has populated the intestinal tract (Van Kruiningen, 1995). After colonization of the enterocyte surface, C. jejuni can produce an enterotoxin that causes a secretory diarrhea. Campylobacterjejuni can also cause an erosive enterocolitis by invasion of the ileal and colonic epithelium along with production of a cytotoxic agent; this may be the mechanism that causes hematochezia. In addition, C. jejuni can produce illness by translocation, i.e., multiplication in the lamina propria and transportation to regional lymph nodes by macrophages. This causes mesenteric lymphadenitis.

Diagnosis and differential diagnosis.

Fresh feces (per rectum) are best for ensuring an adequate diagnostic sample. Presumptive diagnosis may be made by demonstration of highly motile curved or spiral organisms with dark-field or phase-contrast microscopy. Gram-stained C. jejuni appear as gull-winged rods. Definitive diagnosis requires isolation of the organism (Sherding and Johnson, 1994). Culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of 42~ Any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis, including canine parvovirus, coronavirus, distemper virus, Giardia, and Salmonella infections; helminth infestations; and hemorrhagic gastroenteritis.

Proper environmental sanitation, waste disposal, and food storage can prevent campylobacteriosis. In enzootic situations, group housing should be avoided. Outbreaks are controlled by isolation and treatment of affected individuals.

Dogs with clinical campylobacteriosis have altered intestinal histology and temporary derangements to digestive and absorptive functions.

e. Monocytic Ehrlichiosis Etiology.

Monocytic ehrlichiosis is caused by Ehrlichia canis, a small, pleomorphic, gram-negative bacteria in the family Rickettsiaceae. Monocytic ehrlichiosis has been historically referred to as canine tropical pancytopenia, tracker dog disease, canine hemorrhagic fever, or canine typhus.

Clinical signs. Based on experimental infections in dogs, three phases to the disease have been described: acute, subclinical, and chronic. Clinical signs observed vary with the phase of the disease, and the acute and subclinical phases are often missed or misdiagnosed (C. G. Couto, personal communication, 1993; Waddle and Littman, 1988; Woody and McDonald, 1985). A history of tick exposure may be noted prior to onset of signs. In the acute phase, clinical signs range from mild to severe and may last 1-2 weeks. They include inappetance, lethargy, fever, generalized lymphadenopathy, hepatosplenomegaly, exercise intolerance or dyspnea, petechial or ecchymotic hemorrhages, and peripheral edema. Central nervous system (CNS) signs may also be present such as hyperaesthesia, myoclonus, and cranial nerve deficits. Clinical laboratory abnormalities noted during the acute phase include thrombocytopenia, anemia, neutropenia or neutrophilia, and bicytopenia or pancytopenia. Hyperplastic bone marrow, mild hyperglobulinemia, and elevated hepatic enzymes may be noted during this phase (Kuehn and Gaunt, 1985). Clinical signs are generally absent during the subclinical phase. Mild thrombocytopenia, anemia, or leukopenia may be seen. The chronic phase develops 1-4 months after the initial infection, and signs may be subclinical to severe. An extremely varied clinical picture can emerge during this time and can mimic several other clinical syndromes. The following constellation of clinical signs may be observed: chronic lethargy, weight loss, inappetance or anorexia, fever, generalized lymphadenopathy, hepatosplenomegaly, petechial or ecchymotic hemorrhages, epistaxis, hematuria, melena, pallor, anterior or

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posterior uveitis, chorioretinitis, peripheral edema, ataxia, upper and lower motor neuron deficits, altered mentation, cranial nerve deficits, and seizures. Persistent thrombocytopenia is the most consistent laboratory abnormality noted for all three stages. Many other hematologic abnormalites may be found, such as regenerative or nonregenerative anemia (more frequently the latter), positive Coombs' test, bicytopenia or pancytopenia, and splenic plasmacytosis or lymphocytosis. On bone marrow evaluation, plasmacytosis along with hypoplasia of erythroid, myeloid, and/or megakaryocyte lines may be seen. Hyperglobulinemia as a result of polyclonal or occasionally monoclonal gammopathy has been noted in 50-100% of E. canis seropositive or infected dogs (Kuehn and Gaunt, 1985; Breitschwerdt et al., 1987; Shimon et al., 1996). Proteinuria and/or hypoalbuminemia have also been seen.

Epizootiology and transmission. Ehrlichia canis is an obligate intracellular parasite that infects mononuclear cells. The definitive hosts are arthropods; domestic and wild canids are parasitized secondarily. The primary vector and reservoir is the brown dog tick, Rhipicephalus sanguineus. Ehrlichia canis is found worldwide and follows the distribution of the vector. Infection in dogs is most prevalent in tropical and subtropical areas (Greene, 1991). In the United States, cases are concentrated in the southeastern and southwestern states but have been reported in almost every state (Breitschwerdt, 2000). Transmission is primarily by tick bites, but it can also occur via blood transfusions from dogs infected for as long as 5 years. Ticks become infected by feeding on an infected dog that is in the first 10-15 days of an acute infection (Lewis et al., 1977), and ticks can shed the organisms for up to 5 months. Within the tick population, E. canis is transmitted transstadially (within developmental stages) but not transovarially (from female to offspring) (Groves et al., 1977). Pathogenesis. In experimental infections, the incubation period prior to the onset of the acute phase is 7-21 days. During the acute phase, which can last from 2 - 4 weeks, the bacteria replicate within circulating and tissue monocytes, resulting in lymphoreticular hyperplasia in affected tissues. Infected monocytes then spread hematogenously to other organs in the body, in particular the lungs, kidney, and meninges. Infected cells adhere to the vascular endothelium and induce vasculitis, which is the primary mechanism whereby the organism causes disease. The thrombocytopenia during the acute phase is due to both sequestration and destruction, and the development of anemia is a result of red blood cell destruction and suppression of erythrocyte production. The subclinical phase of the disease occurs 6 - 9 weeks after initial infection. During this stage, dogs that can mount an effective immune response clear the infection. Those that cannot mount such a response progress to the chronic stage. Infection

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does not confer protective immunity in dogs that recover. German shepherds and Doberman pinschers seem to be more severely affected than other breeds.

Pathologic findings. Gross lesions are varied and change, depending on the phase of the disease. The most common findings are petechial and ecchymotic hemorrhages and edema of dependent tissues (Woody and Hoskins, 2000). The most common histologic abnormality noted is lymphocytic-plasmacytic inflammation of numerous organs. Mononuclear phagocytic system hyperplasia, extramedullary hematopoiesis, and splenic erythrophagocytosis may also be seen. Diagnosis and differential diagnosis. The most sensitive, specific, and commonly employed method for diagnosing E. canis infections is the indirect fluorescent antibody (IFA) test. Antibodies can be detected as early as 7 days postinfection, although some dogs may not seroconvert until 28 days postinfection (Buhles et al., 1974). Cross-reaction may occur between E. canis, E. chaffeensis, and E. ewingii. Titers greater than 1:10 are considered positive and indicative of infection and may persist for up to 1 year. Effective treatment typically produces seronegative results in 6 - 9 months. In some cases, asymptomatic dogs may remain seropositive for years after treatment or may be seropositive with a persistent hematologic abnormality (Bartsch and Greene, 1996). The exact mechanism for this finding has not been elucidated. Ehrlichia canis morulae can be demonstrated in circulating monocytes of Giemsa-stained blood smears. However, this method is labor-intensive and has low sensitivity, as morulae are present transiently and in low numbers. Using buffy coat smears from capillary blood may increase the diagnostic yield. Polymerase chain reaction (PCR) assays are also available to identify E. canis. Differential diagnoses include immune-mediated hemolytic anemia/thrombocytopenia, multiple myeloma, chronic lymphocytic leukemia, and lymphoma. Prevention. Preventing laboratory animals from contacting ticks is the primary means to avoid monocytic ehrlichiosis in research dogs. Avoid exercising dogs in areas infested with ticks. Use topical acaricides to prevent tick infestations. Keep kennel areas tick-free. Dogs used as blood donors and dogs from unproven sources should be tested for E. canis. Treatment. Doxycycline is the drug of choice for treating monocytic ehrlichiosis. Oral doses of either 2.5-5 mg/kg q12 hr or 10 mg/kg q24 hr for 21 days are very effective at eliminating the organism. Tetracycline, chloramphenicol, and enrofloxacin are also effective antibiotics; however, chloramphenicol should not be used in animals with cytopenias. In chronic cases, antibiotic treatment should be extended for an additional 1-2 weeks.

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Research complications. The most significant research complication is the thrombocytopenia that persists for all stages of the disease. Additionally, there is probable alteration in immune function and increased susceptibility to infectious agents. For these reasons, dogs positive for antibodies to E. canis should not be used in research. f

Thrombocytic Ehrlichiosis

Etiology. This disease, caused by Ehrlichia platys, was first described as cyclic thrombocytopenia by Harvey et al. in 1978. Clinical signs. In most cases, infection with E. platys results in subclinical disease. A generalized lymphadenopathy may be noted. Epizootiology and transmission. The vector for E. platys is assumed to be a tick; however, this mode of transmission has not been established. Experimental studies by Simpson et al. ( 1991) failed to demonstrate Rhipicephalus sanguineus as a vector for E. platys. Coinfection with E. canis has been reported, which suggests a common vector for both organisms (French and Harvey, 1983; Kordick et al., 1999). Dogs have been experimentally infected by inoculation with infected blood or infected platelets from other dogs (Harvey et al., 1978; Gaunt et al., 1990). The geographic distribution of thrombocytic ehrlichiosis is assumed to follow that of other Ehrlichia organisms. The highest concentration of cases seems to be in southeastern states, but isolated cases have been reported as far north as Michigan and as far west as Oklahoma (Wilson, 1992; Mathew et aL, 1997). The prevalence of seropositive dogs can be high in some parts of the country. A study by Bradfield et al. (1996) reported that 74% of the dogs entering a research institute's quarantine facility from sources in eastern North Carolina were seropositive for E. platys. Hoskins et al. (1988) reported a 54.2% seropositive prevalence in healthy dogs from kennels in Louisiana. Pathologic findings. Gross and histopathologic findings during experimental E. platys infection in dogs have been described by Baker et al. (1987). Generalized lymphadenopathy was the only gross lesion noted. Follicular hyperplasia and plasmacytosis were the predominate findings in lymphoreticular tissues. All dogs also had extramedullary hematopoiesis, erythrophagocytosis, and crescent-shaped hemorrhages in the spleen. Multifocal Kupffer's cell hyperplasia was noted in the liver, and mild multifocal lymphocytic-plasmacytic interstitial inflammation was seen in the kidneys. Pathogenesis. The pathogenesis of E. platys in dogs has primarily been determined through experimental infection (Harvey et al., 1978). After inoculation the organism directly infects platelets. Thrombocytopenia occurs by day 10-14 and fluctu-

ates, along with parasitemia, at 10 to 14 day intervals. In some cases the rebound may be within the normal range for thrombocyte counts. The nadir can be lower than 20,000 platelets/~d. Concurrent with low platelet counts is the development of megakaryocytic hyperplasia in the bone marrow. Interestingly, despite extremely low platelet counts, spontaneous bleeding has not been reported in cases of E. platys infection. The mechanism responsible for the cyclic nature of the infection has not been elucidated.

Diagnosis and differential diagnosis. Ehrlichia platys infection may be diagnosed on stained blood smears by visualization of the organisms within platelets. However, this method is very unreliable due to the cyclic nature of the parasitemia and the low numbers of infected thrombocytes. Available IFA assays are much more sensitive and specific, and there is reportedly no serologic cross-reaction with other Ehrlichia species. Dogs usually develop detectable titers 2 - 3 weeks postinfection. PCR assays for E. platys have now been developed as well (Chang and Pan, 1996; Mathew et al., 1997). Differential diagnoses for thrombocytic ehrlichiosis include E. canis infection, immunemediated thrombocytopenia, and disseminated intravascular coagulation (DIC). Prevention and treatment. Prevention and treatment for E. platys is the same as described for E. canis, above. Research complications. Ehrlichia platys infection may increase the risk of bleeding during surgical or traumatic procedures. Coinfection with E. platys may potentiate the pathogenicity of other infectious agents, in particular E. canis (Breitschwerdt, 2000). g.

Borreliosis (Lyme disease)

Etiology. Lyme disease is caused by Borrelia burgdorferi sensu lato, a microaerophilic spirochete that is primarily an extracellular pathogen. Clinical signs. Clinical signs may be highly variable; lameness due to polyarthritis has been reported as the most common sign. The onset of lameness may be acute or chronic, shift from limb to limb, and be accompanied by swelling and joint pain. Synovial fluid analysis from affected joints is consistent with a diagnosis of suppurative arthritis. Other clinical signs include fever, anorexia, lethargy, lymphadenopathy, and weight loss. Over the course of the disease, signs may wax and wane over a period of weeks to months. Dogs rarely develop erythema chronicum migrans (the characteristic rash seen in infected people) and do not exhibit the severe arthritis and neurologic sequelae seen in human beings (Greene, 1991; Manley, 1994). Hematologic and biochemical profiles are generally unremarkable.

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Epizootiology and transmission. Lyme disease is thought to be the most common arthropod-borne disease of human beings (and possibly of dogs) in the United States. It affects humans and dogs worldwide. The geographic distribution of canine borreliosis is assumed to follow that of the human disease and is related to the range of the arthropod vectors. Three major endemic foci that have been identified in the United States account for 94% of reported human cases (Appel and Jacobson, 1995). The distribution of these cases is as follows: Northeast/midAtlantic focus, 85%; midwestern focus (Michigan, Wisconsin, Minnesota, Iowa, Illinois, and Missouri), 10%; and California and Oregon, 4%. For the most part, dogs in the remainder of the country are not at risk for contracting Lyme disease. Borrelia burgdorferi is transmitted exclusively by Ixodes ticks. Other arthropod hosts may carry the organism but have not as yet been implicated in the transmission of disease. Ixodes scapularis, a three-host tick with a 2 to 3 year life cycle, is the prototypical vector for North America. The spirochetes are spread by tick bites from both nymphs and adults. Ticks become infected by feeding on an infected mammal and by transstadial transmission (transovarial passage is rare). In endemic areas, 5 0 - 8 0 % of adult ticks may be infected (Appel and Jacobson, 1995). The primary reservoir for the organism is the whitefooted deer mouse, Peromyscus ieucopus, which can carry spirochetes for its life span without becoming ill. Evidence also indicates that the eastern chipmunk, Tamias striatus, is an important reservoir (Slajchert et al., 1997), and birds may also be a significant reservoir. Deer, however, serve only as hosts for the tick vectors and not as a reservoir for the spirochete. Pathogenesis. The pathogenesis of Lyme disease is poorly understood, primarily because of a lack of good animal models and the chronic nature of the disease. Infection can be induced experimentally by the bite of a single infected tick. Clinical signs develop 6 0 - 9 0 days postinfection. Some evidence points to the host's inflammatory response to the organism as etiologic for disease (Pershing et aL, 1994; Greene, 1991). Seroconversion in dogs occurs 4 - 6 weeks after infection with B. burgdorferi. Antibody titers may remain extremely elevated for at least 18 months. IgM titers also remain elevated for several months and are indicative of neither acute nor active infection (Appel and Jacobson, 1995). Because antibiotic treatment may not eliminate the organism, persistent infections in dogs (treated for 30 days with antibiotics) can be reactivated by steroid treatment up to 420 days postinfection (Straubinger et al., 1998). Diagnosis and differential diagnosis. Appel and Jacobson (1995) recommend that three of the following four criteria be met to establish a diagnosis of Lyme disease in dogs: (1) history of exposure to Ixodes ticks in an endemic area, (2) characteristic clinical signs, (3) positive serology, and (4) rapid resolution of clinical signs with antibiotic therapy. IFA or ELISA tests for Borrelia antibodies are the assays of choice. It should be re-

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membered, however, that a positive titer in an endemic area indicates exposure and not necessarily disease and that vaccinated dogs will also have a positive titer. Responses to vaccine versus infection may be distinguished by Western blot. Culture or identification of the organism provides a definitive diagnosis but is very difficult to perform. Differential diagnoses include immune-mediated polyarthritis and septic arthritis from other etiologic agents.

Prevention and control. Prevention and control are the same as for the other tick-borne diseases (see discussion of monocytic ehrlichiosis, Section III,A,l,e above). A vaccine against B. burgdorferi is available but should not be necessary in a research setting. Treatment. Doxycycline is the drug of choice for treating Lyme borelliosis. A typical dosing regimen is 10 mg/kg q12 hr for 3 - 4 weeks. Amoxicillin, tetracycline, and the quinolones are also effective. Of significant note is that antibiotic treatment results in resolution of clinical signs but may not result in elimination of the organism. Research complications. No complications to research have been reported. However, clinically infected dogs are probably not appropriate models for orthopedic or rheumatological research. h.

Helicobacteriosis

Etiology. Helicobacters are gram-negative, microaerophilic, spiral bacteria that typically reside in and infect the gastrointestinal tract. The following species are considered to naturally infect dogs: Helicobacter felis, "H. heilmannii," H. bizzozeronii, H. (Flexobacter) rappini, H. canis, and H. bilis (Fox and Lee, 1997). "Helicobacter heilmannii" and H. bizzozeronii are thought be the same species, with the latter being the updated nomenclature. This species, as well as H. rappini and H. canis, is considered to be zoonotic (Fox and Lee, 1997). Clinical signs. Most infections are subclinical in the dog. Clinical infections may present with vomiting, diarrhea, fever, and anorexia, pica, or polyphagia. Epizootiology and transmission. The epizootiology and transmission of Helicobacter spp. in the dog remains to be elucidated. The prevalence of canine Helicobacter infections in colony or shelter situations has been reported to range from 82% to almost 100% (Fox, 1995; Hermanns et al., 1995). Both oral-oral and fecal-oral routes for transmission have been suggested. Pathologic findings. No gross lesions are noted; the primary lesion is that of histologic gastritis. This is typically characterized by reduced mucus content of the surface epithelium; vacu-

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olation, swelling, karyolysis, and karyorrhexis of parietal cells; and multifocal infiltrates of plasma cells and neutrophils into the subepithelium, primarily around blood vessels and between the gastric pits (Hermanns et al., 1995). Focal areas of lymphocytic inflammation and lymphoid follicles may also be seen. Pathogenesis. Some Helicobacter spp. colonize the gastric epithelium exclusively and other species colonize lower parts of the gastrointestinal tract. Helicobacter felis and "H. heilmannii" infections have been linked to gastric lesions in laboratoryraised beagles (Fox and Lee, 1997). The mechanism by which these organisms cause disease may be related to the host's inflammatory response to colonization and the Helicobacter's ability to produce urease. Urease splits urea into ammonia and bicarbonate; ammonia is toxic for the epithelial cells, and bicarbonate may help the organism survive the acidic environment (Marshall et al., 1990; Shimoyama and Crabtree, 1998). Diagnosis and differential diagnosis. Any of the numerous causes of acute or chronic vomiting and diarrhea in the dog (including canine distemper, viral or bacterial gastroenteritis, and ingested toxicants) should be considered as differential diagnoses. Definitive diagnosis for dogs requires either endoscopic or surgical biopsy. Confirmation of infection with Helicobacter spp. requires demonstration of the organism in biopsy samples by histopathology, culture, or recognition by PCR. A positive urease test on a biopsy sample may give a presumptive diagnosis, but only for those species that produce urease. The use of Warthin-Starry silver stain may increase the sensitivity for histopathologic diagnosis. Prevention and control. Until more is known about the epizootiology and transmission of Helicobacter spp. in the dog, specific recommendations cannot be made about prevention and control in this species. Treatment. Combination therapy has proven to be the most effective method for treating Helicobacter spp. infections in dogs. Combination therapy of amoxicillin (10 mg/kg q12 hr), metronidazole (30 mg/kg q24 hr), and sucralfate (0.25-0.5 mg/kg q8 hr) for 21 days has been suggested for dogs (Hall and Simpson, 2000). Replacing the sucralfate with famotidine (0.5 mg/kg q24 hr), omeprazole (0.3 mg/kg q24 hr), or bismuth subsalicylate (0.2 ml/kg q 4 - 6 hr) may also be effective (Marks, 1997; Jenkins and Bassett, 1997; DeNovo and Magne, 1995). The benefits of antimicrobial therapy in dogs still need to be established by controlled therapeutic studies. Research complications. Helicobacter spp. infections could result in altered gastrointestinal responses to drugs and toxic or carcinogenic compounds. Therefore, dogs used in gastric physiology or oral pharmacology studies should be free from helicobacteriosis.

2.

Viral and Chlamydial Diseases

a.

Canine Parvovirus

Clinical signs. Clinical signs of canine parvovirus usually appear 5 days after inoculation by the fecal-oral route and are characterized by anorexia, fever, depression, and vomiting. Profuse, intractable diarrhea ensues, which may become hemorrhagic. Approximately 85% of affected dogs develop severe leukopenia, with a total granulocyte/lymphocyte count ranging from 500-2000 WBC/~d or less. Repeated hemograms may provide prognostic value, because rebounds in leukocyte counts are indicative of impending recovery. Terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. Epizootiology and transmission. Parvovirus can infect dogs of any age, but puppies between 6 and 20 weeks of age appear to be particularly susceptible. Puppies less than 6 weeks of age are generally protected from infection by passive maternal antibody. Adult dogs probably incur mild or inapparent infections that result in seroconversion. Pathogenesis. Canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes an acute, highly contagious enteritis with intestinal crypt necrosis and villus atrophy. The virus also has tropism for the bone marrow and lymphoid tissues; thus leukopenia and lymphoid depletion accompany the intestinal destruction. Diagnosis and differential diagnosis. Parvovirus can be detected in fecal samples with a commercially available ELISA from CITE. At necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. Other lesions include myeloid degeneration and widespread lymphoid depletion. Parvovirus can also be demonstrated in frozen sections by fluorescent antibody techniques. Differential diagnoses should include other viral enteritides, salmonellosis, and small intestinal obstruction. Prevention and control. Prevention of transmission begins with isolation of affected animals and quarantine for 1 week after full recovery. Disinfection of potentially infected kennel and diagnostic areas with diluted bleach (1:30) or commercially prepared disinfectant (such as Kennesol, available from AlphaTech, Lexington, Massachusetts) is essential for elimination of the virus. Six-week-old puppies should be vaccinated every 2 - 4 weeks with a commercially available modified live vaccine until 16-18 weeks of age. Young Rottweilers and Doberman pinschers appear to be predisposed to parvoviral enteritis and should be vaccinated every 3 weeks (5 times) from 6-18 weeks of age. Treatment. Treatment is largely supportive and is aimed primarily at restoring fluid and electrolyte balance.

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Research complications. Infection with parvovirus obviously precludes the use of a particular dog in an experimental protocol. Given the potential for significant discomfort of the affected animal, and the cost of therapy, humane euthanasia is usually the option chosen in a research setting. b.

Canine Coronavirus

Canine coronavirus infection is usually inapparent or causes minimal illness. This epitheliotropic virus preferentially invades the enterocytes of the villous tips, resulting in destruction, atrophy, and fusion and subsequent diarrhea of varying severity. Subclinical infections are most common, but abrupt gastrointestinal upset accompanied by soft to watery, yelloworange feces is possible. Definitive diagnosis by virus isolation or paired sera is usually not made, because supportive therapy generally results in rapid resolution of the diarrhea. Inactivated coronavirus is present in commercially available combination vaccines, which are administered immunoprophylactically at 6 - 8, 10 - 12, and 12-14 weeks of age and then annually thereafter. The role of these vaccines in protection from coronaviral infection is unknown, because the virus typically causes inapparent or mild illness (Hoskins, 1998).

c.

Canine Distemper

Etiology. Canine distemper virus (CDV) belongs to the family Paramyxoviridae, within the genus Morbillivirus, which includes human measles virus and rinderpest virus of ruminants. Clinical signs. Although there is only one serotype of CDV, there is a wide difference in strain virulence and tissue tropism. Some strains produce mild clinical signs that are similar to tracheobronchitis, whereas other strains cause generalized infections of the gastrointestinal tract, integument, and central nervous system, resulting in enteritis, digital hyperkeratosis, and encephalitis, respectively. Other factors contributing to the severity and progression of clinical signs include environmental conditions, immune status, and age of the host. A transient subclinical fever and leukopenia occur 4 - 7 days after exposure, with a subsequent fever spike 7-14 days later, accompanied by conjunctivitis and rhinitis. Other clinical signs associated with acute distemper include coughing, diarrhea, vomiting, anorexia, dehydration, and weight loss. Secondary bacterial infections can cause progression to mucopurulent oculonasal discharge and pneumonia. An immune-mediated pustular dermatitis may develop on the abdomen; this is usually a favorable prognostic sign (Greene and Appel, 1998), because dogs that develop skin lesions often recover. Neurologic complications of distemper infection may occur weeks to months after recovery from an acute infection. Dogs that develop late-onset disease are usually immunocompetent hosts, suggesting that the virus may have escaped complete

elimination by the immune system, possibly because of protective effects by the blood-brain barrier. Classic neurologic signs that may occur in acute or chronic CDV infection include ataxia, incoordination, vocalization, "chewing gum" seizures, and myoclonus with or without paresis of the affected limb. Canine distemper is the most common cause of seizures in dogs less than 6 months of age. Dogs with extensive neurologic involvement often have residual clinical deficits, including flexor spasm and olfactory dysfunction. CDV has also been associated with two forms of chronic encephalitis in mature dogs: multifocal encephalitis and "old dog encephalitis."

Epizootiology and transmission. The virus is highly prevalent and contagious to dogs and other carnivores, especially at the age of 3 - 6 months, coincident with the waning of maternal antibody. Transmission is primarily by aerosolization of infective droplets from body secretions of infected animals. Pathologic findings. The predominant histopathologic lesion in neurologic forms of distemper is demyelination, which may be accompanied by gliosis, necrosis, edema, and macrophage infiltration. Acidophilic cytoplasmic inclusions can be found in epithelial cells of mucous membranes, reticulum cells, leukocytes, glia, and neurons, while intranuclear inclusions are often present in lining or glandular epithelium and ganglion cells. .

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Diagnosis and differential diagnosis. Diagnosis Of CDV is based on history of exposure and clinical signs. Young dogs who have not received routine immunoprophylaxis (or similarly, mature dogs with a questionable vaccination history) and present with rhinitis, mucopurulent oculonasal discharge, plus or minus hyperkeratosis of the footpads and neurologic signs, are highly likely to have CDV. Ophthalmologic examination may reveal chorioretinitis with acute disease or retinal atrophy in chronic cases. Definitive diagnosis of acute infection can be made by fluorescent antibody testing of intact epithelial cells from conjunctival and mucous membranes. Attenuated strains of CDV, found in modified live vaccines, are not disseminated from lymphoid tissue to epithelial cells and thus are not detected by the fluorescent antibody. Serologic testing is usually not useful, because dogs frequently fail to mount a measurable immunologic response. Because of the variety of clinical signs, there are many differential diagnoses for canine distemper. An important differential diagnosis for respiratory illness is infectious tracheobronchitis (kennel cough). Bacterial, viral, and protozoal causes of gastroenteritis must be considered for cases presenting with vomiting and diarrhea, and rabies, pseudorabies, bacterial meningitis, and poisonings are differential diagnoses for dogs with central nervous system disorder. Prevention and treatment. A series of three immunizations from 6 to 14 weeks of age, followed by yearly boosters, is a recommended preventative. Treatment is largely supportive, but

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because of the profound immunologic effects and significant morbidity of CDV, humane euthanasia is usually undertaken in the research setting.

d.

Canine Herpesvirus

fected neonates. In general, adult bitches that have multiple abortions, stillbirths, or persistent infertility should be culled from the breeding colony. Examination of these animals may reveal raised vesicular lesions on the vaginal mucosa. Adult male dogs that have vesicular lesions on the base of the penis and preputial mucosa should be similarly culled.

Etiology.

Canine herpesvirus (CHV) infection causes a generalized hemorrhagic disease with a high mortality rate in newborn puppies less than 2 weeks of age. In adult dogs, CHV causes a persistent, latent infection of the reproductive tract with recrudescence and shedding during periods of physiologic stress.

Clinical signs. Clinically affected puppies do not suckle, cry persistently, become depressed and weak, and fail to thrive. Petechial hemorrhages of the mucous membranes and erythema of sparsely haired regions such as the caudal abdomen and inguinal area are evident. Older puppies, aged 3 - 5 weeks, develop less severe clinical signs and are likely to survive with neurologic sequelae such as ataxia and blindness resulting from reactivation of latent infection. Infection in adult dogs may result in stillbirths, abortions, and infertility. Lesions in adult bitches include raised vesicular foci in the vaginal mucosa, accompanied by mild vaginitis. Adult males have preputial discharge due to vesicular lesions at the base of the penis and on the preputial mucosa. Epizootiology and transmission.

Transmission occurs during passage of puppies through the birth canal or venereally in adult dogs. Puppies can also be horizontally infected by littermates. Entire primiparous litters may be lost, with subsequent litters protected by colostral antibody.

Research complications.

Canine herpesvirus infection in adult dogs would obviously interfere with production operations, and affected animals should be culled based on the criteria noted above in the discussion of prevention and treatment. Because of the severity of clinical illness in puppies, such animals should be humanely euthanatized.

e.

Rabies

Etiology.

Rabies virus is a member of the rhabdovirus family and is essentially contagious to all species of warm-blooded animals.

Clinical signs. Clinical progression of neurologic disease occurs in three stages. The first, or prodromal, stage is characterized by a change in species-typical behavior. The loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. In the second, or furious, stage animals are easily excited or hyperreactive to external stimuli and will readily snap at inanimate objects. The third, or paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. Death usually occurs within 2 - 7 days of the onset of clinical signs, due to respiratory failure. Epizootiology and transmission.

Pathologic findings.

Pathologic findings include multifocal ecchymotic hemorrhages of the kidneys, liver, lungs, and gastrointestinal tract. Basophilic intranuclear inclusions in necrotic areas of parenchymal organs are characteristic findings.

Wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. Transmission occurs primarily by contact of infected saliva from a rabid to a naive animal (or human), usually via bite wounds.

Diagnosis and differential diagnosis.

Diagnosis of canine herpesvirus infection in adult dogs is based on a history of reproductive infertility and the presence of genital vesicular lesions. Differential diagnoses for stillbirths, abortions, and infertility include canine brucellosis, canine distemper virus and parvovirus infections, and pyometra. The diagnosis in infected puppies is usually made based on clinical history and characteristic lesions (multifocal systemic hemorrhages) (Carmichael and Greene, 1998). Differential diagnoses for the disease in neonates would include canine ehrlichiosis and causes of disseminated intravascular coagulation, including bacterial endotoxemia.

The incubation period for rabies is generally 3 - 8 weeks from the time of exposure to the onset of clinical signs but can range from 1 week to 1 year. Bites of the head and neck typically result in shorter incubation periods because of the proximity to the brain. Following infection, the virus migrates centripetally via peripheral nerve fibers to the central nervous system and eventually to neurons within the brain, resuiting in neurologic dysfunction. On reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission.

Prevention and treatment.

Diagnosis and differential diagnosis.

A vaccine is not available, and there is no effective curative treatment. Supportive therapy is unrewarding, and death usually ensues within 48 hours in in-

Pathogenesis.

Diagnosis of rabies is based on clinical signs; differential diagnoses include pseudorabies, canine distemper, bacterial meningitis, and toxicants that

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affect neurologic function. Definitive diagnosis is based on fluorescent antibody demonstration of the virus in Negri bodies of hippocampal cells. Prevention and treatment. Puppies should be vaccinated at 4 6 months of age, "boostered" in 1 year, then vaccinated annually or triennially, depending on state and local laws and which vaccine product is used. Treatment of rabies is not recommended, because of the risk of human exposure. Research complications. In a research setting, dogs are often not vaccinated for rabies, because of the low incidence of exposure to wild-animal reservoirs. A healthy, purpose-bred dog that bites a human in a research facility should be quarantined for 10 days and observed for signs of rabies. This quarantine interval is based on the knowledge that dogs do not shed rabies in the saliva for more than a few days before the onset of neurologic disease. A random-source dog with an unknown vaccination history that bites a human should be immediately euthanized. The brain should be examined for rabies virus to determine if the dog was infected, and if the test is positive, postexposure immunization should be initiated for the human patient. A rabies vaccine licensed for use in humans is available, and immunoprophylaxis is recommended for animal care and research personnel who may have high work-related risks of exposure. 3.

Parasitic Diseases

a.

Protozoa

i. Giardiasis Etiology. Giardiasis is a small-intestinal disease of the dog caused by Giardia duodenalis (lamblia), a binucleate flagellate protozoan. Clinical signs. Most Giardia infections are subclinical. When dogs are clinically affected, diarrhea is the most prominent sign. The diarrhea is a result of intestinal malabsorption and is often characterized as voluminous, light-colored, foul-smelling, and soft to watery. Weight loss has also been associated with clinical infection. Clinical illness is more often seen in young animals. Epizootiology and transmission. Giardia has a direct life cycle. Dogs (and people) typically become infected when they consume water (or food) contaminated with Giardia cysts. The pH change from the stomach (acid) to duodenum (neutral) causes excystation. Trophozoites migrate to the distal duodenum and proximal jejunum and attach to the villus surface. Eventually the trophozoites encyst and pass in the feces to perpetuate the life cycle.

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Pathologic findings. Giardiasis is rarely fatal. On histopathology of duodenal or jejunal specimens, Giardia trophozoites can be seen attached to enterocytes. Mucosal inflammation and ulceration, and villous atrophy, have been observed. Pathogenesis. The exact pathogenesis of Giardia-induced illness is unknown. It is thought that tissue invasion, although occasionally observed, is unimportant for pathogenesis. It is suspected that illness is caused by physical obstruction of enteric absorption, enterotoxicity, competition for nutrients, excess mucus production, and/or secondary bacterial overgrowth. Diagnosis and differential diagnosis. Definitive diagnosis requires observation of the organism in fecal or intestinal samples. Direct fecal smears are considered best for observing trophozoites, and zinc sulfate flotation is preferred for detection of cysts. Commercial ELISA kits and direct immunofluorescent tests are available to detect fecal Giardia antigens, but the diagnostic specificity and/or sensitivity of these tests may not be sufficient to warrant substitution for the less expensive direct fecal examination or zinc sulfate preparation (Barr, 1998). Differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. Prevention. High-quality water sources will eliminate the possibility of infection developing within an animal research facility. Use of dogs with a known husbandry and medical background will minimize the chances of giardiasis developing in a research colony. Control. Once giardiasis has been diagnosed in a canine population, segregation of infected animals will help to reduce further infection (provided other dogs were not preinfected at the same source location as the signal case). Disinfection with quaternary ammonium compounds, bleach, or steam is usually successful in eradication of Giardia cysts. Treatment. The most common treatment for giardiasis is metronidazole (Flagyl) at 25-30 mg/kg per os twice per day for 5-10 days. Quinacrine hydrochloride (Atabrine) at 9 mg/kg per os once per day for 6 days, furazolidone (Furoxone) at 4 mg/kg per os twice per day for 7-10 days, and the anthelmintics albendazole and fenbendazole have been proposed for use against metronidazole-resistant strains of Giardia. A1bendazole is recommended at 25 mg/kg per os q12 hr for 2 days, and fenbendazole at 50 mg/kg per os q24 hr for 3 days. Fenbendazole was thought to be safer for both puppies and pregnant females (nonteratogenic) (Barr, 1998). Research complications. Typical asymptomatic infections probably have no consequence on research protocols, with the exception of intestinal physiology or immunology studies.

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Clinical diarrhea would clearly need to be treated before a dog could be used as a research subject.

crowding, and providing as stress-free an environment as possible.

ii. Coccidiosis Etiology. Intestinal coccidia that have been associated with enteropathy in dogs include Cystoisospora canis, C. ohioensis, C. burrowsi, and C. neorivolta.

Treatment. Treatment for the presence of coccidial oocysts may often not be necessary, because Cystoisospora infections are typically self-limiting and clinically insignificant. Treatment may, however, help to limit the number of oocysts shed in a kennel housing situation and may be necessary in cases of protracted clinical illness. Possible choices for treatment include daily administration of sulfadimethoxine (25-30 mg/lb per os for 10 days), trimethoprim sulfa (15 mg/lb per os for 10 days), or quinacrine (5 mg/lb per os for 5 days). Amprolium, which is not labeled for dogs, can also be used as a coccidiostat. It can be given in gelatin capsules for 7-12 days at a daily dose of 100 mg for small-breed pups and 200 mg for larger breeds.

Clinical signs. Dogs are typically asymptomatic when infected with intestinal coccidia, and oocysts are an incidental finding on fecal flotation or direct smear. Dogs that are clinically infected usually develop diarrhea, which can vary from soft to watery and may contain blood or mucus. Vomiting, dehydration, lethargy, and weight loss can also be seen. Epizootiology and transmission. Cystoisospora oocysts are typically spread by fecal-oral transmission, usually by ingestion of fecal-contaminated food or other objects in the environment. An indirect form of transmission is also possible, whereby the dog consumes a rodent or other animal that is serving as a transport host. Once inside the small intestine, the cyst releases sporozoites that infect enteric epithelium. Several generations of asexual reproduction can occur in the enterocyte before sexual reproduction produces gamonts. The gamonts fuse to become a zygote, which encysts, ruptures the enterocyte, and passes in the feces. Once in the environment the cyst sporulates and is now an infective stage for ingestion by another host. Pathologic findings. Dogs with coccidiosis may have hyperemia or fluid retention at affected intestinal segments. The mucosa may appear normal, raised, or ulcerated. Histologically, there may be necrosis of enterocytes, hyperemia, and submucosal inflammation. The oocysts are usually readily apparent within the epithelial cells (Van Kruiningen, 1995). Pathogenesis. Intestinal coccidia are opportunistic organisms; they do not typically cause illness unless other predisposing factors are present. Such factors include immunodeficiency, malnutrition, and/or concurrent disease. Overcrowding and unsanitary conditions can also promote clinical coccidiosis by providing a high population of infective oocysts to stressed animals. Diagnosis and differential diagnosis. Diagnosis is somewhat difficult, as coccidian oocysts (of both Cystoisospora and nonCystoisospora spp.) can be seen on fecal examinations of clinically healthy dogs, as well as animals with diarrhea. Other causes for diarrhea (e.g., parvovirus, roundworms, Giardia spp., Campylobacter jejuni, and inflammatory bowel disease) should be excluded before a coccidial etiology is implicated. Prevention. Clinical coccidiosis can be readily prevented by adhering to proper sanitation guidelines, reducing any over-

Research complications. As with any enteric disease, the presence of clinical coccidiosis can cause aberrations in gastrointestinal physiological parameters. Dogs used in intestinal pharmacokinetic studies should be confirmed to be free of Cystoisospora infections. b.

Nematodes

i. Ascarids Etiology. The most common ascarid of dogs is Toxocara canis. Toxascaris leonina can also infect both dogs and cats. Clinical signs. Ascarid infestations are most commonly subclinical. However, large worm burdens can cause diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization. Puppies may have a classical "potbellied" appearance and dull hair coat. Heavy infestations can cause intussusception and/or intestinal obstruction, in which case the young dogs may be found dead. Visceral larval migrans caused by Toxocara canis can cause pneumonia. Epizootiology and transmission. Toxocara canis typically infects puppies. In fact, a unique characteristic of T. canis is its ability to infect prenatal puppies by transplacental migration, and neonatal puppies by transmammary migration. Ingestion of infective eggs that have been shed in the feces is another common route of transmission, and infection by ingestion of a transport or intermediate host is also possible. Pathologic findings. Puppies that die from ascarid infestations typically have large worm populations in the lumen of the small intestine. Such populations can cause intestinal obstruction and may also result in intussusception or intestinal perforation. Puppies that experience lung migrations of large larval worm populations can have severe pulmonary parenchymal damage and develop fatal pneumonia.

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Pathogenesis. The infective stage of T. canis is the third-stage larva (L3). Infections initiated by ingestion of infective eggs have three possibilities for larval migration: liver-lung migration (which leads to intestinal infection), somatic tissue migration, and intestinal wall migration. Older dogs that become infected typically have an age-related resistance to liver-lung migration and instead experience the other two migratory patterns. These larval migrations are often asymptomatic, and progression of the L3 larvae is arrested in the tissues. It is these larvae that become reactivated in a pregnant bitch, thus establishing the transplacental and transmammary routes of transmission. If the source of infection is transplacental, puppies may be born with L3 larvae in their lungs, because larval migration is already in progress (Sherding, 1989). Diagnosis and differential diagnosis. The characteristic large (70-85 ~tm in diameter) and relatively round ascarid eggs can be readily diagnosed by standard fecal flotation methods. Prevention and control. Monthly administration of milbemycin or ivermectin plus pyrantel pamoate (Heartgard Plus) is recommended for prevention and control of canine ascarid infestation (Hall and Simpson, 2000). Treatment. Most anthelmintics are effective for treatment of ascariasis. Pyrantel pamoate (Nemex) and fenbendazole (Panacur) are commonly used. Treatment should be started early in puppies (2, 4, 6, and 8 weeks) because of the possibility of prenatal or neonatal infection. Pyrantel pamoate, dosed at 5 mg/kg per os, is safe for puppies and is also effective in treatment of hookworms (see Section III,A,3,b,ii). In breeding colonies in which ascarid infestation is a known problem, treatment of the pregnant and nursing bitch may be advantageous. Extended fenbendazole therapy (50 mg/kg per os twice per day for 14 days or once per day from day 40 of gestation through day 14 of lactation) has been shown to be experimentally safe and effective in decreasing ascarid burdens in puppies. Research complications. Puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. ii. Hookworms Etiology. The most common and most pathogenic hookworm of dogs is Ancylostoma caninum. Other, less pathogenic canine hookworms found in North America are A. braziliense, which can be found in the American tropics and southern United States, and Uncinaria stenocephala, which is distributed in the northern United States and Canada. Clinical signs. Only A. caninum infestation typically results in clinical illness, because of the amount of blood that it con-

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sumes. Puppies with A. caninum infestations are typically pale and weak (from anemia), with bloody diarrhea or melena. Other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain.

Epizootiology and transmission. Infective larvae (L3) are typically ingested by puppies and develop directly in the intestinal tract. Ingestion can be from the bitch's milk (transmammary migration occurs with A. caninum), from food or objects contaminated with infective larvae, or from ingestion of a paratenic host. Transplacental migration does occur with A. caninum, but to a much lesser extent than is seen with Toxocara canis. Larvae can also penetrate intact skin, migrate to the lung via somatic or circulatory routes, and be coughed and swallowed to reach the intestine. The prepatent period is 3 weeks. Pathologic findings. Infected puppies often have severe anemia and eosinophilia. The anemia can be from acute blood loss or can also be an iron-deficiency anemia caused by chronic blood loss coupled with limited iron reserves. On gross necropsy, the small-intestinal tract contains worms admixed with intestinal contents containing fresh or digested blood (Fig. 3a). Ulcerative enteritis caused by hookworm attachment is evident on histopathologic examination, and worms with mouthparts embedded in the mucosa can be identified in some sections (Fig. 3b). Pathogenesis. The severe pathogenicity of A. caninum is a direct result of its voracious consumption of blood and body fluids. Each adult hookworm can consume 0.01-0.2 ml of blood; thus an extensive infection could deplete a puppy of 20 ml of blood per day, which is approximately 15% of the blood volume of a 2.0 kg animal. In contrast, A. braziliense and U. stenocephala consume 0.001 and 0.0003 ml per worm, respectively. Diagnosis and differential diagnosis. Diagnosis of ancylostomiasis is made by identification of eggs or larvae from fecal samples by either flotation or direct smear. Parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in the diagnosis of a young dog with anemia. Prevention and control. Purchase of purpose-bred animals will limit the exposure to hookworm larvae, and effective sanitation programs will easily eradicate the infective larvae. Unlike ascarid eggs, hookworm eggs are readily killed by drying, sunlight, or cold; however, they do survive readily in warm, moist environments. Monthly administration of milbemycin or ivermectin plus pyrantel pamoate (Heartgard Plus) is recommended for prevention and control of canine ascarid infestation (Hall and Simpson, 2000).

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Fig. 3. (a) Gross necropsy presentation of hookworm infestation in a research dog. Intestinal lumen contains fresh and digested blood and adult worms. (b) Photomicrograph of hookworm infestation in a research dog. Longitudinal and cross sections of worms can be seen within the intestinal lumen, surrounded by erythrocytes, cellular debris, and fecal material. Magnification: X 10.

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Treatment. Pyrantel pamoate (Nemex) is the anthelmintic of choice because it is safest in young ill animals and is also effective against ascarids and other enteric helminths. Because of the possibility of transplacental or milk-borne infection, puppies should be treated every 2 weeks from weeks 2-8. A follow-up treatment at 11 weeks is recommended to kill any larvae that have migrated and matured since the initial therapy. Severely ill puppies may require supportive fluid therapy and possibly whole blood transfusions and iron supplementation.

Epizootiology and transmission.

Research complications.

Pathologic findings.

iii. Strongyloides Etiology. Strongyloides stercoralis is a small strongyle that

Pathogenesis.

Anemic puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study.

can cause hemorrhagic enteritis in puppies. It is found in warm, humid climates such as the southeastern United States.

Epizootiology and transmission.

Strongyloides stercoralis infects dogs and other animals by third-stage larval penetration of the skin or mucous membranes. Larvae migrate via the circulatory system to the lung and then are coughed and swallowed to initiate the intestinal parasitism. The eggs of S. stercoralis hatch within the gut lumen, and so it is the first-stage larvae that pass in the feces and need to be identified by diagnostic examination. Once passed, the larvae can either develop into the infectious third-stage larvae or mature into free-living, nonparasitic adults. Diagnosis and differential diagnosis.

The Baermann procedure is usually performed on fresh feces in order to detect the motile first-stage larva (280-310 ~tm x 3 0 - 8 0 ~tm). The larvae must be distinguished from larva of Filaroides hirthi and hatched Ancylostoma caninum.

Treatment. The usual treatment for S. stercoralis is fenbendazole (Panacur) at 50 mg/kg per day for 5 days. iv. Whipworms Etiology. Trichuris vulpis, the canine whipworm, can cause acute or chronic large-intestinal diarrhea. The adult whipworm typically resides in the cecum or ascending colon.

Clinical signs.

Most whipworm infections are subclinical. In symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. Abdominal pain, anorexia, and weight loss are also seen. Dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. Severe dehydration with electrolyte imbalance has occurred occasionally as an acute crisis episode.

Trichuris vulpis has a direct life cycle. Adult worms residing in the canine large intestine intermittently release eggs that pass in the feces. The eggs are very hardy and can persist for years. In optimal conditions, the eggs develop into an infective embryo within 10 days. After ingestion by a dog, the larvae hatch in the small intestine, burrow into the small-intestinal mucosa, and then reemerge several days later to travel and burrow into the cecal and colonic mucosa. The prepatent period is typically 2 - 3 months long. Dogs do not typically die from whipworm infestations. Lesions seen as incidental findings feature adult worms embedded into the colonic and cecal mucosae, causing local granulomatous inflammatory reactions and mucosal hyperplasia. The penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to the clinical development of diarrhea. Factors that influence the possible.development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host.

Diagnosis and differential diagnosis.

Whipworm infestation is diagnosed by the presence of characteristic trichurid eggs on fecal flotation. These eggs are barrel-shaped, with thick walls and bipolar plugs. Because of the intermittent release of eggs by the adult female worms, negative fecal flotation does not exclude the possibility of clinical whipworm infection. Adult worms can be seen on colonoscopy (Jergens and Willard, 2000). Differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis.

Prevention and control.

Trichuris eggs are resistant to disinfection, making control difficult. Dessication or incineration is the only completely effective means to eradicate whipworm eggs from the environment. Treatment. Fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. Treatment for whipworm infestation should be at monthly intervals for 3 months (Jergens and Willard, 2000). Treatment is also suggested in cases wherein whipworm infestation is suspected but not confirmed by multiple fecal flotation. Rapid response to treatment would be indicative of a correct diagnosis; lack of response should prompt further diagnostic efforts. Research complications.

Whipworm infestation has not been documented to interfere with research protocols, although one would anticpate that aberrations in local enteric immune function and absorptive functions of the large intestine could result from trichuriasis.

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v. Dirofilariasis (heartworm infestation) Etiology. Heartworm disease of dogs is caused by the filarial worm, Dirofilaria immitis. Adult heartworms reside in the pulmonary artery; severe infestations can result in the presence of worms in the right ventricle and atrium. Microfilariae, the immature worms produced by the adults, circulate in the bloodstream until a mosquito (intermediate host) ingests them.

Clinical signs. Most heartworm infestations are asymptomatic. The most common clinical signs observed are coughing and dyspnea. Clinical signs of exercise intolerance and rightsided heart failure can be seen in severe infestations. Epizootiology and transmission.

Successful heartworm transmission requires the presence of mosquitoes. For this reason, random-source dogs or dogs housed in outdoor kennels are much more likely to have heartworm infestations than indoor, purpose-bred dogs. Mosquitoes become infested with heartworm microfilariae when they take a blood meal from the dog. The microfilaria progress through several larval stages within the mosquito, eventually terminating at the third stage. This stage is then returned to the canine bloodstream during feeding. This stage matures within the dog's circulatory system, and the adults reside in the pulmonary artery. Male and female heartworms will then sexually reproduce to create more microfilariae and propagate the parasitic life cycle. In the United States, transmission of heartworm by mosquitoes occurs over a 6 month or shorter period, except for the southeastern and Gulf Coast states. Here, climatic conditions enable longer survival of the mosquitoes (possibly year-round), thus resulting in the highest prevalence of heartworm infestation (Knight, 2000).

Pathologic findings.

On necropsy, the small, slender worms can be seen in the pulmonary artery, right ventricle, and/or right atrium (Fig. 4a). There may be no histologic abnormalities associated with a minor worm burden, although typically the arterial endothelium in these areas is hyperplastic (Fig. 4b). Endothelial cell hyperplasia, vascular smooth muscle hyperplasia, inflammation, and thrombosis of the pulmonary arteries and arterioles characterize more significant infestations. Severe infestations can lead to right-sided heart failure and its pathologic sequelae of ascites, pleural effusion, hepatomegaly, and right heart and pulmonary artery enlargement. Verminous pulmonary embolism can result from treatment of dogs with anthelmintics when a worm burden is present. Immune responses to circulating microfilariae can cause pathologic lesions, most commonly glomerulonephritis.

Pathogenesis.

The physical presence of the worms in the pulmonary artery is partially responsible for clinical signs observed in severe cases. However, the host immunologic response to this infestation, coupled with secretion by the heart-

worms of physiomodulative factors, contributes significantly to the complications seen with this disease. Endothelial cell proliferation, damage, and sloughing stimulates periarteritis and proliferation of the vascular media of pulmonary arteries and arterioles. These changes lead to thrombosis of these vessels and the arterial truncation that can be seen radiographically in severe infestations. The heartworms also release circulating factors that affect vascular tone and can promote bronchoconstriction (Dillon, 2000). These factors are discussed in more detail below, under "Research complications."

Diagnosis and differential diagnosis.

For dogs used in biomedical research, diagnosis of asymptomatic heartworm disease is important, especially if the dogs are used in cardiovascular, pulmonary, or long-term studies. A diagnosis of dirofilariasis is typically made by detection of adult heartworm antigens in a blood sample. Use of adult heartworm antigen tests has virtually eliminated the historical status of "occult" heartworm disease, which was caused by infestation of adult worms without corresponding microfilarial circulation. Commercial test kits that assay for the presence of adult heartworm antigens, and designed for use by veterinary practitioners, are readily available. False-negative results can occur during the prepatent period after initial infection (first 6 - 7 months), and when the adult worm burden is light or predominantly male. Infections consisting of more than three mature female worms are usually detected by antigenic serology (Knight, 2000). A significant feature of these tests for circulating antigen is that they have a very high specificity (low rate of false-positive resuits). If a dog were negative on initial testing because of prepatency or small worm burden, it will more than likely be detected on a follow-up test 7 months later. Examination for circulating microfilariae could be used to confirm an antigenic diagnosis of dirofilariasis or to establish that microfilarial production had occurred. Microfilarial detection can be done by microscopic examination of the buffy coat of a microhematocrit tube or by concentration techniques, such as the modified Knott test and filter tests. Tests that examine for microfilariae have the inherent problem of false positives caused by microfilariae of Dipetalonema reconditum, a nonpathogenic filarial worm. Other serologic diagnostic tests that were more common historically, and that may still be useful, include detection of antibodies to either adult heartworm antigens or microfilarial antigens. These same techniques can be used to diagnose clinical heartworm disease. Additional diagnostic tests that can augment a diagnosis of clinical heartworm disease include thoracic radiography (pulmonary artery and right-heart enlargement), electrocardiography (right-heart enlargement), and hematology (eosinophilia). Differential diagnoses for symptomatic heartworm disease (coughing, dyspnea, and exercise intolerance) include canine distemper, canine infectious tracheobronchitis

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Fig. 4. (a) Canine lung with adult heartworms occluding the pulmonary artery. (b) Intimal proliferation in the pulmonary artery of a dog, caused by dirofilariasis. Magnification: x25.

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(complicated), streptococcal or other bacterial pneumonia, nocardiosis, and congestive heart failure.

Prevention and control. For dogs used in biomedical research, prevention is primarily via insect control and housing of the dogs in a controlled, indoor environment. Purpose-bred dogs reared in such an environment are usually free from dirofilariasis. However, any dog (random-source or purposebred) exposed to mosquitoes could become inoculated with infective larvae and, if untreated, could develop adult heartworm disease. There are many commercial anthelmintic preparations used to prevent heartworm infestation by killing the larval stages in the canine bloodstream before they become adult worms (e.g., ivermectin, milbemycin, and diethylcarbamazine). These could be used in a research setting in which heartwormnegative dogs are housed outdoors and thus could potentially be infected through mosquito bites. If a research facility is conditioning random-source dogs for long-term use, the presence of circulating adult heartworm antigen should disqualify an animal from the conditioning program. Treatment. Treatment for eradication of heartworms (adults, juveniles, and microfilaria) is a long process that can pose a significant risk to the patient with regard to both drug side effects (Hoskins, 1989) and immunologic reactions to dead worms lodged in the pulmonary vasculature. For this reason, medical treatment of heartworm disease is not usually attempted in research dogs. In a rare instance when such treatment was in the best interest of a long-term canine experiment, thiacetarsamide (Caparsolate) and ivermectin (Ivomec) were used to eradicate adults and microfilariae, respectively (authors' personal experience). Alternative choices include melarsomine (Immiticide) as an adulticide and milbemycin (Interceptor), levamisole (Levasol), or fenthion (Spotton) as microfilaricidal agents. Dosing regimens for these agents are detailed in Dillon (2000). Research complications. The physiomodulative properties of heartworm infection have been studied. Such studies have looked at factors released by adult heartworms, as well as changes in the function of host tissues in response to the worm presence. Probably the most consistent finding is that endothelial cell-dependent relaxation of pulmonary arterial smooth muscle is depressed in heartworm-infected dogs as compared with control dogs, indicative of alterations in local endothelial cell behavior (Maksimowich et al., 1997; Matsukura et al., 1997; Mupanomunda et al., 1997). The extension of this effect on peripheral arteries (in vivo and in vitro) has been supported in some studies (Kaiser et al., 1992) but refuted in others (Tithof et al., 1994). It is thought that the endothelium is perturbed by a factor released from the adult Dirofilaria, possibly a cyclooxygenase product such as prostaglandin D2 (Kaiser et al., 1990, 1992). These products have also been demonstrated to

cause constriction in in vitro rat tracheal ring preparations (Collins et al., 1994), suggesting that bronchoconstriction could be an aspect of the pathogenesis of the infestation. Platelet reactivity was also been found to be enhanced in dogs naturally infected with Dirofilaria, when compared with uninfected controis (Boudreaux and Dillon, 1991). Based on these data, dogs that are positive for adult heartworm antigen should be considered inappropriate for use as research subjects and, if used, should be restricted to nonsurvival preparations that do not require physiological measurements.

c.

Cestodes

Etiology. Several species of cestodes (tapeworms) parasitize the small intestine of dogs. The most common is Dipylidium caninum. Other species include Taenia pisiformis and, more rarely, Echinococcus granulosus, Multiceps spp., Mesocestoides spp., and Spirometra spp. Clinical signs. Most cestode infestations are subclinical. Severe infestations with Dipylidium can be associated with diarrhea, weight loss, and poor growth. Epizootiology and transmission. The cestode life cycle requires an intermediate host. For Dipylidium caninum, the intermediate hosts are fleas and lice. Thus this species of tapeworm can be readily transmitted by ingestion of arthropods that are canine parasites in and of themselves. Taenia pisiformis requires small ruminants, rabbits, or rodents for intermediate hosts, so spread is less likely, especially in a research setting. Echinococcus granulosus uses not only sheep as an intermediate host but also human beings, and thus the zoonotic potential of this cestode must be considered. Pathologic findings. Adult cestodes in the small intestine are usually an incidental finding at necropsy. Diagnosis and differential diagnosis. Definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. Dipylidium egg packets are large (100 X 150 Bm) and contain 1-63 eggs per packet (Hall and Simpson, 2000). Prevention and control. The most significant means to limit cestode infestation is to control the population of fleas and/or lice infesting the colony. See the sections on these ectoparasites for effective means to treat infested dogs and kennels. Treatment. Praziquantel at 5-12.5 mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially Taenia or Echinococcus species. Fenbendazole, mebendazole, or oxfendazole may also be effective against Dipylidium caninum (Hall and Simpson, 2000).

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Research complications. Unless the infestation is severe, canine tapeworms probably have minimal impact on experimental studies. However, dietary and nutritional studies would require dogs to be free of all intestinal parasites. d.

Trematodes

i. Pulmonary trematodiasis Etiology. Canine lung fluke infestation is caused by Paragonimus kellicotti in the Western Hemisphere and P. westermani in Asia. Clinical signs. Most lung fluke infestations are inapparent, but coughing can develop in cases that prompt a strong inflammatory response. Pneumothorax has been a sequela of cyst rupture, in which case dyspnea with reduced lung sounds would be the typical presentation. Epizootiology and transmission. The lung fluke life cycle requires two intermediate hosts: a snail and then a crayfish. Dogs become infested after eating crayfish, which essentially limits this disease to random-source dogs. On ingestion, the immature flukes (metacercariae) migrate to the lungs and encyst in the pulmonary parenchyma. Eggs produced by adult flukes are passed into the bronchioles, coughed up, swallowed, and passed in the feces to complete the life cycle. Pathologic findings. Grossly, the trematode cysts containing adult flukes can be seen in the lung parenchyma. Areas of eosinophilic inflammation surround the cysts, and eosinophilic granulomas can also be seen encircling released eggs. Pleural hemorrhages may also be caused by the migrating metacercariae (Lopez, 1995). Pathogenesis. Clinical illness is usually a result of a severe eosinophilic inflammatory response, pneumothorax caused by cyst rupture, or secondary bacterial pneumonia. Diagnosis and differential diagnosis. Definitive diagnosis of Paragonimus infestation requires identification of the characteristic ovoid eggs (80-115 ~tm long) with a single operculum in either the feces or a transtracheal wash. Identification from fecal samples requires sedimentation techniques. Other causes of coughing in dogs (e.g., infectious tracheobronchitis, dirofilariasis, congestive heart failure) need to be considered. Radiographically, the appearance of (multi)focal densities within the air-filled lung field needs to be differentiated from pulmonary neoplasia (primary or metastatic) or systemic fungal pneumonias. Prevention. Use of purpose-bred dogs virtually eliminates the chance of pulmonary trematodiasis in a research animal.

Treatment. Praziquantel (at 25 mg/kg q8 hr X 3 days) or fenbendazole (25-50 mg/kg q12 hr X 10-14 days) are recommended for treatment of canine Paragonimus infestation (Hawkins, 2000). Effectiveness is monitored by fecal sedimentation tests for eggs and resolution of radiographic lesions (which may never resolve entirely). Early diagnosis of pulmonary trematodiasis should warrant discontinuation of a dog from a long-term study because of the possibility of more serious clinical sequelae, such as pneumothorax. Research complications. Experimental studies involving the immune system, especially eosinophilic or local pulmonary responses, would be significantly affected by even minor infestations. Clinical illness would complicate almost any research project and makes dogs poor anesthetic risks. Radiographic lesions may confound diagnostic evaluation for pulmonary metastasis of tumors. e.

Mites

i. Demodicosis Etiology. Canine demodicosis is caused by Demodex canis, a commensal mite that lives in the hair follicles. It is considered to be normal fauna of dog skin, but certain conditions (i.e., immunosuppression) cause development of clinical illness. Clinical signs. Demodex canis infestation is typically asymptomatic. Clinical demodicosis presents with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. It can occur anywhere on the body but is often seen on the feet and the face and around the ears (DeManuelle, 2000a). Secondary bacterial pyoderma is a common complication. Epizootiology and transmission. Demodex canis mites pass to nursing pups from the dam. They live their entire lives on one dog and are not considered contagious to other dogs or humans. Certain breeds are predisposed to the generalized form of Demodex dermatitis (see "Pathogenesis," below). Beagles are among the predisposed breeds, as are German shepherds, Doberman pinschers, Old English sheepdogs, collies, boxers, and shorthair brachycephalic breeds (Muller et al., 1983). Pathologic findings. Histologically, Demodex infections are characterized by perifolliculitis and folliculitis with mites and keratin debris visible in the hair follicles. Cases with generalized demodicosis (see "Pathogenesis," below) may have a minimal cellular response with no eosinophils, indicative of severe immunosuppression (Scott et al., 1995). Pathogenesis. When clinical demodicosis develops, it is classified into "localized" or "generalized" (e.g., more than one

11. BIOLOGY AND DISEASES OF DOGS foot affected, or five or more small areas, or one large body area). Localized demodicosis is typically seen in juvenile dogs (< 18 months) and usually resolves without treatment as natural immunological control develops. Generalized demodicosis can develop in juvenile or adult populations. Juvenile-onset generalized demodicosis occurs in dogs with a genetic predisposition, thought to be an inherited T-lymphocyte dysfunction. Adult-onset generalized demodicosis is usually indicative of an underlying endocrine (hyperadrenocorticism, diabetes mellitus, hypothyroidism) or neoplastic disorder or can develop as a result of immunosuppressive therapy (such as corticosteroid administration).

Diagnosis and differential diagnosis. Demodex is readily identified from deep skin scrapings of lesioned areas (Campbell, 2000; Noli, 2000). Demodex canis has a characteristic "cigar shape," with short, stubby legs on a body 100-400 ~tm long. Differential diagnoses for local demodicosis include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. The primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma; remember, however, that bacterial pyoderma is a common secondary complication of the generalized form of this parasitism. Prevention and control. Dogs with generalized demodicosis should not be maintained in a breeding colony. Treatment: Ivermectin (Ivomec) at 200-600 ~tg/kg and oral milbemycin (Interceptor) at 1-2 mg/kg/day have been found to be effective treatments. These parasiticides are probably the most practical to use in a research setting, although they are not labeled for treatment of Demodex canis. Amitraz (Mitaban) dips (250 ppm every 14 days) can be used for more problematic cases. Treatment duration can be extensive and must be accompanied by repeated skin scrapings. Research complications. Dogs with generalized demodicosis should not be used in research studies, because this disease is indicative of another underlying disorder (endocrine or immunological). Dogs that receive immunosuppressive agents or paradigms could develop generalized demodicosis as an unexpected consequence of the experimentation. ii. Sarcoptic mange Etiology. Canine sarcoptic mange is caused by Sarcoptes scabiei var. canis. Clinical signs. The most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas such as the ear pinnae, elbows, and ventral thorax and abdomen. Lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. These lesions may be exacerbated by excoriation due to the pruritic nature of the condition.

425 Epizootiology and transmission. Sarcoptes mites live their entire lives in the stratum corneum of the host animal; however, they can survive for 1-3 weeks away from the host, and it is this ability that enables them to spread from dog to dog. Sarcoptes scabiei var. canis can also infect cats and humans. Pathologic findings. Histologic examination can be unrewarding because mites are rarely seen on tissue sections, and the associated dermatitis is nondiagnostic: perivascular and interstitial dermatitis with hyperkeratosis, with or without eosinophilic infiltration. Suggestive histopathologic lesions are epidermal "nibbles," small foci of edema, exocytosis, degeneration, and necrosis (Scott et al., 1995). Pathogenesis. Lesions and illness are a result of the female mites burrowing through the epidermal layers to deposit eggs, and the larvae migrating back to the surface. The typical locations of mange lesions are a result of the mite's preference for relatively hairless areas. Diagnosis and differential diagnosis. Sarcoptic mange can be difficult to diagnose because multiple skin scrapings can yield negative results with this parasitic disorder. Hopefully, adult mites, mite eggs, or mite feces can be observed on superficial skin scrapings. Even if scrapings are negative, however, a therapeutic trial should be initiated if the clinical signs and history suggest a Sarcoptes etiology. Demonstration of anti-mite IgE in either the serum or via an intradermal antigen test can be used as a diagnostic aid (Campbell, 2000). An important differential diagnosis is flea allergy dermatitis; in contrast, mange is nonseasonal and contagious. Prevention and control. Use of purpose-bred dogs limits the possibility of having research animals with sarcoptic mange. For random-source dogs, an ectoparasite control program should be in place to limit possible infestations. Many institutions use ivermectin as a means to control both endoparasites and ectoparasites. Treatment. Unless treatment would interfere with research objectives, all dogs with sarcoptic mange (no matter how minor the lesions) and their kennel mates should be treated because of the contagious nature of the disease and its zoonotic potential. In research colonies, the usual means of treatment is either ivermectin (Ivomec) at 200-400 ~tg/kg q14 days or milbemycin (Interceptor) at 3 oral doses of 2 mg/kg q7 days (Scott et al., 1995). Neither of these agents is approved for treatment of sarcoptic mange, but they are considered to be effective. Acaricidal dips (e.g., lime sulfur, organophosphates, amitraz) can also be used. Research complications. The local skin inflammation and systemic immune response to sarcoptic mange probably make

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infected dogs poor subjects for dermatologic and immunologic studies.

f

Lice and Ticks

i. Lice Etiology. Dogs can be infested by one species of sucking louse (Linognathus setosus) and two species of biting lice (Trichodectes canis and Heterodoxus spiniger). Clinical signs. Mild cases of pediculosis may be asymptomatic or may cause pruritic areas of dry skin. More severe infestations can cause significant pruritus and produce alopecia, papules, and crusts. These lesions lead to excoriation and secondary bacterial dermatitis. Severe Linognathus infestations could cause anemia, because this species feeds on blood. Epizootiology and transmission.

Louse infestations are uncommon in both pet animal practice and the research setting. They would most likely be seen in random-source dogs that were obtained from a pound or shelter. Transmission is usually by direct contact, for lice spend their entire lives on the host species. Lice are host-specific and not zoonotic.

Pathogenesis.

The biting lice usually cause more local irritation than the sucking louse and therefore are more apt to induce clinical dermatologic signs. Trichodectes canis can serve as vector for the canine tapeworm Dipylidium caninum. The most severe complication of infestations by the sucking louse is the potential anemia.

Diagnosis and differential diagnosis.

Pediculosis is diagnosed by direct observation of the lice or nits (eggs) on the dog's skin. Cellophane tape can be used to pick up surface debris from skin lesions, which may include nits or immobilized lice (Muller et al., 1983). Differential diagnoses include dermal acariasis, flea allergy dermatitis, and seborrhea.

Prevention.

Use of high-quality conditioned dogs for research should prevent pediculosis from ever being seen within a research facility. Random-source dogs should be shampooed or treated prophylactically with topical insecticide before being permitted to enter the research colony.

Treatment.

Most commercially available insecticide shampoos and dips readily treat louse infestations. Treatment should be repeated in 10-14 days, because any nits that were not killed would have hatched by that time (Muller et al., 1983).

Research complications.

There is probably minimal interference with research, unless severe Linognathus infestations cause anemia.

ii. Ticks Etiology. Ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. Except for the brown dog tick (Rhipicephalus sanguineus), ticks have a wide host range and are not especially host-specific; so any number of tick genera and species can be found on dogs. Genera that more commonly infest dogs in the United States include species of Rhipicephalus, Dermacentor, and Ixodes. The primary significance of tick infestation is the tick's ability to be a vector for many other infectious diseases, including Rocky Mountain spotted fever (caused by Rickettsia rickettsii), Lyme disease (Borrelia burgdorferi), and the canine forms of ehrlichiosis (Ehrlichia canis and E. platys), babesiosis (Babesia canis), haemobartonellosis (Haemobartonella canis), and hepatozoonosis (Hepatozoon canis).

Clinical signs. As an entity unto itself, tick infestation causes minimal clinical signs. Most infestations are subclinical, although some dogs may lick and bite at the site, aggravating the local lesion. Some dogs can develop a hypersensitivity reaction after several tick bites; these dogs develop a more granulomatous response at the location of the bite (Merchant and Taboada, 1991). Some species of ticks (primarily Dermacentor andersoni and D. variabilis) produce a salivary neurotoxin that can cause an ascending flaccid paralysis (Malik and Farrow, 1991). The paralysis develops within 5 - 9 days of tick attachment and can result from a single tick. This paralysis is fatal once the respiratory musculature is affected. Epizootiology and transmission.

In dogs used for biomedical research, tick infestation may occasionally be seen in randomsource dogs, because these dogs are more likely to have been in tick habitats than purpose-bred dogs. Ticks commonly reside in wooded areas until they contact a suitable host for a blood meal. The brown dog tick may reside within kennels (attics, bedding, wall insulation) (Garris, 1991).

Pathologic findings.

Under most circumstances, tick infestation will be an incidental finding on necropsy (unless tick paralysis was the cause of death).

Pathogenesis.

Tick-bite paralysis is caused by the presence of a salivary neurotoxin released by female ticks of certain genera (e.g., Dermacentor) while consuming a blood meal (Malik and Farrow, 1991). Interestingly, dogs seem to be most affected by this condition, whereas cats appear to be resistant. The primary dysfunction appears to be at the neuromuscular junction, as stimulation of the motor nerves fails to elicit a response, but direct stimulation of the muscle tissue results in contractions. Tick bites can also transmit pathogen microorganisms to the dog, because ticks serve as vectors for several infectious diseases, including Lyme borreliosis, ehrlichiosis, babesiosis, and Rocky Mountain spotted fever.

11. BIOLOGY AND DISEASES OF DOGS

427

Diagnosis and differential diagnosis.

well as the flanks, thighs, and abdomen (Muller et al., 1983). The lesions are typically worse in the summer and autumn months and are progressively more severe as the dog ages.

For uncomplicated tick bites and tick-bite paralysis, definitive diagnosis is made by identification of the offending arachnid (and improvement of paralysis after removal). Differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (Malik and Farrow, 1991).

Prevention.

Purpose-bred dogs should be free from all ectoparasites, but ticks can occasionally be seen on randomsource animals. Research dogs should not be exercised in outdoor areas infested with ticks, and kennels must be cleaned properly and regularly so as to remain free of ticks and other parasites.

Treatment. Removal of the offending tick is the primary treatment for both local inflammation as well as tick-bite paralysis. Dogs with tick-bite paralysis usually show improvement within 24 hr, with complete recovery within 72 hr (Malik and Farrow, 1991) To remove an attached tick from a dog, forceps should be used to grasp the tick as close to the dog's skin as possible. The tick should not be grabbed by the body, as this may cause the parasite to either rupture or inject its body contents into the dog. The tick should be pulled away from the dog with steady pressure. Many of the diseases transmitted by ticks are zoonotic so precautions, such as wearing gloves, should be taken. Use of topical acaricide/insecticides on newly arrived random-source dogs should help to limit infestations. Research complications.

Simple uncomplicated tick bites probably have minimal impact on research variables. The significant concern for tick infestation is the possible development of tick-bite paralysis or of any one of a number of systemic diseases spread by ticks (see Sections III,A,l,e-g).

g.

Other

i. Flea infestation Etiology. Fleas are laterally flattened wingless insects that feed on animal blood. The most common flea to infest dogs is Ctenocephalides felis, the cat flea. Other fleas that can affect dogs are Ctenocephalides canis, Pulex irritans, and Echidnophaga gallinacea. The fleas are speciated by the shape of their head and by the presence or absence of ctenidae (spiny combs on or behind the head) (Campbell, 2000).

Clinical signs. Flea infestations usually cause foci of alopecia and pruritus. Dogs that are hypersensitive to antigenic proteins in flea saliva develop the more severe "flea allergy dermatitis," which features papules and crusting. Acute moist dermatitis ("hot spots") can also be seen in these cases, and secondary pyoderma or seborrhea can develop. Lesions from flea allergy dermatitis generally appear in the dorsal lumbosacral region, as

Epizootiology and transmission.

Fleas are readily transmitted between animals and even between host species. They move readily between the host and the environment, making transmission easy and control difficult. Because fleas require host blood for food, they can survive off of a host for only 1-2 months (Muller et al., 1983).

Pathologic findings.

Biopsy samples are usually nondiagnostic in cases of flea allergy dermatitis. Lesions are typically characterized by perivascular eosinophilic inflammation and may feature pustules and folliculitis if secondary pyoderma develops (Muller et al., 1983).

Pathogenesis.

Fleas are parasites that require animal blood for their meals. When they bite host animals, they inject some saliva into the host's skin. If the host develops an allergic response to the flea saliva, it will develop the more pruritic flea allergy dermatitis. Fleas can also transmit or serve as vectors for other pathogens (e.g., Dipylidium tapeworms).

Diagnosis and differential diagnosis.

Flea infestations and flea allergy dermatitis are definitively diagnosed by observing the fleas on the host's skin. Given that this may be difficult because of the mobility of the flea and the majority of the time it spends off of the host, diagnosis is often based on clinical signs, history, and lesion distribution. Sometimes the presence of flea excrement ("flea dirt") on the dog's skin can support a presumptive diagnosis (DeManuelle, 2000b). Circulating eosinophilia is seen in some dogs with flea allergy dermatitis. Differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs (e.g., food, drug, or contact hypersensitivity).

Prevention.

Most dogs obtained from high-quality purposebred facilities should be free from flea infestations. Dogs received from pounds, shelters, or licensed dealers would be more likely to be affected by fleas (or any ectoparasitism). Thorough knowledge of prevention, control, and treatment measures at these facilities should be obtained, and dogs from sources where proper prevention and/or therapy are not practiced should be evaluated and/or empirically treated upon arrival at the facility.

Control. Thorough cleaning of the dog's housing environment should remove the risk of perpetuating or transmitting flea infestation in the colony. Treatment. Treatment for fleas needs to address treatment of both the dog and the environment. Many insecticide formulations such as shampoos, sprays, dips, powders, and oral

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systemics can be used for initial treatment of the individual dog. The active ingredients include pyrethrins, pyrethroids, carbamates, and organophosphates. Flea control in the kennel may need to include outdoor areas in warm climates. Typically combinations of adult insecticides and juvenile growth regulators are used for environmental treatment. Directed sprays are the most effective means of treating housing areas, because flea "bombs" or foggers do not penetrate adequately into tight areas where fleas might hide (DeManuelle, 2000b). In addition to insecticide therapy, dogs with flea allergy dermatitis may also require anti-inflammatory medication to relieve clinical signs. Oral prednisone or prednisolone at 0.5 mg/kg q12 hr for 5 - 7 days has been proposed as a starting therapy (Muller et al., 1983). The use of hyposensitization with flea-bite antigens is controversial and not practical for the research setting.

Research complications. Mild flea infestation probably has minimal impact on most research protocols, and treatment measures may in fact be more detrimental to the experimental objective than the actual ectoparasitism. In a research setting, the residual effects of insecticides may preclude their use in experimental animals. Such treatments should be used judiciously to ensure that experimental results are not more seriously affected by the therapy rather than the infestation. Dogs with flea-allergy dermatitis are more severely affected by the flea infestation and should be treated apigropriately; however, systemic corticosteroids may also interfere with experimental objectives, especially in studies involving functions of the immune system. The ability of fleas to transmit other parasitic diseases must also be considered. 4.

Fungal Diseases

a.

Superficial Dermatophytoses

Etiology. Dermatophytoses ("ringworm") are fungal skin infections, which in dogs in the United States are usually caused by either Microsporum canis, M. gypseum, or Trichophyton mentagrophytes (Muller et al., 1983).

Pathologic findings. On close inspection of skin samples, broken hair shafts (and not complete hair loss) would be seen with uncomplicated dermatophytosis. Histologically, fungal elements can be seen within the stratum corneum or in and around the hair and hair follicles (Muller et al., 1983). Stains that facilitate visualization of fungal elements include periodic acidSchiff (PAS) or Gomori methenamine-silver. The pattern of inflammation in the affected foci is very variable and can feature folliculitis, perivascular dermatitis, hyperkeratosis, and/or vesicular dermatitis. Pathogenesis. The dermatophytes typically infect the hair shaft itself, the hair follicle, and possibly the skin around the affected hair. The hair follicle is not destroyed (unless by secondary bacterial infection), but the hair itself becomes brittle and breaks. This causes short stubbly hair to be seen within the lesion. As the lesion progresses, the hairs in the center recover from the infection, thus leading to the classic "ringworm" appearance of the alopecic areas. It is postulated that the inflammatory process produces an environment that is unfavorable for dermatophyte survival, whereas the periphery of the lesion still enables continued fungal growth (Muller et al., 1983). Diagnosis and differential diagnosis. Diagnosis of dermal fungal infection is typically made by scraping the affected area to obtain hair and superficial epidermal cells. These scrapings are then digested with potassium hydroxide to facilitate observation of fungal elements. Fungal elements can also be seen on skin biopsy samples. For speciation of a fungus, skin scrapings can also be inoculated onto agars that promote fungal growth, such as Sabouraud's medium or dermatophyte test medium (DTM). Incubation should be at 30~ with 30% humidity for 14-30 days. Lesions caused by M. canis may fluoresce when inspected using a Wood's (253.7 nm ultraviolet) light. Unfortunately, some strains of M. canis do not fluoresce, and neither does M. gypseum or T. mentagrophytes. Differential diagnoses for dermatomycosis include seborrhea, localized demodecosis, folliculitis, histiocytoma, and acral lick dermatitis (Muller et al., 1983).

Clinical signs. Uncomplicated superficial dermatophytoses are characterized by circumscribed circular areas of alopecia, usually with minimal to no inflammation. These skin lesions are usually seen around the face, neck, and forelimbs but can be found anywhere on the body. Secondary bacterial infections can develop; these lesions are called kerions and are selflimiting, for the fungus cannot survive in inflamed skin (Muller et al., 1983).

Prevention. Purpose-bred dogs are typically free of infectious dermatophytes, but ringworm may be diagnosed on randomsource animals.

Ep&ootiology and transmission. The fungi that cause skin infections are very contagious and readily transmissible between dogs and other species (including human beings), but they can also be obtained from the soil.

Treatment. Topical antifungal therapy is most commonly used. Shampoos, rinses, and creams containing miconazole, ketoconazole, enilconazole, or chlorhexidine are commercially available to treat ringworm (Stannard et al., 2000). Severe cases

Control. In cases of dermatophytosis, isolation of the affected animal(s) is prudent, because the fungi are easily spread to other dogs, as well as to people. Treatment, if acceptable, should be started immediately.

11. BIOLOGYANDDISEASESOF DOGS may require systemic therapy with griseofulvin, ketoconazole, itraconazole, or fluconazole. However, these systemic antifungal agents may have considerable side effects (such as vomiting and teratogenicity with griseofulvin). Many of the newer agents are also expensive and not labeled for use in dogs.

429 1968; Manning 1979). Rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (Peterson and Ferguson, 1989; Kemppainen and Clark, 1994).

Clinical signs. Because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referResearch complications. The infection itself probably has no able to many organ systems. An individual dog with hypothyimpact on most research applications for dogs. Unfortunately, roidism may have one or any combination of clinical signs. the zoonotic implications of dermatophytoses force the issue of Hypothyroidism reduces the dog's metabolic rate, which then aggressive treatment, and many antifungal agents may not be produces such signs as obesity, lethargy, cold intolerance, and compatible with biomedical research studies. constipation. Additionally, hypothyroidism can produce several dermatologic abnormalities, including alopecia, hyperpigmentation, seborrhea, and pyoderma (Peterson and Ferguson, 1989; b. Systemic Fungal Infections Panciera, 1994). Several clinicopathologic abnormalities have Systemic fungal infections disseminate to multiple organ sys- also been reported in a large percentage of hypothyroid dogs. tems from a single mode of entry (usually through the respi- These aberrations include increased serum cholesterol and ratory tract). Dogs are susceptible to several fungi that charac- triglycerides due to a decrease in lipolysis and decreased numteristically cause systemic mycosis, including Blastomyces bers of low-density lipopolysaccharide receptors (Peterson and dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Ferguson, 1989; Panciera, 1994). Normocytic normochromic and Cryptococcus neoformans var. neoformans. These diseases nonregenerative anemia and increased serum alkaline phosare not typically seen in the research setting, because of the low phatase and creatine kinase have also been reported in a sigoverall incidence and noncontagious nature of these disorders nificant number of hypothyroid dogs (Peterson and Ferguson, and because of the use of purpose-bred animals. These condi- 1989; Panciera 1994). Neurologic signs of hypothyroidism, tions could, however, present in the rare random-source dog that which include lameness, foot dragging, and paresis, may be was subclinical at its point of origin, especially if the animal be- caused by several mechanisms such as segmental nerve decomes immunosuppressed (either naturally or by virtue of ex- myelination or nerve entrapment secondary to myxedema (Peperimental manipulation). Typical clinical signs include weight terson and Ferguson, 1989). Mental impairment and dullness loss, fever, lymphadenopathy, and cough and dyspnea (if the have also been reported in hypothyroid dogs, secondary to athlungs are affected). The reader is advised to read veterinary erosclerosis and cerebral myxedema (Peterson and Ferguson, medical text chapters (e.g., Taboada, 2000) for more complete 1989). Hypothyroidism has been implicated in other neurologiinformation on these disorders and their possible treatments. cal abnormalities such as Horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (Bischel B. Metabolic and Nutritional Diseases et al., 1988; Panciera, 1994), and so the relationship between hypothyroidism and these problems has not been completely 1. Genetics defined (Panciera, 1994). Myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of a. Hypothyroidism type II muscle fibers and can present with signs similar to neuAlthough the incidence of hypothyroidism in the canine pop- rological disease (Peterson and Ferguson, 1989). Hypothyulation is not high (Kemppainen and Clark, 1994), deficiency in roidism can also cause bradycardia as a result of decreased thyroid hormone can significantly affect basal metabolism and myocardial conductivity. Abnormalities that may be detected by immune function. Because these factors are important in many ECG include a decrease in P and R wave amplitude (Peterson biomedical research studies, it is imperative that laboratory ani- and Ferguson, 1989) and inverted T waves (Panciera, 1994). mal veterinarians be able to recognize, diagnose, and treat this These electrocardiographic abnormalities are caused by lowproblem. ered activity of ATPases and calcium channel function. Several reports have suggested that hypothyroidism is associated with Etiology. The majority of cases of canine hypothyroidism are von Willebrand's disease and bleeding abnormalities. However, due to lymphocytic thyroiditis, an autoimmune disorder, or id- the relationship is probably one of shared breed predilection iopathic atrophy of the thyroid gland. Both of these causes re- and not a true correlation. It has been demonstrated that dogs sult in a gradual loss of functional thyroid tissue (Kemppainen with hypothyroidism are not deficient in von Willebrand's and Clark, 1994). Lymphocytic thyroiditis is the major cause of factor when compared with other dogs. In addition, the rehypothyroidism in laboratory beagles and appears to be famil- placement of thyroid hormone in dogs did not increase the levial in that breed (Tucker, 1962; Beierwaltes and Nishiyama, els of vWF:Ag in naturally occurring (Panciera and Johnson,

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1994) or experimentally induced (Panciera and Johnson, 1996) hypothyroidism.

Epizootiology.

The prevalence of hypothyroidism in the general canine population has been reported to be less than 1% (Panciera, 1994). The disorder occurs most often in large-breed dogs but has been reported in several other breeds as well as mongrels. Doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism when compared with other breeds (Panciera, 1994; Peterson and Ferguson, 1989; Scarlett, 1994). There have been several reports about hypothyroidism in laboratory colonies of beagles (Manning, 1979; Tucker, 1962; Beierwaltes and Nishiyama, 1968). In general, the problem is usually recognized in middle-aged animals, and some reports state that there is a higher incidence of hypothyroidism in spayed female dogs (Panciera, 1994; Peterson and Ferguson, 1989).

Diagnosis and differential diagnosis.

Because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. Likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. The tests currently available and in popular use will be discussed further. However, a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. For additional information, the reader is referred to one of several manuscripts available (Peterson and Ferguson, 1989; Ferguson, 1994). Currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total T 4 (thyroxine) and free T 4 (Peterson and Ferguson, 1989; Ferguson, 1994). T 4 s e r v e s primarily as a precursor for T 3 in the body and is heavily proteinbound. Free T4 represents the unbound fraction that is available to the tissues (Peterson and Ferguson, 1989). Using the measurement of serum total T 4 and free T4, hypothyroidism can usually be ruled out if the values are within the normal range or higher. If both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial is in order (Peterson and Ferguson, 1989). However, it must be noted that nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (Peterson and Ferguson, 1989; Ferguson, 1994). Therefore, low values do not always indicate that hypothyroidism is present, and animals should not be treated solely on the basis of serum hormone levels if clinical signs are absent. If the clinical signs are equivocal or if only total T 4 or free T 4 is decreased, further diagnostic testing is warranted (Peterson and Ferguson, 1989). Although T 3 is the most biologically active form of thyroid hormone in the body, the measurement of serum T 3 levels is an unreliable indicator of hypothy-

ROBERT C. DYSKO ET AL.

roidism (Peterson and Ferguson, 1989; Ferguson, 1994). Like T4, s e r u m T 3 can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). In addition, T 3 may be preferentially released, and conversion of T 4 to T 3 may be enhanced in the hypothyroid dog (Peterson and Ferguson, 1989; Ferguson, 1994). T 3 was within normal limits in 15% of the hypothyroid dogs in one study (Panciera, 1994). Autoantibodies can be responsible for false elevations in the concentrations of T 3 and T 4 found in these respective assays. It has been recommended that free T4, measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high T 3 and T 4. Autoantibodies have been found in less than 1% of the samples submitted to one laboratory (Kemppainen and Behrend, 2000). Other means of diagnosing hypothyroidism have been described. In humans, endogenous TSH (thyroid-stimulating hormone) levels provide reliable information on thyroid status, and an assay for endogenous TSH is now available in dogs. However, TSH levels can be normal in some dogs with hypothyroidism, and high TSH levels have been noted in normal dogs. Therefore, it is recommended that TSH levels be considered along with other information (clinical signs, T4) prior to diagnosis and treatment (Kemppainen and Behrend, 2000). TSH stimulation testing using exogenous bovine TSH provides a good and reliable method for establishing a diagnosis. Unfortunately, the availability and expense of TSH limit the use of this diagnostic tool (Peterson and Ferguson, 1989; Ferguson, 1994). Another drawback of TSH testing is that the test must be postponed for 4 weeks if thyroid supplementation has been given (Peterson and Ferguson, 1989). When TSH is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. One recommendation is 0.045 U of TSH per pound of body weight (up to a maximum of 5 U) to be administered IV. For this protocol, blood samples are taken prior to administration of TSH and 6 hours after. A normal response to the administration of TSH should create an increase of T 4 levels at least 2 ktg/dl above the baseline levels or an absolute level that exceeds 3 ~tg/dl (Peterson and Ferguson, 1989; Wheeler et al., 1985).

Treatment. The treatment of choice for hypothyroidism in the dog is L-thyroxine (sodium levothyroxine). A recommended dosing regimen is 0.02 mg/kg once a day or 0.05 mg/m 2 (body surface area)/day for very small or very large dogs. If drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. After the supplementation has begun, the thyroid hormone level should be rechecked in 6 - 8 weeks, and blood samples should be drawn 4 - 8 hours after the morning pill. A clinical response is usually seen in 6 - 8 weeks and would include weight loss, hair regrowth, and resolution of other signs (Panciera, 1994). ECG abnormalities also return to normal

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11. BIOLOGY AND DISEASES OF DOGS

(Peterson and Ferguson, 1989). For dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (Panciera, 1994).

Epizootiology.

Obesity affects up to 40% of pet dogs (Mac-

Ewen, 1992).

Diagnosis and differential diagnosis. 2.

Management-Related Issues

a.

Obesity

Weight gain and eventual obesity are also frequent findings in dogs in the research environment. Because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must be aware of the development of obesity and the potential effect that it can have on research.

Etiology.

Obesity is defined as a body weight 20-25% over the ideal. In general, obesity occurs when the intake of calories exceeds the expenditure of energy. Excessive caloric intake resuits from overeating or eating an unbalanced diet. Overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (MacEwen, 1992). In addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. In the laboratory animal setting, overeating is less likely than in a household, because access to food is more restricted and diets are usually a commercially prepared balanced ration. However, obesity can still be a problem if specific guidelines for energy requirements are not followed. In addition, the necessary caging of dogs in the research environment and thus the limitation to exercise reduces energy expenditure and predisposes dogs to weight gain. It is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy expenditure are followed (Butterwick and Hawthorne,. 1998). As in humans, genetics plays an important role in the development of obesity in dogs. It has been established that certain breeds are more predisposed toward obesity. In a study of dogs visiting veterinary clinics in the United Kingdom, Labrador retrievers were most likely to be obese. Other breeds affected included Cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. The beagle was also listed as a breed predisposed to obesity in the household environment (Edney and Smith, 1986). In addition to genetics, several metabolic or hormonal changes are associated with obesity. It has been well established that neutering promotes weight gain. In one study, spayed female dogs were twice as likely to be obese when compared with intact females (MacEwen, 1992). The authors proposed that the absence of estrogen promotes an increase in food consumption. A similar trend toward obesity was found in castrated male dogs (Edney and Smith, 1986). In addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (MacEwen, 1992).

The diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. The ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. When the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. There are few objective, quantifiable methods for establishing this diagnosis. Ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict total body fat (Wilkinson and McEwan, 1991). After a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. A complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. Serum thyroid hormones should be evaluated (see Section III,B,l,a), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism.

Treatment. Restricting food intake readily treats obesity, and this is easily done in the research setting. It has been suggested that a good weight loss program involves restriction of intake to 60% of the calculated energy requirement to maintain ideal body weight. It has been shown that restriction of calories down to 50% produces no adverse health effects. However, T 3 levels will decrease in direct proportion with caloric intake. Ideally, weight loss will occur at a rate of 1-2% of body weight per week (Laflamme et al., 1997). With more severe calorie restriction and more rapid weight loss, the individual is more likely to rebound and gain weight after restrictions are relaxed. There has been agreat deal of attention in humans as to the correct diet to be fed to encourage weight loss. Likewise, the type of diet fed to dogs has been examined. As mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. Alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (MacEwen, 1992). There has been much concern about the addition of fiber to the diet in both humans and animals as a method for reducing caloric intake while maintaining the volume fed. Studies in dogs have examined the addition of both soluble and insoluble fibers to calorierestricted diets. These studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (Butterwick and Thorne, 1994; Butterwick and Thorne, 1997). Research complications.

It is important to control weight gain in research animals, because of the association of obesity and

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several metabolic changes. Although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (MacEwen, 1992), this relationship is not consistently apparent (Edney and Smith, 1986). Obesity in dogs over 10 years of age appears to be related to an increase in cardiovascular problems (Edney and Smith, 1986), and obesity has been linked to hypertension. Joint problems including osteoarthritis and hip dysplasia have also been related to obesity (MacEwen, 1992; Kealy et al., 1997). In addition, diabetes mellitus has been linked to obesity, and obesity induces hyperinsulinism in several experimental models (MacEwen, 1992).

C. I.

T r a u m a t i c Disorders

Generalized Traumatic Wound Care

In the laboratory setting, the majority of traumatic wounds will be small in size. In facilities with good husbandry practices and a diligent staff, traumatic wounds will generally be observed quickly and attended to promptly. Under these conditions, proper initial treatment will lead to uncomplicated wound healing. Complications such as infection and delayed healing arise when wounds are not noticed immediately or. when the basic principles of wound management are not followed. To aid in the description of wounds and in decision making about wound therapy, several classification systems have been developed for traumatic injuries. At one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a "golden period." It is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the capability of the host's defense systems (Swaim, 1980; Waldron and Trevor, 1993). One of the most widely used classification systems is based upon wound contamination and categorizes wounds as either clean, clean-contaminated, contaminated, or dirty (see Table V). The vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. These wounds may be treated with the basic wound care described below and primary closure of the wound. Contaminated and dirty wounds, which are seen infrequently in the laboratory setting, require more aggressive therapy. Dirty wounds can occur as postsurgical infections or complications of initial wound therapy. When one is in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. The initial treatment of a wound is the same regardless of the wound's classification. When first recognized, the wound should be covered' with a sterile dressing until definitive treatment is rendered. Bleeding should be controlled with direct

Table V

Classification and Treatmentof TraumaticWounds

a

Classification Clean

Description

Treatment options

Aseptic, immediate closure CleanRecent wound Simplelaceration, Woundlavage, contaminated withminimal, brokentoenail debridement, easily removed ___immediate contamination closure Contaminated Severalhours Bite wounds, old Woundlavage, since wounding; lacerations,fecal debridement grossly contamination ___drain contaminated placement, +_delayed closure Dirty Purulent exudate Infectedbite Woundlavage, and infection wound, anal sac debridement, already present abscess,postsurgical drainplaceinfection ment, delayed or no closure a

Aseptic wound

Examples Surgical incision

Modified fromWaldronand Trevor(1993).

pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (Swaim, 1980). It is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (Swaim, 1980). When the treatment of a wound begins, anesthesia or analgesia may be necessary, and the choice of anesthetic regimen will depend on the size and location of the wound as well as the preference of the clinician. If the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed at this time. Then a water-soluble lubricant gel may be applied directly to the wound. A wide margin of hair should then be clipped from around the wound, using a #40 blade. After the clipping, a surgical scrub is performed around the edges of the wound. Povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (Osuna et al., 1990a,b). Simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. Full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. Two solutions, 0.05% chlorhexidine diacetate in water (Lozier et al., 1992) and 1% povidone-iodine in saline, are most often recommended for wound lavage (Waldron and Trevor, 1993). The chlorhexidine solution may offer the advantage of greater bactericidal activity but does not significantly alter wound healing when compared with povidone-iodine (Sanchez et al., 1988). Actually, the type of solution chosen

433

11. BIOLOGYAND DISEASESOF DOGS may not be as important to wound care as the volume and pressure at which the solution is delivered. It has been suggested that 8 psi is required to obtain adequate tissue irrigation, and this may be achieved by using a 35 ml syringe and an 18- or 19gauge needle (Waldron and Trevor, 1993). For wounds that are contaminated or dirty, debridement is an important part of initial therapy. Debridement usually proceeds from superficial to deeper layers. Skin that is obviously necrotic should be removed. Although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. In addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (Waldron and Trevor, 1993). If one is unsure about tissue viability in areas that are devoid of extra skin, the tissue may be left (Swaim, 1980; Waldron and Trevor, 1993), and nonviable areas will demarcate within 2 - 3 days (Waldron and Trevor, 1993). Necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. Often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. Damaged muscle should also be liberally resected (Swaim, 1980). The wound should be irrigated several times during debridement and again after completion. After initial wound treatment, the options concerning wound closure must be weighed. The principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. Primary wound closure is defined as closure of the wound at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. Closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. If dead space will remain in the wound, a drain should be place d. Subcutaneous closure should be performed with absorbable suture such as polydioxanone (PDS), polyglactin 910 (Vicryl), or polyglycolic acid (Dexon). It is best to use interrupted sutures and avoid leaving excess suture material in the wound. It may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. Skin closure is generally performed with nylon (3-0 or 4-0). In situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. After debridement and irrigation, the wound should be bandaged. Initially, the wound can be covered by gauze sponges soaked in saline or chlorhexidine to create a wet-to-dry bandage. When the sponges are later pulled from the wound, dried exudates will also be removed. When the wound appears clean, the layer in contact with the wound may be changed to a nonadherent dressing such as vaseline-impregnated gauze (Swaim, 1980). The contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. The bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound.

Wound closure within 3 - 5 days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. When the wound is closed after 5 days, this is considered secondary closure (Waldron and Trevor, 1993). Secondintention healing involves allowing the wound to heal without surgical intervention. This type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (Swaim, 1980). It is important to note that second-intention healing will take longer than surgical repair of a wound, and in the case of large wounds it will be more expensive because of the cost of bandaging materials. Several factors must be weighed concerning the use of antibiotics in traumatic wounds, including the classification and site of the wound, host defenses, and concurrent research use of the animal. When wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. When wounds have been severely contaminated or are dirty, antibiotics are indicated, and the type of antibiotic will ultimately depend on culture and sensitivity results. Until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. In skin wounds, Staphylococcus spp. are generally of concern, whereas Pasteurella multocida should be considered in bite wounds. Cephalosporins, amoxicillin-clavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (Waldron and Trevor, 1993). 2. Pressure Sores (Decubital Ulcers)

Etiology.

Pressure sores (decubital ulcers) can be a problem in long-term studies that require extended periods of recumbency. Decubital ulcers usually develop due to continuous pressure from a hard surface contacting a bony prominence such as the elbow, the tuber ischii, tarsus, or carpus. The compression of the soft tissues between the hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (Swaim and Angarano, 1990). Several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores. These factors include poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting casts or bandages (Swaim and Angarano, 1990).

Clinical signs. At first, the skin at the developing site will appear red and irritated. Over time, constant trauma can result in full-thickness skin wounds and can progress to necrosis of underlying structures such as bone. The severity of the sores may be graded from I to IV, according to the depth of the wound and the tissues involved, from superficial skin irritation to bone necrosis. Epizootiology.

The problem usually occurs in large-breed dogs, but any type of dog can be affected.

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Prevention and control. Minimizing or eliminating those factors that can predispose to decubital ulcers is important to both the prevention and the control of this condition. If the dogs are going to experience long periods of recumbency, adequate bedding or padding must be provided. Skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. The skin should be kept clean and dry at all times. If urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. Finally, an appropriate diet to maintain good flesh and adequate healing is also important (Swaim and Angarano, 1990). Treatment. The treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. The extent of initial wound management will largely depend on the depth of the wound. For simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. When wounds involve deeper tissues, including fat, fascia, or bone, more aggressive therapy must be performed. The affected area should be radiographed to assess bone involvement, and the wound should be cultured. All of the damaged tissue should be debrided, and wound management guidelines should be followed (see Section III,C,1). When a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (Swaim and Angarano, 1990). With extensive lesions, reconstruction with skin flaps may be necessary. Bandaging should be performed on all full-thickness wounds; however, it is important to remember that ill-fitting or inadequately padded bandages or casts may worsen the problem. The area over the wound itself should not be heavily padded, because this will increase the pressure over the wound. The wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound. This will displace the forces acting on the wound over a larger area and over healthier tissue. Then the doughnut is incorporated into the bandage. If a cast has been applied to the area for treatment or for research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (Swaim and Angarano, 1990). Bandages should be removed at least once or twice a day to allow wound care. After wound care has been initiated the causative factors for the pressure sore must be addressed (see "Prevention and control," above). Recumbent animals should be moved frequently to prevent continuous compression on the wound. If the dog tends to favor a position that aggravates the problem, splinting the body part to reduce contact with hard surfaces may be necessary. 3.

Acral Lick Granuloma

Etiology. Acral lick granuloma is a psychodermatosis, a skin lesion caused by self-trauma. In a few cases, self-trauma begins

because of identifiable neurologic or orthopedic causes (Tarvin and Prata, 1988). However, the majority of the cases begin because of repetitive licking by dogs that are confined and lack external stimuli (Swaim and Angarano, 1990). It has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (Dodman et al., 1988). The laboratory setting is an environment that could promote this abnormal behavior and lead to acral lick granuloma.

Epizootiology. The lesions associated with acral lick granuloma are seen most often in large-breed dogs, but any type of dog can be affected (Walton, 1986). Clinical signs. At first, lesions appear as irritated, hairless areas usually found on the distal extremities (Swaim and Angarano, 1990). The predilection for the limbs may be due to accessibility or possibly may be caused by a lower threshold for pruritus in these areas. As the lesions progress, the skin becomes ulcerated, and the wound has a hyperpigmented edge. The wounds may partially heal and then be aggravated again when licking resumes. Diagnosis and differential diagnosis. Acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. In addition, mast-cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. Many of these problems can be ruled out by the history of the animal. When in doubt, a biopsy should be taken. An uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (Walton, 1986). Prevention and control. Behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. The environment of a dog with this problem can be enriched with exercise and the introduction of toys. In addition, the relief of boredom or anxiety can be attempted through the use of drugs such as phenobarbital, megestrol acetate, and progestins. These drugs may produce side effects, however (Swaim and Angarano, 1990), and may interfere with experimental results. Treatment. Several treatments have been reported for acral lick granuloma, and none of them have been proven to be successful in aH cases. One of the most important aspects of treatment is to break the cycle of self-trauma. Mechanical restraint with an Elizabethan collar is one of the easiest methods to accomplish this goal. Several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (Swaim and Angarano, 1990; Walton, 1986). Opioid antagonists have been used in an attempt to treat acral lick granuloma by blocking endogenous opioids. In one study, either naltrexone (1 mg/kg SQ)

435

11. BIOLOGYAND DISEASESOF DOGS or nalmefene ( 1 - 4 mg/kg SQ) successfully reduced the excessive licking behavior in 7 of 11 dogs; however, lesions returned after the drug was discontinued (Dodman et al., 1988). The use of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide (3 ml of Banamine [Schering] mixed with 8ml of Synotic [Diamond Laboratories]) applied topically twice daily has also been shown to be effective (Walton, 1986). The prognosis for acral lick granuloma should be considered guarded, because the lesions often recur or new lesions develop when treatment is discontinued. 4.

Elbow Hygroma

Etiology.

Hygromas are fluid-filled sacs that develop as a result of repeated trauma over a bony prominence. The area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (Newton et al., 1974).

treatment (Johnston, 1975). Likewise, simple surgical excision of elbow hygromas can be associated with complications such as wound dehiscence and ulceration (Johnston, 1975). A technique that has been used successfully involves placement of multiple Penrose drains. The drains are kept in place for 2 - 3 weeks, and the limb remains bandaged for 4 weeks with this technique (Bellah, 1993). Another technique has been described that involves the removal of a crescent-shaped piece of the skin and capsule. The remaining dead space is closed with mattress sutures over stents, and then the wound is closed in a routine fashion. The stents are removed in 5 - 7 days, and the wound is bandaged until suture removal in 10-14 days (Newton et al., 1974). Regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. 5.

Corneal Ulcers

Etiology. Epizootiology.

Elbow hygromas are most frequently reported in large and giant breeds of dogs around 6-18 months of age (Johnston, 1975; Bellah, 1993). Elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. However, the housing environment for research dogs predisposes them to hygromas, because these animals spend a large amount of time on hard surfaces such as cage bottoms or cement runs. For this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition.

Clinical signs. A dog with an elbow hygroma presents with a unilateral or bilateral, painless, fluctuant swelling over the point of the elbow. The animals are not usually lame. Over a long period of time, elbow hygromas may become inflamed and ulcerated. If the hygroma is secondarily infected, the animal may exhibit pain and fever (Johnston, 1975; Bellah, 1993). Pathology.

The fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. Hygromas lack an epithelial lining and therefore are not true cysts. The fluid within the cavity is yellow or red and is a serous transudate. This fluid is less viscous than joint fluid, and elbow hygromas do not communicate with the joint (Johnston, 1975).

Treatment.

The treatment of elbow hygromas should be conservative whenever possible, and surgical options should be reserved for complicated or refractory cases. Conservative management of the elbow hygroma is aimed at relieving pressure at the point of the elbow by providing a padded cage surface and/or bandaging the elbow in a manner similar to that used to treat pressure sores (see Section III,C,2). More aggressive therapy, including needle drainage and the injection of corticosteroid into the hygroma, has been described but is not recommended, because infection is a serious complication of this

In the research environment, corneal ulcers are most often associated with either direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. Chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these cases would be rare in the laboratory setting.

Clinical signs. The signs of corneal ulceration are blepharospasm, epiphora, and photophobia. The eye may appear irritated and inflamed. In minor cases, the cornea may not appear abnormal; however, in cases of deeper ulceration, the cornea may appear roughened or may have an obvious defect. In addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. Diagnosis.

A tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. A definitive diagnosis of corneal ulcers may be made by the green appearance of the cornea when stained with fluorescein dye. When a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies or abnormal eyelids or cilia.

Treatment. The treatment of corneal ulcers will depend on the depth and size of the affected area. Deep ulcers may require debridement and primary repair. In such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. Superficial abrasions are generally treated with topical application of antibiotics. A triple antibiotic ointment that does not contain steroids given 3 times a day for 2 - 3 days usually provides adequate treatment. Ointments are preferred over drops, because use of the former requires less frequent. Simple corneal ulcers are restained with fluorescein after 3 days and should show complete healing at that time. If the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. Topical anesthetic should

ROBERT C. DYSKO ET AL.

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be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. This will remove the unattached edge of the cornea and healing should progress normally after debridement. In all cases, an Elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye.

D. 1.

latrogenic Diseases

Indwelling Intravascular Catheter Infections

Indwelling intravascular catheters, including intracaths and vascular access ports, often play a vital role in research protocols. The catheters are most often placed in a central vein or artery where they may be used for repeated blood sampling, administration of anesthetics and experimental compounds, or measurement of hemodynamic parameters. Although catheters vary in composition, number of ports, and port placement, the basic principles of their implantation and maintenance are similar. It is important that the laboratory animal veterinarian be familiar with these principles and the potential complications of catheter use. When appropriately maintained, indwelling catheters may remain functional for months without serious complication. The actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. This is due largely to the fact that many of the problems may be incidental findings or related to a particular research protocol. One study (Hysell and Abrams, 1967) examined the lesions found at necropsy in animals with chronic indwelling catheters (exact

vascular locations not specified). The lesions found were categorized as traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. The traumatic cardiac lesions consisted primarily of masses of fibrin and inflammatory cells on the heart valves. The visceral infarcts were noted in the spleen, kidney (Fig. 5), and brain and resulted from fibrin embolization from either the valvular lesions or the catheter tip. Fatal hemorrhages were most often found in animals with experimentally induced hypertension. These animals developed clinical signs of sepsis and later ruptured a major vessel associated with mycotic infection and aneurysm.

Etiology. The leading complication associated with the use of indwelling vascular catheters is infection, either systemic or local at the point of entry through the skin. Septicemia may develop from bacterial colonization of either the tract around the catheter or the catheter lumen. Clinical signs. The signs and treatment of systemic infection are covered in Section III,D,3. Problems with the skin defect associated with the catheter port vary from mild skin irritation to obvious infection. The signs may include redness and swelling of the skin around the external port, discharge from the skin wound, or even abscess formation. Prevention. Because indwelling catheters play an important role in many research protocols, it is highly desirable to prevent catheter complications that may result in loss of the device. The catheter should be made of nonthrombogenic material. In addition, it is recommended that catheters be as simple as possible. A catheter with extra ports or multiple lumens requires addi-

Fig. 5. Chronicrenal infarct in the kidneyof a dog that had an indwellingaortic catheter.

11. BIOLOGYAND DISEASES OF DOGS tional management and supplies more routes for infection. The use of vascular access ports that lie entirely under the skin eliminates many problems with infection. It has also been found that a long extension of tubing connected to the port may actually reduce the potential for infection of the catheter (Ringler and Peter, 1984). The initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. The placement of the catheter should be verified by radiography. Catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. After catheter placement, the animals should be observed daily for signs of either local or systemic infection. The catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. Once a month, the catheter line may be disinfected with chlorine dioxide, as described below (see "Treatment"). Throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. Treatment. The treatment of catheter infections almost invariably involves removal of the catheter, as demonstrated in both dogs and monkeys (Ringler and Peter, 1984; DaRif and Rush, 1983). Superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. Systemic antibiotic therapy should be initiated for a 10-day period. The choice of drug will ultimately be based on previous experience and culture results. Aerobic and anaerobic cultures of blood and locally infected sites should be performed (Ringler and Peter, 1984). Localized abscesses or sinus tracts may be managed by establishing drainage and flushing with chlorhexidine. Again, the catheter should be removed. If retention of a catheter is important, the catheter lumen may be disinfected by filling with chlorine dioxide solution. It has been shown that there are no adverse effects from the use of chlorine dioxide in catheters (Dennis et al., 1989). The solution is removed after 15 min and replaced with heparinized saline. All of the extension lines and fluids used in the catheter should be discarded. The blood cultures should be repeated 3 days after the antibiotic therapy has ceased. If bacteria are still cultured, the catheter must be removed. 2.

Intestinal Access Ports

Intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. These catheters are usually vascular access ports with several modifications to allow secure placement in bowel (Meunier et al., 1993). When placed and managed correctly, these ports may remain in place for months without complications.

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The most frequently reported complication associated with these catheters is infection around the port site (Meunier et al., 1993, Kwei et al., 1995). These infections lead to removal of the catheters despite treatment with local lavage and systemic antibiotics. There have also been reports of catheters dislodging from the intestinal tract and resulting in peritonitis. This complication has largely been eliminated with the improved security afforded by a synthetic cuff added to the end of the catheter (Meunier et al., 1993). The chapter authors have also seen migration of the catheter end within the lumen of the intestine (caused by peristaltic motion to egest the catheter), extensive intra-abdominal adhesions, and intestinal torsion (Figs. 6a,b) as complications of intestinal access ports. The procedures for placement and maintenance of the catheters are similar to those outlined previously for indwelling vascular catheters. It is important that the catheters be firmly secured to the intestine to prevent migration or dislodgment. An omental patch placed over the site of entry may help form a firm adhesion. In addition, it is important to place the proper length of catheter within the peritoneal cavity; excess catheter length can promote adhesion formation, whereas insufficient catheter length to account for visceral organ motion can result in detachment. The placement and patency of the catheters can be verified periodically by contrast radiography using iodinated contrast material or by fecal occult blood testing after a small amount of blood has been injected through the catheter (Meunier et al., 1993).

3.

Sepsis

Etiology. Sepsis is defined as the systemic response to infection. Most often, sepsis is a result of infection with gramnegative bacteria; however, sepsis may also be associated with gram-positive bacteria and fungi. In laboratory animals, sepsis is seen as a complication of surgical procedures or associated with chronic implants. Sepsis may also be seen as a complication of infectious diseases such as parvovirus. Clinical signs. The signs of sepsis can vary, depending on the source of the infection and the stage of the disease. Early in the course of sepsis, dogs will present with signs of a hyperdynamic response, including an increased heart rate, increased respiratory rate, red mucous membranes, and a normal to increased capillary refill time. Systemic blood pressure and cardiac output will be increased or within the normal range. The animals will often be febrile. Later in the course of the syndrome, the animals will show the classic signs of septic shock, including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. Cardiac output and blood pressure are decreased as shock progresses. Peripheral edema and mental confusion have also been reported (Hauptman and Chaudry, 1993).

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Fig. 6. (a) Jejunal volvulus in a research dog caused by the presence of surgically implanted intestinal ports. The point of insertion of the jejunal catheter (on the right in the photograph) is coincident with the distal margin of the ischemic bowel. The catheter on the left in the photograph enters the duodenum. (b) Another view of the dog in (a), highlighting the swollen and ischemic segment of jejunum affected by the torsion.

Pathogenesis. The pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such ~as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. The body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. However, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse.

Diagnosis.

In general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. However, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. One study

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examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. That study found that septic individuals had higher temperatures, WBC counts, and percentage of bands than nonseptic individuals, whereas platelet counts were lower in the septic dogs. There were no differences in respiratory rate or glucose levels between the groups. Using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (Hauptman et al., 1997). Ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. If the signs of sepsis are evident but the focus is not, several systems should be evaluated for infection, including urinary tract, reproductive tract, abdominal cavity, respiratory tract, teeth, and heart valves (Kirby, 1995).

Treatment. The treatment of sepsis has three aims. The first aim is to support the cardiovascular system. All septic animals should be treated with fluids to replace deficits and to maximize cardiac output. Crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. Colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. Acid-base and electrolyte imbalances should also be addressed. After the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. Obvious sources of infection should be drained or surgically removed. If an implant is associated with the source of infection, the implant should be removed. Antibiotic therapy should also be instituted. The choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and Gram stains. The organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. Ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. Second- or third-generation cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. Finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. Several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. Consequently, there is no "magic bullet" for the treatment of sepsis at this time. Successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. 4.

Aspiration Lung Injury

Etiology. In research animals, aspiration into the lungs may occur accidentally during the oral administration of various

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substances or by the misplacement of gastric tubes. Aspiration of gastric contents may also occur as a complication of anesthesia. In pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting.

Clinical signs. The signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. When respiration is severely affected, the oxygen saturation of blood will be decreased. The diagnosis of this problem is based on a history consistent with aspiration and the physical findings. Classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. However, these lesions may not appear for several hours after the incident of aspiration. In addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. Pathogenesis. Aspiration of gastric contents or other compounds can create lung injury of variable severity, depending upon the pH, osmolality, and volume of the substance. The compounds aspirated can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response probably mediated by cytokines. The result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. The inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. If the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. It should be noted that infection is not present in the early stages of this condition but may complicate the problem after 2 4 - 4 8 hr. Treatment. The treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. In cases in which a small amount of a relatively innocuous substance (e.g., barium) has been aspirated, treatment may not be necessary. When severe inflammation is present, systemic fluid therapy should be instituted. Support of the cardiovascular system should be performed judiciously; fluid overload could lead to an increase in pulmonary edema. The use of colloids is controversial because of the increase in vascular permeability that occurs in the lungs. Oxygen therapy is also controversial, because it may increase lung injury if administered at high concentrations for long periods of time (Nader-Djahal et al., 1997). Several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. In humans, antibiotics are reserved for use in cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. It has been suggested that dogs should be treated with antibiotics immediately when the

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aspirated material is either not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. Amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (Hawkins, 2000). The presence of pneumonia should be verified by tracheal wash and cultures. 5.

Burn Wounds

Etiology.

In laboratory animals, accidental burns usually result from thermal injury (heating pads, water bottles) or harsh chemicals (strong alkalis, acids, disinfectants). The insult to the skin results in desiccation of the tissue and coagulation of proteins. In addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. Even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. The proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research.

Clinical signs. The clinical signs vary with the type and degree of burn injury. Initially, the injury may not be noticed. The first signs may be oozing from the skin and matting of the overlying hair. Within a couple of days, progressive hair and skin loss may be observed (Johnston, 1993). The wounds may vary in severity from very superficial (involving only the epidermis) to those in which the epidermis and dermis are completely destroyed. Superficial wounds appear as red, inflamed skin similar to sunburn in humans. The pain associated with these injuries usually subsides in 2 - 3 days, and the wound reepithelializes without complications in 3 - 5 days. Deeper burns develop a thick covering, or eschar, composed of the coagulated proteins and desiccated tissue fluid. The wound heals by granulation under the eschar, which eventually sloughs or is removed to allow further healing by contraction and reepithelialization. Within 2 - 3 days of injury, the burn wound will be colonized by grampositive bacteria that rapidly cover the entire wound. Several days later, gram-negative organisms can appear in the burn wound (Johnston, 1993). At this point, signs of wound infection and sepsis may occur (see Section III,D,3). Treatment. Appropriate and timely treatment of a burn wound will reduce the extent of the injury. Thermal injuries should be immediately cooled to reduce edema and pain (Demling and Lalonde, 1989). Chemical burns should be thoroughly lavaged for 60 min after wounding. The damaged tissues may be unable to mount appropriate responses to changes in temperature; therefore, the lavage should be performed with warm water to prevent hypothermia. After the initial treatment, all burn wounds should be gently cleansed 2 - 3 times a day (Demling and Lalonde, 1989). Burns involving the epidermis and part of

the dermis can be extremely painful, and analgesia should be addressed throughout the treatment period. Systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. Therefore, topical wound dressings are recommended in the early stages of treatment. A thin film of a water-soluble broad-spectrum antibiotic ointment should be applied to the wound surface after each cleaning. Silver sulfadiazine has a broad spectrum, penetrates eschar well, and is often the preparation of choice for burn wound therapy. Povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. Mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it is associated with pain upon application (Demling and Lalonde, 1989). When signs of wound or systemic infection are present, systemic antibiotics should be employed, and their ultimate selection should be based on culture and sensitivity results. After the topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. Burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds. When the eschar over a burn wound has formed and become fully defined, a small or moderately sized wound may be completely resected.

Prevention.

Obviously, prevention of burn wounds is preferable to a long course of treatment. Care should be taken to prevent direct exposure to harsh chemicals. Tables, floors, and other surfaces should be rinsed thoroughly after chemical use, prior to allowing any animal contact. Electric heating pads should be avoided, and only heated water blankets or circulating warm-air devices should be used to provide warmth to the animals. In rare instances, heated water blankets have also caused burns; therefore these devices should be carefully monitored. As a precaution, a thin towel may be placed between the animal and the water blanket. 6.

Perivascular Extravasation of Drugs

Etiology.

Research and/or anesthetic protocols may require the intravenous injection of various solutions. When these substances have a pH or osmolarity significantly different from that of the surrounding tissues, the accidental perivascular extravasation of the solutions may result in tissue damage. Several drugs have been shown to cause problems when injected perivascularly, including pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin (Swaim and Angarano, 1990; Waldron and Trevor, 1993).

Clinical signs. The immediate signs of perivascular injection are swelling at the injection site and withdrawal of the limb or other signs of discomfort. Later, the area may appear red,

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swollen, and painful as inflammation progresses. Often there will be eventual necrosis of the skin around the injection site. In cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a 1 to 4 month period. This is because the drug is released over time from the dying cells (Swaim and Angarano, 1990).

Prevention.

Because the degree of injury and extensive treatment associated with perivascular extravasation of a drug can be detrimental to research protocols and can cause severe discomfort to the dog, prevention of these injuries is preferred. Prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. If a potentially caustic compound is to be used in a fractious subject, sedation of the dog is warranted if this will not interfere with the research protocol. Whenever possible, insertion of an indwelling catheter is extremely important. Access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. When peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. Additional injections are best given through newly placed catheters in previously unused vessels. The repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. Prior to use, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. Any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used.

Treatment. The treatment of perivascular injections will depend on the amount and type of substance injected. In most cases, dilution of the drug with subcutaneous injections of saline is recommended. In addition, steroids may be infiltrated locally to reduce inflammation. Topical application of dimethyl sulfoxide (DMSO) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions (Swaim and Angarano, 1990). The addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (Hoskins, 1989), and local infiltration of hyaluronidase accompanied by warm compresses has been suggested for use in cases of vinblastine injection (Waldron and Trevor, 1993). Despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. In cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (Swaim and Angarano, 1990). In all cases, the condition can be painful, and analgesia should be addressed.

7.

Hepatic Encephalopathy

Etiology.

Hepatic encephalopathy is the result of the derangements in metabolism associated with abnormal liver function. This condition may be seen in young dogs with congenital portosystemic shunting of blood flow. However, in the research setting, encephalopathy occurs more often in canine models of hepatic disease that lead to liver failure. A well-developed knowledge of the pathophysiology of liver disease is necessary for the initial treatment and long-term management of hepatic encephalopathy.

Pathogenesis.

When the liver function is severely impaired because of either portosystemic shunting of blood flow or loss of metabolically active hepatic tissue, the result is an accumulation of ammonia, toxic amines, aromatic amino acids, and short-chain fatty acids (Hardy, 1989; Center, 1998). These compounds have several toxic effects that result in a decrease in cerebral energy metabolism and a decrease in excitatory neurotransmitter synthesis. Concurrently, there is an increase in the concentration of false neurotransmitters and the inhibitory substance 7-aminobutyric acid (GABA).

Clinical signs. The signs of hepatic encephalopathy include lethargy, depression, muscle tremors, and convulsions. Diagnosis and differential diagnosis.

A presumptive diagnosis of hepatic encephalopathy may be based on the appearance of clinical signs following experimental manipulation of the liver. Additional diagnostic tests to verify the loss of liver function can be performed to confirm the diagnosis. Serum glucose and protein levels may be low if hepatic function is severely impaired. A low serum urea nitrogen level suggests that the normal hepatic metabolism of ammonia into urea has been impaired. Elevated levels of serum bile acids and blood ammonia also verify the loss of liver function (Hardy, 1989). Measurement of serum hepatic leakage enzymes are nondiagnostic, because they can be low, high, or normal.

Treatment. Because of the severity of hepatic encephalopathy, treatment may be initiated based on a presumptive diagnosis. During initial treatment, supportive care with fluids and electrolytes should be instituted, based on the results of serum chemistry and blood gas analysis. The majority of animals with hepatic dysfunction will be hypokalemic, alkalotic, and hypernatremic; therefore, either 0.45% sodium chloride or 0.45% sodium chloride with 2.5% dextrose, supplemented with potassium chloride, is recommended (Hardy, 1989). The type of drug to be used for seizure control is controversial. The short halflife of diazepam makes it an attractive choice compared with barbiturates, which have prolonged metabolism when hepatic function is impaired (Maddison, 1995). However, endogenous benzodiazepines mediate some of the CNS signs seen with

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hepatic encephalopathy. Therefore, the use of diazepam has been discouraged in favor of phenobarbital (Johnson, 2000). The drug selected for seizure control should be titrated carefully, given the altered liver metabolism. Most importantly, the treatment of dogs with hepatic encephalopathy must be aimed at reducing the levels of toxic metabolites in the bloodstream. Because protein metabolism is a major source of ammonia, all oral food intake should cease until the signs of hepatic encephalopathy have abated. Because gastrointestinal bleeding may occur in individuals with liver failure and this is also a source of protein, the use of H2 blockers such as cimetidine or ranitidine is suggested (Swalec, 1993). In addition, lactulose retention enemas should be performed (10-15 ml/lb of a 30% solution in water, retained for 2 0 30 min) (Hardy, 1989). Lactulose is an indigestible semisynthetic sugar that is metabolized in the gut to lactic and other acids. The decrease in colonic pH reduces ammonia levels in the bloodstream by converting intestinal ammonia into less diffusible ammonium ions. Lactulose will also cause an osmotic diarrhea. Antibiotics such as neomycin (10 mg/lb, 3 - 4 times/ day) or metronidazole (9 mg/lb, 3 times/day) should also be used to reduce the intestinal load of urease-producing bacteria responsible for splitting urea into ammonia (Hardy, 1989). When the signs of hepatic encephalopathy have resolved, the dog may be fed a low-protein diet. Diets suitable for dogs with renal insufficiency are recommended initially. This type of diet is not suitable for long-term use, however, because it appears that individuals with some types of hepatic disease actually have increased protein requirements. These requirements may be met by slowly increasing protein in the diet as long as signs of hepatic encephalopathy do not recur. To maintain the appropriate balance of aromatic and branched-chain amino acids, the diet should be based on vegetable and dairy protein instead of meat or fish protein (Center, 1998). In addition, the antibiotics suggested above should be continued to reduce the effects of increasing dietary protein levels.

E.

Neoplastic Diseases

1. Introduction

The prevalence of cancer in the general canine population has increased over the years (Dorn, 1976). This can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. Because of these changes, cancer has become a major cause of death in dogs (Bronson, 1982). In a lifetime cancer mortality study of intact beagles of both sexes, Albert et al. (1994) found death rates similar to the death rate of the at-large dog population (Bronson, 1982). Approximately 22% of the male beagles died of cancer. The majority of

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the tumors were lymphomas (32%) and sarcomas (29%), including hemangiosarcomas of the skin and fibrosarcomas. Of the female beagles dying of cancer (26% of the population studied), three-quarters had either mammary cancer (40%), lymphomas (18%), or sarcomas (15%). Of the sarcomas in females, one-third were mast cell tumors. In addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (Hayes and Fraumeni, 1975; Benjamin et al., 1996). Because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. 2.

Biopsy Techniques

Fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. This technique allows for rapid differentiation of benign and neoplastic processes. In cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. Needle-punch or core biopsies can also be performed in awake patients but typically require local anesthesia. An instrument such as a Tru-Cut needle (Travenol Laboratories, Inc., Deerfield, Illinois) is used to obtain a 1 mm X 1 to 1.5 cm biopsy of a solid mass. A definitive diagnosis may be limited by the size of the sample acquired using this technique. Incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. Excisional biopsies are the treatment of choice when surgery is necessary, because the entire mass is removed. Surgical margins should extend at least 1 cm around the tumor, and 3 cm if mast cell tumors are suspected (Morrison et al., 1993). Incisional biopsies are performed when large soft-tissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. When performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. 3.

Lymphomas

Etiology. Lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. Whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. In humans, data implicate the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as a cause of nonHodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (MacEwen and Young, 1991). Clinical signs. Multicentric and alimentary lymphomas account for most cases of canine lymphoma. In multicentric lymphoma, animals usually present with enlarged lymph nodes and

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11. BIOLOGYAND DISEASESOF DOGS nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, and lethargy. When the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. Alimentary lymphoma is associated with vomiting and diarrhea, in addition to previous clinical signs. Less commonly, dogs develop mediastinal, cutaneous, and extranodal lymphomas. Dogs with mediastinal lymphoma often present with respiratory signs secondary to pleural effusion. Hypercalcemia is most frequently associated with this form of lymphoma and may result in weakness. Cutaneous lymphoma varies in presentation from solitary to generalized and may mimic any of a number of other skin disorders. The tumors may occur as nodules, plaques, ulcers, or dermatitis. Approximately half of the cases are pruritic. A number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. Clinical presentation varies, depending on the site of involvement.

Diagnosis and differential diagnosis.

Epizootiology.

Research complications.

The incidence of lymphoma is highest in dogs 5-11 years old, accounting for 80% of cases. Although the neoplasm generally affects dogs older than 1 year, cases in puppies as young as 4 months have been reported (Dorn et al., 1967).

Pathologic findings.

Enlarged neoplastic lymph nodes vary in diameter from 1 to 9 cm and are moderately firm. Some may have areas of central necrosis and are soft to partially liquefied. The demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. The spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. The enlarged liver may have disseminated pale foci or multiple large, pale nodules. In the gastrointestinal tract, both nodular and diffuse growths are observed. These masses may invade through the stomach and intestinal walls. Histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. The neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas.

Differential diagnoses for multicentric lymphoma include systemic mycosis; salmonpoisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. Alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. In order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections are frequently needed.

Treatment. Therapy for lymphoma typically consists of one or a combination of several chemotherapeutic agents. The treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. MacEwen and Young (1991) provide a thorough discussion of therapeutic options for the treatment of lymphomas in the dog. Given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with signifcant clinical illness. 4.

Tumors of the Skin and Soft Tissue

a.

Fibrosarcomas

Etiology.

The fibrosarcoma group of tumors encompasses not only malignant tumors of fibroblasts but also a number of indistinguishable tumors, all of which are capable of collagen production (Pulley and Stannard, 1990). Frequently classified in this group are undifferentiated leiomyosarcomas, liposarcomas, malignant melanomas, and malignant schwannomas.

Clinical signs. Although these neoplasms can arise throughout the body, they are most commonly found in the skin, subcutaneous tissues, and oral cavity. Fibrosarcomas are extremely variable in size and can grow to be quite large. In general, they are irregular and nodular, poorly demarcated, and nonencapsulated, and they frequently invade deeper tissues. Epizootiology.

Most fibrosarcomas develop in adult and aged animals but can affect dogs as young as 6 months or less.

Pathogenesis.

All lymphomas regardless of location should be considered malignant. A system for staging lymphoma has been established by the World Health Organization. The average survival time for dogs without treatment is 4 - 6 weeks. Survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (MacEwen and Young, 1991). Hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. The pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes.

Pathologic findings.

Histologically, fibrosarcomas appear as interwoven bundles of densely packed spindle-shaped fibroblasts. The cells show large numbers of mitotic figures, while undifferentiated tumors may exhibit multinucleated giant cells and cells with bizarre shapes.

Pathogenesis.

Fibrosarcomas exhibit rapid, invasive growth, recurring frequently after excision. Metastasis occurs in only one-fourth of cases, usually by the bloodstream to the lungs. Less frequently, spread to local lymph nodes is observed.

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Diagnosis and differential diagnosis.

Differential diagnoses for fibrosarcomas vary with the location of the tumor. Histopathologic exam should be used to distinguish these tumors from round cell tumors (mast cell tumors, histiocytomas, transmissible venereal tumors), papillomas, and other neoplasms.

Treatment. Treatment of any soft-tissue sarcoma would begin with wide surgical excision. If the tissue margins indicate incomplete resection, radiotherapy could be used. For any highgrade tumors, adjuvant chemotherapy would be recommended (see MacEwen and Withrow, 1991a, for a complete discussion). Research complications.

Because fibrosarcomas are locally invasive and often recur, dogs with these neoplasms should not be considered good subjects for long-term studies.

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c. Histiocytomas Etiology.

Histiocytomas are benign skin growths that arise from the monocyte-macrophage cells in the skin. Some debate exists as to whether this growth is actually a neoplasm or a focal inflammatory lesion (Pulley and Stannard, 1990).

Clinical signs. The most frequent sites for histiocytomas are the head (especially the pinna) and the skin of the distal forelegs and feet. The masses are usually domelike or buttonlike (often referred to as "button tumors") and usually measure 1-2 cm in diameter. Epizootiology.

Histiocytomas are the most common tumors of young dogs, mostly occurring in dogs less than 2 years of age.

Pathologic findings. b. Lipomas Etiology.

Neoplasms of lipocytes and lipoblasts are welldifferentiated tumors referred to as lipomas.

Clinical signs. These growths can be found as single or multiple round, ovoid, or discoid masses in the subcutaneous tissues of the lateral and ventral thorax, abdomen, and upper limbs. Generally they are well circumscribed, encapsulated, and soft on palpation. Further, the skin is freely movable over the tumor. Epizootiology.

Lipomas occur principally in aged animals (average 8 years), and the incidence increases with age (Pulley and Stannard, 1990). The tumors are most commonly seen in overweight female dogs, but no breed predisposition is observed.

Pathologic findings.

Histologically, lipomas are indistinguishable from normal adipose tissue except when a fibrous capsule is present.

Histologically, these tumors contain round to ovoid cells with pale cytoplasm and large nuclei. The cells infiltrate the dermis and subcutis, displacing collagen fibers and skin adnexa. Despite being benign lesions, histiocytomas characteristically have a high mitotic index.

Pathogenesis.

This tumor typically exhibits rapid growth (14 weeks) but does not spread. Most histiocytomas will spontaneously regress in less than 3 months.

Diagnosis and differential diagnosis.

Histiocytomas must be distinguished from potentially metastatic mast cell tumors. This is accomplished by staining with toluidine blue, which would stain the cytoplasmic granules of mast cells red or purple.

Treatment. Although most histiocytomas will spontaneously resolve, conservative surgery or cryosurgery will provide an expeditious resolution. Research complications.

Histiocytomas should not interfere

with most studies.

Pathogenesis:

Lipomas are typically slow-growing and do not recur after complete surgical excision.

d. Mast Cell Tumors

Diagnosis and differential diagnosis.

Lipomas are not frequently confused with other tumors but can sometimes be difficult to distinguish from normal adipose tissue. Generally, the distinction can be made from the clinical history.

Etiology.

Treatment. Treatment for lipomas is not usually necessary unless the mass is causing problems with normal ambulation. In such cases, surgical excision is usually curative.

Clinical signs.

Research complications.

Lipomas usually do not complicate research studies unless they are interfering with other systemic functions or ambulation.

Neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog (Bostock, 1986). Mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin. Well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, 1 to 10 cm nodules in the skin. Alopecia may be observed, but ulceration is not usual. Poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema. Mast cell tumors can be found on any portion of the dog's skin but frequently affect the hindquarters, especially the thigh and in-

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11. BIOLOGY AND DISEASES OF DOGS

These tumors tend to affect middle-aged dogs but have been observed in dogs ranging from 4 months to 18 years (Pulley and Stannard, 1990).

hypo-osmotically lysing any mast cells left behind. This technique has recently been refuted by Jaffe et al. (2000). For systemic mastocytosis, and nonresectable or incompletely excised mast cell tumors, chemotherapy can be used. Treatment options would include oral prednisolone, intralesional triamcinalone, and the combination of cyclophosphamide, vincristine, and prednisolone (Graham and O'Keefe, 1994).

Pathologic findings.

Research complications.

guinal and scrotal areas. Mast cell tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues.

Epizootiology.

Because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by Patnaik et al. (1986) has become widely accepted. In this system, grade I has the best prognosis, and grade III the worst prognosis. Grade I tumors are well differentiated, with round to ovoid uniform cells. The nuclei are regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. Grade II (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. Grade III (anaplastic or undifferentiated) mast cell tumors have large, irregular nuclei with multiple prominent nucleoli. The cytoplasmic granules are few, but mitotic figures are much more frequent. In addition to skin lesions, mast cell tumors have been associated with gastric ulcers. These lesions are most likely secondary to tumor production of histamine. Histamine stimulates the H2 receptors of the gastric parietal cells, causing increased acid secretion. Gastric ulcers have been observed in large numbers (>75%) of dogs with mast cell tumors (Howard et al., 1969). The ulcers can be found in the fundus, pylorus, and/or proximal duodenum.

Pathogenesis.

Although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. Grade III tumors are most likely to disseminate internally. This spread is usually to regional lymph nodes, spleen, and liver and less frequently to the kidneys, lungs, and heart.

Diagnosis and differential diagnosis.

Mast cell tumors can be distinguished histologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue, which metachromatically stains the cytoplasmic granules of the mast cells red or purple.

Treatment. Initial treatment for mast cell tumors is generally wide surgical excision (1 to 3 cm margins). Even with wide surgical margins, approximately 50% of mast cell tumors may recur. If the site is not amenable to wide surgical excision, debulking surgery and radiation therapy may be used. Other alternatives include amputation (if on a limb) or radiation therapy alone. As an adjunct to surgery, Grier et al. (1990, 1995) found that deionized water injected into surgical margins reduced tumor recurrence by

Because of the possibility of systemic histamine release and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. Grade I mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed on a regular basis (monthly). Grade II tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every 3 - 6 months for detection of systemic mastocytosis. Because of the poor prognosis for grade III tumors, treatment is unwarranted in the research setting.

e.

Hemangiosarcomas

Etiology.

Hemangiosarcomas are malignant tumors that originate from endothelial cells.

Clinical signs. These tumors may arise in the subcutis but are more commonly found in the spleen and the right atrium. Clinical signs are associated with the site of involvement. Vascular collapse is frequently observed secondary to rupture and hemorrhage from splenic masses. Heart failure can be observed secondary to tumor burden or hemopericardium. When found in the skin, hemangiosarcomas are poorly circumscribed, reddish black masses that range in size from 1 to 10 cm in diameter. The most common cutaneous sites are the ventral abdomen, the prepuce, and the scrotum. Epizootiology:

Hemangiosarcomas occur most frequently in 8- to 13-year-old dogs. The German shepherd dog is most commonly affected.

Pathologic findings.

Grossly, splenic hemangiosarcomas resemble nodular hyperplasia or hematomas (Fig. 7). The masses are spherical and reddish black and can range in size up to 1520 cm in diameter. On cut section the masses may appear reddish gray or black and have cavernous areas of clotted blood. When the masses are found in the heart, the endocardium may be covered by a thrombus, giving the 2 to 5 cm tumors a reddish gray or yellow appearance. Histologically, hemangiosarcomas are composed of immature endothelial cells that form vascular channels or clefts. These spaces may be filled with blood or thrombi. The neoplastic cells are elongated with round to ovoid, hyperchromatic nuclei and frequent mitotic figures.

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Fig. 7. Splenichemangiosarcomain a random-sourceresearch dog. Pathogenesis.

Hemangiosarcomas can be found in one or many sites. In cases where multiple sites are involved, it may be impossible to identify the primary tumor. This neoplasia is highly malignant and spreads easily. Metastasis occurs most frequently to the lungs but can be found in any tissue.

Diagnosis and differential diagnosis.

Splenic hemangiosarcoma may resemble nodular hyperplasia or some manifestations of lymphoma. When the heart is affected, other causes of heart failure must be ruled out. Echocardiography is a valuable tool for identifying the primary lesion. Histopathology should be used to differentiate dermal hemangiosarcoma from hemangiomas and other well-vascularized tumors.

Treatment. Surgery is generally the first choice of treatment for hemangiosarcoma. Dermal tumors are treated with radical resection, splenic tumors by total splenectomy, and heart tumors by debulking and pericardiectomy. Because of the high likelihood of metastasis, adjunct chemotherapy should always be considered. Research complications.

Dogs with dermal hemangiosarcoma may be cured after complete resection with margins, but monitoring should be done regularly for recurrence. The other forms of hemangiosarcoma have a much poorer long-term prognosis, and treatment is typically unwarranted in the research setting.

5.

Transmissible Venereal Tumors

Etiology.

Also known as infectious or venereal granuloma, Sticker tumor, transmissible sarcoma, and contagious venereal tumor, the transmissible venereal tumor is transmitted to the genitals by coitus (Nielsen and Kennedy, 1990). The origin of this tumor is still unknown but has been described as a tumor of lymphocytes, histiocytes, and reticuloendothelial cells. Although this tumor has been reported in most parts of the world, it is most prevalent in temperate climates (MacEwen, 1991).

Clinical signs. The tumors are usually cauliflower-like masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. These friable masses vary in size up to 10 cm, and hemorrhage is frequently observed. In male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis (Fig. 8). Less frequently, the tumor is found on the prepuce. Females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. When located around the urethral orifice, the mass may protrude from the vulva. These tumors have also been reported in the oral cavity, skin, and eyes. Epizootiology and transmission. Transmissible venereal tumors are most commonly observed in young, sexually active dogs. Transmission takes place during coitus when injury to the genitalia allows for transplantation of the tumor. Genital to oral

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11. BIOLOGY AND DISEASES OF DOGS

Fig. 8. Transmissiblevenerealtumor on the penis of a dog.

to genital transmission has also been documented (Nielsen and Kennedy, 1990). Extragenital lesions are believed to be a result of trauma prior to exposure to the tumor.

mg/m 2) IV once weekly for 4 - 6 treatments will induce remission and cure in greater than 90% of the cases (MacEwen, 1991).

Pathologic findings.

Research complications.

Pathogenesis.

6.

Histologically, cells are arranged in compact masses or sheets. The cells are round, ovoid, or polyhedral and have large, round hyperchromic nuclei. Also present are large nucleoli and numerous mitotic figures. The cytoplasm is eosinophilic, and cell borders are indistinct.

Tumor growth is rapid after implantation but later slows. Metastasis is rare (<5% of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites.

Experimental implantation of transmissible venereal tumors has been shown to elicit formation of tumor-specific IgG (Cohen, 1972). This response may occur in natural infections and could possibly interfere with immunologic studies. Mammary Gland Tumors

Etiology.

Dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones.

Diagnosis and differential diagnosis.

Transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. Cytology may be of benefit in making a definitive diagnosis, so impression smears should be made prior to processing for histopathology.

Prevention.

Thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony.

Clinical signs. Single nodules are found in approximately 75% of the cases of canine mammary tumors. The nodules can be found in the glandular tissue or associated with the nipple. Masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. Benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger and invasive and coalesce with adjacent tissues. Epizootiology.

Control.

Removing affected individuals from a breeding program should stop further spread through the colony.

Treatment. Surgery and radiation can be used for treatment, but chemotherapy is the most effective. Vincristine (0.5-0.7

Mammary tumors are uncommon in dogs under 5 years of age with the incidence rising sharply after that. Median age at diagnosis is 10-11 years. Mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting Sertoli cell tumors.

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Pathologic findings.

Based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (Bostock, 1977). Extensive discussions of classification, staging, and histopathologic correlations can be found in MacEwen and Withrow (199 lb) and Moulton (1990).

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tinue on study. If removed early enough, malignant masses could yield the same results. All dogs should be monitored regularly for recurrence and new mammary tumors. 7.

Thyroid Carcinomas

Etiology.

Beagles are among the breeds with the highest prevalence of thyroid carcinomas. Benjamin et al. (1996) reported a correlation between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in the beagle.

Pathogenesis.

Mammary tumors of the dog develop under the influence of hormones. Receptors for both estrogen and progesterone can be found in 60-70% of tumors. Futher, Schneider et al. (1969) showed that the risk of developing mammary tumors increased greatly after the first and second estrus cycles. Dogs spayed prior to the first estrus had a risk of 0.8%, whereas dogs spayed after the first and second estrus had risks of 8% and 26%, respectively. Malignant mammary tumors typically spread through the lymphatic vessels. Metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. The fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. Many mammary carcinomas will eventually metastasize to the lungs.

Diagnosis and differential diagnosis.

Both benign and malignant mammary tumors must be distinguished from mammary hyperplasia and mastitis.

Clinical signs. Thyroid carcinomas generally present as palpable cervical masses. Affected animals may experience dysphagia, dyspnea, and vocalization changes. Precaval syndrome resulting in facial edema is also observed in some cases. Epizootiology and transmission.

The mean age of dogs presented with thyroid carcinomas is 9 years, with equal distribution of cases between the sexes.

Pathologic findings.

Grossly, thyroid carcinomas are multinodular masses, frequently with large areas of hemorrhage and necrosis. They tend to be poorly encapsulated and invade local structures such as the trachea, esophagus, larynx, nerves, and vessels. The masses are unilateral twice as often as bilateral (Capen, 1990). Histologically, thyroid carcinomasare divided into follicular, papillary, and compact cellular (solid) types (see Capen, 1990, for complete discussion).

Pathogenesis. Prevention.

Mammary tumors can effectively be prevented by spaying bitches prior to the first estrus. This is commonly done in the general pet population at 6 months of age. Recently, the topic of spaying sexually immature dogs (8-16 weeks of age) has received much attention for the control of the pet population. Kustritz (1999) reviewed the techniques for anesthesia and surgery, as well as possible pros and cons of spaying at this young age.

Thyroid carcinomas tend to grow rapidly and invade local structures. Early metastasis is common and occurs to the lungs by invasion of branches of the thyroid vein.

Diagnosis and differential diagnosis.

Nonpainful cervical swellings such as seen with thyroid tumors are also consistent with abscesses, granulomas, salivary mucoceles, and lymphomas. Often a preliminary diagnosis can be made by fineneedle aspirate.

Treatment. Surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. The extent of the surgery is dependent on the area involved. Lumpectomy or nodulectomy should be elected in the case of small discrete masses, while mammectomy and regional or total mastectomies are reserved for more aggressive tumors. At the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. Inguinal lymph nodes should be removed any time the fourth and fifth glands are excised (MacEwen and Withrow, 199 lb).

Treatment. Surgery is the treatment of choice for thyroid carcinomas that have not metastasized. When the tumor is freely movable, surgery may be curative. Surgical excision may be difficult for tumors that adhere to local structures, requiring excision of the jugular vein, carotid artery, and associated nerves. When bilateral tumors are observed, preservation of the parathyroid glands may not be possible. In such cases, treatment for hypoparathyroidism will be necessary. Both chemotherapy and radiation therapy have been suggested for extensive bilateral tumors and/or after incomplete excision, but no controlled trials have been performed (Ogilvie, 1991).

Research complications.

Research complications.

Because 50% of mammary tumors are benign, treatment may be rewarding, allowing dogs to con-

In the research setting, treatment of this tumor may not be rewarding. Only freely movable tumors

11. BIOLOGY AND DISEASES OF DOGS can be practically treated without seriously affecting research efforts. Euthanasia is warranted in the more advanced cases when clinical illness is apparent.

F.

Miscellaneous Diseases

1. Congenital Disorders

Beagles are subject to many of the inherited and/or congenital disorders that affect dogs in general. In a reference table on the congenital defects of dogs (Hoskins, 2000b), disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor VII deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, GM1 gangliosidosis, globoid cell leukodystrophy, XX sex reversal, and cutaneous asthenia (EhlersDanlos syndrome). In addition, there are defects that affect so many breeds that the author simply lists "many breeds" for the breeds affected by those disorders. Thus these defects could also affect beagles and include pectus excavatum, polydactyly, radial and ulnar dysplasia, hypoadrenocorticism, entropion, lens coloboma, factor VIII deficiency (von Willebrand's disease), renal agenesis or ectopia, and developmental defects of the reproductive and lower urinary tracts. At a commercial breeder of purpose-bred beagles, the most common birth defects were umbilical hernia (1.82% of births) and open fontanelle (1.44% of births) (R. Scipioni and J. Ball, personal communication, 1999). Other defects observed include cleft palate and cleft lip, cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. Each of these other congenital defects occurred at less than 1.0% incidence. 2. Age-Related Diseases

a.

Cataracts and Nuclear Sclerosis

Etiology. Cataract is an opacification of the lens or the lens capsule. It is the pathologic response of the lens to illness or injury, because the lens has no blood supply. Cataracts can be caused by metabolic, inflammatory, infectious, or toxic causes and can be congenital, juvenile, or degenerative. Nuclear sclerosis is an apparent opacification of the lens caused by the compression of older lens fibers in the center of the lens (nucleus) as a consequence of the production of new fibers. Because the nucleus increases in size as the animal ages, the sclerosis is more apparent in older animals and may be mistaken as a senile cataract. The ability to see the fundus during ophthalmoscopy persists with nuclear sclerosis but is obstructed by a true cataract.

449 Clinical signs. The first clinical sign is typically the ability to visualize the opaque lens through the pupil of the dog's eye. Dogs have an impressive ability to tolerate bilateral lens opacity (especially when development is gradual), and often visual impairment is detected late in the development of the condition (Helper, 1989). Moderate vision loss may cause the dog to be hesitant in moving in new surroundings or unable to locate movable objects (such as a toy). Rapid cataract development can result in a sudden vision loss, such as can occur with diabetic cataracts. Epizootiology and transmission. Certain dog breeds can be predisposed to the development of juvenile or senile cataracts or to metabolic disorders that result in cataract development, such as diabetes mellitus. Dogs in studies for diabetes mellitus should be observed regularly for cataract development. Toxicological studies may also induce formation of cataracts. Pathogenesis. Lens fibers respond to all biological or chemical insults by necrosis and liquefaction (Render and Carlton, 1995), because they have no blood supply with which to recruit an inflammatory and repair process. Disruption of these fibers by any means, therefore, leads to opacification. The exact processes by which the varieties of congenital and juvenile cataracts are produced have not been determined. In diabetic cataracts, the excess glucose is metabolized to sorbitol and fructose. As these alcohols and sugars accumulate in the lenticular cells, they produce an osmotic imbalance, which brings fluid into the cells, causing swelling and degeneration of lens fibers and resultant opacity (Capen, 1995). Diagnosis and differential diagnosis. The ability to visualize the retina and fundus during ophthalmoscopy differentiates true cataracts from nuclear sclerosis. Dogs with cataracts should be evaluated for possible causes, especially diabetes mellitus. Diabetes mellitus will typically affect middle-aged dogs and feature rapid cataract formation, whereas juvenile and senile cataracts are slow to develop and affect younger and older dogs, respectively. Progressive retinal atrophy can also cause secondary cataract formation; pupillary light response is maintained with primary cataracts (even if the lens is completely opaque), whereas this reflex is obtunded by retinopathy. Prevention. Most forms of cataracts cannot be prevented, for their exact etiologic pathogenesis is unknown. Diabetic cataracts, however, can be prevented by proper regulation of blood glucose concentrations with insulin therapy and proper diet. Treatment. Because dogs do not need to focus visual images as accurately as human beings, proper lens clarity and function are not necessary for an adequate quality of life. Many dogs adjust quite well to the visual impairment caused by persistent cataracts. Lens removal can be performed for dogs seriously

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affected by cataracts, but this would not be anticipated for dogs in the research setting. Information on surgical lens extraction procedures can be found in Helper (1989) or other veterinary ophthalmology textbooks.

Research complications. Research complications would be minimal with cataracts, unless the dogs were intended for use in ophthalmologic or visual acuity-based studies. b.

Hip Dysplasia

Etiology. Hip dysplasia is a degenerative disease of the coxofemoral joint. A specific etiology is unknown, but the development of hip dysplasia has a strong genetic component (Pedersen et al., 2000), modified by age, weight, size, gender, conformation, rate of growth, muscle mass, and nutrition (Smith et al., 1995). Clinical signs. The initial clinical abnormality caused by hip dysplasia is laxity of the coxofemoral joint. This may present as a gait abnormality without any indication of lameness or stiffness. Eventually, affected dogs will have periods of lameness and, in protracted cases, will be rendered immobile by severe pain. Epizootiology and transmission. Hip dysplasia has been seen in most dog breeds, but it typically affects larger breeds of dogs. In the research setting, it is primarily a condition of randomsource large-breed dogs used for surgical research. Diagnosis and differential diagnosis. Hip dysplasia is classically diagnosed by radiography of the pelvis and hip joints. Radiographic abnormalities consistent with hip dysplasia include shallow acetabula with remodeling of the acetabular rim, flattening of the femoral head, subchondral bone sclerosis (caused by erosion of articular cartilage and exposure of underlying bone), and osteophyte production around the joint (Pedersen et al., 2000). Hip dysplasia needs to be differentiated from other musculoskeletal or neurological conditions that can cause unusual gaits and/or lameness. This may be somewhat difficult, because clinical signs of hip dysplasia may develop before radiographic abnormalities. Radiographic calculation of the distraction index (DI) to measure joint laxity has proven to be a good means to predict future hip dysplasia before other radiographic changes are evident (Smith et al., 1995). Prevention. Because of the genetic component, dogs with hip dysplasia should not be used in breeding colonies. Dogs should be provided a good plane of nutrition but not be allowed to become overweight. Dogs that were limit-fed at 75% of the food amount eaten by ad libitum-fed dogs had lower body weights and decreased severity of radiographic lesions of hip dysplasia (Kealy et aL, 1997).

Treatment. In the stages when clinical signs are episodic, cage rest and analgesics for several days can be used to treat the symptoms. More advanced cases may require continuous analgesia. Sectioning of the pectineus muscle or tendon may provide some pain relief but does not affect the progression of the disease (Pedersen et al., 2000). Surgical treatments for hip dysplasia include femoral head ostectomy and total hip replacement. Neither surgical treatment is likely in a research setting. Research complications. Long-term studies using large-breed dogs may be affected by the eventual development of hip dysplasia. In studies where hip dysplasia would be a serious complication or confounding variable (e.g., orthopedic research), dogs should be radiographed upon arrival to assess possibility of early coxofemoral joint degeneration and suitability for use in the study. c.

Benign Prostatic Hyperplasia

Etiology. Benign prostatic hyperplasia (BPH) is an agerelated condition in intact male dogs. The hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. Clinical signs. BPH is often subclinical. Straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum as it passes through the pelvic canal. Urethral discharge (yellow to red) and hematuria can also be presenting clinical signs for BPH. Epizootiology and transmission. BPH typically affects older dogs (>4 years), although it has been seen as early as 2 years of age. Pathologic findings. In its early stages, canine B PH is hyperplasia of the prostatic glandular tissue. This is in contrast to human BPH, which is primarily stromal in origin. Eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. The prostate becomes more vascular (resulting in hematuria or hemorrhagic urethral discharge), and BPH may be accompanied by mild chronic inflammation. Pathogenesis. BPH occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. The presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to 5a-dihydrotestosterone (Kustritz and Klausner, 2000). Diagnosis and differential diagnosis. BPH is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland

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in intact male dogs, with normal hematologic profiles and urinalysis characterized by hemorrhage, at most. Differential diagnoses include squamous metaplasia of the prostate, paraprostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). These differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. As such, these conditions do not necessarily abate or resolve when castration is used for treatment of prostatic enlargement.

Prevention.

Castration is the primary means for prevention of benign prostatic hyperplasia.

Treatment. The first and foremost treatment for B PH is castration. In pure cases of B PH, castration results in involution of the prostate gland detectable by rectal palpation within 7-10 days. For most dogs in research studies this is a viable option to rapidly improve the animal's condition. The alternative to castration is hormonal therapy, primarily with estrogens. This may be applicable in cases in which the dog is a valuable breeding male (e.g., genetic diseases), and semen collection is necessary. If the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. Newer drugs marketed for human males have also shown promise in treating canine BPH. Finasteride (Proscar) is a 5a-reductase inhibitor that limits metabolism of testosterone to 5a-dihydrotestosterone. Treatment at daily doses of 1-5 mg/kg has been effective in causing prostatic atrophy without affecting testicular spermatogenesis (Kustritz and Klausner, 2000). Dogs given 1.0 mg/kg were proven to still be fertile. There are also indications that lower doses may be effective in relieving B PH. Androgen receptor antagonists (flutamide and hydroxyflutamide) have also been studied in the dog and found to be effective for treatment of BPH while maintaining libido and fertility (Kustritz and Klausner, 2000). Unfortunately, both the 5areductase inhibitors and the androgen receptor antagonists are not presently labeled for use in male dogs in the United States.

breeds). The lesion consistent with the syndrome is systemic necrotizing vasculitis. The cause of the vasculitis has not been established, but it appears to have an autoimmune-mediated component and may have a hereditary predisposition.

Clinical signs. Clinical signs of JPS include fever, anorexia, lethargy, and reluctance to move the head and neck. The dogs tend to extend the neck ventrally. Most dogs seem to be in pain when touched, especially in the neck region. The syndrome typically has a course of remissions and relapses characterized by 3 - 7 days of illness and 2 - 4 weeks of remission (ScottMoncrieff et al., 1992). There may be a component of this condition that is subclinical, given that a vasculitis has been diagnosed postmortem in beagles that had no presenting signs. Epizootiology and transmission.

JPS typically affects young beagles ( 6 - 4 0 months), with no sex predilection.

Pathologic findings.

On gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (Snyder et al., 1995). Local lymph nodes may be enlarged and hemorrhagic. Histologically, necrotizing vasculitis and perivasculitis of small to medium-sized arteries are seen. These lesions are most noticeable in the three locations where gross lesions are observed, but they may be seen in other visceral locations. The perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries (Fig. 9a). The cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (Snyder et al., 1995). Fibrinous thrombosis of the affected arteries is also seen (Fig. 9b). A subclinical vasculitis has also been diagnosed in beagles postmortem; it is not known whether this subclinical condition is a different disorder or part of a JPS continuum. This subclinical vasculitis often affects the coronary arteries (with or without other sites).

Pathogenesis. Research complications.

BPH can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. It is presently unknown whether the use of the newer antihyperplastic agents systemically alters physiologic parameters outside of the prostate itself. The development of tenesmus as a clinical sign may also affect studies of colorectal or anal function.

3.

Other Miscellaneous Diseases

a.

Juvenile Polyarteritis Syndrome

Etiology.

Juvenile polyarteritis syndrome (JPS) is a painful disorder seen in young beagles (occasionally reported in other

The initiating factors for JPS are unknown. It was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with JPS. Immune mediation of JPS is strongly suspected, because the clinical signs have a cyclical nature and respond to treatment with corticosteroids, and the affected dogs have elevated a2-globulin fractions and abnormal immunologic responses. There may be hereditary predisposition, given that pedigree analysis of some affected dogs has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resuited in 1/7 affected pups (Scott-Moncrieff et al., 1992).

Diagnosis and differential diagnosis.

Differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. In the research facility,

Fig. 9. (a) Epicardium and coronary artery from a research beagle that exhibited shifting leg lameness and nonlocalized pain. The coronary artery is surrounded by rings of inflammatory cells, consistent with juvenile polyarteritis syndrome. Magnification: X 10. (b) Higher magnification of the coronary artery with perivascular inflammation shown in (a). Inflammatory cells have infiltrated the tunica media, and fibrinous thrombosis occludes the arterial lumen. Magnification: X 100.

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11. BIOLOGY AND DISEASES OF DOGS

Fig. 10. Hyperplasiaof the gland of the nictitating membrane ("cherryeye") in a researchbeagle.

the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. Beagles with JPS that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience).

Prevention and control. are known at this time.

No prevention and control measures

brane (third eyelid). This is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. A specific etiology is not known.

Clinical signs. The glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit (Fig. 10). Excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion.

Treatment. Clinical signs can be abated by administration of corticosteroids. Prednisone administered orally at 1.1 mg/kg, q12 hr, was associated with rapid relief of clinical symptoms. Maintenance of treatment at an alternate-day regimen of 0.250.5 mg/kg was shown to relieve symptoms for several months. However, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks.

Pathologic findings. Typically the glandular tissue is hyperplastic, possibly with inflammation. Rarely is the tissue neoplastic.

Research complications. Because of the potentially severe clinical signs and the need for immunosuppressive treatment, JPS is often incompatible with use of the animal as a research subject. It is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses.

Prevention. Hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. Preventative surgical measures might be warranted.

b.

Hyperplasia of the Gland of the Nictitating Membrane

Etiology. "Cherry eye" is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating mem-

Pathogenesis. Prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (Helper, 1989).

Treatment. Corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. However, surgical reduction or excision of the affected gland is typically required to resolve the condition. In the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (Helper, 1989). If reduction is not possible (as

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with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. Such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. The gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane.

Research complications. In most cases, research complications would be minimal, especially if treated adequately. Either the presence of the hyperplastic gland, or its removal, might compromise ophthalmologic studies. c.

Interdigital Cysts

Etiology. Interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes (Fig. 11). The cause for most interdigital cysts is usually not identified unless a foreign body is present. Bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym "sterile pyogranuloma complex"). Clinical signs. Dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. Exudation may be noticed at the site of the lesion. The lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. Epizootiology and transmission. Interdigital cysts are common in a variety of canine breeds, including German shepherds. Beagles have been affected in the research setting. Interdigital cysts usually occur in the third and fourth interdigital spaces (Bellah, 1993). Pathologic findings. Histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. Pathogenesis. Initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. Diagnosis and differential diagnosis. Bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection, and radiopaque foreign bodies or bony lesions, respectively. A biopsy should be taken if neoplasia is suspected. Treatment. If a foreign body is associated with the lesion, then removal is the first order of treatment. If biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at 1 mg/kg ql2h) can be initiated and then tapered once the lesion heals. Interdigital cysts that are refractory to medical therapy require

Fig. 11. Interdigitalcyst between the third and fourth digits of the forelimb of a research beagle. surgical removal. Excision includes removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (Bellah, 1993). The foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weightbearing. The prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (Bellah, 1993).

Research complications. Research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. Treatment with systemic steroids could be contraindicated with some experimental designs.

REFERENCES

Albert, R. E., Benjamin,S. A., and Shukla,R. (1994). Life span and cancer mortality in the beagle dog and human. Mech. Ageing Devel. 74, 149-159. Appel, M. J. G., and Jacobson,R. H. (1995). CVT update: CanineLymedisease. In "Kirk's Current Veterinary Therapy 12: Small Animal Practice" (J. D. Bonagura, ed.), pp. 303-309. Saunders, Philadelphia. Arthur, G. H., Noakes,D. E., and Pearson, H. (1989). "VeterinaryReproduction and Obstetrics (Theriogenology),"6th ed. Bailliere Tindall, London. Baker, D. C., Simpson, M., Gaunt, S. D., and Corstvet, R. E. (1987). Acute Ehrlichia platys infection in the dog. Vet. Pathol. 24(5), 449-453. Barr, S. C. (1998). Enteric protozoal infections. In "Infectious Diseases of the Dog and Cat" (C. E. Greene, ed.), pp. 482-491. Saunders, Philadelphia.

11. BIOLOGY AND DISEASES OF DOGS Bartsch, R. C., and Greene, R. T. (1996). Post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on 68 patients treated primarily with tetracycline and/or doxycycline. J. Vet. Intern. Med. 10, 271. Beierwaltes, W. H., and Nishiyama, R. H. (1968). Dog thyroiditis: occurrence and similarity to Hashimoto's struma. Endocrinology 83, 501-508. Bellah, J. R. (1993). Surgical management of specific skin disorders. In "Textbook of Small Animal Surgery" (D. Slatter, ed.), 2nd ed., pp. 341-354. Saunders, Philadelphia. Bemis, D. A. (1992). Bordetella and mycoplasma infections in dogs and cats. Vet. Clin. North Am. Small Anim. Pract., 22(5), 1173-1186. Benjamin, S. A., Stephens, L. C., Hamilton, B. E, Saunders, W. J., Lee, A. C., Angelton, G. M., and Mallinckrodt, C. H. (1996). Associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles. Vet. Pathol. 33, 486-494. Bergdall, V. K., Eng, V., and Rush, H. G. (1996). Diagnostic exercise: peracute death in a research dog. Lab. Anim. Sci. 46(2), 226-227. Birchard, S. J., and Sherding, R. G. (1994). "Saunders Manual of Small Animal Practice." Saunders, Philadelphia. Bichsel, P., Jacobs, G., and Oliver, J. E. (1988). Neurologic manifestations associated with hypothyroidism in four dogs. J. Am. Vet. Med. Assoc. 192(12), 1745-1417. Bostock, D. E. (1986). Neoplasms of the skin and subcutaneous tissues in dogs and cats. Br. Vet. J. 142, 1-19. Bostock, R. (1977). Neoplasia of the skin and mammary glands of dogs and cats. In "Current Veterinary Therapy 6: Small Animal Practice" (R. W. Kirk, ed.), pp. 493-496. Saunders, Philadelphia. Boudreaux, M. K., and Dillon, A. R. (1991). Platelet function, antithrombin-III activity, and fibrinogen concentration in heartworm-infected and heartworm-negative dogs treated with thiacetarsamide. Am. J. Vet. Res. 52(12), 1986-1991. Brabb, T., Maggio-Price, L., Dale, D., Deeb, B., and Liggitt, D. (1995). Pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis. Lab. Anim. Sci. 45(4), 357-362. Bradfield, J. E, Vore, S. J., and Pryor, W. H. (1996). Ehrlichia platys infection in dogs. Lab. Anim. Sci. 46(5), 565-568. Breitschwerdt, E. B. (2000). The rickettsioses. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 400408. Saunders, Philadelphia. Breitschwerdt, E. B., Woody, B. J., Zerbe, C. A., et al. (1987). Monoclonal gammopathy associated with naturally occurring canine ehrlichiosis. J. Vet. Intern. Med. 1, 2.

Bronson, R, T. (1982). Variation in age at death of dogs of different sexes and breeds. Am. J. Vet. Res. 43, 2057-2059. Brown, C. A., Roberts, W. A., Miller, M. A., et al. (1996). Leptospira interrogans serovar grippotyphosa infection in dogs. J. Am. Vet. Med. Assoc. 209(7), 1265-1267. Brown, J. (1984). Efficacy and dose titration study of mibolerone for treatment of pseudopregnancy in the bitch. J. Am. Vet. Med. Assoc. 184, 1467. Buhles, W. C., Huxsoll, D. L., and Ristic, M. (1974). Tropical canine pancytopenia: clinical, hematologic, and serologic response of dogs to Ehrlichia canis infection, tetracycline therapy, and challenge inoculation. J. Infect. Dis. 130, 357-367. Burnens, A. P., Angeloz-Wick, B., and Nicolet, J. (1992). Comparison of Campylobacter carriage rates in diarrheic and healthy pet animals. Zentralbl. Veterinarmed. 39, 175. Butterwick, R. F., and Hawthorne, A. J. (1998). Advances in dietary management of obesity in dogs and cats. J. Nutr. 128, 2771S-2775S. Butterwick, R. E, and Thorne, C. J. (1994). Effect of level and source of dietary fiber on food intake in the dog. J. Nutr. 124, 2695S-2700S. Butterwick, R. E, and Thorne, C. J. (1997). Effect of amount and type of dietary fiber on food intake in energy-restricted dogs. Am. J. Vet. Res. 58, 272-276. Campbell, K. L. (2000). External parasites: identification and control. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 58-62. Saunders, Philadelphia.

455 Capen, C. C. (1990). Tumors of the endocrine glands. In "Small Animal Clinical Oncology" (S. J. Withrow and E. G. MacEwen, eds.), 2nd ed., pp. 553639. Saunders, Philadelphia. Capen, C. C. (1995). Endocrine system. In "Thomson's Special Veterinary Pathology" (W. W. Carlton and D. M. McGavin, eds.), 2nd ed., pp. 247284. Mosby-Year Book, St. Louis. Carmichael, L. E., and Greene, C. E. (1998). Canine herpesvirus infection. In "Infectious Diseases of the Dog and Cat" (C. E. Greene, ed.), 2nd ed., pp. 28-32. Saunders, Philadelphia. Center, S. A. (1998). Nutritional support for dogs and cats with hepatobiliary disease. J. Nutr. 128, 2733S-2746S. Chang, W. L., and Pan, M. J. (1996). Specific amplification of Ehrlichia platys DNA from blood specimens by two step PCR. J. Clin. Microbiol. 34(12), 3142-3146. Cohen, D. (1972). Detection of humoral antibody to the transmissible venereal tumor of the dog. Int. J. Cancer 10, 207-212. Collins, J. M., Williams, J. E, and Kaiser, L. (1994). Dirofilaria immitis: heartworm products contract rat trachea in vitro. Exp. Parasitol. 78, 76-85. Committee on Dogs, Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council. (1994). Dogs: Laboratory Animal Management. National Academy Press, Washington, D.C. DaRif, C. A., and Rush, H. G. (1983). Management of septicemia in rhesus monkeys with chronic indwelling catheters. Lab. Anim. Sci. 33, 90-94. DeManuelle, T. C. (2000a). Client information series: canine demodicosis. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., p. 1970. Saunders, Philadelphia. DeManuelle, T. C. (2000b). Client information series: fleas and flea allergy dermatitis. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., p. 1971. Saunders, Philadelphia. Demling, R. H., and Lalonde, C. (1989). Management of the burn wound. In "Burn Trauma," pp. 42-65. Thieme Medical Publ., Philadelphia. Dennis, M. B., Jones, D. R., and Tonover, E C. (1989). Chlorine dioxide sterilization of implanted right atrial catheters in rabbits. Lab. Anim. Sci. 39, 5155. DeNovo, R. C., and Magne, M. L. (1995). Current concepts in the management of Helicobacter associated gastritis. In "Proceedings of the 13th Annual ACVIM Forum," pp. 57-61. Lake Buena Vista, Florida. Dillon, R. (2000). Dirofilariasis in dogs and cats. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 937963. Saunders, Philadelphia. Dodman, N. H., Shuster, L., White, S. D., Court, M. H., Parker, D., and Dixon, R. (1988). Use of narcotic antagonists to modify stereotypic self-licking, self-chewing, and scratching behavior in dogs. J. Am. Vet. Med. Assoc. 193(7), 815-819. Dorn, C. R. (1976). Epidemiology of canine and feline tumors. Compend. Contin. Educ. Pract. Vet. 12, 307-312. Dorn, C. R., Taylor, D. O. N., and Hibbard, H. H. (1967). Epizootiologic characteristics of canine and feline leukemia and lymphoma. Am. J. Vet. Res. 29, 993-1001. Dungworth, D. L. (1985). In "Pathology of Domestic Animals" (K. V. E Jubb, P. C. Kennedy, and N. Palmer, eds.) 3rd ed., p. 483. Academic Press, London. Edney, A. T. B., and Smith, P. M. (1986). Study of obesity in dogs visiting veterinary practices in the United Kingdom. Vet. Rec. 118, 391-396. Evans, H. E., and Christensen, G. C. (1993). "Miller's Anatomy of the Dog," 3rd ed. Saunders, Philadelphia. Ferguson, D. C. (1994). Update on diagnosis of canine hypothyroidism. Vet. Clin. North Am. 24(3), 515-539. Fox, J. G. (1995). Helicobacter-associated gastric disease in ferrets, dogs, and cats. In "Kirk's Current Veterinary Therapy 12: Small Animal Practice" (J. D. Bonagura, ed.), pp. 720-723. Saunders, Philadelphia. Fox, J. G., and Lee, A. (1997). The role of Helicobacter species in newly recognized gastrointestinal tract disease of animals. Lab. Anim. Sci. 47(3), 222-255.

456 French, T. W., and Harvey, J. W. (1983). Serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test. Am. J. Vet. Res. 44, 2407-2411. Garnett, N. L., Eydelloth, R. S., Swindle, M. M., et al. (1982). Hemorrhagic streptococcal pneumonia in newly procured research dogs. J. Am. Vet. Med. Assoc. 181, 1371-1374. Garris, G. I. (1991). Control of ticks. Vet. Clin. North Am. Small Anim. Pract. 21(1), 173-183. Gaunt, S. D., Baker, D. C., and Babin, S. S. (1990). Platelet aggregation studies in dogs with acute Ehrlichia platys infection. Am. J. Vet. Res. 51(2), 290293. Gay, W. I. (1984). Health benefits of animal research: the dog as a research subject. Physiologist 27(3), 133-141. Graham, J. C., and O'Keefe, D. A. (1994). Soft tissue sarcomas and mast cell tumors. In "Manual of Small Animal Practice" (S. J. Birchard and R. G. Sherding, eds.), 1st ed., pp. 200-217. Saunders, Philadelphia. Greene, C. E. (2000). Bacterial diseases. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 390-400. Saunders, Philadelphia. Greene, C. E., and Appel, M. J. (1998). Canine distemper. In "Infectious Diseases of the Dog and Cat" (C. E. Greene, ed.), 2nd ed., pp. 9-22. Saunders, Philadelphia. Greene, R. T. (1991). Canine Lyme borreliosis. Vet. Clin. North Am. Small Anita. Pract. 21(1), 51-64. Grier, R. L., Di Guardo, G., Schaffer, C. B., et al. (1990). Mast cell tumor destruction by deionized water. Am. J. Vet. Res. 51, 1116-1120. Grier, R. L., Di Guardo, G., Myers, R., and Merkley, D. E (1995). Mast cell tumour destruction in dogs by hypotonic solution. J. Small Anim. Pract. 36, 385-388. Groves, M. G., Dennis, G. L., Amyx, H. L., et al. (1977). Transmission of Ehrlichia canis to dogs by ticks (Rhipicephalus sanguineus). Am. J. Vet. Res. 36, 937-940. Hall, E. J., and Simpson, K. W. (2000). Diseases of the small intestine. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 1182-1237. Saunders, Philadelphia. Hardy, R. M. (1989). Diseases of the liver and their treatment. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger, ed.), pp. 1479-1527. Saunders, Philadelphia. Harvey, J. W., Simpson, C. E, and Gaskin, J. M. (1978). Cyclic thrombocytopenia induced by a rickettsia-like agent in dogs. J. Infect. Dis. 137, 182-188. Hauptman, J. G., and Chaudry, I. H. (1993). Shock. In "Disease Mechanisms in Small Animal Surgery" (M.J. Bojrab, ed.), pp. 17-20. Lea and Febiger, Philadelphia. Hauptman, J. G., Walshaw, R., and Olivier, N. B. (1997). Evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs. Vet. Surg. 26(5), 393-397. Hawkins, E. C. (2000). Pulmonary parenchymal disease. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 1061-1091. Saunders, Philadelphia. Hayes, H. M., Jr., and Fraumeni, J. E, Jr. (1975). Canine thyroid neoplasms: epidemiologic features. J. Natl. Cancer Inst. 55, 931-934. Helper, L. C. (1989). "Magrane's Canine Ophthalmology," 4th ed. Lea and Febiger, Philadelphia. Hermanns, W., Kregel, K., Breuer, W., et al. (1995). Helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats. J. Comp. Pathol. 112, 307-318. Hoskins, J. D. (1989). Thiacetarsamide and its adverse effects. In "Current Veterinary Therapy 10: Small Animal Practice" (R. W. Kirk, ed.), pp. 131134. Saunders, Philadelphia. Hoskins, J. D. (1998). Canine viral enteritis. In "Infectious Diseases of the Dog and Cat" (C. E. Greene, ed.), 2nd ed., pp. 40-49. Saunders, Philadelphia. Hoskins, J. D. (1999). Pediatrics: puppies and kittens. Vet. Clin. North Am. Small Anim. Pract. 29(4), 837-1027. Hoskins, J. D. (2000a). Canine viral diseases. In "Textbook of Veterinary Inter-

ROBERT C. DYSKO E T AL.

nal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 418423. Saunders, Philadelphia. Hoskins, J. D. (2000b). Congenital defects of the dog. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 1983-1996. Saunders, Philadelphia. Hoskins, J. D., Breitschwerdt, E. B., Gaunt, S. D., et al. (1988). Antibodies to Ehrlichia canis, Ehrlichia platys, and spotted fever group rickettsia in Louisiana dogs. J. Vet. Intern. Med. 2, 55. Howard, E. B., Sawa, T. R., Nielson, S. W., et al. (1969). Mastocytoma and gastroduodenal ulceration. Vet. Pathol. 6, 146-158. Hysell, D. K., and Abrams, G. D. (1967). Complications in the use of indwelling vascular catheters in laboratory animals. Lab. Anim. Care 17, 273-280. Institute of Laboratory Animal Research, Commission of Life Sciences, National Research Council. (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. Jaffe, M. H., Hosgood, G., Kerwin, S. C., Hedlund, C. S., and Taylor, H. W. (2000). Deionised water as an adjunct to surgery for the treatment of canine cutaneous mast cell tumours. J. Small Anim. Pract. 41, 7-11. Jenkins, C. C., and Bassett, J. R. (1997). Helicobacter infection. Compend. Contin. Educ. Pract. Vet. 19, 267-279. Jergens, A. E., and Willard, M. D. (2000). Diseases of the large intestine. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 1238-1256. Saunders, Philadelphia. Johnson, S. E. (2000). Chronic hepatic disorders. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 1298-1325. Saunders, Philadelphia. Johnston, D. E. (1975). Hygroma of the elbow in dogs. J. Am. Vet. Med. Assoc. 167, 213. Johnston, D. E. (1993). Thermal injuries. In "Disease Mechanisms in Small Animal Surgery" (M. J. Bojrab, ed.), 2nd ed., pp. 170-177. Lea and Febiger, Philadelphia. Kaiser, L., Lamb, V. L., Tithof, P. K., Gage, D. A., Chamberlin, B. A., Watson, J. T., and Williams, J. F. (1992). Dirofilaria immitis: Do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta? Exp. Parasitol. 75(2), 159-167. Kaiser, L., Tithof, P. K., and Williams, J. F. (1990). Depression of endotheliumdependent relaxation by filarial parasite products. Am. J. Physiol. 259(2, Part 2), H648-H652. Kalin, M., Devaux, C., Di Fruscia, R., et al. (1999). Three cases of canine leptospirosis in Quebec. Can. Vet. J. 40(3), 187-191. Kemppainen, R. J., and Behrend, E. N. (2000). CVT update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease. In "Kirk's Current Veterinary Therapy 13: Small Animal Practice" (J. D. Bonagura, ed.), pp. 321-324. Saunders, Philadelphia. Kemppainen, R. J., and Clark, T. P. (1994). Etiopathogenesis of canine hypothyroidism. Vet. Clin. North Am. 24(3), 467-476. Kealy, R. D., Lawler, D. F., Ballam, J. M., Lust, G., Smith, G. K., Biery, D. N., and Olsson, S. E. (1997). Five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs. J. Am. Vet. Med. Assoc. 210(2), 222-225. Keil, D. J., and Fenwick, B. (1998). Role of Bordetella bronchiseptica in infectious tracheobronchitis in dogs. J. Am. Vet. Med. Assoc. 212(2), 200-207. Kirby, R. (1995). Septic shock. In "Kirk's Current Veterinary Therapy 12: Small Animal Practice" (J. D. Bonagura, ed.), pp. 139-146. Saunders, Philadelphia. Kleiber, M. 1975. Food as fuel. In "The Fire of Life," pp. 257-258. Robert E. Krieger Publ. Co., Huntington, New York. Knight, D. H. (2000). CVT update: heartworm testing and prevention in dogs. In "Kirk's Current Veterinary Therapy 13: Small Animal Practice" (J. D. Bonagura, ed.), pp. 777-782. Saunders, Philadelphia. Kordick, S. K., Breitschwerdt, E. B., Hegarty, B. C., et al. (1999). Coinfection with multiple tick-borne pathogens in a Walker hound kennel in North Carolina. J. Clin. Microbiol. 37(8), 2631-2638. I

11. BIOLOGY AND DISEASES OF DOGS Kornegay, J. N., Sharp, N. J. H., Scheuler, R. O., and Betts, C. W. (1994). Tarsal joint contracture in dogs with golden retriever muscular dystrophy. Lab. Anim. Sci. 44(4), 331-333. Kuehn, N. E, and Gaunt, S. D. (1985). Clinical and hematological findings in canine ehrlichiosis. J. Am. Vet. Med. Assoc. 186, 355. Kustritz, M. V. (1999). Early spay-neuter in the dog and cat. Vet. Clin. North Am. Small Anim. Pract. 29, 935-943. Kustritz, M. V. R., and Klausner, J. S. (2000). Prostatic diseases. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 1687-1698. Saunders, Philadelphia. Kwei, G. Y., Gehret, J. R., Novak, L. B., Drag, M. D., and Goodwin, T. (1995). Chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs. Lab. Anim. Sci. 45, 683-685. Laflamme, D. P., Kuhlman G., and Lawler, D. E (1997). Evaluation of weight loss protocols for dogs. J. Am. Anim. Hosp. Assoc. 33(3), 253-259. Lewis, G. E., Ristic, M., Smith, R. D., et al. (1977). The brown dog tick Rhipicephalus sanguineus and the dog as experimental hosts of Ehrlicha canis. Am. J. Vet. Res. 38, 1953 - 1955. Lewis, L. D., Morris, M. L., and Hand, M. S. (1987). "Small Animal Clinical Nutrition," Vol. 3. Mark Morris Associates, Topeka, Kansas. Loeb, W., and Quimby, F. (1999). "The Clinical Chemistry of Laboratory Animals," 2nd ed. Taylor and Francis, Philadelphia. Logas, D., and Kunkle, G. A. (1994). Double-blinded crossover study with marine-oil supplementation containing high-dose eicosapentaenoic acid for the treatment of canine pruritic skin disease. Vet. Dermatol. 5(3), 99. Lopez, A. (1995). Respiratory system. In "Thomson's Special Veterinary Pathology" (W. W. Carlton and D. M. McGavin, eds.), 2nd ed., pp. 116174. Mosby-Year Book, St. Louis. Lozier, S., Pope, E., and Berg, J. (1992). Effects of four preparations of .05% chlorhexidine diacetate on wound healing in dogs. Vet. Surg. 21(2), 107112. MacEwen, E. G. (1991). Transmissible venereal tumors. In "Small Animal Clinical Oncology" (S. J. Withrow and E. G. MacEwen, eds.), 2nd ed., pp. 533-538. Saunders, Philadelphia. MacEwen, E. G. (1992). Obesity. In "Kirk's Current Veterinary Therapy 11: Small Animal Practice" (R. W. Kirk and J. D. Bonagura, eds.), pp. 313318. Saunders, Philadelphia. MacEwen, E. G., and Withrow, S. J. (1991a). Soft tissue sarcomas. In "Small Animal Clinical Oncology" (S. J. Withrow and E. G. MacEwen, eds.), 2nd ed., pp. 211-226. Saunders, Philadelphia. MacEwen, E. G., and Withrow, S. J. (1991b). Tumors of the mammary gland. In "Small Animal Clinical Oncology" (S. J. Withrow and E. G. MacEwen, eds.), 2nd ed., pp. 356-372. Saunders, Philadelphia. MacEwen, E. G., and Young, K. M. (1991). Canine lymphoma and lymphoid leukemias. In "Small Animal Clinical Oncology" (S. J. Withrow and E. G. MacEwen, eds.), 2nd ed., pp. 451-479. Saunders, Philadelphia. Maddison, J. E. (1995). Medical management of chronic hepatic encephalopathy. In "Kirk's Current Veterinary Therapy 12: Small Animal Practice" (J. D. Bonagura, ed.), pp. 1153-1158. Saunders, Philadelphia. Maksimowich, D. S., Mupanomunda, M., Williams, J. E, and Kaiser, L. (1997). Effect of heartworm infection on in vitro contractile responses of canine pulmonary artery and vein. Am. J. Vet. Res. 58(4), 395-397. Malik, R., and Farrow, B. R. H. (1991). Tick paralysis in North America and Australia. Vet. Clin. North Am. Small Anim. Pract. 21(1), 157-171. Manley, P. (1994). Lyme disease. In "Saunders Manual of Small Animal Practice" (S. J. Birchard and R. G. Sherding, eds.), 1st ed., pp. 1108-1109. Saunders, Philadelphia. Manning, P. J. (1979). Thyroid gland and arterial lesions of beagles with familial hypothyroidism and hypedipoproteinemia. Am. J. Vet. Res. 40(6), 820828. Marks, S. L. (1997). Bacterial gastroenteritis in dogs and cats: more common than you think. In "Proceedings of the 15th Annual ACVIM Forum," May 22-25, pp. 237-239. Lake Buena Vista, Florida. Marshall, B. J., Barrett, L. J., Prakash, C., et al. (1990). Urea protects Heli-

457 cobacter (Campylobacter) pylori from the bactericidal effect of acid. Gastroenterology 99, 697-702. Mathew, J. S., Ewing, S. A., Murphy, G. L., et al. (1997). Characterization of a new isolate of Ehrlichia platys using electron microscopy and polymerase chain reaction. Vet. Parasitol. 68(1-2), 1-10.

Matsukura, Y., Washizu, M., Kondo, M., Motoyishi, S., Itoh, A., Nakajyo, S., Shimizu, K., and Urakawa, N. (1997). Decreased pulmonary arterial endothelium-dependent relaxation in heartworm-infected dogs with pulmonary hypertension. Am. J. Vet. Res. 58(2), 171-174. McCandlish, I. A. P., Thompson, H., and Wright, N. G. (1978). Vaccination against canine bordetellosis: protection from contact challenge. Vet. Rec. 104, 51- 54. Merchant, S. R., and Toboada, J. (1991). Dermatologic aspects of tick bites and tick-transmitted diseases. Vet. Clin. North Am. Small Anim. Pract. 21(1), 145-155. Meunier, L. D., Kissinger, J. T., Marcello, J., Nichols, A. J., and Smith, P. L. (1993). A chronic access port model for direct delivery of drugs into the intestine of conscious dogs. Lab. Anim. Sci. 43, 466-470. Miller, W. (1989). Clinical trial of DVM Derm Caps in the treatment of allergic diseases in dogs: a nonblinded study. J. Am. Anim. Hosp. Assoc. 25, 163. Morrison, W. B., Hamilton, T. A., Hahn, K. A., Richardson, R. C., and Janas, W. (1993). Diagnosis of neoplasia. In "Textbook of Small Animal Surgery" (D. Slatter, ed.), 2nd ed., pp. 2036-2048. Saunders, Philadelphia. Moulton, J. E. (1990). Tumors of the mammary gland. In "Tumors in Domestic Animals" (J. E. Moulton, ed.), 3rd ed., pp. 518-552. Saunders, Philadelphia. Muller, G. H., Kirk, R. W., and Scott, D. W. (1983). "Small Animal Dermatology," 3rd ed. Saunders, Philadelphia. Mupanomunda, M., Williams, J. E, Mackenzie, C. D., and Kaiser, L. (1997). Dirofilaria immitis: Heartworm infection alters pulmonary artery endothelial cell behavior. J. Appl. Physiol. 82(2), 389-398. Murphy, J., Lavach, J., and Severin, G. (1978). Survey of conjunctival flora in dogs with clinical signs of external eye disease. J. Am. Vet. Med. Assoc. 172, 66-68. Nader-Djahal, N., Knight, P. R., Davidson, B. A., and Johnson, K. (1997). Hyperoxia exacerbates microvascular injury following acid aspiration. Chest 112, 1607-1614. National Research Council. (1985). "Nutrient Requirements of Dogs." National Academy Press, Washington, D.C. Newton, C. D., Wilson, G. P., Allen, H. L., and Swenberg, J. A. (1974). Surgical closure of elbow hygroma in the dog. J. Am. Vet. Med. Assoc. 164(2), 147-149. Nielsen, S. W., and Kennedy, P. C. (1990). Tumors of the genital system. In "Tumors in Domestic Animals" (J. E. Moulton, ed.), 3rd ed., pp. 479-517. Saunders, Philadelphia. Noli, C. (2000). Practical laboratory methods for the diagnosis of dermatologic diseases. In "Kirk's Current Veterinary Therapy 13: Small Animal Practice" (J. D. Bonagura, ed.), pp. 526-536. Saunders, Philadelphia. Nowak, R. M. (1999). "Walker's Mammals of the World," 6th ed., pp. 669-672. Johns Hopkins Univ. Press, Baltimore. Ogilvie, G. K. (1991). Tumors of the endocrine system. In "Small Animal Clinical Oncology" (S. J. Withrow and E. G. MacEwen, eds.), 2nd ed., pp. 316-346. Saunders, Philadelphia. Olson, P. N., Hilgren, J. D., and Brooke, R. J. (1973). Beta hemolytic streptococcus isolated from the canine vagina. J. Am. Vet. Med. Assoc. 163, 200. Osuna, D. J., DeYoung, D. J., and Walker, R. L. (1990a). Comparison of three skin preparation techniques in the dog. Part 1: Experimental trial. Vet. Surg. 19(1), 14-19. Osuna, D. J., DeYoung, D. J., and Walker, R. L. (1900b). Comparison of three skin preparation techniques in the dog. Part 2: Clinical trial in 100 dogs. Vet. Surg. 19(1), 20-23. Overall, K. L. (1997). "Clinical Behavioral Medicine for Small Animals." Mosby-Year Book, St. Louis. Panciera, D. L. (1994). Hypothyroidism in dogs: 66 cases (1987-1992). J. Am. Vet. Med. Assoc. 204(5), 761-767.

458 Panciera, D. L., and Johnson, G. S. (1994). Plasma von Willebrand factor antigen concentration in dogs with hypothyroidism. J. Am. Vet. Med. Assoc. 206(5), 594-596. Panciera, D. L., and Johnson, G. S. (1996). Plasma von Willebrand factor antigen concentration and bleeding time in dogs with experimental hypothyroidism. J. Vet. Intern. Med. 10(2), 60-64. Patnaik, A. K., Ehler, W. J., and MacEwen, E. G. (1986). Canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs. Vet. Pathol. 21, 469-474. Pedersen, N. C., Morgan, J. P., and Vasseur, P. B. (2000). Joint diseases of dogs and cats. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 1862-1886. Saunders, Philadelphia. Pershing, D. H., Rutledge, B. J., and Rys, P. N. (1994). Target imbalance: disparity of Borrelia burgdorferi genetic material in synovial fluid from Lyme arthritis patients. J. Infect. Dis. 25, 441. Peterson, M. E., and Ferguson, D. C. (1989). Thyroid diseases. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger, ed.), 3rd ed., pp. 1632-1675. Saunders, Philadelphia. Pulley, L. T., and Stannard, A. A. (1990). Tumors of the skin and soft tissue. In "Tumors in Domestic Animals" (J. E. Moulton, ed.), 3rd ed., pp. 23-87. Saunders, Philadelphia. Render, J. A., and Carlton, W. W. (1995). Pathology of the eye and ear. In "Thomson's Special Veterinary Pathology" (W. W. Carlton and D. M. McGavin, eds.), pp. 561-605. Mosby-Year Book, St. Louis. Rentko, V. T., Clark, N., Ross, L. A., and Schelling, S. H. (1992). Canine leptospirosis: a retrospective study of 17 cases. J. Vet. Intern. Med. 6(4), 235244. Ringler, D. H., and Peter, G. K. (1984). Dogs and cats as laboratory animals. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 241-271. Academic Press, Orlando, Florida. Sanchez, I. R., Swaim, S. F., Nusbaum, K. E., Hale, A. S., Henderson, R. A., and McGuire, J. A. (1988). Effects of chlorhexidine diacetate and povidoneiodine on wound healing in dogs. Vet. Surg. 17(6), 291-295. Scarlett, J. (1994). Epidemiology of thyroid diseases of dogs and cats. Vet. Clin. North Am. 24(3), 477-486. Schneider, R., Dorn, C. R., and Taylor, D. O. N. (1969). Factors influencing canine mammary cancer development and postsurgical survival. J. Natl. Cancer Inst. 43, 1249-1261. Scott, D. W., Miller, W. H., Jr., and Griffin, C. E. (1995). "Muller and Kirk's Small Animal Dermatology," 5th ed. Saunders, Philadelphia. Scott-Moncrieff, J. C. R., Snyder, P. W., Glickman, L. T., Davis, E. L., and Felsburg, P. J. (1992). Systemic necrotizing vasculitis in nine young beagles. J. Am. Vet. Med. Assoc. 201(10), 1553-1558. Searcy, G. P. (1995). Hemopoietic system. In "Thomson's Special Veterinary Pathology" (W. W. Carlton and D. M. McGavin, eds.), 2nd ed., pp. 285331. Mosby-Year Book, St. Louis. Sherding, R. G. (1989). Diseases of the small bowel. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger, ed.), 3rd ed., pp. 1344-1350. Saunders, Philadelphia. Sherding, R. G. (1994). Canine infectious tracheobronchitis (kennel cough complex). In "Saunders Manual of Small Animal Practice" (S. J. Birchard and R. G. Sherding, ed.), 1st ed., p. 104. Saunders, Philadelphia. Sherding, R. G., and Johnson, S. E. (1994). Diseases of the intestines. In "Saunders Manual of Small Animal Practice" (S. J. Birchard and R. G. Sherding, ed.), 1st ed., p. 702. Saunders, Philadelphia. Shimon, H., Trevor, W., Yaakov, A., et al. (1996). Serum protein alterations in canine erhlichiosis. Vet. Parasitol. 66(3-4), 241-249. Shimoyama, T., and Crabtree, J. E. (1998). Bacterial factors and immune pathogenesis in Helicobacter pylori. Gut 43(Suppl. 1), $2-$5. Simpson, R. M., Gaunt, S. D., Hair, J. A., et al. (1991). Evaluation of Rhipicephalus sanguineus as a potential biologic vector of Ehrlichia platys. Am. J. Vet. Res. 52(9), 1537-1541. Slajchert, T., Kitron, U. D., Jones, C. J., and Mannelli, A. (1997). Role of the eastern chipmunk (Tamias striatus) in the epizootiology of Lyme borreliosis in northwestern Illinois, U.S.A.J. Wildl. Dis. 33(1), 40-46. Smith, G. K., Popovitch, C. A., Gregor, T. P., and Shofer, E S. (1995). Evalua-

ROBERT C. DYSKO E T AL.

tion of risk factors for degenerative joint disease associated with hip dysplasia in dogs. J. Am. Vet. Med. Assoc. 206(5), 642-647. Snyder, P. W., Kazacos, E. A., Scott-Moncrieff, J. C., HogenEsch, H., Carlton, W. W., Glickman, L. T., and Felsburg, P. J. (1995). Pathologic features of naturally occurring juvenile polyarteritis in beagle dogs. Vet. Pathol. 32, 337-345. Stannard, A. A., Cannon, A. G., and Olivry, T. (2000). Scaling and crusting dermatoses. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 47-51. Saunders, Philadelphia. Straubinger, R. K., Staubinger, A. E, Summers, B. A., et al. (1998). Clinical manifestations, pathogenesis, and effect of antibiotic treatment on Lyme borreliosis in dogs. Wien. Klin. Wochenschr. 110(24), 874-881. Sundberg, J. P., Hill, D., Wyand, D. S., et al. (1981). Streptococcus zooepidemicus as the cause of septicemia in racing greyhounds. Vet. Med. Small Anim. Clin. (June), 839-842. Swaim, S. F. (1980). Trauma to the skin and subcutaneous tissues of dogs and cats. Vet. Clin. North Am. Small Anim. Pract. 10(3), 599-618. Swaim, S. E, and Angarano, D. W. (1990). Chronic problem wounds of dog limbs. Clin. Dermatol. 8(3-4), 175-186. Swalec, K. M. (1993). Portosystemic shunts. In "Disease Mechanisms in Small Animal Surgery" (M. J. Bojrab, D. D. Smeak, and M. S. Bloomberg, eds.), pp. 298-305. Lea and Febiger, Philadelphia. Taboada, J. (2000). Systemic mycoses. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), 5th ed., pp. 453-476. Saunders, Philadelphia. Tarvin, G., and Prata, R. G. (1980). Lumbosacral stenosis in dogs. J. Am. Vet. Med. Assoc. 177, 154-159. Thompson, H., McCandlish, I. A. P., and Wright, N. G. (1976). Experimental respiratory disease in dogs due to Bordetella bronchiseptica. Res. Vet. Sci. 20, 16-23. Tithof, P. K., Schwartz, A. J., Mupanomunda, M., Schwartz, N. R., Lamb, V. L., Williams, J. E, and Kaiser, L. (1994). Dirofilaria immitis: Depression of endothelium-dependent relaxation of canine femoral artery seen in vivo does not persist in vitro. Exp. Parasitol. 79(2), 159-165. Tucker, W. E. (1962). Thyroiditis in a group of laboratory dogs: a study of 167 beagles. Am. J. Clin. Pathol. 38(1), 70-74. U.S. Dept. of Agriculture, Animal and Plant Health Inspection Service. (1998). "Animal Welfare Report: Fiscal Year 1997," Table 6, p. 36. Van Kruiningen, H. J. (1995). Gastroinstestinal system. In "Thomson's Special Veterinary Pathology" (W. W. Carlton and D. M. McGavin, eds.), 2nd ed., pp. 1-80. Mosby-Year Book, St. Louis. Waddle, J. R., and Littman, M. P. (1988). A retrospective study of 27 cases of naturally occurring canine ehrlichiosis. J. Am. Anim. Hosp. Assoc. 24, 615620. Wagener, J. S., Sobonya, R., Minnich, L., et al. (1994). Role of canine parainfluenza virus and Bortedella bronchiseptica in kennel cough. Am. J. Vet Res. 45, 1862-1866. Waldron, D. R., and Trevor, P. (1993). Management of superficial skin wounds. In "Textbook of Small Animal Surgery" (D. Slatter, ed.), 2nd ed., pp. 269280. Saunders, Philadelphia. Walton, D. K. (1986). Psychodermatosis. In "Current Veterinary Therapy 9: Small Animal Practice" (R. W. Kirk, ed.), pp. 557-559. Saunders, Philadelphia. Wheeler, S. L., Husted, P. W., Roscychuk, R. A. W., Allen, T. A., Nett, T. M., and Olson, P. N. (1985). Serum concentrations of thyroxine and 3,5,3'-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs. Am. J. Vet. Res. 26(12), 2605-2608. Wilkinson, M. J. A., and McEwan, N. A. (1991). Use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs. J. Nutr. 121, $47-$50. Wilson, J. (1992). Ehrlichia platys in a Michigan dog. J. Am. Anim. Hosp. Assoc. 28, 381. Woody, B. J., and Hoskins, J. D. (2000). Ehrlichial diseases of dogs. Vet. Clin. North Am. Small Anim. Pract. 21(1), 75-98. Woody, B. J., and McDonald, R. K. (1985). Canine ehrlichiosis. Miss. Vet. J. 1, 2-5.

Chapter 12 Domestic Cats as Laboratory Animals Brenda Griffin and Henry J. Baker

I.

II.

III.

IV.

V.

VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

460

A.

Unique Contributions of Cats to Biomedical Research . . . . . . . . . . . .

460

B.

Feline Genomics and Inherited Feline Diseases as Models

C.

of H u m a n Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Infectious Disease Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sources of Cats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

462

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Directories of Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

462

B.

Random Sources

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C.

Commercial Purpose-Bred Colonies . . . . . . . . . . . . . . . . . . . . . . . . . .

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D.

Institutional Breeding Colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

462

Housing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A. B. C.

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Caging Design and Operating Procedures . . . . . . . . . . . . . . . . . . . . . . Animal Care Staff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Feline Social Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D.

Housing to Exclude Pathogens

E.

Environmental Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..............................

466 466

Breeding Colony Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

466

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Estrous Cycle and Mating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

466

B.

Pregnancy and Parturition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

469

C. Infertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Neonatal Care and Weaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nutrition and F e e d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

470 471 474

A. Commercial Diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Energy Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Feline Lower Urinary Tract Disease . . . . . . . . . . . . . . . ........... Infectious Disease Exclusion and Control . . . . . . . . . . . . . . . . . . . . . . . . . A. Preventive Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Pathogen Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Eliminating Parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

474 474 475 475 475 476 478

D.

478

Personnel Health Risks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

LABORATORYANIMALMEDICINE,2nd edition

460 460

480

Copyright 2002, Elsevier Science (USA).All rights reserved. ISBN 0-12-263951-0

460

BRENDA GRIFFIN AND HENRY J. BAKER

I.

A.

INTRODUCTION

U n i q u e Contributions of Cats to Biomedical Research

Of the 1,267,828 nonrodent animals used in research in the United States in 1997, only 26,091, or 2%, of these were cats. Furthermore, use of cats in research in this country has declined dramatically during the past two decades from a high of 74,000 in 1974, amounting to a 65% reduction (Animal Care division of APHIS, USDA). If the importance of cats as a research species was judged entirely on these numbers, one might conclude incorrectly that cats do not contribute significantly to biomedical research. In fact, cats contribute uniquely to science, and their special biological characteristics and diseases rank them as the favored species for several disciplines, including experimental neurology, some aspects of ophthalmology, retrovirus research, inherited diseases, and immunodeficiency diseases.

B. Feline G e n o m i c s and Inherited Feline Diseases as M o d e l s of H u m a n Diseases

The domestic cat (Felis cattus) is one of only a few mammals other than humans and mice for which extensive information has been generated on its genome. This unusual emphasis on feline genomics is driven partially by the substantial number of naturally occurring inherited diseases that are useful as models of their human counterpart and partially by the interest of the National Cancer Institute's Laboratory of Genomic Diversity in host factors that determine susceptibility to feline leukemia virus and feline immunodeficiency virus. Scientists of this laboratory have made major contributions to characterization of the feline genome. At their website, , they provide primer sequence and other information on 711 loci in the cat genome, many of which are homologous to human anchor loci or coding gene sequences. They also provide a genetic map for the cat with comparisons with syntenic chromosomal locations in humans, which shows an extraordinarily homology between gene locations of these diverse species. For example, of the 9 loci that map to autosome D 1 of the cat, all are located on human chromosome 1 lp. With the development of powerful tools of molecular biology that permit careful dissection of genomes, there has been a explosive interest in human inherited diseases and their animal counterparts. There was an unrealistic expectation that knockout transgenic mice would provide all models needed to study specific gene mutations. The reality is that mice with induced malfunction of many genes have no apparent disease, have lethal disease, or have clinical diseases that do not mimic the corresponding human disease. This is in sharp contrast to many inherited diseases of cats, which are virtually identical to the

analogous human disease with respect to clinical presentation, patterns of inheritance, histopathology, and biochemistry (see Table I). These models continue to be exceedingly important in research on pathogenic mechanisms and potential therapeutic modalities.

C.

Infectious Disease M o d e l s

Several naturally occurring infections of cats have been used experimentally for research on analogous human diseases. The three that are highlighted here were selected because of recent discoveries that have led to development of these models and because of the importance of the human disease for which these unique cat diseases provide excellent experimental models. 1.

Feline Leukemia Virus Disease as a Model of Viral Oncogenesis

Domestic cats have the highest incidence of naturally occurring lymphoid malignancies of any nonrodent mammal. Feline leukemia virus (FeLV) is an oncornavirus that causes lymphosarcoma, leukemia, and aplastic anemia in cats and is similar to leukemia viruses of mice (murine leukemia virus) and chickens (avian leukemia virus). The feline disease is considered to be an important model for several characteristics of retrovirally induced disease, particularly hematopoietic tumors such as acute lymphoblastic leukemia and lymphoma. Paradoxically, the virus also causes immunodeficiency and myelosuppression. After infection, cats are persistently viremic and virus is excreted, particularly through saliva and nasal secretions. A regressive, nonviremic form is also recognized. Serological tests are based on detection of the major viral core protein of FeLV (p27 gag) in serum or plasma by enzyme-linked immunosorbent assay (ELISA). Strengths of this model include substantial information on FeLV, pathogenesis of the disease, responses of the immune system, availability of FeLV strains of known virulence, and the ease of inducing infection and disease in cats (Hoover and Mullins, 1991). 2.

Feline Immunodeficiency Virus Disease as a Model of Human AIDS

Valid animal models of human acquired immunodeficiency syndrome (AIDS) are essential for research on pathogenesis, therapy, and vaccine development. Immunodeficiency disease of cats caused by the lentivirus feline immunodeficiency virus (FIV) is considered by many to be one of the most relevant naturally occurring models of AIDS (Gardner, 1989). The advantages of the feline disease model include the similarities with HIV (the human lentivirus), similarities in pathogenesis and clinical signs, ease of experimental infection, and predictable disease progression. The weakness of the model relates to the

461

12. DOMESTIC CATS AS LABORATORY ANIMALS Table I

Feline Inherited Diseases a Disease b

Protein affected

Gene affected

Mutation

Reference(s)

Amyloidosis Chediak-Higashi syndrome Chylomicronemia Ehlers-Danlos syndrome, type II Endocardial fibroelastosis Barth's syndrome Feline spongiform encephalopathy Globoid cell leukodystrophy, or Krabbe's disease Glycogenosis II Glycogenosis IV GM1 gangliosidosis

AA amyloid Nidogen? Lipoprotein lipase Procollagen peptidase Nk

Nk Nk LPL PLOD Nk

Nk Nk A412G exon 8 Nk Nk

Gruys et al. (1998), Boyce (1984) Kramer et al. (1977) Jones et al. (1998) Patterson and Minor (1977) Paasch and Zook (1980)

Prion protein Galactocerebroside 13-galactosidase et-1,4-Glucosidase Glycogen branching enzyme 13-Galactosidase

Prn

Nk Nk

Prusiner (1995) Johnson (1970)

GM2 gangliosidosis

Hexosaminidase B

HEXB

Sandstrom et al. (1969) Fyfe and Kurzhals (1998) Baker et al. (1971), Baker et al. (1998) Muldoon et al. (1994), Martin et al. (1999)

Gyrate atrophy of choroid and retina Hageman trait bleeding disorder Hemophilia A Hemophilia B Hurler syndrome, or MPS I Hypertrophic cardiomyopathy Klinefelter's syndrome et-Mannosidosis Maroteaux-Lamy syndrome, or MPS VI

Ornithine ~-aminotransferase Factor XII Factor VIII Factor IX et-L-Iduronidase Nk X chromosome chimerism et-Mannosidase N-Acetylgalactosamine-4sulfatase, or arylsulfatase B

OAT

Nk 172-bp deletion 1-base sub CGTACCT, 1486 fHEXKorat, 1-bp del fHEXBaker, 25-bp inversion Nk Nk Nk Nk Nk Nk Nk 1748 del 4 IA76P D520N

Methemoglobinemia MPS II, or I-cell disease Muscular dystrophy Neuroaxonal dystrophy Neuronal ceriod lipofuscinosis Sphingomyelin lipidosis, or Niemann-Pick disease, type C Pyruvate kinase deficiency Polycystic kidney disease Porphyria Progressive retinal atrophy Retinal degeneration Waardenburg's syndrome

NADH-methemoglobin reductase Phosphotransferase Dystrophin Nk Nk Sphingomyelinase

Nk Nk DMD Nk CLN-1 NP-C

Nk Nk Nk Nk Nk Nk

Erythrocytic R-type pyruvate kinase Nk Porphyrin Nk Nk Homeobox?

R-PK PKD1 Nk Nk Nk PAX3?

13 del exon 6 FCA476 linkage Nk Nk Nk Nk

Kier et al. (1980) Cotter et al. (1978) Maggo-Price and Dodds (1993) Haskins et al. (1979) Kittleson et al. (1998) Jones (1969) Berg et al. (1997) Jezyk et al. (1977), Hopwood et al. (1998), DiNatale et al. (1992) Giger et al. (1998) Haskins et al. (1998) Gaschen et al. (1998) Woodard et al. (1974) Green and Little (1974) Baker et al. (1987), Lowenthal et al. (1990) Giger et al. (1998) DiBartola et al. (1998) Glenn et al. (1968) Narfstrom et al. (1998) Bellhorn (1973) Bergsma and Brown (1971)

Nk GAA GBE GLB1

Nk Nk Nk IDUA Nk

Nk MANB Nk

Valle et al. (1981)

aFor a current summary description of the relevant human diseases, see Scriver et al. (1995). bMPS, mucopolysaccharidosis; Nk, not known.

limited variety of reagents available for identifying cells of the cat i m m u n e system. FIV has been molecularly cloned and resembles HIV in tissue and cell tropism but is antigenically distinct. Experimental transmission is achieved readily with infected blood or cultured cells. Cell-associated viremia occurs within 1 - 2 weeks and remains persistent, even after development of antibodies. Characteristic changes in the i m m u n e system include lymphadenopathy, neutropenia, decreased lymphocyte proliferative response, and increased susceptibility to

opportunistic infections. B-cell l y m p h o m a s and myeloproliferative disease are seen in some infected cats.

3. Helicobacterfelis Infection as a Model of H u m a n Helicobacter Diseases Helicobacterpylori is the etiologic agent responsible for a sequence of degenerative changes in the h u m a n gastric mucosa, starting with gastritis, progressing to peptic ulcers, and ending

BRENDA GRIFFIN AND HENRY J. BAKER

462

in gastric carcinoma. Animal models are critically important for research on this prevalent and important human disease. Of several Helicobacter species infecting animals, H. felis is one of the most interesting and useful because of its wide host range, its ability to induce many, if not all, of the lesions found in human Helicobacter disease, and its adaptability to experimental induction in mice and cats. Helicobacterfelis is a naturally occurring pathogen in cats that appears to be prevalent in some colonies, but its prevalence or significance as an agent of clinical diseases in the general cat population is not clear (Lee et al., 1988; Perkins et al., 1996). Fox et al. (1993) have studied this organism and the disease that it induces in mice and cats. It is clear from their work that H. felis contributes importantly to Helicobacter research as experimental infections of both mice and cats. In fact, these investigators have demonstrated that H. felis infection can faithfully reproduce all of the lesions found in the human disease (except ulcers), particularly those associated with the chronic infection (Enno et al., 1995; Wang et al., 2000). In addition to H. felis infection, cats appear also to be naturally infected with H. pylori, raising the possibility that domestic cats could serve as a reservoir for this human pathogen (Perkins et al., 1996).

II.

SOURCES OF CATS

A.

Directories of Sources

cause they frequently incubate or are actively infected with a variety of pathogens that are lethal or cause extended morbidity and that may be zoonotic. Intensive quarantine and conditioning procedures may or may not minimize these problems or be economical. Addition of cats of this type into facilities with stable colonies of cats introduces an unacceptable risk because, even after long periods of quarantine, inapparent or latent diseases such as feline leukemia, feline immunodeficiency disease, and feline infectious peritonitis may be transmitted to healthy cats. When the risks of morbidity and mortality from infectious diseases (including zoonoses), unknown reproductive status, and variable tractability of random-source cats are compared with those of cats derived from breeding colonies, use of purposebred cats clearly becomes a wise investment. An additional important consideration is public objection to use of cats in research after being surrendered to shelters. The sum of these important issues argues strongly against the use of randomsource cats. One exception is the discovery of an interesting inherited disease or other diseases in cats that come from less than optimal environments and must be maintained in the laboratory but that may present some risks due to unknown or poor health history. In such cases it is important to impose a prolonged (8 to 12 week) isolation and observation period and to adopt intensive procedures to identify diseases, eliminate parasites, and vaccinate in order to prevent pathogen transmission (see Table II).

C.

Directories of sources for purchase of cats for use in biomedical research are available from the following organizations: (1) The Institute for Laboratory Animal Research is a unit of the National Research Council of the National Academy of Sciences and an excellent source of information about laboratory animal species. Its website provides an International Index of Laboratory Animals, which gives the location and status of 21 sources of laboratory cats worldwide. (2) The American Association for Laboratory Animal Science also maintains a website with a Reference Directory that provides details about commercial vendors of cats and an online search for papers published in Laboratory Animal Science that may pertain to use of cats in research. (3) Animal Care, the U.S. Department of Agriculture division that administers the Animal Welfare Act (AWA), maintains a website that provides access to a variety of documents relating to the AWA, including a listing of licensed animal dealers.

B.

Random Sources

Random-source cats derived from animal control agencies and dealers usually are not satisfactory research subjects be-

Commercial Purpose-Bred Colonies

Several commercial sources provide minimal-disease purpose-bred cats. Some of these colonies originated from cesarean-derived stock and have been maintained using strict barrier procedures to exclude pathogens (Festing and B leby, 1970). In addition to assurance of good health and immunizations, vendor selection should be based on cats that are well socialized. Referrals from previous customers of these vendors will provide an indication of the health and behavioral characteristics of cats from a particular source. Vendors should be able to provide reports of health examinations and vaccine protocols. Serological test results are helpful to indicate exclusion of some pathogens, but these results may not be entirely reliable. Except for the higher cost involved, purpose-bred cats are preferred over cats from random sources.

D.

Institutional Breeding Colonies

Projects that require a regular source of substantial numbers of normal cats or that depend on special characteristics such as perpetuation of an inherited trait can be satisfied best by establishment of an institutional breeding colony. Although this should not be undertaken lightly, it is within the capability of most organizations, as long as prescribed procedures are fol-

463

12. DOMESTIC CATS AS LABORATORY ANIMALS

Table II Basic Principles of Feline Infectious Disease Control 1. Establish the colony with disease-free stock and close the colony to any additions that do not meet or exceed the health status of the original stock 2. Regardless of the presumed health status of new additions, they should be subjected to the following before entering a closed colony: a. Isolation for at least 4 weeks, and longer if disease problems are suspected b. Thorough physical examination, including laboratory testing for FeLV, FIV, endoparasites, and ectoparasites. Other health screening such as hematology and clinical chemistry tests can be informative. Serologies should be repeated before termination of the quarantine c. Continued surveillance for clinical signs of infectious diseases that may be incubating at time of arrival d. Administration of vaccines, vermifuges, and ectoparasiticides as indicated. Repeat parasite control measures as recommended by the manufacturer 3. Vaccinate kittens at 8, 12, and 15 weeks of age against feline viral rhinotracheitis, calicivirus, and panleukopenia (FVRCP). Perform physical examinations of kittens at this time. Perform annual formal physical examinations, and administer FVRCP booster vaccines to adults once every three years. Breeding queens should be immunized after delivering kittens or before rebreeding to avoid infection of kittens in utero with modified live virus vaccines. Only killed panleukopenia vaccines should be used, because they confer highly protective immunization and live products may result in cerebellar hypoplasia if inadvertently given to pregnant queens 4. Conduct annual hematology, biochemical profile, and urinalysis on cats over 5 years of age. Kidney function is of particular concern, especially for breeding males 5. Repeat random serological screening for FeLV and FIV of at least 10% of the population annually 6. Instruct personnel to perform and report daily observation of all cats for changes in appetite, behavior, activity, or body condition. Changes require professional examinations 7. Immediately isolate any suspected sick cat, and conduct intensive diagnostic procedures, including necropsy examination if indicated

lowed. Careful analysis of cost and complexity should be undertaken to determine if this approach is justified. Thoughtful planning of facilities, operational procedures, and personnel assignments is essential for success. In this chapter we provide details on housing and reproduction that constitute basic information useful for establishing an institutional breeding program. When possible, breeders should be derived from minimaldisease stock, and a rigorous program of vaccination and health testing must be followed to assure continued good health. A knowledgeable and experienced professional should oversee the breeding colony operation and be responsible for training personnel. Animal care personnel must be given adequate time to interact with these cats, particularly young kittens, to assure proper socialization, which will lead to tractable cats that will be suitable for routine handling and experimental manipulations. Periodic assessment Of reproductive success, ability to meet the needs of research projects, and colony health status is useful in making corrective adjustments and assuring that the breeding colony effort is economical and serves its intended purpose.

lIl.

HOUSING

A. CagingDesign and Operating Procedures Success or failure of virtually every facet of laboratory cat management depends on housing design and operation. Although a significant advantage of using cats is their adaptability to high-density housing, such housing conditions can introduce potentially serious problems, including abnormal behavior, infectious disease transmission, and reproductive failure. Careful planning of facility design, adoption of strict management pro-

tocols, thorough training and supervision of personnel, and oversight by a knowledgeable professional are essential for Success.

Cats are housed commonly in three basic arrangements: single cages, multiple runs within a room, and free ranging in a room. Space recommendations for each of these arrangements are suggested in the "Guide for the Care and Use of Laboratory Animals," but these should be regarded only as guidelines, because the specific needs of an experiment or a breeding colony may vary from these recommendations (Institute for Laboratory Animal Research, 1996). Substantial variance from these guidelines should be approved by the institutional animal care and use committee. Requirements of the Animal Welfare Act for space and density restrictions also should be consulted, because housing must comply with these regulations or exceptions sought for good cause (U.S. Department of Agriculture, 1995). Domestic cats develop highly structured interactive social groups, and most cats do not thrive in isolation (Fig. 1). Therefore, individual housing should be avoided unless particular experimental objectives dictate the use of single-cage housing or if caging is needed for short periods to permit collection of specimens, to administer material individually, or to accomplish treatment and/or observation. If caged, cats should be allowed out of their cages daily to exercise. At a minimum, cats should be housed in compatible pairs (Fig. 2) or, preferably, in small groups of the same sex or, if breeding, a few females with a tom. It is advantageous to house compatible pregnant queens together before they deliver because they usually share nursing and neonatal care. After delivery, pairing becomes more problematic. Installation of multiple runs within a room is the most economical use of floor space. Depending on the dimensions of the room, runs can be 3 to 4 feet wide and 4 to 6 feet long and 6 feet high (12-24 ft 2 of floor area). The smaller runs are

464

Fig. I. Catshoused singly often display anxiety-relatedstereotypic behaviors, including pacing, circling, and pawing (Overall, 1997). The authors strongly recommendgroup housing of cats to ensure social companionshipand well-being as well as reliable research data.

adequate for pregnant or lactating queens and their litters or 2 3 juveniles. The larger runs are best for breeding groups of toms and queens, postweaning family groups, and single-sex adult groups. The "Guide for the Care and Use of Laboratory Animals" recommends 3 ft 2 of floor area for each cat weighing less than 4 kg and 4 ft 2 for cats over 4 kg (Institute for Laboratory Animal Research, 1996). Galvanized wire panels with 1-inch mesh fence wire and a top panel are inexpensive, durable materials for run construction. As indicated in Section III,E, one or more high-density plastic resting boards should be installed approximately 2 ft above the floor. When a free-ranging room

BRENDA GRIFFIN AND HENRY J. BAKER

arrangement is used, a chain-link fence "foyer" is usually constructed at the door inside the room to allow personnel entry into the room without giving any opportunity for a cat to escape into the hallway. Freestanding vertically oriented shelf structures provide environmental enrichment and opportunities to escape from socially dominant members of the group (Fig. 3). Regardless of the cage arrangement used, wall, floor, and ceiling surfaces must be easily sanitized to achieve the pathogen control measures described below. Litter pans and utensils for food and water may be durable plastic or stainless steel and should be able to withstand 180~ wash water. Litter can be any clean, dust-free, absorbent material, including extruded corncob pellets. A minimum of one box per two cats should be provided. Soiled litter must be removed and replaced daily to minimize personnel exposure to infective Toxoplasma oocysts, to minimize cat-to-cat transmission of enteric pathogens, and to control odors. Room illumination must be controlled to provide duration, intensity, and spectrum of light that is optimal for specific needs of an experiment. In general, daylight-spectrum fluorescent tubes and daylight-dark cycles of 12:12 or 14:10 hr are useful and are required for successful breeding. Nesting boxes can be made of cardboard boxes, which are discarded when soiled. Enclosed boxes 24 inches square with a doorway cut into one side are useful for pregnant and lactating queens and their litters. Open boxes of the same size with walls 12 inches high are preferred for juveniles and adults. Boxes also serve to enhance the comfort of housed cats by providing places to hide and substrates for scratching, behaviors that are both fundamental needs of cats. B.

A n i m a l Care Staff

Animal care staff must enjoy working with cats and be willing to interact with them to assure socialization and tractability. The staff members become aware of the personalities of "their" cats, which is necessary for detection of estrous cycling, potential health problems, or incompatibility of runmates caused by social dominance. The staff must be instructed thoroughly and must adhere to the prescribed sanitation protocol. Freshly washed garments such as surgical scrub suits should be worn and changed between rooms. Dedicated shoes should be used for each room, or disposable shoe covers should be worn in housing rooms. Face masks may be indicated, especially to prevent allergy or irritation from inhaling cat hair and dander. Individuals with established cat-related allergy must use tightfitting face masks if they are required to enter cat housing rooms, which typically have high concentrations of allergen. Disposable gloves should be worn when handling soiled litter. C.

Fig. 2. If cats must be kept in cages, they should be housed in compatible pairs. Resting boards and hiding places shouldbe providedand serve to reduce stress.

Feline Social Behavior

As natural predators, cats possess keen senses and heightened fight-or-flight responses, making them particularly susceptible

12. DOMESTIC CATS AS LABORATORY ANIMALS

465

Fig. 3. Grouphousing of cats is preferred. Whereas single-housed cats tend to withdraw from handling, cats in a group environmenttend to be more outgoing and interactivewith human caretakers (Overall, 1997). Installation of shelving allows cats to utilize both vertical and horizontal space, functionallyreducing overcrowding.

to environmental stress (Greco, 1991). In a laboratory setting, cats become readily entrained to daily activity patterns and respond strongly to their surroundings as well as to their human caretakers (Carlstead et al., 1993). Unpredictable caretaking and handling are potent stressors in cats and may result in activity depression and withdrawal behavior. Overcrowding and insufficient resting and hiding places also increase stress (Carlstead et al., 1993). In cats, the ability to control aversive stimuli through hiding profoundly decreases cortisol concentrations when measured over time or in response to adrenocorticotropic hormone (ACTH) (Carlstead et al., 1993). As in many species, persistence of stress may compromise both immune and reproductive function (Griffin, 1989). In our experience, provision of proper social housing, exercise, environmental enrichment, and a predictable routine dramatically reduces the incidence of behavioral problems, including urine spraying, fighting, hiding, and silent heat. With the exception of being solitary hunters, free-roaming cats are social creatures (Crowell-Davis et al., 1997). The majority of their activities are performed within stable social groups in which cooperative defense, cooperative care of young, and a variety of affiliative behaviors are practiced. Affiliative behaviors are those that facilitate proximity or contact. Cats within groups commonly practice mutual grooming and allorubbing, in which cats rub their heads and faces against one another. This may serve as a greeting or as an exchange of odor for recognition, familiarization, marking, or development of a communal scent. Although both males and females exhibit

affiliative behavior, these behaviors are more common in females. Play behavior and food sharing are common in kittens and adolescent cats. Adult queens form social groups along with their kittens and juvenile offspring (Crowell-Davis et al., 1997). Adult toms reside within one group or roam between a few established groups. The formation of social hierarchy occurs within groups of cats. Establishment of ranking order is a social adaptation that minimizes agonistic behavior between individuals within a group. Signals of dominance and submission may be subtle or obvious and include vocalization (growling, hissing), visual cues (facial expression, posturing of the body, ears, and tail), and scent marking (urine, feces, various glands of the skin). Maternal behavior is the primary social pattern of the female cat. Queens exhibit strong maternal instincts. They nest communally and care for each other's kittens. Cooperative nursing is common. Kittens raised in communal nests develop faster and leave the nest sooner than kittens raised by solitary mothers. Although they are social animals also, tomcats commonly exhibit aggressive behavior toward one another during the establishment of dominance in relationships and during competition for territory, breeding, food, and other resources (CrowellDavis et al., 1997). Urine spraying and fighting are the most common undesirable male behaviors. In contrast to their interaction with other males, tomcats commonly display affiliative or "friendly" behavior with females regardless of their reproductive status. For these reasons, tomcats should be housed with spayed females when not breeding. If not used for breeding,

BRENDA GRIFFIN AND HENRY J. BAKER

466

toms should be castrated. Neutering before puberty is the best for prevention of undesirable male behaviors such as urine spraying and fighting. If the sexually mature tomcat is neutered, these behaviors will usually subside within a few weeks, facilitating intersex group housing. Neutered males display more affiliative behavior toward both other males and females. In the laboratory setting, once social order is established, particularly in a free-ranging room group, introduction or removal of individuals requires a period of adjustment that is usually stressful, induces fighting, and may disrupt breeding until a new social hierarchy and territorial limits are established (Hawthorne et al., 1995; Overall, 1997; B. Griffin and H. J. Baker, unpublished observations, 1999). Even in multiple-run housing in a single room, rearrangement of run groups or even relocation of an intact group within the room may induce imbalance of the social order and anxiety. Therefore, every effort should be made to minimize reorganization of groups once they are established, and if restructuring is necessary, ample time should be allowed for restabilization of social order before experimental interventions are attempted.

D.

Housing to Exclude Pathogens

As with other laboratory species, infectious disease control for cats should be based on exclusion. This requires that members of the colony are free from specific pathogens when the group is established, that vaccines are used where indicated to minimize susceptible populations, that rapid diagnosis and removal of ill cats be practiced rigorously, and, most of all, that the colony be closed to any new individuals that do not meet the rigorous health standards of the group. Disease transmission in breeding colonies is particularly problematic because there is usually a large proportion of juveniles in the group that are susceptible to infection between the lapse of maternal immunity and induction of protection by vaccines. Facility design that encourages a high level of sanitation and operational policies that assure cleanliness are essential to minimize infectious disease transmission. Daily operations should include vacuuming and mopping floors, disposal of soiled litter, replacing soiled cardboard nesting boxes, and washing utensils for water and food as needed. Weekly procedures should include washing litter boxes and food/water utensils in 180~ water, scrubbing soiled areas, and replacing nesting boxes. At least monthly, the room should be vacated, washed with hot water plus detergent, followed by disinfection with 10% hypochlorite. The premises should be dried thoroughly before cats are returned. Cats can be housed in airline-type carriers for the few hours required for this operation and become acclimated to the procedure. Individual cages should be accorded the same level of sanitation and processed through a mechanical cage washer weekly, because soiling in these closely confined cages is unavoidable, and daily hand washing is usually inadequate to maintain sanitation. Food and water should be separated from litter as much as possible.

E.

Environmental Enrichment

Environmental enrichment is essential for behavioral health of closely confined cats. The most effective environmental enrichment is a staff that enjoys interacting with cats and is willing to spend adequate time to ensure their socialization. Rest boards are required for comfort and contentment of cats because cats instinctively feel more secure when they can perch at a high point. These also provide an opportunity for lactating females to have rest periods away from their young. Boards should be constructed of dense plastic and anchored in such a way that crevices that accumulate hair and debris are avoided. Metal is too cold for comfort, and wood cannot be sanitized adequately. Play items that stimulate activity such as plastic balls, rings, hanging ropes, and scratching boards are recommended as long as they are sanitizable or can be discarded if soiled.

IV.

BREEDING COLONY MANAGEMENT

Because optimal conditions for exclusion of infectious diseases depend on use of purpose-bred cats, breeding colony management becomes exceedingly important for the use of cats in research. Fortunately, domestic cats are very prolific, and if reasonably uncomplicated guidelines are followed, as described here, high rates of production can be achieved in a laboratory environment with minimal complications. However, certain characteristics of feline reproduction are unique and must be recognized to achieve optimal breeding performance.

A.

Estrous Cycle and Mating

On average, queens reach puberty or experience their first estrous cycle between 5 and 9 months of age, although the onset may range from 3.5 to 18 months of age. In addition to age, factors that affect the onset of puberty include breed, time of year or photoperiod, social environment, health, physical condition, and nutritional status. With proper health maintenance, nutrition, and control of light cycles, adolescent queens begin to cycle after attaining a body weight of 2 kg or more. Group housing, especially the introduction of a tomcat or estral queen, provides social stimuli that hasten the onset of estrus (Michel, 1993). Free-roaming queens are seasonally polyestrous. In the Northern Hemisphere, the season begins in January or February after the winter solstice, as the days get longer, and lasts until fall. Anestrus persists from October through December until the next breeding season begins in January or February. Cats are extremely sensitive to photoperiod. In an environmentally controlled laboratory setting, 10 or more hours of light in a 24 hr period is required for reproductive cycling (Shille and Sojka,

12. DOMESTIC CATS AS LABORATORY ANIMALS

467

1995). Maintaining a 14 hr light photoperiod and the use of natural daylight spectrum fluorescent bulbs assures the maximum fertility period and estrous cycling (H. J. Baker, unpublished observations, 1999). Estrous cycling typically occurs within 7 10 weeks of instituting such a light cycle (Dawson, 1941; Scott and Lloyd-Jacob, 1959); however, this period can be shortened if preceded by a nonstimulatory light cycle of 8 or fewer hours of light (Hurni, 1981), or if a tomcat or queen in estrus is introduced at the time of increasing the duration of light (Michel, 1993). Peak sexual activity occurs between 1.5 and 7 years of age, with an average of 2 - 3 litters per year, with 3 - 4 kittens per litter (range 1-15 kittens per litter). Queens can bear 50-150 kittens in a breeding life of approximately 10 years if allowed to mate naturally. Like tomcats, queens are polygamous and rarely form long-term bonds with a mate, although they often display preferences for particular mates. If allowed, a female may accept a number of males, and therefore litters may have multiple sires. Adolescent queens (queens less than 1 year of age) and queens greater than 8 years of age tend to cycle irregularly and to have smaller litters, more abortions, more stillbirths, and more kittens with birth defects. Following a normal lactation and weaning, queens return to estrus in 2 - 8 weeks (average 4 weeks) (Feldman and Nelson, 1996). Many queens, however, return to fertile cycling while nursing their kittens (L6fstedt, 1982). If a queen aborts or if her litter is removed by 3 days postpartum, she will return to estrus in approximately 1 week. Queens may experience estrus during pregnancy. In fact, 10% of females experience estrus between the third and sixth weeks of pregnancy (Beaver, 1992). Fertile cycles are rare in the pregnant queen, but the development of different-age fetuses as a result of separate matings in different estrous cycles, known as superfetation, can occur. Although it is possible for a female to be continuously pregnant, nursing, or both, this high intensity of breeding is not recommended, because queens need a period of rest to regain body condition before the next period of pregnancy and lactation. Providing a period of short days (8 hr of light or less) for 4 - 6 weeks each year ensures anestrus, and

reproductive rest and may ultimately enhance reproductive performance. The estrous cycle of the queen consists of five phases: proestrus, estrus, interestrus, diestrus, and anestrus (Table III). Proestrus is the first phase of the feline estrous cycle and is defined as the time when queens attract toms but are not sexually receptive to them. Queens typically vocalize; rub their faces against objects, other cats, or human companions; and act "friendly." Rubbing usually progresses to rolling, and many queens will stretch and squirm in lateral recumbency, opening and closing their paws. Queens may assume a lordosis stance and tread with their hindlimbs, but upon introduction to a tom, they are not sexually receptive and may aggressively turn on the male, hissing and striking out with their claws. Proestrus lasts as little as 12 hours or up to 3 days. Signs may be overt or subtle. In fact, proestrus is not observed regularly in all queens. Many queens abruptly shift from anestrus behavior (no display of sexual behavior) to standing, receptive heat (estrus). Estrus is the phase of sexual receptivity that lasts 4 - 7 days on average, with a range of 1-21 days. Coital contact does not shorten estrus. During this period, queens commonly vocalize and call to toms when approached. They crouch and posture in a lordosis stance, treading in place with their hindlimbs. In this position, the queen's ventral thorax and abdomen touch the floor, her perineum is elevated, and her tail is displaced laterally as she presents herself to her mate (Fig. 4). This stance can usually be induced by stroking the queen's back or dorsal rump. Occasionally, queens in estrus may exhibit urine spraying and marking. Behavioral estrus is more readily detectable in grouphoused cats than in individually cage-housed cats. Silent heat may occur in "shy" or low-social-order queens. Behavioral signs of estrus are absent in these queens despite normal hormonal cyclicity. Decreasing housing density or housing groups of lowsocial-order queens separately from more dominant queens often results in normal displays of estrous behavior (B. Griffin and H. J. Baker, unpublished observations, 1999). There are various means of suppressing estrus in the queen, but ovariohysterectomy is the preferred method of contraception because it is safe,

Table llI

Five Phases of the Feline Estrous Cycle Phase

Duration

Proestrus

~A-3days

Estrus

4-7 days (range 1-21 days)

Interestrus Diestrus

1-3 weeks average (range 3 days-7 weeks) 45-50 days average (range 30-100 days)

Anestrus

October-January (free-roaming)

Signs

Hormone activity

Rubbing and rolling, "friendly,"vocalization lordosis, treading, not receptive to tom Sexually receptive, lordosis, treading, tail deflection, vocalization occasional urine spraying None

Ovarian follicular growth and estrogen synthesis Follicular phase, sharp rise in estradiol concentrations

None

Formation of corpora lutea, progesterone-dominant phase Baseline estradiol and progesterone concentrations

None

Baseline estradiol concentrations

468

BRENDA GRIFFIN AND HENRY J. BAKER

Fig. 4. Mating sequence of the domestic cat. (a) An interested male approaches a queen in estrus. (b) As the tom grasps her neck, the queen exhibits lordosis and tail deflection. (c) The tom mounts the queen. Intromission and ejaculation occur in 5 15 sec.

469

12. DOMESTIC CATS AS LABORATORY ANIMALS

effective, and not associated with deleterious side effects. Progesterone compounds and androgenic compounds can suppress estrus but are not recommended, because of the wide range of severe side effects associated with their use. Because cats are polyestrous and do not ovulate following every estrous period, an interestrous period or nonestrous interval commonly follows estrus. Interestrus is the interval of sexual inactivity between waves of follicular function in cycling queens. During this period, all breeding behaviors cease. Queens typically return to proestrus within 1-3 weeks, although this period is variable and may range from 3 days to 7 weeks. If ovulation occurs during estrus, diestrus follows. Corpora lutea form within 2 4 - 4 8 hr of ovulation and begin secreting progesterone. They remain functional for 30-50 days in the nonpregnant queen, at which time regression occurs. An interestrous interval follows such that diestral queens cease breeding behavior for 33-100 days before proestrus/estrus resumes. Because breeding displays cease in diestrus, this phase is behaviorally indistinguishable from anestrus or interestrus. Anestrus is the period of sexual rest that occurs between October and January in most free-roaming queens. Anestrus queens are sexually noninviting and nonreceptive. They may hiss or strike out at toms that make sexual advances. The domestic cat is an induced ovulator. Until recently, queens were believed to require copulation or mechanical stimulation of the vagina and cervix for release of luteinizing hormone (LH) and induction of ovulation. Ovulation induced by noncopulatory stimulation also occurs in the cat and is much more common than previously believed. Numerous studies have demonstrated progesterone concentrations consistent with ovulation in nonbred queens (Lawler et al., 1993; Potter et al., 1991; B. Griffin, unpublished observations, 1999). Luteal-phase diseases, including feline inflammatory uterine disease and pyometra, occur in individually housed or nonbred queens. Noncopulatory stimulation capable of inducing ovulation may include the stroke of a hand down the back, other physical stimulation, and visual, auditory, or olfactory cues from a nearby tomcat. Vaginal cytology may be used to assess the stage of estrous in queens, but any method used to obtain a smear may result in sufficient vaginal stimulation to induce ovulation. Pseudopregnancy occurs when a queen ovulates but does not become pregnant. Clinical signs of pseudopregnancy are rare in the cat but, when present, may include lactation, nesting, and tending kittens. If present, pseudocyesis is usually mild and short-lived and does not require therapy. Courtship usually occurs at night. Receptive queens sit at a distance from competing males and crouch, roll, and tread in place. The male may approach the female and rub his chin and face against hers in courtship. When the male imitates the female's "heat cry," this is a signal that he is ready to mate. This courtship lasts 10 s e c - 5 min and the duration decreases with repeated breedings. Mating is accomplished as the tom grasps the female by the neck with his teeth, grips her forequarters with

his front legs, and straddles her with his hindlimbs (Fig. 4). Intromission and ejaculation occur in a few seconds. After the tom releases his grip, the female displays postcoital "after-reaction," which lasts up to several minutes and is characterized by a scream, vigorous rolling and rubbing on the floor, and licking of the vulva. During this time she is unreceptive to the male. Additional mating resumes in 2 0 - 3 0 minutes. Several matings (10-30) occur during the next 24 hr and continue over several days, with the interval between matings becoming increasingly longer. Tomcats reach puberty between 8 and 13 months of age. They are sexually active year-round, are polygamous, and rarely form long-term bonds with queens. Most tomcats experience peak reproductive function between 2 and 8 years of age. Docile, tractable, easy-to-handle tomcats are ideally suited for breeding, given that studies relate these behavioral traits in kittens, at least in part, to paternity (Reisner et al., 1994 and Turner et al., 1986). Blood type A toms should not be bred to type B queens, to prevent neonatal isoerythrolysis (Casal et al., 1996). Blood type B is rare in domestic shorthairs, but common in certain purebreds.

B.

Pregnancy and Parturition

The queen's gestation period is 6 5 - 6 6 days on average with a range of 60-70 days. Large litters typically have a longer gestation period than smaller ones. Serum progesterone concentrations do not significantly differ in pregnant and pseudopregnant cats and therefore are not useful for pregnancy diagnosis. Relaxin is the only pregnancy-specific hormone in cats. Plasma relaxin assays (Witness | Relaxin, Synbiotics Corp., San Diego, CA) may be used to diagnose pregnancy in dogs after days 22-24 of pregnancy and are expected to become available for use in cats. Relaxin is secreted by the placenta. Concentrations increase from days 2 0 - 3 0 post-mating and remain elevated throughout pregnancy. This hormone helps maintain pregnancy and results in relaxation of the connective tissue of the pelvis (Verstegen, 1998). Abdominal palpation is the most common method for diagnosing pregnancy in the queen. Fetuses may be palpated first at 17 days (2.5 weeks) as discrete, firm, spherical nodules (2-2.5 cm in diameter). By day 25, fetuses are no longer discretely palpable. Instead, generalized uteromegaly is evident and remains palpable through parturition. Beyond the 45th day of gestation, fetal heads can be palpated. With experience, palpation is a very reliable method of pregnancy detection and serves as the most economical and practical method in a laboratory setting. Behavioral changes may aid in pregnancy diagnosis, but they typically remain subtle during the first 2 trimesters, when some queens become increasingly docile. By the third trimester, behavioral changes are usually obvious and

470

BRENDA GRIFFIN AND HENRY J. BAKER

include excessive grooming of the mammary glands and perineum and nesting behavior. Occasionally, queens become irritable or defensive during their last week of pregnancy. Physical changes become apparent beginning at 2.5-3 weeks of pregnancy. The queen's nipples become pinker, larger, and more erect. Abdominal distension becomes evident by the fourth week and obvious by the sixth week. Imaging methods used for pregnancy diagnosis include radiography and ultrasound. Calcification of the fetal skeletons may occur as early as day 38 of gestation but is not a reliable finding until day 43; therefore, to ensure diagnostic study, radiography should be performed after day 43 of gestation. Uteromegaly may be seen before this but cannot be distinguished from pyometra or other inflammatory uterine disease. Abdominal radiographs are most useful for evaluating litter size prepartum. Ultrasound is a quick, easy, accurate, safe, and reliable method of pregnancy detection in the cat. Ultrasonographic evidence of pregnancy may be seen as early as 11-14 days, and fetal heartbeats can be recognized at 3.5-4 weeks (Mattoon and Nyland, 1995). Pregnant cats should be allowed moderate exercise and fed a high-quality feline diet designed for growth or lactation (see Section V,B). Caloric intake increases by approximately onethird by mid-gestation. Stress should be avoided in the pregnant queen, and a quiet, warm, dark nesting area should be provided during the last trimester. Parturition usually occurs at night. Behavioral and physical changes accompany impending parturition (Beaver, 1992). One week prior to parturition, queens seek out dark, dry areas suitable for a nest. An increase in selfgrooming and irritability may be noted. Two to 3 days prior to parturition, mammary glands enlarge, and milk may be expressed. Twelve to 24 hr prior to parturition, the queen often exhibits nesting behavior characterized by restlessness, digging at the floor, vocalization, posturing to defecate, and failure to eat. Most queens prefer seclusion at parturition. A decrease in body temperature usually precedes delivery by 12 hr, however, this is not a reliable indicator of labor in the queen. Most litters are delivered within 2 hr, with 15 to 30 min intervals between kittens, but intervals may range from seconds to hours. Occasionally, a delay of 12-48 hr may be noted between kittens. This is usually secondary to disturbances, which may result in delayed parturition and/or moving of the kittens by the queen. Alternatively, the queen may elect to rest during parturition. This should not be confused with dystocia, which is rare in the cat.

C.

Infertility

Reproductive failure in domestic cats is uncommon. When present, it is most commonly associated with disease and/or environmental stress. If reproductive failure occurs, affected cats should receive thorough physical examinations, including careful inspection of the external genitalia. Infectious diseases such as feline leukemia virus should be ruled out by performing the

appropriate serological testing. Husbandry practices should be critically assessed. Are proper housing, nutrition, and exercise being provided? Are environmental or social stressors evident? For example, has there been a recent environment change, such as pairing a dominant queen with an inexperienced tom? Low-social-order queens may exhibit silent heat (Shille, 1979; Griffin, unpublished observations, 1999). Some queens show aversion to certain toms and preferences for others (Voith, 1980). Early embryonic death and fetal resorption may occur secondary to inherited or infectious diseases, including feline viral rhinotracheitis, feline leukemia virus, feline infectious peritonitis virus, panleukopenia, toxoplasmosis, and a variety of bacterial infections. In addition, fetal defects may contribute to early embryonic death and fetal resorption. Abortion may go unrecognized, because the queen may consume fetal tissues before they are seen. Because female infertility may be due to embryonic death and resorption or unobserved abortion, early pregnancy diagnosis is needed to assess possible contribution of infertility of the tom to reproductive failure. If the male is suspected and physical examination and infectious disease screening are normal/negative, semen evaluation should be performed. Although some tomcats can be trained to ejaculate into an artificial vagina with the use of a teaser queen, most require general anesthesia and electroejaculation for semen collection. Postcoital vaginal cytology is the easiest and most practical method of semen evaluation. Accurate breeding records are essential to evaluate breeding performance. The following information should be recorded for each queen: parents, birth date, date estrus is observed, breeding dates (copulation if observed or exposure to male), identification of breeding tom, results of ultrasound examination (if performed), dates of delivery of each litter, litter size, numbers of male and female kittens, live births, number and cause of stillbirths or neonatal mortality (if known), number of kittens weaned, and date of recurrence of estrus. Periodic review of these records will reveal infertility problems, fecundity, lactation problems, and abnormal viability of kittens in utero and postnatally. Queens or toms with a history of recurring poor production should be eliminated from a breeding colony. Inbreeding is a common cause of reduced fecundity, birth defects, and infertility. Breeding records should indicate clearly whether inbreeding is likely to be the cause of reproductive failure, and outbreeding to unrelated cats from minimal-disease stock may solve this problem. Methods for assisted reproduction in the cat are not as advanced as for mice, cattle, and dogs. Semen can be collected under anesthesia with custom-designed electroejaculators. Cat semen is very small in volume (0.1 ml) and concentrated. Once collected, semen can be diluted in preservative and frozen. Defrosted semen has been used successfully to fertilize ova in vivo. Preservation of semen from special stocks, such as important inherited-disease models, can be valuable to ensure against loss due to disease and to reduce the cost of maintenance over long periods (Swanson et al., 1998).

12. DOMESTIC CATS AS LABORATORY ANIMALS D.

Neonatal Care and Weaning

The queen and her new family require warmth, peace, and solitude. Whenever possible, the same personnel should care for queens to prevent stress associated with unfamiliar handlers. This is especially important in the case of new and inexperienced mothers, which may become nervous and trample, injure, or even cannibalize their kittens. Care should be taken to ensure that all kittens nurse as soon as possible after birth. If necessary, kittens should be placed on a teat to suckle. All kittens should be examined individually shortly after birth. Small, cold, or less vigorous kittens should be identified. Such kittens commonly are rejected by the queen, and their survival will require extra assistance in the first few days of life. Feeding supplemental milk replacer to them in addition to the queen's milk may be necessary (see Table IV). The umbilical area of each kitten should be examined. Some mothers may sever the umbilical cord too vigorously or groom the umbilical stump excessively, creating hernias in their kittens. Occasionally, if the umbilical cord is left too long, it may become wrapped around the limb of a kitten, and if not discovered and removed in time, it will dry and shrink, causing strangulation of the limb, with resulting edema and necrosis. Physical examination of newborn kittens should include an oral exam for cleft palate, which is one of the most common developmental defects in kittens. Affected kittens have difficulty nursing and are predisposed to aspiration pneumonia. Kittens should also be examined for atresia ani, a less common anomaly in which the anus is absent and feces cannot be passed. Surgical correction is possible but usually not practical, necessitating humane euthanasia of affected kittens. Because newborn kittens are unable to regulate their body temperature, they require a warm environment during the first 3 weeks of life. A local (nesting box) environmental temperature of 90~ should be maintained during the first week of life. After this, kittens are able to shiver, and an environmental temperature of 80~ is adequate during the next 2 weeks. Normally,

Table IV General Guidelines for Hand-Feeding Orphan Kitten Week of life

Frequency to f e e d

Volumeof warm milk replacer per feeding

First

Every 2 hours

1.5-2 ml

Second

Every 2 hours

3-4 ml

Third

Every 4 hours

8-10 ml

Fourth

Every 4 hours

10-12 ml

Fifth-Sixth

Every 4-6 hours

Decreasevolume and frequency of milk replacer feedings as intake of solid food increases

471

the queen can provide warmth to the kittens if an insulated nesting box is provided and the room temperature is 7 5 ~ 1 7 6 If the room temperature is too low, an external source of heat, such as a recirculating hot-water blanket under the nest box, may be necessary. At 3 - 4 weeks of age, kittens are old enough to start eating solid food on their own. Canned food that is softened with additional water should be used to start kittens and offered for 1-2 weeks. The addition of commercially available kitten milk replacer or powdered whole milk to the gruel may stimulate the kittens' interest and appetite. The food should be placed in a shallow pan. Some kittens will eat readily, while others will walk through the food and introduce themselves to the food as they groom. A few kittens will require prompting by opening their mouths and inserting a small bit of food. This simple assistance proves essential for some kittens to begin eating on their own. Healthy kittens that have reached 5 5 0 - 6 0 0 gm body weight may be weaned at 6 weeks of age (Lawler, 1997); however, weaning should not be rushed. The stress of weaning is less if kittens are already consuming adequate quantities of solid food. Kittens should be weaned fully and removed from their mothers by 8-10 weeks of age. After this time, they should be fed a high-quality food formulated for growth. Ideally, kittens should be fed free-choice; however, if meal feeding is necessary, a minimum of 3 or 4 meals per day must be fed. Kittens grow rapidly, attaining 75% of their adult body weight by 6 months of age. By 10-12 months of age, they are full-grown and can be changed to a diet formulated for adult maintenance. Although most queens are excellent mothers, certain circumstances arise occasionally in breeding colonies that necessitate care of orphan kittens. A queen may die following parturition, reject her kittens, become too ill to care for them, fail to produce sufficient milk, or develop postparturient hypocalcemia or mastitis. Fortunately, most queens will readily foster kittens of another queen. An experienced queen whose own kittens are less than 1 week old or are nearly weaned is the ideal foster mother (Beaver, 1992). Housing together two pregnant queens due to deliver at approximately the same time facilitates shared care of kittens. Newborn kittens rely on passive transfer of maternal antibodies for protection against infection. In kittens, passive immunity results primarily from translactational immunoglobulin transfer rather than transfer in utero. Intestinal absorption of immunoglobulins ceases after the first 16 hr of life, so newborns must begin nursing within 12 hr of birth in order to receive protective quantities of immunoglobulins (Casal et al., 1996). Kittens not receiving colostrum within 12 hr of birth should be isolated and vaccinated (using a killed product) at 4 weeks of age. Alternatively, injection of newborns with 15 ml serum from a Felv/FIV negative, well-vaccinated adult cat SQ or IP over 24 hours provides adequate passive immunity (Levy, 2000). If a foster mother is unavailable, kittens may be hand-raised.

472

BRENDA GRIFFIN AND HENRY J. BAKER

12. DOMESTIC CATS AS LABORATORY ANIMALS

473

Fig. 5. Commonly used types of identification of individual cats include tattoos, ear tags, and implantable microchips. (a) Permanent tattoo on the inner pinna of the ear of a cat. The medial aspect of the thigh is another common site for tattoo placement; however, growth of hair often makes reading difficult. Heavy sedation is required prior to tattoo placement. (b) Ear tag. Stainless steel bands are manufactured for wing banding of birds and are ideal for identification of young kittens. Skillful placement is necessary to prevent loss or ingrowth by placing the band too far out or into the pinna. (c) Instrument for microchip implantation with close-up views. Microchip is 12 x 2 mm. Cats can quickly and easily be scanned for positive identification.

Hand-raising kittens is time-consuming and sometimes difficult. Kittens should be kept together in a warm nesting box (80~176 Because commercial milk replacement formulas for human infants and puppies do not supply the high levels of fat and protein that kittens require, formulas designed for rearing orphan kittens should be used. Warmed milk replacer (98 ~ 99 ~F) should be fed via bottle or gastric tube, according to the schedule detailed in Table IV. The kittens and their environment must be kept clean. After each feeding, the anogenital area of each kitten is gently stroked with a soft cotton ball or tissue to stimulate urination and defecation. Several methods of individual identification are commonly used, including tattooing, placement of ear tags, and microchip implantation (see Fig. 5). Each method has attributes and limitations that recommend their use for specific applications. Tattoos are permanent and may be applied to the inside of the ear

or medial aspect of the thigh by using a tattooing machine with multiple needles. Care must be taken to disinfect the needles between individuals. India ink can be used and causes minimal inflammation at the site. Disadvantages of tattooing are that reading is limited by the presence of hair, and the characters can fade over time. Tattooing requires heavy sedation. Ear tags manufactured for wing banding of birds and made of inert stainless steel are especially useful for individual identification of kittens. They must be placed skillfully to provide enough space to accommodate ear growth, while being deep enough to prevent loss. Other complications include inflammation at the puncture site and secondary infection. Ear tags should be considered to be an interim measure until tattooing or microchip placement can be performed. Ear tagging requires sedation except in kittens less than 2 weeks of age. The most secure method is implantation of a microchip subcutaneously in the

BRENDA GRIFFIN AND HENRY J. BAKER

474

space between the shoulder blades. Migration is usually not a complication. Identification does require use of a reader held above the implanted chip site (see Fig. 5). Because of the large diameter of the implantation needle, sedation and/or local anesthesia should be used, and the wound sealed with surgical glue.

V.

bitum feeding because they can be left out overnight without

spoiling. Canned foods tend to be highly palatable and energydense, although they are more expensive, more labor-intensive to use, and may spoil if left for more than 8-12 hr. Supplementing ad libitum dry rations with canned food is ideal in growing kittens and pregnant or nursing queens, who have high energy requirements that are more easily satisfied by a calorically dense canned product.

NUTRITION AND FEEDING B.

As obligate carnivores, cats are nutritionally and metabolically unique, and their dietary requirements differ considerably from those of most other species. They require diets high in protein and fat but low in carbohydrate. Cats lack the ability to synthesize sufficient quantities of essential nutrients such as taurine, arginine, vitamin A, niacin, and arachidonic acid, which in the wild were present in tissues of their prey. Because their intestinal tract is short relative to that of dogs and other monogastrics, highly digestible diets are preferred (Laflamme, 1994; Buffington, 1991).

A.

Commercial Diets

To avoid nutritionally incomplete rations, it is best to select commercially prepared feline diets. The ideal cat food is highly palatable and precisely formulated to provide optimal levels of readily bioavailable nutrients. The nutrients are balanced to the caloric content of the diet, ensuring appropriate intake of each. For best results, cats should be fed a high-quality nutritionally complete diet appropriately formulated for their life stage. When high-quality commercial feline diets are fed, nutritional supplements are unnecessary. Commercially prepared diets should be approved by the Association of American Feed Control Officials (AAFCO), and the method of approval should be considered when selecting a product to feed (Buffington, 1991). AAFCO approval may be based on calculation of the nutritional content of a product's ingredient list, on chemical analysis of its nutritional content, or on feeding trials. In feeding trials, diets are tested by being fed to cats in the life stage for which the product claims to be nutritionally adequate. Unlike nutrient calculation or analysis, feeding trials assess the digestibility, bioavailability, and palatability of a diet, making them the best test of a product's performance. Because labels do not always state the basis of AAFCO approval, one may need to call the manufacturer to ascertain if feeding trials were conducted to validate the nutritional claims of a product. High-quality commercial feline diets are available in both wet (canned) and dry formulations. Dry foods are relatively inexpensive, convenient, and promote good dental hygiene through the action of chewing. Dry foods are ideal for continuous ad li-

Energy Requirements

Age, life stage, activity level, reproductive status, and environment all affect energy requirements. The energy needs of adult cats at maintenance are 6 0 - 8 0 kcal/kg body weight per day (Laflamme, 1994). Inactive and obese cats require 4 0 50 kcal/kg body weight per day. Individual cats may vary considerably (up to 20%) in the amount of food necessary to maintain their optimal body weight (Buffington, 1991). Properly fed adult cats should be well muscled but not overweight and should appear well proportioned. The ribs should be readily palpable beneath a thin layer of fat. Viewing the cat from the side, the waist should be moderately tucked up behind the last rib, and the inguinal fat pad should be modest. Cats may be fed ad libiturn unless portion control becomes necessary to avoid excessive weight gain and obesity. Whereas most sexually intact cats tend to self-regulate their intake, neutered cats tend to eat all food available to them (Flynn et al., 1996). In addition, neutered cats may have lower metabolic rates and require less food than sexually intact cats. If adult cats tend to overeat, portioned feedings twice a day are recommended. The energy requirements of growing and pregnant animals are greater than those of other adults. Breeding queens should be fed a high-quality feline diet designed for reproduction or growth. Queens gain weight throughout parturition in a linear fashion, with their energy requirements increasing by 25-30% by mid-gestation (Buffington, 1991). After parturition, energy requirements continue to rise to 3 - 4 times those of maintenance, as queens nurse their kittens (Lawler and Bebiak, 1986). Peak lactation occurs 2 - 3 weeks postpartum. Maintaining adequate nutrition during this time is extremely important to ensure production of sufficient quantities of high-quality milk, particularly in queens with large litters. Exclusive use of canned foods during the third trimester of pregnancy and during lactation helps ensure adequate energy intake, because these products are more calorically dense than dry foods. After weaning, milk production and mammary congestion can be decreased by fasting queens for 24 hr before returning to maintenance feeding. As always, a continuous supply of fresh, clean drinking water must be available. Kittens require 250 kcal/kg body weight per day. This gradually decreases over the first year of life until they reach their

12. DOMESTIC CATSAS LABORATORYANIMALS adult maintenance requirement. During the first 3 - 4 weeks of life, kittens receive all nutrients from the queen's milk. Normal birth weight for kittens is 100 ___ 10 gm (Lawler and Bebiak, 1986). Healthy kittens should double their weight in the first week of life and continue to gain 5-10% of their body weight daily during the first 3 weeks of life. If there are concerns about adequacy of growth, kittens should be weighed every 2 - 3 days, and records should be maintained to assess growth (see Section IV,D). Abrupt diet changes should be avoided. Although some cats readily eat novel diets, others may fast rather than accepting unfamiliar food. Anorexia and rapid weight loss, particularly in obese cats, may result in a life-threatening condition known as hepatic lipidosis, in which severe hepatocellular lipid accumulation leads to impairment of liver function and a variety of other serious metabolic consequences. If changes in diet or feeding schedules are necessary, they should be introduced gradually. Weight reduction, particularly in obese cats, should be accomplished gradually over a period of months to prevent the induction of hepatic lipidosis. If a change is made in a diet that is fed ad libitum, the new diet should be mixed in gradually or the old diet should continue to be offered for a limited period of time twice daily until the new diet is accepted. Whenever diet changes are made, food intake should be monitored closely.

C.

Feline Lower Urinary Tract Disease

Feline lower urinary tract disease (FLUTD), formerly known as feline urologic syndrome, or FUS, is a complex condition with multiple etiologies. Although the precise etiology or etiologies have yet to be defined, diet may be one contributing factor. Clinical signs associated with FLUTD include hematuria, dysuria, and pollakiuria. Cats may be observed entering the litter box frequently to strain, passing only small amounts of bloody urine each time. In males, urethral obstruction is a common sequela and, without rapid medical intervention, will result in death. The formation of magnesium ammonium phosphate crystals (struvite) is a complicating factor in FLUTD and can be controlled with dietary manipulation. Widely reported research in the 1970s concluded that dietary magnesium was the cause of FLUTD in cats, and low-magnesium diets were recommended to prevent recurrence of the disease (Buffington, 1991). However, recent studies indicate that the most important factor in the development of struvite urolithiasis is urine acidity. Struvite crystals do not form in acid urine (pH --< 6.5). In the last decade, cat food manufacturers have included ingredients in their diets to maintain urine acidity, including "digest," a product formed from the hydrolysis of animal tissues and by-products that contains phosphoric acid, which serves as a urinary acidifier. Digest is commonly sprayed on the outside of dry cat foods at 4-10% of the weight of the final finished product. It enhances the

475

palatability of food as much as 2- to 3-fold. Chronic acidification of urine may result in a whole new set of problems for cats, including potassium depletion, renal dysfunction, and formation of calcium oxalate stones. When clinical signs of FLUTD are present, a complete diagnostic evaluation including urinalysis should be performed to aid in selection of treatment and dietary management. Diet has been shown to be important in the management of FLUTD, especially when struvite urolithiasis is involved. Because FLUTD tends to be a recurrent problem in affected cats, practical recommendations for long-term dietary management usually include feeding diets that acidify the urine and avoiding diets containing excessive dietary magnesium (--- 20-25 mg magnesium/100 kcal) (Laflamme, 1994). Because precipitation of struvite crystals is intensified by a low volume of urine, use of canned foods is also recommended, because canned foods contain 7 0 - 8 0 % water and promote higher urine volume.

VI.

INFECTIOUS DISEASE EXCLUSION AND C O N T R O L

Veterinary graduates are well versed in the breadth of infectious diseases affecting cats, including pathogenesis, diagnosis, and therapy. Additionally, abundant texts and journal references are available on practice management of these diseases. Therefore, this chapter will emphasize infectious disease issues that apply uniquely to colonies of cats and that are critically important to health management of cats used in research.

A.

Preventive Medicine

Preventive health care involves recognizing and managing factors that affect disease transmission, including genetics, environmental stress, immunization, disease surveillance, nutrition, and housing design, maintenance, and sanitation (for a description of housing to control infectious diseases, see Section III,D) (Knowles and Gaskell, 1991; Hoskins, 1994; Lawler and Evans, 1997). Selection for disease resistance and docile temperaments that cope better with being housed and handled should be considered. For example, queens repeatedly producing kittens who experience upper respiratory infections (URI) should be removed from breeding stock. Stress has a profound influence on disease transmission. Stress commonly reactivates latent viral respiratory infections, leading to increased virus shedding and even recurrence of clinical disease (Hawthorne et al., 1995). Simply moving cats from one room to another can precipitate virus shedding. Overcrowding is one of the most potent stressors recognized in cats

476

(Carlstead et al., 1993; Hoskins, 1994; Lawler, 1997). Overcrowding increases both the number of susceptible animals and the number of asymptomatic carriers in a given group, while increasing the likelihood of disease transmission between group members through both direct contact and exposure to contaminated fomites. Other stressors include irregular schedules of feeding and cleaning, unpredictable daily manipulations, and infrequent or indifferent human contacts (Carlstead et aL, 1993). Therefore, effective preventive health care starts with minimization of stress through regular daily activity patterns and environmental enrichment. Group housing of cats ensures social companionship. Installation of shelving allows cats to utilize both horizontal and vertical space, functionally reducing overcrowding by increasing the area available to each cat. Such structures also provide resting and hiding areas and allow cats to escape from aversive stimuli. The provision of toys and cardboard boxes ensures adequate opportunity for scratching and exercise and helps prevent circling, pacing, and other anxiety-related behaviors. As maternal antibodies wane at 9-14 weeks of age, kittens become increasingly susceptible to a variety of infections, particularly URI and enteric coronavirus, which are commonly enzootic in some cat populations despite optimal preventive medicine and husbandry. To prevent such enzootic infections, early weaning, along with segregation and isolation of litters, has been advocated as a method of minimizing disease transmission (Hawthorne et al., 1995). Although this method m a y serve to prevent contact of kittens with adults that may be asymptomatic carriers or with acutely infected kittens from other litters, one must consider that early weaning may be inherently stressful (Lawler and Evans, 1997; H. J. Baker, unpublished observations, 1999). Some kittens experience marked separation anxiety, and smaller kittens may benefit from continued nursing. The mother-kitten relationship is extremely important for normal social and emotional development of kittens, particularly singletons. Exclusion of infectious diseases should be the goal of cat health management because several feline pathogens have the capacity to decimate a cat colony. Procedures to exclude infectious diseases include design of facilities to segregate the colony into subunits that are physically separate, management procedures that prevent or minimize entrance of personnel into more than one room, adequate ventilation and air pressure gradients that prevent recirculation of air or exchange of air between rooms, initiating the colony with disease-free stock, selective use of vaccines, regular health examinations (including serology), daily observations for illness, removal and quarantine of any suspected ill individuals, thorough necropsy examination of seriously ill cats, and, if addition of new cats is required, introduction of only cats of known health history following isolation and quarantine.

BRENDA GRIFFIN AND HENRY J. BAKER B.

Pathogen Control

Different strategies should be employed for control of specific infections, depending on their potential threat to the colony, importance as a human health hazard, and availability of effective vaccines. Table II lists basic principles of infectious disease control. For a more comprehensive discusion of control for each of the major pathogens of cats, see Pederson (1995) and Greene (1998). Although domestic cats are susceptible to a large number of viral diseases, only a few viral diseases are significant for colony-reared cats. Although they are common infections of pet cats, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) diseases can be excluded from research colonies by preventive measures described in Section VI,A (see Table V). Feline panleukopenia, caused by a parvovirus, is highly contagious and causes serious clinical disease but fortunately can be controlled easily by vaccination. Kittens should receive modified live-virus vaccines for panleukopenia at 8, 12, and 15 weeks of age. Multivalent vaccines containing feline viral rhinotracheitis, calicivirus, and panleukopenia virus are preferred. Boosters should be administered once every three years. Killed products are recommended in breeding queens. The most serious infectious-disease threats to colony-reared cats are the upper respiratory viruses and feline infectious peritonitis, which are discussed in detail below. 1.

Upper Respiratory Infection

Feline viral rhinotracheitis caused by feline herpesvirus (FHV-1) and feline calicivirus (FCV) are the primary etiologic agents in 80% of all upper respiratory infections (URI) in cats (Knowles and Gaskell, 1991; Lawler and Evans, 1997). Other agents, including Chlamydia, Mycoplasma, reovirus, and Bordetella may be primary, concurrent, or secondary to the viral diseases. Chlamydia and Mycoplasma commonly cause primary conjunctivitis. Bordetella has been implicated recently as a cause of acute bronchitis and pneumonia. Its significance in the pet population is not known, but it can be a serious infection of colonies. A vaccine is available and may be warranted in colonies with a history of Bordetella infection. Clinical signs of URI commonly include depression, inappetence, pyrexia, sneezing and nasal discharge. Conjunctivitis and keratitis are common in FHV-1 infections. Oral and lingual ulceration are common in FCV infections. The severity of clinical signs seen with these diseases is dependent on the age of the cat at infection, the quality and duration of acquired maternal immunity, the duration of exposure, the challenge dose of the virus, and the cat's nutritional plane, stress level, and general health. Once enzootic in a population of cats, upper respiratory viruses manifest mostly as acute disease in young kittens as passive immunity is lost and, at that point, may be difficult to control. As many as 80% of cats that recover from FHV- 1 become car-

12. DOMESTIC CATS AS LABORATORY ANIMALS

477

Table V

Feline Retrovirusesa Disease Feline leukemia virus (FeLV)

Feline immunodeficiency virus (FIV)

Incubation

Transmission

Weeks to years Bothvertical and horizontal, direct and close contact; mutual grooming; sharing food, water, and litter containers; bite wounds. Virus is continually present in large amounts in saliva, respiratory secretions, and blood in lesser amounts in urine and feces Weeks to years Bothvertical and horizontal (primarily bite wounds).Virus is continuallypresent in saliva and blood

Diagnosis

Clinical signs

Treatment

Screening: antigen test (ELISA). Highly sensitive indicatorof viremia. Confirmation with IFA

Wide range of manifestations: aplastic anemia, leukopenia, pancytopenia, lymphadenopathy, immunosuppression, secondary infections, reproductive failure, urinary incontinence, lymphosarcoma, and others

None (supportive care only)

Screening: antibody test (ELISA). Confirmationby Western blot

Acquired None immunodeficiency (supportive syndrome and AIDS- careonly) related complex

Prevention Test and remove. Note:Infected cats

may not test positive until several weeks postexposure; incoming cats should be tested on entry, isolated for 4 weeks, and retested before joining a colony

Test and remove

aSee Cotter (1990). riers and intermittently shed virus in oronasal and conjunctival secretions for life (Knowles and Gaskell, 1991; Lawler and Evans, 1997). Under natural conditions, approximately 50% of latently infected cats shed virus following stress. Virus shedding usually begins within 1 week after a stressful episode and continues for approximately 2 weeks. Following FCV infection, cats may shed virus continually for months to years. FHV-1 and FCV persist in the environment for 1-2 days and 8 - 1 0 days, respectively. Spread is by direct viral contact through nasal and ocular secretions and via fomites. Transplacental transmission does not occur. The incubation period is 2 - 6 days, and mortality is low except in young kittens. Clinical signs persist from two days to two weeks in individual cats. In many cats, infections are self-limiting. Treatment is supportive. Eyes and noses should be kept clean of discharge. Strong-smelling, moist canned foods stimulate the appetite, aid in maintenance of hydration and are gentler on sore throats than dry products. If secondary bacterial infection develops, administration of antibiotics may be necessary. After an enzootic episode, complete elimination of URI is not a realistic goal. Both parenteral and intranasal vaccine are available for FHV-1 and FCV. Vaccines are safe, but protection may not be complete. Parental vaccines are available in both modified live and inactivated products. Parental modified live vaccines may produce stronger immunity than killed products but should not be given to pregnant queens, to avoid prenatal infection of fetuses. Modified live intranasal vaccines have the advantage of producing rapid local immunity within 2 - 4 days; however, they frequently induce mild clinical disease. Intranasal vaccination

should be strongly considered when URI is a major problem in preweanling or newly weaned kittens. These vaccinations are effective as early as 3 - 4 weeks of age, and mild vaccine-associated disease is preferable to serious morbidity and mortality from natural disease. 2.

Feline Infectious Peritonitis

Feline infectious peritonitis is a potentially important infection of colony cats because it may arise in otherwise healthy cats, cannot be detected serologically, and causes recurring appearance of disease, which is fatal. Two types of coronaviruses infect cats: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). They are antigenically and morphologically indistinguishable. FECV is ubiquitous and avirulent. FIPV frequently coexists with FECV and is virulent. Most researchers believe that FIPV is a mutant of FECV. FECV is endemic in nearly all environments where a large number of cats share close quarters. It is associated with subclinical or self-limiting gastrointestinal signs, especially diarrhea. FECV infection commonly approaches 100%, but disease is almost always insignificant and no mortality results. FECV is transmitted via the fecal-oral route. After the virus is ingested, it binds to enterocytes, replicates, and kills the cell. If enough enterocytes are destroyed, a transient diarrhea ensues. Humeral immunity is stimulated as FECV is taken up by mesenteric lymph nodes. Local antibody production blocks further infection of enterocytes. FIPV differs from FECV in that it is capable of replicating in macrophages. The mutation that allows this to occur confers

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potent virulence because the virus is capable of replicating in macrophages that leave the mesenteric lymph nodes and migrate peripherally. FIPV is a systemic intracellular pathogen, but systemic antibodies are not protective. In fact, antibody production may actually enhance disease because complexes formed when antibodies bind with virus result in increased uptake of the virus by macrophages, where further replication occurs. Clinical FIPV disease is most common in young (< 18 months old) and old cats (> 13 years old). It may manifest as an acute vasculitis with pleural and/or peritoneal effusions or as a chronic pyogranulomatous disease. There is no effective treatment once clinical disease is present. Serological testing does not differentiate FECV from FIPV and therefore is not an effective diagnostic tool. The only definitive diagnosis is postmortem exam. Cats suspected of infection should be humanely euthanized, and a postmortem exam should be performed. Dried virus may survive at room temperature for weeks to months. It is readily destroyed by disinfectants. Litter has been cited as the most important fomite in transmission of this disease. The phenomenon of antibody-dependent enhancement makes vaccination controversial. However, an intranasal vaccine using a temperature-sensitive mutant is available. Early weaning and barrier raising may also be useful management practices to prevent infection of kittens. In addition, proper maintenance and husbandry, including vacuuming of litter particles and cleaning and frequent changing of litter boxes, are important to limit transmission of coronaviruses.

C.

Eliminating Parasites

Although cats are susceptible to a wide range of parasites, effective antiparasitic drugs are available, and the high level of sanitation that should be practiced in research colonies makes these easily eliminated. The most common parasites include fleas, ear mites, cestodes, ascarids, hookworms, and coccidia. Fleas cause marked allergic dermatitis in many adults and serve as vectors for transmission of infectious diseases and tapeworms (Dipylidium caninum). Several very effective commercial products are available for flea control. Because both cats and kittens are extremely sensitive to toxic effects from insecticides, products should be selected carefully and used only on animals of the age for which they are intended. After eliminating fleas on adult cats, eradication can be achieved because sanitation eliminates opportunities for larval development. Ear mites (Otodectes cynotis) are the most common cause of otitis externa in the cat. They live in the external ear canal, feeding on tissue fluids and producing irritation. Their presence results in the formation of a thick, dark brown exudate consisting of cerumen and exfoliated debris. Infested cats shake their heads, scratch their ears, and often excoriate their pinnae. Untreated infestations may result in permanent damage to the ear. Diagnosis is made on close visual inspection of aural exudate where the

mites are barely visible with the naked eye or by microscopic examination of exudate in mineral oil at x 10 magnification with a light microscope. If ear mites are diagnosed in a colony, all cats, whether infected or not, should be treated. Although not labeled for this use, ivermectin (200-300 Bg/kg SQ q2 weeks x 2 treatments) is safe, practical, inexpensive, and extremely effective. Endoparasites include ascarids or roundworms (Toxocara cati and Toxascaris leonina), hookworms (Ancylostoma and Uncinaria), and coccidia. Transmammary transmission is the most common route of transmission for both roundworms and hookworms, although cats may become infested by ingesting contaminated soil. Larvae ingested by adult cats migrate to body tissues and persist for years. During pregnancy, these larvae are reactivated and travel to the mammary glands, where they are shed into the milk and ingested by nursing neonates. Infested kittens may develop diarrhea as early as 2 - 3 weeks of age. Hookworms cause blood loss and anemia. Female worms produce eggs that pass in the feces and may persist in the soil for years. Control is readily achieved through proper sanitation and routine deworming of kittens. Pyrantel pamoate (810 mg/kg PO q3 weeks x 3 treatments) is highly effective against both roundworms and hookworms and is cost-effective and easy to administer. Adult cats acquire immunity and rarely experience reinfestation. In humans, hookworms and ascarids are associated with cutaneous larval migrans and visceral larval migrans, respectively. Protozoal parasites (coccidia and, less commonly, giardia) may occur in conditions of poor sanitation, particularly in kittens. Parasitization of the small intestine may result in diarrhea. Although uncommon, giardiasis is potentially zoonotic. Eradication consists of treatment of all cats with giardiacidal drugs (metronidazole at 50 mg/kg PO daily for 5 days or fenbendazole at 50 mg/kg PO daily for 5 days) and proper sanitation.

D.

Personnel Health Risks

A selected list of infections of cats with zoonotic potential are listed in Table VI. More complete lists are available in the literature (Lappin, 1993; Evans, 1997). Although no potential human health risk should be underestimated, in fact there are only a few of these infections that should be of any concern for a minimal-disease, closed cat colony. Infections of concern include cat scratch disease, dermatophytosis, and toxoplasmosis. There is little that can be done about cat scratch disease except to avoid cat scratch and bite injuries and to be aware of the potential for this infection in a wound that does not respond to the usual treatment. Dermatophytosis can be diagnosed by culture of the organism. It can be a difficult disease to treat in large groups of cats, and if treatment is attempted, the risk of human exposure must be considered (Moriello, 1995; Moriello and DeBoer, 1995). Toxoplasmosis is an obligate intracellular

Table VI Selected Feline Zoonotic Diseases ,

Clinical signs Disease

Etiologicagent

Cat scratch disease (CSD)

Bartonella henselae (intracellular bacterial parasite)

Transmission From cat to cat through close contact and possibly fleas

Diagnosis Serology

Humans

Cats

Typical CSD: erythematous papular eruptions at site of inoculation within 2 days-

Subclinical

2 weeks of cat scratch, followed by regional lymphadenopathy in 1-7 weeks; systemic signs: fever, malaise, anorexia in approximately 30% of cases

From cat to human through scratches and bites (poorly understood)

Control and prevention Flea control Avoid scratch and bite wounds Wounds should be cleaned and disinfected immediately

Atypical CSD: (rare) CNS signs, focal or multifocal Dermatophytosis (ringworm)

Microsporum canis, Trichophyton mentagrophytes

Direct contact with infected cats or humans, with carriers, or with fomites (spores live 18 + months)

Wood's lamp, fungal culture

Focal or multifocal dermatitis characterized by any combination of hair loss, scaling, crusting, erythema, and miliary dermatitis with variable pruritis

Focal or multifocal dermatitis of variable appearance, classic appearance: circular erythematous lesions

Environmental sanitation (thorough vacuuming followed by bleach at 1:10). For cats, topical therapy with lime sulfur dips twice a week plus systemic therapy with griseofulvin (50mg/kg PO q day) or itraconazole (10 mg/kg POq day). All exposed cats should be treated, whether infected or not

Pasteurellosis

Pasteurella multocida

Normal flora of nasal and oral cavities of cats Cat to cat or cat to human, via bite wounds or scratches

All cat bite wounds should be considered contaminated with

Cellulitis and abscessation at site of wound(s)

May become rapidly systemic and life-threatening in humans

Prevent bites and scratches through proper handling of cats. Immediate disinfection of all wounds. Antibiotic therapy for all puncture wounds. Amoxicillin (10mg/lb PO BID) is the drug of choice in cats. Humans with cat bite wounds should consult a physician!

Ingestion of oocysts in raw or poorly cooked meats; environmental contamination from sporulated oocysts in feces is possible in human beings

Serology

Usually subclinical; severe multifocal systemic disease may occur in immunocompromised individuals

Usually subclinical; may have transient flu-like symptoms. Abortion, stillbirths, or birth defects may occur in pregnant women

Avoid undercooked and raw meat; change litter boxes daily to prevent sporulation, which occurs in 1-5 days

(gram-negative bacillus)

Toxoplasmosis

Toxoplasma gondii (obligate intracellular coccidian parasite)

,,

Pasteurella

,

,

,

,

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BRENDA GRIFFIN AND HENRY J. BAKER

p r o t o z o a n parasite that can be t r a n s m i t t e d to cats and h u m a n s by in g e s t i o n of infected feces/soil or u n d e r c o o k e d meat. Diagnosis is difficult, but the s i m p l e e x p e d i e n t of c h a n g i n g litter daily, using gloves w h e n h a n d l i n g litter and litter pans, and w a s h i n g hands will essentially e l i m i n a t e risk. Rabies vaccination of cats should be c o n s i d e r e d b e c a u s e of legal obligations and interstate shipping regulations; o t h e r w i s e there is little or no risk to cats m a i n t a i n e d in a c l o s e d c o l o n y derived f r o m diseasefree stock. Cat salivary and urine proteins are p o t e n t allergens, and m a n y p e o p l e e x p e r i e n c e severe allergic reactions w h e n e x p o s e d to cats. B e c a u s e of h i g h - d e n s i t y h o u s i n g in the laboratory, special p r e c a u t i o n s m u s t be taken to e x c l u d e allergic p e r s o n n e l f r o m w o r k i n g with cats and to r e d u c e the potential for i n d u c t i o n of allergy. Pe rs o n n e l with k n o w n allergy to cats should not w o r k with t h e m unless they take special p r e c a u t i o n s such as using face m a s k s and gloves.

REFERENCES

Baker, H. J. (1987). Sphingomyelin lipidosis in a cat. Vet. Pathol. 24, 386-391. Baker, H. J., Lindsey, J. R., McKhann, G. M., and Farrell, D. E (1971). Neuronal GM1 gangliosidosis in a Siamese cat with [3-galactosidase deficiency. Science 174, 838-839. Baker, H. J., Smith, B. E, Foureman, P., Varadarajan, G. S., Varadarajan, U., Martin, D. R., and Castagnaro, M. (1998). The molecular bases of feline GM~ and GM2 gangliosidoses. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Beaver, B. V. (1992). Female feline sexual behavior. In "Feline Behavior: A Guide for Veterinarians," pp. 141-169. Saunders, Philadelphia. Bellhorn, R. W., and Fischer, C. A. (1970). Feline central retinal degeneration. J. Am. Vet. Med. Assoc. 157, 842-849. Berg, T., Tollersrud, O. K., Walkley, S. U., Siegel, D., and Nilssen, O. (1997). Purification of feline lysosomal tx-mannosidase, determination of its cDNA sequence, and identification of a mutation causing a-mannosidosis in Persian cats. Biochem. J. 328, 863-870. Bergsma, D. R., and Brown, K. S. (1971). White fur, blue eyes, and deafness in the domestic cat. J. Hered. 62, 171-185. Boyce, J. T., DiBartola, S. P., Chen, D. J. and Gasper, P. W. (1984). Familial renal amyloidosis in Abyssinian cats. Vet. Pathol. 21, 33. Buffington, C. A. (1991). Meeting the nutritional needs of your feline patients. Vet. Medi. 86, 720-727. Carlstead, K., Brown, J. L., and Strawn, W. (1993). Behavioral and physiological correlates of stress in laboratory cats. Appl. Anim. Behav. Sci. 38, 143158. Casal, M. L., Jezyk, P. E, and Giger, U. (1996). Transfer of colostral antibodies from queens to their kittens. Am. J. Vet. Res. 57(11), 1653-1658. Cotter, S. M. (1990). Feline viral neoplasia. In "Infectious Diseases of the Dog and Cat" (C. E. Greene, ed.), pp. 316-345. Saunders, Philadelphia. Cotter, S. M., Brenner, R. M., and Dodds, W. J. (1978). Hemophilia A in three unrelated cats. J. Am. Vet. Med. Assoc. 172, 166-168. Crowell-Davis, S. L., Barry, K., and Wolfe, R. (1997). Social behavior and aggressive problems of cats. Vet. Clin. North Am. Small Anim. Pract. 27(3), 549-568. Dawson, A. B. (1941). Early estrus in the cat following increased illumination. Endocrinology 28, 907-910.

DiBartola, S. E, Eaton, K. A., Menotti-Raymond, M. A., Biller, D. S., Wellman, M. L. and Radin, M. J. (1998). Autosomal dominant polycystic kidney disease in Persian cats. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. DiNatale, P., AnneUa, T., Daniele, A., Spaqnuolo, G., Cerundolo, R., DeCapraiis, D., and Gravino, A. E. (1992). A new case of feline MPS VI. J. Inherit. Metab. Dis. 15, 17. Enno, A., O'Rourke, J. L., Howlett, C. R., Jack, A., Dixon, M. E, and Lee, A. (1995). MALToma-like lesions in the murine gastric mucosa after longterm infection with Helicobacterfelis. Am. J. Path. 147, 217-222. Evans, R. H. (1997). Public health and important zoonoses in feline populations. In "Consultations in Feline Internal Medicine 3" (J. R. August, ed.), pp. 634-646. Saunders, Philadelphia. Feldman, E. C., and Nelson, R. W. (1996). Feline reproduction. In "Canine and Feline Endocrinology and Reproduction," 2nd ed., pp. 741-767. Saunders, Philadelphia. Festing, M. F. W., and Bleby, J. (1970). Breeding performance and growth of SPF cats. J. Small Anim. Pract. 11, 533-542. Flynn, M. F., Hardie, E. M., and Armstrong, P. J. (1996). Effect of ovariohysterectomy on maintenance energy requirement in cats. J. Am. Vet. Med. Assoc. 209(9), 1572-1580. Fox, J. G., Blanco, M., Murphy, J. C., Taylor, N. S., Lee, A., Kabok, Z., and Pappo, J. (1993). Local and systemic immune responses in murine Helicobacterfelis active chronic gastritis. Infect. lmmun. 61, 2309-2315. Fyfe, J. C., and Kurzhals, R. L. (1998). Glycogen storage disease type IV in Norwegian forest cats: molecular detection of carriers. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Gardner, M. B., and Luciw, P. A. (1998). Animal models of AIDS. FASEB J. 3, 2593 -2606. Gaschen, E P. (1998). Dystrophin deficient hypertrophic feline muscular dystrophy in the cat. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Giger, U., Rajpurohit, Y., Skelly, B., Wqang, P., Ford, S., Kohn, B., Niggemeier, A., Patterson, D. F., and Henthorn, P. S. (1998a). Erythrocyte pyruvate kinase deficiency in cats. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Giger, U., Wang, P., and Boyden, M. (1998b). Familial methemoglobin reductase deficiency in domestic shorthair cats. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Glenn, B. L., Glenn, H. G., and Omtvedt, I. T. (1968). Congenital porphyria in the domestic cat: preliminary investigations on itiheritance pattern. Am. J. Vet. Res. 29, 1653. Greco, D. S. (1991). The effect of stress on the evaluation of feline patients. In "Consultations in Feline Internal Medicine" (J. R. August, ed.), pp. 13-17. Saunders, Philadelphia. Green, P. D., and Little, P. B. (1974). Neuronal ceroid lipofuscin storage in Siamese cats. Can. J. Comp. Med. 38, 207-212. Greene, C. E., ed. (1998). Infectious Diseases of the Dog and Cat. W. B. Saunders Co., Philadelphia. Griffin, J. E T. (1989). Stress and immunity: a unifying concept. In "Veterinary Immunology and Immunopathology," Vol. 20, pp. 263-312. Elsevier Science Publ. B. V., Amsterdam. Gruys, E., Van de Stadt, M., Blok, J. J., Tooten, P. C. J., Van der Linde-Sipman, J. S., and Niewold, T. A. (1998). Feline amyloidosis. "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Haskins, M. E. (1998). Lysosomal storage diseases in cats: an overview. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Haskins, M. E., Jezyk, P. E, Desnick, R. J., McDonough, S. K., and Patterson, D. F. (1979). a-L-Iduronidase deficiency in a cat: a model of mucopolysaccharidosis I. Pediatr. Res. 13, 1294-1297. Hawthorne, A. J., Loveridge, G. G., and Horrocks, L. J. (1995). Housing design

12. DOMESTIC CATS AS LABORATORY ANIMALS and husbandry management to minimise transmission of disease in multicat facilities. In "Waltham Feline Medicine Symposium 1995," pp. 97-107. Hoover, E. A., and Mullins, J. I. (1991). Feline leukemia virus infection and disease. J. Am. Vet. Med. Assoc. 199, 1287-1297. Hopwood, J. J., Crawley, A. C., Byers, S., Yogalingam, G., and Bielicki, J. (1998). Feline MPS VI as a model to study pathology and evaluate efficacy of therapy for Maroteaux-Lamy syndrome patients. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Hoskins, J. D. (1994). Surveillance, prevention, and control of viral diseases in catteries. In "Consultations in Feline Internal Medicine 2" (J. R. August, ed.), pp. 615-619. Saunders, Philadelphia. Hurni, H. (1981). Day length and breeding in the domestic cat. Lab. Anim. 15, 229-233. Institute for Laboratory Animal Research. (1996). "Guide for the Care and Use of Laboratory Animals." National Academy Press, Washington D.C. Jezyk, P. E, Haskins, M. E., Patterson, D. E, Mellman, W. J., and Greenstein, M. (1977). Mucopolysaccharidosis in a cat with arylsulfatase-B deficiency: a model of Maroteaux-Lamy syndrome. Science 198, 834-936. Johnson, K. H. (1970). Globoid cell leukodystrophy in the cat. J. Am. Vet. Med. Assoc. 157, 2057. Jones, B. R., Hayden, M. R., Lewis, S., and Ginzinger, D. G. (1998). Chylomicronemia and inherited lipoprotein lipase deficiency in domestic cats. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Jones, T. C. (1969). Sex chromosome anomaly, Klinefelter's syndrome in tortoiseshell male cats. Comp. Pathol. Bull 5. Kier, A. B., Bresnahan, J. E, White, E J., and Wagner, J. E. (1980). The inheritance pattern of factor XII (Hageman deficiency) in domestic cats. Can. J. Comp. Med. 44, 309- 314. Kittleson, M. D., Meurs, K. M., Kittleson, A., Munro, M., Si-Kwang L., and Towbin, J. A. (1998). Heritable characteristics, phenotypic expression, and natural history of hypertrophic cardiomyopathy in Maine coon cats. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Knowles, J. O., and Gaskell, R. M. (1991). Control of upper respiratory diseases in multiple cat households and catteries. In "Consultations in Feline Internal Medicine" (J. R. August, ed.), pp. 563-569. Saunders, Philadelphia. Kramer, J. W., Davis, W. C., and Prieur, D. J. (1977). The Chediak-Higashi syndrome of cats. Lab. Invest. 36, 554-562. Laflamme, D. P. (1994). Nutritional management and nutrition related diseases in feline populations. In "Consultations in Feline Internal Medicine 2" (J. R. August, ed.), pp. 653-662. Saunders, Philadelphia. Lappin, M. R. (1993). Feline zoonotic diseases. Vet. Clin. North Am. Small Anim. Pract. 23, 57-78. Lawler, D. E (1997). Designing health programs for breeding catteries. In "Consultations in Feline Internal Medicine 3" (J. R. August, ed.), pp. 634646. Saunders, Philadelphia. Lawler, D. E, and Bebiak, D. M. (1986). Nutrition and management of reproduction in the cat. Vet. Clin. North Am. SmalI Anim. Pract. 16(3), 495-519. Lawler, D. E, and Evans, R. H. (1997). Strategies for controlling viral infections in feline populations. In "Consultations in Feline Internal Medicine 3" (J. R. August, ed.), pp. 634-646. Saunders, Philadelphia. Lawler, D. E, Johnston, S. D., Hegstad, R. L., Keltner, D. G., and Owens, S. E (1993). Ovulation without cervical stimulation in domestic cats. J. Reprod. Fertil. Suppl. 47, 57-61. Lee, A., Hazell, S. L., O'Rourke, J., and Kouprach, S. (1988). Isolation of a spiral-shaped bacterium from the cat stomach. Infect. Immun. 56, 28432850. Levy, J. K., and Crawford, P. C. (2000). Failure of passive transfer in neonatal kittens: Correction by administration of adult cat serum. In "ACVIM Proceedings," p. 137. L6fstedt, R. M. (1982). The estrous cycle of the domestic cat. Compend. Contin. Educ. 4(1), 52-58.

481 Lowenthal, A. C., Cummings, J. F., Wenger, D. A., Thrall, M. A., Wood, E A., and de Lahunta, A. (1990). Feline sphingolipidosis resembling NiemannPick disease type C. Acta Neuropathol. (Berlin) 81, 189. Maggo-Price, L., and Dodds, W. J. (1993). Factor IX deficiency (hemophilia B) in a family of British shorthair cats. J. Am. Vet. Med. Assoc. 203, 17021704. Martin, D. R., Varadarajan, G. S., Varadarajan, U., Smith, B. E, and Baker, H. J. (1999). A unique mutation of the beta subunit of hexosaminidase causes feline GM 2 gangliosidosis variant 0. Proceedings of the Tenth North American Collaquium on Gene Mapping and Cytogenetics of Domestic Species, Apalachicola, Florida. Mattoon, J. S., and Nyland, T. G. (1995). Ultrasonography of the genital system. In "Veterinary Diagnostic Ultrasound" (T. G. Nyland and J. S. Mattoon, eds.), pp. 141-164. Saunders, Philadelphia. Michel, C. (1993). Induction of oestrus in cats by photoperiodic manipulations and social stimuli. Lab. Anim. 27, 278-280. Moriello, K. A. (1995). Treatment of feline dermatophytosis: revised recommendations. "Waltham Feline Medicine Symposium 1995," 31- 37. Moriello, K. A., and DeBoer, D. J. (1995). Feline dermatophytosis: recent advances and recommendations for therapy. Vet. Clin. North Am. Small Anim. Pract. 25(4), 901-921. Muldoon, L. L., Pagel, M. A., Neuwelt, E. A., and Weiss, D. L. (1994). Characterization of the molecular defect in a feline model for type II GM 2 gangliosidosis. Am. J. Pathol. 144, 109. Narfstrom, K. (1998). Progressive retinal atrophy in Abyssinians. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Overall, K. L. (1997). Recognizing and managing problem behavior in breeding catteries. In "Consultations in Feline Internal Medicine 3" (J. R. August, ed.), pp. 634-646. Saunders, Philadelphia. Paasch, L. H., and Zook, B. C. (1980). The pathogenesis of endocardial fibroelastosis in Burmese cats. Lab. Invest. 42, 197-204. Patterson, D. E, and Minor, R. R. (1977). Hereditary fragility and hyperextensibility of the skin of cats: a defect in collagen fibrillogenesis. Lab. Invest. 37, 170. Pederson, N. C. (1995). An overview of feline enteric corona viruses and infectious peritonitis virus infections. Feline Pract. 23, 7-19. Perkins, S. E., Yan, L. L., Shen, Z., Hayward, A., Murphy, J. C., and Fox, J. G. (1996). Use of PCR and culture to detect Helicobacter pylori in naturally infected cats following triple antimicrobial therapy. Antimicrob. Agents Chemother. 40, 1486-1490. Potter, K., Hancock, D. H., and GaUina, A. M. (1991). Clinical and pathologic features of endometrial hyperplasia, pyometra, and endometritis in cats: 79 cases (1980-1985). J. Am. Vet. Med. Assoc. 198(8), 1427-1431. Prusiner, S. B. (1995). Prion diseases. In "The Metabolic and Molecular Bases of Inherited Diseases" (C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, eds.). McGraw-Hill, New York. Reisner, I. R., Houpt, K. A., Erb, H. N., and Quimby, E W. (1994). Friendliness to humans and defensive aggression in cats: the influence of handling and paternity. Physiol. Behav. 55(6), 1119-1124. Sandstrom, B., Westman, J., and Ockerman, E A. (1969). Glycogenosis of the central nervous system in the cat. Acta Neuropathol. (Berlin), 14, 194. Scott, E E, and Lloyd-Jacob, M. A. (1959). Reduction in the anoestrus period of laboratory cats by increased illumination. Nature 184, 2022. Scriver, C. R., Geaudet, A. L., Sly, W. S., and Valle, D., eds. (1995). "The Metabolic and Molecular Bases of Inherited Diseases," 7th ed. McGraw-Hill, New York. Shille, V. M., and Sojka, N. J. (1995). Feline reproduction. In "Textbook of Veterinary Internal Medicine" (S. J. Ettinger and E. C. Feldman, eds.), p. 1690. Saunders, Philadelphia. Shille, V. M., Lundstrom, K. E., and Stabenfeldt, G. H. (1979). Follicular function in domestic cats as determined by estradiol 17~ concentrations in plasma: Relation to estrous behavior and cornification of vaginal epithelium. Biol. of Reprod. 21, 953-963.

482 Swanson, W. E, Haskins, M. E., Thrall, M. A., Baker, H. J., and Howard, J. (1989). Assisted reproductive technology for facilitating management of feline hereditary disease models. In "Proceedings of the First International Feline Genetic Disease Conference," Univ. of Pennsylvania. Turner, D. C., Feaver, J., Mendl, M., and Bateson, P. (1986). Variation in domestic cat behavior towards humans: a paternal effect. Anim. Behav. 34, 1890 - 1892. U.S. Dept. of Agriculture, Animal and Plant Health Inspection Serv. (1995). Animal Welfare Act. "Code of Federal Regulations," Title 9 (Animals and Animal Products), Chap. 1, Subchap. A (Animal Welfare). Valle, D. L., Boison, A. P., Jezyk, P., and Aguirre, G. (1981). Gyrate atrophy of the choroid and retina in a cat. Invest. Ophthalmol. Vis. Sci. 20, 251-255. Verstegen, J. P. (1998). Physiology and endocrinology of reproduction in female

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cats. In "Manual of Small Animal Reproduction and Neonatology" (G. C. England and M. Harvey, eds.), pp. 11-16. British Small Animal Veterinary Assoc. Pub., Cheltenham, UK. Voith, V. L. (1980). Feline reproductive behavior. In "Current Therapy in Theriogenology (D. E. Morrow, ed.), pp 839-843. Saunders, Philadelphia. Wang, T. C., Dangler, C. A., Chen, D., Goldenring, J. R., Koh, T., Raychowdhury, R., Coffey, R. J., Ito, S., Varro, A., Dockray, G. J., and Fox, J. G. (2000). Synergistic interaction between hypergastrinemia and helicobacter infection in a mouse model of gastric cancer. Gastroenterology 118, 36-47. Woodard, J. C., Collins, G. H., and Hessler, J. R. (1974). Feline hereditary neuroaxonal dystrophy. Am. J. Pathol. 74, 551-560.

Chapter 13 Biology and Diseases of Ferrets Robert P. Marini, Glen Otto, Susan Erdman, Lori Palley, and James G. Fox

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. TaxonomicConsiderations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Use in Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Availability and Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. LaboratoryManagement and Husbandry . . . . . . . . . . . . . . . . . . . . . . II. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. UniqueAnatomic and Physiologic Characteristics . . . . . . . . . . . . . . . B. NormalValues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. InfectiousDiseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Metabolicand Nutritional Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . C. TraumaticDisorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. IatrogenicDiseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. NeoplasticDiseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. MiscellaneousDiseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

A.

Introduction

Taxonomic Considerations

Ferrets (Mustela putorius furo) belong to the ancient family Mustelidae, which is believed to date back to the Eocene period, some 40 million years ago. The taxonomic groups in the family Mustelidae, as recognized by Corbet and Hill (1980), include 67 species from North, Central, and South America, Eurasia, and Africa. No other carnivore shows such diversity of adaptation, being found in a wide variety of ecosystems ranging from LABORATORY ANIMAL MEDICINE, 2nd edition

483 483 484 484 484 485 485 486 486 487 490 490 506 507 507 507 512 513

arctic tundra to tropical rain forests. Mustelids have retained m a n y primitive characteristics, which include relatively small size, short stocky legs, five toes per foot, elongated braincase, and short rostrum (Anderson, 1989). The Mustelinae is the central subfamily of the Mustelidae. The best-known members of the Mustelinae are the weasels, mink, and ferrets (genus Mustela) and the martens (genus Martes) (Anderson, 1989). The genus Mustela is divided into five subgenera: Mustela (weasels), Lutreola (European mink), Vison (American mink), Putorius (ferrets), and Grammogale (South American weasels). According to one author, ferrets (Mustela putoriusfuro) have been domesticated for more than 2000 years (Thomson, 1951). Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

ROBERT P. MARINI ET AL.

484

Earlier references to ferrets are probably the basis of the belief that ferrets originated in North Africa (Thomson, 1951). Evidently they were bred specifically for rabbiting (rabbit hunting) and were muzzled before being sent into rabbit burrows. This practice was later introduced into Europe, Asia, and the British Isles, where the sport is still practiced today. Although the ferret has been historically used for hunting, more recently it has been increasingly used in biomedical research and is popular in North America as a pet. It is most likely a domesticated version of the wild European ferret or polecat (M. putorius or M. furo) (Thomson, 1951). Alternatively, it may be related to the steppe polecat (M. eversmanni), which it closely resembles in skull morphology (Walton, 1977). The domesticated ferret, although introduced to North America by the early English settlers some 300 years ago, has not established feral colonies on this continent.

rets from fur operations or may make arrangements with commercial vendors in the United States. Even though the ferret is nonstandardized with regard to exact genotype and pedigree, its routine availability in a clinically healthy state has aided immeasurably its acceptance as a research animal. Readily available commercial stocks, based on coat color, are albino, sable (or fitch), Siamese, silver mitt, and Siamese-silver mitt (Siamese with white chest and feet) (McLain et al., 1985). The fitch or so-called wild coat color is the most common, recognized by yellow-buff fur with patches of black or dark brown, particularly on the tail and limbs (Andrews and Illman, 1987). The production of ferrets by large commercial operations has raised concern by some that inbreeding of these animals has made the ferret more susceptible to diseases, e.g., endocrinerelated disorders.

D. B.

Laboratory Management and Husbandry

Use in Research This topic is covered in more detail in Chapter 21.

The ferret was not recognized as having potential as an animal model for biomedical research until the 1900s. Early studies utilized the ferret in classic experiments with influenza virus pathogenesis (Pyle, 1940). Its use was cited infrequently; an article published in 1940, detailing the use of ferrets in research, cited only 26 publications (Pyle, 1940). Literature reviews undertaken in 1967, 1969, 1973, and 1985, however, revealed an increasing appreciation for the ferret's usefulness and versatility in the study of human physiologic, anatomic, and disease mechanisms (Hahn and Wester, 1969; Marshall and Marshall, 1973; Shump et al., 1974; Frederick and Babish, 1985). In 1991, a bibliography containing "selected" literature citations on the ferret and its use in biomedical research was published (Clingerman et al., 1991). The document was designed to serve as a reference tool for individuals involved in the care or use of ferrets in the laboratory setting. Although not comprehensive, the document provides extensive coverage of ferret biology, diseases, and use as an animal model. The domesticated ferret has been and continues to be used extensively in studies involving virology, reproductive physiology, anatomy, and endocrinology, as well as other areas of biomedical research (Morgan and Travers, 1998). The ferret is also being used to replace the cat in some types of neuroendocrinology, neuroanatomy, and cardiology experiments.

C.

Availability and Sources

The ferret's increasing popularity in research and as a pet is mainly a result of large-scale commercial production. For example, commercial farms have been raising ferrets for almost 50 years. Biomedical researchers in the United States can request animals of a specific sex, weight, and age for individual experiments. Investigators in other countries may acquire fer-

1. Housing and Husbandry Housing of ferrets in a research facility is similar to that of other small carnivores such as cats (Fox, 1998c). Ferrets tolerate low temperatures well and high temperatures poorly; the recommended temperature range for juvenile and adult animals is 4 - 1 8 ~ (Hammond and Chesterman, 1972). Ferrets less than 6 weeks of age should be housed at > 15 ~C. Kits under this age require a heat source if separated from the dam; older kits that are group-housed do not. Elevated temperatures (>30~ cannot be tolerated by ferrets, because they have poorly developed sweat glands and are susceptible to heat prostration. Signs of hyperthermia include panting, flaccidity, and vomiting. The preferred humidity is 40-65%. For nonbreeding animals that will remain in the facility for a short time, a conventional dark-light cycle at 12:12 hr is adequate. Lighting may be altered to control breeding cycles. Breeding and lactating jills should be exposed to 16 hr of light daily. Ferrets that are maintained for breeding or for use beyond 6 months should be exposed to "winter" l i g h t w 6 weeks per year of 14 hr of dark dailywto maintain physiologic normalcy. It is also essential that researchers receiving time-pregnant jills preserve the photoperiod to which jills were exposed prior to shipment. Failure to do so may cause inappetence, with subsequent negative energy balance and pregnancy toxemia. Similar to other laboratory animal species, ferrets should be housed with 10-15 air changes per hour (USDHHS, 1996). It is important to use nonrecirculated air because of the strong odor of ferrets and the susceptibility to respiratory tract infections. The ferret odor should not overlap into any rodent housing areas, because rodents have an instinctive fear of ferrets, and the ferret scent can disrupt rodent breeding and physiology (Fox, 1998c).

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2. Caging Female ferrets can be housed singly or in groups, but estrous females that are cohoused may become pseudopregnant (Beck et al., 1976). Males should be housed individually after 12 weeks of age. Molded plastic caging used to house rabbits works very well for ferrets. The solid bottom is perforated with holes and is readily sanitizable. An absorbable paper liner may be used in the pan beneath the cage to facilitate daily disposal of urine and feces. In a research setting, the plastic caging should be washed weekly to avoid excessive soiling. The spacing of grid walls should be 1.0 X 0.5 inches apart, or 0.25 inch if using wire mesh. Ferrets like to lick and bite at their enclosures, so sharp edges and galvanized metal should be avoided. Zinc toxicosis has been reported from licking galvanized bars from which metals had leached during steam sterilization (Straube and Walden, 1981) (Table I). Ferrets can be trained to use a litter box because they repeatedly urinate or defecate in one corner of the cage. Clay litters have been reported to cause chronic upper respiratory irritation

Table I Housing Ferrets in Research Parameter Cage size Grid size Temperature range Humidity range Air handling Animals amenable to group/pair housing

Photoperiod (hours light: hours dark)

Diet (protein source: meat)

Feeding schedule Quantity consumed (dry-weight basis) Water consumption (adults)

Comment 24 X 24 X 18 inches (adequate for two adult ferrets) 1 X 0.5 inches (0.25 inch if wire mesh or slated flooring) 4~176 (40~176 animals less than 6 weeks (> 15~ 60~ 40 -65% 10-15 complete air changes/hr (nonrecirculated) Female ferrets Anestrous Nonlactating Weanling ferrets 4 -12 weeks old Males separated at 12 weeks Breeding; lactation (16: 8) Winter cycle (10:14) Nonbreeders housed for <6 months (12:12) Nonbreeding adult males and females: 18-20% fat, 30-40% protein Breeding males and females: Minimum 25% fat, minimum 35% protein Peak lactation: 30% fat minimum, 35% protein Ad libitum 43 gm/kg body weight 75-100 ml daily

from inhaled dust (Jenkins and Brown, 1993). Ferrets prefer sleeping in a soft isolated area, and in a research facility this can be accomplished by providing a washable "snooze tube" (Fox, 1998c).

II.

A.

BIOLOGY

Unique Anatomic and Physiologic Characteristics

The thorax of the ferret is narrow and elongated, and as a result the trachea is proportionally long. This makes the ferret an ideal species for studies of tracheal physiology. The tracheal size and laryngeal anatomy make endotracheal intubation somewhat challenging, and as a result the ferret has been advocated as a species suitable for use in pediatric intubation training (Powell et al., 1991). The lungs are relatively large, and the total lung capacity is nearly 3 times that which would be predicted based on body size, as compared with other mammals. This characteristic, together with a higher degree of bronchiolar branching and more extensive bronchial submucosal glands (as compared with the dog), makes the ferret an attractive model for pulmonary research studies (Vinegar et al., 1985). Although a previous report (Willis and Barrow, 1971) commented that the carotid arterial branching pattern in the ferret is unusual, it is actually typical for a carnivore. As is the case in the dog and the cat, the paired common carotid arteries arise from the brachiocephalic trunk (sometimes called the innominate artery) at the level of the thoracic inlet (Andrews et al., 1979b). The ferret's gastrointestinal tract is specialized to fit its carnivorous nature. The simple monogastric stomach is similar to that of the dog. There is no cecum present, and the indistinct ileocecal transition makes it difficult to identify the junction of the small and large intestines during a gross examination. The overall length of the alimentary tract is very short relative to the body size, resulting in a gastrointestinal transit time as short as 3 hr (Bleavins and Aulerich, 1981). As in other mustelids, the paired anal scent glands of the ferret are well developed. Although not as potent as those of the skunk, the secretions of the ferret are sufficiently odoriferous that many pet or research ferrets are descented. Surgical techniques for this procedure have been described (Creed and Kainer, 1981; Mullen, 1997). Ferrets, especially intact males and estrous jills, may possess a distinctive musky odor even after a successful descenting, because of normal sebaceous secretions. Ferrets lack well-developed sweat glands for use in thermal regulation, and as a result they are predisposed to heat prostration when ambient temperatures reach 32~ (90 ~F) (Ryland et al., 1983). Extramedullary hematopoiesis is commonly found during histological examination of the spleen, and in some cases it may

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ROBERT P. MARINI E T AL.

result in a grossly evident splenomegaly (Erdman et al., 1998). This must be differentiated from splenomegaly that can arise from a variety of pathologic conditions or from isoflurane administration (see Section III,E). Experimental evidence suggests that ferrets have no naturally occurring antibodies against unmatched erythrocyte antigens, and that none develop even in the face of repeated transfusions (Manning and Bell, 1990b). Ferrets are seasonal breeders, and the resulting pronounced physiological variations in body weight, behavior, and gametogenesis are utilized in scientific studies of photoperiod responses and neuroendocrine control. Prolonged estrus in unbred females can cause an aplastic anemia, an effect that can be reproduced with exogenous estrogen administration (Bernard et al., 1983). The male has a radiographically evident os penis, and, contrary to some earlier reports, a prostate gland is present in males (Evans and An, 1998).

B.

Normal Values

Newborn ferret kits weigh 6-12 gm at birth and will grow to 400 gm by the time they are weaned at 6 - 8 weeks (Shump and Shump, 1978). In sexually intact populations, males ( 1 . 0 -

2.0 kg) can be twice the size of females (0.5-1.0 kg). The adult weight of nonobese male and female ferrets that have been gonadectomized prior to weaning and raised in captivity will generally fall between 0.8 and 1.2 kg (Brown, 1997a). Adult animals (especially those that are sexually intact) may be subject to seasonal fluctuations in body fat percentage, which can cause body weight to fluctuate by 30-40% (Fox and Bell, 1998). The approximate life span for the ferret is 6 - 8 years, but on rare occasions they may live as long as 11 years (Table II). Normal hematology and serum chemistry values have been reported for the ferret (Thornton et al., 1979; Lee et al., 1982; Fox, 1998e). These values are not greatly dissimilar from those of other domestic carnivores. One distinctive hematological characteristic of the ferret is the presence of a relatively robust erythron, characterized by hematocrit, hemoglobin, and total erythrocyte and reticulocyte counts that are generally higher than those of the dog or cat. Reported neutrophil-lymphocyte ratios range from 1.7:1 to 0.7:1. Representative hematology and chemistry ranges from one of our studies (Fox et al., 1986b) are shown in Tables III and IV, but for diagnostic purposes any laboratory that evaluates ferret samples should develop its own set of specific normal ranges. A low-grade proteinuria may be identified by urinalysis in normal, healthy ferrets (Thornton et al., 1979) (Table V).

Table II Selected Normative Data for the Ferret a Parameter Life span (average) Body temperature Chromosome number (diploid) Dental formula Vertebral formula Age of sexual maturity Length of breeding life Gestation Litter size Birth weight Eyes open Onset of hearing Weaning Water intake Urine volume Urine pH Cardiovascular/respiratory Arterial blood pressure Mean systolic Mean diastolic Heart rate Cardiac output Circulation time Respirations a Adapted

Value 5-11 years 38.8~ (37.8 ~ 40~ 40 2 (I 3/3, C 1/1, P 4/3, M 1/2)

C.

Nutrition

Ferret diets have been formulated both empirically and based upon the nutrient requirements of other mustelids (Fox and McLain, 1998). Specific requirements for various life-cycle stages have not been determined experimentally. Available commercial diets are certainly capable of supporting growth, reproduction, and maintenance in conventional settings. In the

C7T15L5S 3 C14

4 - 1 2 months b 2-5 years 42 ___2 days 8, average (range, 1-18) 6-12 gm 34 days 32 days 6 - 8 weeks 75 - 100 ml/24 hr 26-28 ml/24 hr 6.5-7.5

Female 133, male 161 mmHg (conscious) 110-125 mmHg (anesthetized) 200- 400 beats/min 139 ml/min 4.5-6.8 sec 33-36/min

from Fox, 1998e, Normal Clinical and Biological Parameters. bDependent on photoperiod.

Table III Hematology Values of Normal Ferrets a Parameter (unit)

Observed range

WBC (103/mm3) RBC (103/mm3) Hematocrit (%) Hemoglobin (gm/dl) Total protein (gm/dl) Neutrophils (%) Bands (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) B asophils (%)

1.7-13.4 9.7-13.2 47-59 14.5-18.5 6.2-7.7 22-75 0-2 20 -73 0-4 0- 3 0- 1

aCombined ranges from orbital and cardiac venipuncture of anesthetized male ferrets (Fox et al., 1986b).

487

13. BIOLOGY AND DISEASES OF FERRETS Table IV Serum Chemistry Values of Normal Ferrets Mean ___SEM Observed range a

Serum analyte (unit) Glucose (mg/dl) Urea nitrogen (mg/dl) Creatinine (mg/dl) Sodium (mEq/liter) Potassium (mEq/liter) Chloride (mEq/liter) Calcium (mg/dl) Phosphorus (mg/dl) Alanine aminotransferase (IU/liter) Aspartate aminotransferase (IU/liter) Alkaline phosphatase (IU/liter) Lactate dehydrogenase (IU/liter) Sorbitol dehydrogenase (IU/liter) Protein, total (gm/dl) Albumin (gm/dl) Cholesterol (mg/dl) Triglycerides (mg/dl) Bilirubin, total (mg/dl) Uric acid (mg/dl) Globulin (mg/dl) Carbon dioxide (mmol/liter)

99-135 11-25 0.3-0.8 152-164 4.1-5.2 118-126 7.5-9.9 4.8-7.6 78-149 57-248

Female b 104.9 33.3 0.40 150.4 4.90 117.1 9.0 6.70 150.3

___ 16.4 ___7.6 ___0.10 +__ 1.50 ___0.30 ___ 1.90 _+ 0.30 _ 0.60 ___49.3

ND c

31-66

44.3 ___ 11.3

221-752

ND

ND

2.6 __. 2.2

5.0-6.8 3.3-4.2 119-209 10-32 0-0.1 0.7-2.7 1.8-3.1 16-28

6.0 __. 0.5 3.8 _ 0.2 174.0 +__43.5 ND ND ND ND ND

Male b 104.0 22.0 0.40 154.4 4.90 112.5 9.5 6.70 157.6

+__ 15.0 ___6.3 -4- 0.10 ___3.60 ___0.20 ___9.10 +__0.60 ___ 1.20 ___79.9

101.0 ___35.25 52.4 ___ 11.6 434 ___ 113.5 5.4 ___4.5 5.9 3.7 156.0 18.5 0.55

+__0.3 _ 0.1 ___37.0 ___5.1 ___0.225 ND ND ND

aCombined ranges (Fox et al., 1986b). bFour- to 8-month-old ferrets (Loeb and Quimby, 1999). CND, Not done.

absence of careful analysis, however, it is uncertain whether the proportion and quantity of ingredients in these diets is optimal. Ferrets are strict carnivores with a high requirement for dietary fat and protein. Their short digestive tract and rapid gastrointestinal transit time ( 3 - 4 hr) require protein to be readily digestible. There is general agreement that ferrets should not be

Table V Urine Analytes of Normal Ferrets Urine analyte Volume pH Protein Sodium Potassium Chloride

Units

Female a

Male a

ml/24 hr

8-140 6.5 -7.5 0-32 0.2-5.6 0.9-5.4 0.3 -7.5

8 - 48 6.5 -7.5 7 -33 0 . 4 - 6.7 1.0- 9.6 0.7 - 8.5

mg/100 ml mmol/24 hr mmol/24 hr mmol/24 hr

~Four- to 8-month-old ferrets (Loeb and Quimby, 1999).

given diets high in complex carbohydrates or fiber. Diets that are high in fish products are also not recommended for ferrets (Fox and McLain, 1998). The use of any raw chicken, beef, or other meats is strongly discouraged because of the potential contamination by Campylobacter, Salmonella, Listeria, Mycobacterium, and Streptococcus (Fox, 1998a). Daily maintenance energy consumption for ferrets is 200-300 kcal/kg body weight. Calorie-percent protein ratios have been determined for mink (Mustela vison) kits up to and after 16 weeks of age (Sinclair et al., 1962; Allen et al., 1964). A ratio of 13 and a caloric density of 550 kcal/100 gm of feed, corresponding to 42% protein, provided optimum growth for male kits up to 16 weeks. After 16 weeks, ratios of 17 and 21, corresponding to 36% and 26% protein, respectively, were recommended. Diets containing 9-28% fat and 22-42% carbohydrate have been used successfully to maintain ferrets. One author recommends 3 0 - 4 0 % protein and 18-20% fat for adult, nonbreeding animals and a minimum of 35% protein and 25% fat for reproductively active animals and those that have notreached sexualmaturity (Brown, 1997a). The long-term impact of diets containing high levels of fat and protein are unknown. Ferrets have been used to investigate the absorption, metabolism, and interaction of the dietary micronutrients [3-carotene and vitamin E. Ferrets, like humans, convert [3-carotene to vitamin A in the gut and absorb ~-carotene intact (Fox and McLain, 1998). In intestinal perfusion experiments in ferrets, it was demonstrated that [3-carotene, retinol, and retinyl esters are absorbed intact into lymph and that cleavage products, including [3-apo-12'-carotenal, [3-apo-10'-carotenal, and retinoids, accumulate in the intestinal mucosa (Wang et al., 1992). The intestinal mucosa is capable of converting [3-carotene into retinoic acid and other polar metabolites, which are then transported via the portal vein to the liver (Wang et al., 1993). [3-Carotene absorption is enhanced by co-perfusion with a-tocopherol, and the perfusion of the latter is unaltered by the presence of [3-carotene. The conversion of [3-carotene into retinol is also enhanced by the presence of a-tocopherol (Wang et al., 1995). These and other findings have established the ferret as an important model for the study of these antioxidants. Adult ferrets drink 75-100 ml of water daily, depending on the dry-matter content of the feed (Andrews and Illman, 1987). Fresh water can be provided ad libitum in stainless steel bowls or water bottles with sipper tubes. Ferrets are playful and will overturn bowls or water bottles that are not well secured.

D. Reproduction 1.

Reproductive Physiology

Features of ferret reproduction may be found in Table VI. Female ferrets are seasonal breeders and induced ovulators. The season under natural illumination in the Northern Hemisphere is from March to August for females and from December to July

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Table VI Ferret Reproductive Data a Parameter Age at puberty Female (adult, range 750-1500 gm) Male (adult, range 1500 -2500 gm) Minimum breeding age Estrous cycle b Duration of estrous cycle Type of ovulation Ovulation time Number of ova Copulation time Sperm deposition site Ovum transit time Viability of sperm in female tract Cleavage to formation of blastocoele Implantation Gestation period Implantation -parturition Litter size Size at birth Return to estrus Solid food eaten Breeding life of female Breeding life of male Breeding habits

Value

6-12 months 6-12 months 8-12 months (male); 4 - 5 months (female) Monestrus, March through August c Continuous until intromission Induced by copulation 3 0 - 4 0 hr after mating 12 (range, 5-13) Up to 3 hr Posterior os cervix 5 - 6 days 36-48 hr Uniform rate 12-13 days 42___ 1 days 30 ___ 1 days 8 average (range, 1-18) 6-12gm Next March, b occasionally postpartum estrus 3 weeks, before eyes are open 2-5 years 5+ years One male to several females; in colony production

aAdapted from Fox and Bell, 1998, Growth, Reproduction and Breeding. bDependent on photoperiod. cpolyestrous in this period if a litter is produced.

for males, corresponding temporally to increasing day length. Ferrets born in the late spring or early summer and maintained under natural lighting will not assume an adult pattern of gonadal activity (i.e., puberty) until the following season (Baum, 1998). Under artificial illumination, jills that are maintained at 8 hr light-16 hr dark reach puberty at 10-12 months. Stimulatory photoperiods may be used, however, in the laboratory or intensive production setting, as a method of breeding ferrets out of the natural season. However, the transfer from short to long photoperiods should not occur prior to 90 days of age, because jills that are prematurely transferred will remain anestrous (Hammond and Chesterman, 1972). Management practices in one breeding facility are such that jills commence breeding at 7-10 months, average 3.7 litters a year, and are cycled out of reproduction after 6 litters. In another strategy, ferrets are exposed to a 16:8 hr photoperiod at 12 weeks of age, are bred at 16 weeks during their first estrus, and whelp at 5V2 months. Vulvar swelling is the hallmark of estrus in jills. The ease with

which estrus is detected in the ferret, as well as the size of the ferret and ease of its maintenance in captivity have made the ferret a model for study of neuroendocrine events and their gonadal correlates. Along with the hamster, the ferret has contributed extensively to an understanding of the photoperiodic influences on the hypothalamic-pituitary-gonadal axis (Baum, 1998). As in females of other species, estradiol concentrations are responsible for controlling the development of the female reproductive tract and secondary sexual characteristics, and the tonic inhibition of luteinizing hormone (LH) secretion by the anterior pituitary during both prepubertal life and anestrus. The sensitivity of the hypothalamic gonadostat to negative feedback inhibition by estradiol changes at the time of puberty, and under the influence of increasing light exposure, LH levels rise despite estradiol (Ryan, 1984). Similarly, age differences in the sensitivity of negative feedback inhibition of the hypothalamic secretion of gonadotropin-releasing hormone (GnRH) by testosterone, or to estrogenic compounds derived from the aromatization of testosterone, appear to be essential in determining puberty and seasonality of reproduction in the male (Baum, 1998). 2.

Detection of Estrus and Pregnancy

Estrus in jills is characterized by dramatic vulvar swelling from an anestrous diameter of 5-16 mm to an estrous diameter of 17-33 mm. Changes in vaginal cytology have also been described for the ferret and other mustelid species, but these changes are seldom used to determine onset of estrus or to schedule breeding (Williams et al., 1992). After a 2- to 3-week proestrus, estrus occurs. Estrus onset is not associated with elevated serum FSH in the ferret, as it is in the rodent. Once estrus has occurred, it may terminate in coitus-induced ovulation and pregnancy, pseudopregnancy after infertile mating, pharmacologic termination (by injection of human chorionic gonadotropin (hCG) or GnRH), death due to estrogen-induced aplastic anemia, or spontaneous remission and anestrus due to reduced photoperiod. Waves of follicular development occur in estrus, and 5-13 ova are ovulated approximately 3 0 - 4 0 hr after coitus. Female ferrets are brought to the male approximately 14 days after vulvar enlargement. Females and males copulate many times and for prolonged periods of time; they are typically left together for 2 days. Both intromission and neck restraint by the male are apparently required for induction of ovulation (Baum, 1998). An LH surge accompanies coitus in females, but the same is not true of males (Carroll et al., 1987). Implantation occurs 12 days after mating; both a functional corpus luteum and the anterior pituitary are required for implantation and maintenance of pregnancy. Placentation is typical of carnivores and is zonary and endotheliochorial (Morrow, 1980). Pregnancy may be detected by ultrasonographic demonstration of 3 - 5 discrete nonechogenic structures as early as day 12 (Peter et al., 1990), by palpation as early as day 14, or by radiographic demonstra-

13. BIOLOGY AND DISEASES OF FERRETS tion of calcified fetal skeletons at approximately 30 days of gestation. 3.

Husbandry Needs

Jills within 2 weeks of parturition should be singly housed and provided with a secluded place in which to deliver their kits. When rabbit cages are used for housing, nest boxes may take the form of polypropylene rat cages or other plastic boxes (cat litter box or dishpan). Nest boxes should have bedding provided for warmth and comfort. Materials suitable for bedding include pieces of fabric (towels), ripped cageboard, shredded paper, or cotton batting. The nest box should be at least 6 inches deep and should prevent the kits from wandering from the jill. Entrance to the nest box should be smooth, to avoid injury to the teats and mammary gland. At our institution, jills are provided a stainless steel rectangular box with a smooth-surfaced plastic entrance (Fig. 1). A retractable steel roof panel and a guillotine side panel exposing a Plexiglas sidewall allow access to the jill and permit observation with minimal disturbance. One major supplier of ferrets uses sunken tubs filled with bedding to promote a sense of security and isolation of the jill. Most jills will leave the nest box to eat and drink. If the jill will not leave, however, low-sided food bowls should be placed within the nest box. 4.

Parturition

Parturition occurs rapidly in ferrets and may last as little as 2 3 hr. Primiparous jills typically deliver on day 41 of gestation whereas multiparous jills deliver on day 42. There are few signs

Fig. 1.

489

of impending parturition, although abdominal enlargement and mammary development do occur in the last week or two. Small litters (fewer than three) may result in inadequate stimulus for parturition. Jills that pass their due date without delivery should be palpated for fetuses. Kits remaining in utero beyond the 43rd day typically die; kits with congenital malformations such as cyclopia and exencephaly may also delay the initiation of labor. Dystocia is common in ferrets because of positional abnormalities and fetal oversize and should be treated by cesarean section. Jills tolerate cesareans well and will nurse kits delivered in this way. If small littel: size is responsible for delayed parturition, prostaglandins (0.5-1.0 mg Lutalyse) may be used, followed by 0.3 ml oxytocin (6 U) after 3 hr (Fox and Bell, 1998). Failure to deliver within 8 hr of administration of prostaglandin is an indication for cesarean section. Jills should be provided heat, energy, hydration, and analgesia following cesarean. Kits will attempt to nurse soon after parturition, but jills experiencing difficult labor may not allow them to nurse until all kits are delivered. Jills that are not attentive to their kits should be palpated for the presence of additional, undelivered kits. Oxytocin may be used to facilitate delivery of remaining kits. Offering the jill regular chow mixed with warm water may promote maternal acceptance. Kits should be kept warm pending acceptance by the jill. Jills should be left undisturbed for the first several days postpartum to avoid their cannibalizing the litter. Cross-fostering to other jills may be successfully accomplished, provided that the kits are warm and that the foster jill has kits of similar age. Kits to be fostered should be allowed to mingle with the foster jill's own kits while their dam is absent so that rejection due to olfaction will not occur.

Ferretnesting box. Top and side panels allow inspection without disturbing the jill.

ROBERT P. MARINIET AL.

490 5.

Early Development of the Newborn

Kits are born in an altricial state, covered by lanugo hair and with their eyes closed. By 3 days of age, albino ferrets retain their white hair whereas pigmented ferrets acquire a gray coat. They are completely dependent on the jill for the first 3 weeks of life. Defecation and urination are stimulated by jills through anogenital licking of the kits. Kits are born weighing 6 - 1 2 gm, double their weight in 5 days, and triple it in 10 days to a weight of 30 gm. The 3-week-old male kit should weigh at least 100 gm. Sexual dimorphism in size is apparent by week 7 and persists into adulthood. Developmental landmarks include ability to hear at 32 days, opening of the eyes at 34 days, eruption of deciduous teeth at 14 days, eruption of permanent canines at 47-52 days, and displacement of deciduous canines by 56-70 days (Fox and Bell, 1998). 6.

Sexing

Gender may be distinguished in neonatal ferrets, as in other species, by anogenital distance, with the distance being much shorter in females than in males. In males, the urogenital opening is seen just caudal to the umbilicus. The prominent midline raphe penis overlying the palpable os penis is also a distinctive feature in the male.

can be given at this time. Jills return to estrus approximately 3 weeks after reinstitution of the longer photoperiod. 8.

Artificial Insemination

Artificial insemination is not commonly performed in ferrets but has been studied in the context of providing strategies for species perpetuation of the endangered black-footed ferret (Wildt et al., 1989). 9.

Synchronization

Synchronization of estrus as practiced in rodent production is not used as a tool of reproductive management in the ferret. Synchronization ofjills may be approximated, however, by manipulation of photoperiod. With natural illumination in outdoor housing, jills all come into estrus within a 1- to 2-week period (Baum, 1998). In the laboratory setting, when jills are maintained in a nonstimulatory photoperiod (8 hr light-16 hr dark) for 6 - 8 weeks, followed by reversal of the cycle (16 hr light- 8 hr dark), estrus will follow in 4 weeks (immature jills) or 3 weeks (mature jills) after the change (Carroll et al., 1985). This correlates with follicular development and increased plasma estradiol. Iii.

7.

DISEASES

Weaning

Ferrets are typically weaned at 6 weeks of age. Early weaning may be encouraged by making a slurry of the jill's chow available at 3 - 4 weeks; fat may be added to achieve a fat content of 30%. The fatty acid supplement Linatone (Lambert Kay, Cranberry, New Jersey) is recommended by one author (Brown, 1997a). The diet should contain approximately 30% fat and 40% protein. The slurry should be fed twice daily for a restricted time and then removed to avoid having kits walking through and defecating in the diet. Unthrifty kits over 14 days of age may be supplemented with canine or feline milk replacers administered per os by Tygon-tipped Pasteur pipette (Manning and Bell, 1990a). Weaned ferrets are best housed in groups until sexually mature. Males over 12 weeks old may begin to fight if exposed to greater than 12 hr light per day. Jills may return to estrus during the second or third week of lactation if they have fewer than 5 kits or 2 weeks after weaning if the litter is of normal size. Jills should be rebred or administered hCG to terminate estrus, even if still lactating. A highquality, calorie-dense diet is required for lactation and to maintain pregnancy. If maintained on a stimulatory photoperiod and adequate nutrition, jills may have 2 - 3 liters of 6 or more kits yearly until they are 5 years old (Fox and Bell, 1998). A nonstimulatory photoperiod should be used 6 weeks per year to rest the ferret and preserve maximum fertility; a maintenance diet

A. 1.

Infectious Diseases

Bacterial Infections

The occurrence of infectious disease affects animal health and well-being and may complicate research efforts. A program combining good animal husbandry, optimal nutrition, health monitoring practices, and clinical care is essential to maintaining a healthy ferret colony. a.

Clostridium perfringens Type A

Etiology. The etiologic agent is Clostridium perfringens type A (Clostridium welchii). Epizootiology and transmission. Clostridium perfringens is ubiquitous and is present in the intestinal contents of humans and animals. Clostridium perfringens type A has been associated with the occurrence of acute abdominal distension, dyspnea, and cyanosis in weanling ferrets (Field and Laboratory Service Veterinary Staff, 1984) and an outbreak of gastroenteritis in weanling black-footed ferrets (Schulman et al., 1993). The exact cause of these conditions is uncertain, but predisposing factors such as overeating, sudden changes in diet, the prolifer-

13. BIOLOGY AND DISEASES OF FERRETS

ation of C. perfringens type A, and the production of overwhelming amounts of toxins are suspected (Field and Laboratory Service Veterinary Staff, 1984; Schulman et al., 1993). The alpha toxin is the principal lethal toxin. It is hemolytic and necrotizing and possesses the ability to split lecithin or lecithinprotein complexes, leading to destruction of cell membranes and subsequent necrosis. Reported cases have involved weanling animals exclusively.

Clinical signs. Ferrets may present with acute abdominal distension, dyspnea, and cyanosis or may be found dead and bloated (Field and Laboratory Service Veterinary Staff, 1984; Schulman et al., 1993). Diagnosis. Isolation of C. perfringens type A from gastric and small-intestinal contents is required. Toxin identification may be performed by the use of a mouse protection assay (Smith, 1975). Necropsy findings. Gross findings include markedly distended stomachs and intestines containing a large amount of gas and a moderate amount of brown, semiliquid ingesta, and subcutaneous emphysema with minimal or no putrefaction (Field and Laboratory Service Veterinary Staff, 1984; Schulman et aL, 1993). Histologic findings observed in weanling black-footed ferret cases included the observation of abundant gram-positive bacilli in smears of gastric and intestinal contents. Other findings included varying degrees of gastrointestinal mucosal necrosis, numerous gram-positive bacilli lining the denuded mucosal surface and extending into the gastric glands and intestinal crypts; lymphoid necrosis of lymph nodes, spleen, and thymus; mild to moderate dilatation of central hepatic sinusoids with mild, acute, centrilobular hepatocellular dissociation and multifocal aggregates of small numbers of necrotic neutrophils within portal areas (Schulman et al., 1993). Treatment and control. Prevention through good management and feeding practices is the primary means of control. In the reported cases of C. perfringens type A-associated gastroenteritis in black-footed ferret weanlings, supportive care and gastric trocharization were unrewarding. The occurrence of the condition was eliminated by restricting feeding of weanlings to twice a day instead of 3 times daily. b.

Campylobacteriosis

Etiology. Campylobacteriosis is caused by infection with Campylobacter jejuni. Epizootiology and transmission. Campylobacter jejuni is a gram-negative, spirally curved microaerophilic bacterium that is recognized as a significant cause of human enteritis and is as-

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sociated with diarrheic illness in several animal species, including dogs, cats, cows, goats, pigs, mink, ferrets, and sheep (Carter et al., 1995). It also known to cause mastitis in cows, infectious hepatitis of chickens, and abortion in cattle, sheep, goats, dogs, and mink (Carter et al., 1995). The organism may also be cultured from the feces of normal asymptomatic dogs, cats, and ferrets (Fox et al., 1983; Carter et al., 1995). Transmission occurs by ingestion of organisms through direct contact with feces or contaminated food and water (Carter et al., 1995). There have been reports linking the disease in humans to pets. Many of these outbreaks were associated with dogs, puppies, and kittens recently obtained from animal shelters or pounds and displaying diarrhea before the human illness occurred (Fox et al., 1983). Isolation of Campylobacter jejuni from asymptomatic ferrets also implies a potential for zoonotic transmission (Fox et al., 1982, 1983).

Clinical signs. Experimental oral inoculation of ferret kits with various strains of C. jejuni produced a self-limiting diarrhea that ranged in character from very mild to watery (Fox et al., 1987; Bell and Manning, 1990a, 1991). The presence of mucus and/or blood was also noted in the feces of affected animals. Anorexia, dehydration, and tenesmus with watery diarrhea were also observed. Intravenous inoculation of 4 pregnant mink and 7 pregnant ferrets resulted in reproductive failure, ranging from fetal resorption to expulsion of dead or premature living kits (Bell and Manning, 1990b). Oral inoculation resulted in abortion in a majority of the infected animals (Bell and Manning, 1990b). Diagnosis. Diagnosis is based on history, clinical signs, and culture of affected animals. Reports of spontaneous cases in ferrets require diagnostic confirmation and differentiation from cases of proliferative bowel disease and other infectious and noninfectious causes of diarrhea. Campylobacter jejuni grows slowly and has specific culture requirements that involve the use of selective media or filtration techniques, and a requirement for thermophilic (42~176 and microaerophilic conditions (Fox, 1998a). Cultures should be examined every 48 hours for round, raised, translucent, and sometimes mucoid colonies (Fox, 1998a). Necropsy findings. Studies involving oral inoculation of ferrets with Campylobacter jejuni revealed small focal neutrophilic infiltrates in the lamina propria of the colon of relatively few infected animals (Fox et al., 1987). Bell and Manning (1991) noted mild to moderate enterocolitis with neutrophilic infiltration of the lamina propria, which was most severe in kits with concurrent cryptosporidiosis. Placentitis was the most notable histologic finding in pregnant ferrets and mink after experimental inoculation of a strain of an abortion storm-associated isolate of C. jejuni (Bell and Manning, 1990b).

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Treatment and control. Erythromycin is the drug of choice for treatment of human campylobacteriosis (Fox, 1998a). In a study to eliminate the carrier state in ferrets, erythromycin was ineffective even though in vitro isolates of C. jejuni were sensitive to the antibiotic (Fox et al., 1983). According to the author, reasons for therapeutic failure included dose selection, interspecies differences in pharmacokinetics and possible reinfection. Supportive care should be instituted, and choice of antibiotic therapy in confirmed diarrheic cases should be based on culture and sensitivity. In addition, because of its zoonotic potential, isolation of affected animals and good hygienic practices are recommended. Reculture of animals after treatment to ensure elimination of the organism is recommended.

c.

Helicobacter mustelae

Epizootiology and transmission. In 1985, a gastric Helicobacter-like organism was isolated from the margins of a duodenal ulcer of a ferret and named Helicobacter mustelae (Fox et al., 1986a, 1989a). Subsequently, in the United States, gastritis and peptic ulcers have been routinely reported in ferrets colonized with H. mustelae (Fox et al., 1988b, 1991a). Every ferret with chronic gastritis is infected with H. mustelae, whereas specific pathogen-free (SPF) ferrets not infected with H. mustelae do not have gastritis, gastric ulcers, or detectable IgG antibody to the organism (Fox et al., 1990, 1991a). Helicobacter mustelae has also been isolated from the stomachs of ferrets living in England, Canada, Australia and, most recently, from ferrets in New Zealand (Forester et al., 2000; Tompkins et al., 1988). Koch's postulates have been fulfilled: by oral inoculation of H. mustelae into naive ferrets uninfected with H. mustelae, the infection induced a chronic, persistent gastritis similar to that observed in ferrets naturally infected with H. mustelae (Fox et al., 1991b). It is now known that H. mustelae colonizes nearly 100% of ferrets shortly after weaning. Feces from weanling and adult ferrets have been screened for the presence of H. mustelae to determine whether fecal transmission could explain the 100% prevalence observed in weanling and older ferrets (Fox et al., 1988b, 1992b). Helicobacter mustelae was isolated from the feces of 8 of 74 nine-week-old and 3 of 8 eight-month-old ferrets. Ferrets placed on proton pump inhibitors, which raise gastric pH, have a statistically higher recovery of H. mustelae from feces when compared with age-matched untreated control ferrets (Fox et al., 1993). Clinial signs and pathology. Helicobacter mustelae-infected ferrets examined in our laboratory are usually asymptomatic. Ferrets with gastric or duodenal ulcers can be recognized clinically by vomiting, melena, chronic weight loss, and lowered hematocrit. Clinical signs in ferrets with H. mustelae-associated

gastric adenocarcinoma have consisted of vomiting, anorexia, and weight loss, signs that may be confused with gastric foreign body. Diagnosis. Gastric and duodenal ulcers are observable endoscopically. It is interesting that the ferret is the only domesticated animal to date that has naturally occurring Helicobacterassociated ulcer disease. The H. mustelae isolated from ferrets has similar but not identical biochemical features to those of H. pylori, particularly in regard to the production of large amounts of urease. Gastric samples collected by endoscopy or necropsy are minced with sterile scalpel blades and inoculated onto blood agar plates supplemented with trimethoprim, vancomycin, and polymixin B (Remel, Lenexa, Kansas). The plates are incubated at 37~ or 42~ in a microaerobic atmosphere (80% N2, 10% H2, and 10% CO2) in vented jars for 3 - 7 days. Bacteria are identified as H. mustelae on the basis of Gram-stain morphology; production of urease, catalase, and oxidase; resistance to cephalothin; and sensitivity to nalidixic acid. Necropsy and findings. The histopathological changes occurring in the stomach closely coincided in topography with the presence of H. mustelae (Fox et al., 1990). A superficial gastritis present in the body of the stomach showed that H. mustelae was located on the surface of the mucosa but not in the crypts. Inflammation occupied the full thickness of the distal antral mucosa, the so-called diffuse antral gastritis described in humans (Fig. 2a,b). In this location, H. mustelae was seen at the surface, in the pits, and on the superficial portion of the glands. In the proximal antrum and the transitional mucosa, focal glandular atrophy, a precancerous lesion, and regeneration were present, in addition to those lesions seen in the distal antrum. Also, deep colonization of H. mustelae was observed focally in the affected antral glands. Animals infected with Helicobacter spp. may also be susceptible to gastric cancer (Fox et al., 1994; Yu et al., 1995). There is recent documentation of the presence of argyrophilic bacteria, compatible in location and morphology to H. mustelae, within the pyloric mucosa of 2 male ferrets with pyloric adenocarcinoma (Fox et al., 1997). In humans, epidemiologic data strongly support the association between H. pylori and development of gastric adenocarcinoma. Similarly, we have recently documented a series of H. mustelae-infected ferrets with gastric mucosa-associated lymphoid tissue (MALT) lymphoma that parallels the same syndi'ome found in humans. Lymphoma was diagnosed in the wall of the lesser curvature of the pyloric antrum, corresponding to the predominant focus ofH. mustelaeinduced gastritis in ferrets. Gastric lymphomas demonstrated characteristic lymphoepithelial lesions, and the lymphoid cells were IgG positive in all ferrets (Erdman et al., 1997). These findings and their parallels in H. pylori-infected humans implicate the involvement of H. mustelae in the pathogenesis of gastric cancer in ferrets.

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493

Fig. 2. (a) Diffuse antral gastritis of the Helicobacter mustelae-infected ferret stomach. (b) Helicobacter mustelae organisms colonizing the gastric mucosa (arrowheads, Warthin-Starry stain). (Courtesy of J. G. Fox.)

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Treatment. Studies in ferrets indicate that triple therapy consisting of oral amoxicillin (30 mg/kg), metronidazole (20 mg/ kg), and bismuth subsalicylate (17.5 mg/kg) (Pepto-Bismol original formula, Procter and Gamble) 3 times a day for 3 4 weeks has successfully eradicated H. mustelae (Otto et al., 1990). Clinical improvement, including increased appetite and resolution of melena, may occur within 48 hr of initiation of triple therapy. A new treatment regimen being used to eradicate H. pylori in humans has also been used successfully for eradication of H. mustelae from ferrets (Marini et al., 1999). Ferrets received 24 mg/kg ranitidine bismuth and 12.5 mg/kg clarithromycin per os 3 times daily for 2 weeks. Culture of tissue collected by gastric endoscopic biopsy at 16, 32, and 43 weeks after termination of treatment indicated that long-term eradication was achieved in all 6 ferrets. Eradication was associated with decrease in anti-H, mustelae IgG antibody titers, results that are consistent with findings in humans after H. pylori eradication. Omeprazole in ferrets at an oral dose of 0.7 mg/kg once daily effectively induces hypochlorhydria and may be used in conjunction with antibiotics to treat H. mustelae-associated duodenal or gastric ulcers. Cimetidine at 10 mg/kg TID per os can also be used to suppress acid secretion. Acute bleeding ulcers must be treated as emergencies, and fluid and blood transfusions are essential. d.

that include but are not limited to a complete blood count, chemistry profile, radiographs, and fecal analysis and culture. Necropsy findings. Gross findings include a segmented, thickened lower bowel, usually the terminal colon but occasionally including the ileum and rectum (Fox et al., 1982; Fox, 1998a). Histologic examination consistently reveals marked mucosal proliferation and intracytoplasmic L. intracellularis demonstrated with silver stain within the apical portion of epithelial cells in the hyperplastic epithelial cells (Fox et al., 1982; Fox, 1998a) (Fig. 3a,b). Other common histologic changes observed include the presence of a mixed inflammatory infiltrate that is variable in severity, reduced goblet cell production, hyperplasia of the glandular epithelium, glandular irregularity with penetration of the mucosal glands through the muscularis mucosa, and an increase in thickness of the tunica muscularis (Fox et al., 1982; Fox, 1998a). Translocation of proliferating glandular tissue to extraintestinal sites, including regional lymph nodes and liver, has been described in two ferrets (Fox et al., 1989b). Differential diagnosis. Proliferative bowel disease should be differentiated from other diseases that may cause diarrhea and wasting, including dietary changes, eosinophilic gastroenteritis, gastric foreign bodies, lymphoma, Aleutian disease, and gastric ulcers (Bell, 1997b). A complete physical exam that includes palpation of the abdomen should reveal a palpably thickened intestine in cases of proliferative bowel disease.

Proliferative Bowel Disease

Etiology. Proliferative bowel disease is caused by intracellular campylobacter-like organisms, closely related to Desulfovibrio spp., that are now classified as Lawsonia intracellularis in proliferative enteropathy of swine (Fox, 1998a). The organisms are gram-negative, comma- to spiral-shaped bacteria. Epizootiology and transmission. Proliferative bowel disease is a common clinical disease observed in young ferrets. Fecaloral spread is suspected. The disease typically involves the large bowel, although it has been observed to affect the small bowel (Rosenthal, 1994). Campylobacter species, coccidia, and chlamydia have been isolated from some cases of proliferative bowel disease in ferrets (Li et al., 1996b). The role, if any, of copathogens in this disease is unclear. Clinical signs. Clinical signs include chronic diarrhea, lethargy, anorexia, weight loss (which is often marked), and dehydration. Diarrhea may be blood-tinged, may contain mucus, and is often green in color. Rectal prolapse may be observed in affected animals. Ataxia and muscle tremors have also been observed (Fox et al., 1982). Diagnosis. Diagnosis is based on clinical signs, a palpably thickened colon, and colonic biopsy. It is important to rule out other causes of diarrhea and weight loss through diagnostic tests

Treatment and control. Supportive care, including fluid therapy and nutritional support, should be provided. Treatment with chloramphenicol (50 mg/kg BID PO, SQ, IM) or metronidazole (20 m/kg BID PO) for 2 weeks is reported to be effective (Krueger et al., 1989; Bell, 1997b). Clinical improvement may be apparent within 48 hr. e.

Tuberculosis

Etiology. Tuberculosis can be caused by a variety of Mycobacteria, including Mycobacterium bovis, M. avium, and M. tuberculosis. Epizootiology and transmission. Mycobacteria are aerobic, gram-positive, nonbranching, non-spore-forming, acid-fast rods. Natural infections with Mycobacterium bovis and M. avium have been reported in the ferret. Ferrets are also susceptible to experimental infection with human tubercle bacillus. Most reports of tuberculosis in ferrets are in animals that were used for research in England and the rest of Europe between the years of 1929 to 1953 and were likely related to the feeding of raw poultry, raw meat, and unpasteurized milk to ferrets during this time (Fox, 1998a). The feeding of commercially prepared diets and widespread tuberculosis testing and elimination in livestock and poultry have resulted in the reduced incidence of the disease in ferrets. Mycobacterium avium-infected wild

13. BIOLOGY AND DISEASES OF FERRETS

495

Fig. 3. (a) Proliferativecolitis of the ferret with marked epithelial hyperplasia, mixed inflammatorycell infiltrate, and reduction of goblet cells. (b) Intracytoplasmic microorganismsin hyperplasticcolonic tissue (arrow,Warthin-Starry stain). (Courtesyof J. G. Fox.)

birds shed the organism in feces; prevention of contamination of food and outdoor housing areas of ferrets is warranted. Clinical signs and necropsy findings. Clinical signs and lesions are dependent on the infective strain. Systemic infection with the bovine strain in ferrets results in disseminated disease with weight loss, anorexia, lethargy, death, and miliary lesions involving the lungs and other viscera (Fox, 1998a). Progressive paralysis has also been reported in a case of spontaneously occurring bovine tuberculosis in a ferret (Symmers and Thomson, 1953). Mycobacterium bovis lesions contain numerous acid-fast bacilli within macrophages with little cellular reaction (Fox, 1998a). In contrast, infection of ferrets with the human tubercle bacilli results in localized infection, often confined to the site of injection and adjacent lymph nodes; microscopically few organisms are observed. An impaired cell-mediated response may account for the large number of organisms observed in M. bovis lesions. Vomiting, diarrhea, anorexia, and weight loss were observed in a pet ferret with granulomatous enteritis caused by M. avium

(Schultheiss and Dolginow, 1994). Granulomatous inflammation characterized by large numbers of epithelioid macrophages containing numerous acid-fast bacilli were present in the lamina propria and submucosa of the jejunum and pylorus. Other sites of granulomatous inflammation included peripancreatic adipose tissue, mesenteric lymph nodes, spleen, and liver. A source of infection was not identified in this report. Pulmonary infection with M. avium has also been reported in 3 ferrets in a zoo in France (Viallier et al., 1983). Diagnosis. Definitive diagnosis of tuberculosis requires isolation and identification of the organism from suspect tissue specimens. Great care should be exercised in handling suspect clinical specimens, and an appropriately equipped laboratory should be identified for culture and identification of the organism. Although there has been some experimental work in the area of the intradermal tuberculin skin-test response in ferrets and its apparent use in controlling tuberculosis in a breeding colony of ferrets, a tuberculin skin-testing regimen, including dose and

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type, has not been definitively characterized for clinical use in ferrets (Kauffman, 1981). Treatment and control. Because of the zoonotic risk, ferrets infected with M. bovis and M. tuberculosis should be euthanized (Fox, 1998a). Recurrent M. bovis infection involving the palmar aspect of the wrist of a 63-year-old man, which developed after he was bitten by a ferret at the age of 12, was reported and demonstrates the zoonotic potential (Jones et al., 1993). Mycobacterium avium infection is not reportable but may pose a risk to immunocompromised patients (Fox, 1998a). Personnel at risk should be followed up by a physician for appropriate diagnostic testing (Fox, 1998a). f.

Salmonellosis

Etiology. Salmonellosis is caused by infection with organisms of the genus Salmonella. Epizootiology and transmission. Salmonella is a gram-negative, non-spore-forming, facultative anaerobic rod in the family Enterobacteriaceae (Carter et al., 1995). Infection is by the oral route. Transmission may be direct from infected carrier animals or humans or through contaminated food products or water (Carter et al., 1995). Several Salmonella serovars have been isolated from mink with gastroenteritis and abortion (Gorham et al., 1949). Contaminated raw meat products were suspected as the source in one outbreak. Salmonella typhimurium was isolated in ferrets in an outbreak of clinical disease (Coburn and Morris, 1949) and several serotypes including S. hadar, S. enteritidis, S. kentucky, and S. typhimurium were isolated from the feces of ferrets surveyed in a research colony (Fox et al., 1988a). Clinical signs and necropsy findings. Clinical signs of an outbreak of S. typhimurium in ferrets included conjunctivitis, rapid weight loss, tarry stools, and febrile temperature fluctuations (Coburn and Morris, 1949). Gross findings in 2 ferrets 10 days after inoculation with S. typhimurium of ferret origin included marked tissue pallor, petechiae in the gastric mucosa, and the presence of melena in one and a dark-colored fibrinous exudate in the large intestine of the other ferret (Coburn and Morris, 1949). Studies involving experimental inoculation with S. enteritidis, S. newport, and S. choleraesuis via the oral route to healthy, distemper-infected, and feed-depleted ferrets and mink showed a fairly high resistance to infection (Gorham et al., 1949). Only 2 animals of 29 in the diet-restricted group-- 1 ferret and 1 minknshowed clinical signs of infection after feeding S. newport culture. Signs included lethargy, anorexia, trembling, and fecal blood. The gastrointestinal tract showed a large amount of mucus containing red blood cells; bits of desquamated epithelium and few mononuclear cells overlying the gastric mucosa; an exudate in the small intestine consisting of mu-

coid material, red blood cells, and desquamated small intestinal villi; edematous villi in the ileum; and a diffuse infiltrate of the small intestinal mucosa with lymphocytes and macrophages. Necrotic foci in the liver, spleen, and, less commonly, the kidney, as well as splenomegaly and visceral lymphadenopathy, were observed in chronic fatal infections (Coburn and Morris, 1949). Abortion and gastroenteritis have been reported in mink (Gorham et al., 1949). Diagnosis. Diagnosis is based on history, clinical signs, and isolation of the organism. The organism can be cultured on enrichment and selective media and then characterized serologically. Samples of blood, feces, exudates, tissues, and intestinal material may be cultured. Treatment and control. Coburn and Morris (1949) treated 6 of 12 ferrets experimentally infected with S. typhimurium with sulfathalidine in the feed (Coburn and Morris, 1949). Salmonella typhimurium was isolated in 4 of 6 control animals and none of the treated animals 3 days after the administration of the last dose. Sulfathalidine was administered by the same authors to a colony of 77 ferrets in which an outbreak of salmonella occurred. The group was surveyed 2 days after sulfathalidine treatment and showed weight gain, improvement in condition, and a reduction in the number of Salmonella-infected ferrets (Coburn and Morris, 1949). Salmonella spp. isolated from ferrets may show resistance to a number of antibiotics (Fox, 1998a). Treatment includes appropriate use of antimicrobials and supportive care, which may include fluid therapy, nutritional support, maintenance of electrolyte balance, treatment of concurrent diseases, recognition of and attention to shock, and reduction of stress (Fox, 1998a). g.

Pneumonia

Etiology. Streptococcus zooepidemicus and other group C and G streptococci, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Bordetella bronchiseptica have been reported as primary and secondary bacterial pathogens in pneumonia in ferrets (Fox, 1998a). Epizootiology and transmission. Bacterial pneumonia may occur secondary to megaesophagus in the ferret. An influenza virus-bacteria synergism has been the subject of several studies in ferrets (Fox, 1998a). Debilitated and immunosuppressed animals and animals with concurrent diseases such as influenza may be more susceptible to bacterial pneumonias (Fox, 1998a). Clinical signs. Clinical signs may include nasal discharge, dyspnea, lethargy, anorexia, increased lung sounds, cyanosis, and fever (Rosenthal, 1997). Fulminant pneumonia may progress to sepsis and death (Fox, 1998a).

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Diagnosis. Diagnosis is based on history, clinical findings, a complete blood count, culture and cytology of a tracheal wash or lung wash, and radiographs (Rosenthal, 1997). Differential diagnosis. Diagnostic rule-outs include dilatative cardimyopathy, heartworm disease, mycotic pneumonia, pneumocystis pneumonia in immunosuppressed animals, neoplasia, and influenza. Treatment and control. Treatment should consist of appropriate antimicrobial therapy and supportive care, which may include the administration of oxygen, fluid therapy, and force feeding (Rosenthal, 1997). h.

Abscesses

Etiology. A variety of bacteria have been associated with abscesses and localized infection of the lung, liver, uterus, vulva, skin, mammary glands, and oral cavity. These include Staphylococcus spp., Streptococcus spp., Corynebacterium spp., Pasteurella, Actinomyces, hemolytic Escherichia coli, and Aeromonas spp. (Fox, 1998a). Epizootiology and transmission. Abscesses in ferrets may result from wounds that are inflicted secondary to biting during fighting, playing, mating, or chewing sharp objects. Clinical signs. Localized or subcutaneous abscesses present as swellings with or without draining tracts. The swelling may be fluctuant. In most cases, the abscess is walled off and does not result in systemic signs (Fox, 1998a). Abscesses or infection involving visceral organs may give rise to organ-specific and/or systemic signs. Diagnosis. Cytologic and Gram staining of an aspirate of a suspect subcutaneous swelling will aid in the definitive diagnosis. Culture and sensitivity of the aspirate should also be performed to identify the causative organism and guide appropriate antibiotic therapy. Differential diagnosis. Differential diagnosis of a subcutaneous swelling in a ferret should include myiasis, granuloma, hematoma, and neoplasia. Treatment and control. Prevention of ferrets from exposure to sharp objects in the cage and feed, and limiting the exposure of male and female during breeding, can minimize the occurrence of abscesses. Treatment of localized abscesses should include appropriate antibiotic therapy and establishment of drainage and debridement if necessary. Bacterial culture and sensitivity of the exudate should be performed. A broad-spectrum antimi-

crobial may be used pending results of culture and sensitivity (Orcutt, 1997).

i.

Mastitis

Etiology. Gram-positive cocci such as Streptococcus spp., Staphylococcus aureus, and coliforms such as hemolytic E. coli are the most frequently associated organisms (Bernard et al., 1984; Bell, 1997a). Epizootiology and transmission. Although the exact pathogenesis of mastitis in ferrets is not clear, a number of factors may play a role and include the stress of lactation, injury to mammary glands by the kits' teeth, environmental contamination, and the virulence of the organism. In one report, the causative organism, hemolytic E. coli, was cultured from the feces of mastitic and healthy ferrets and the oral cavity of suckling kits (Liberson et al., 1983). The high level of perineal contamination and the presence of the organism in the oral cavity of suckling kits may enhance transmission and introduction of this organism into mammary tissue. In another outbreak, the causative organisms were cultured from bovine meat fed prior to the outbreak, and the meat was suspected as a possible source. Clinical signs. Mastitis occurs in nursing jills and has been characterized as acute or chronic (Bell, 1997a). The acute form is reported to occur soon after parturition or after the third week of lactation. Examination of affected jills reveals swollen, firm, red or purple, and painful glands. Affected glands may quickly become gangrenous. The chronic form, which may occur when kits are 3 weeks old or as a sequela to the acute form, is characterized by glands that are firm but not painful or discolored. Diagnosis. Diagnosis is based on history, clinical signs, physical examination findings, and isolation of the causative organism. Necropsy findings. In acute mastitis, grossly affected glands are swollen, and the skin overlying the gland may be discolored. Surgical biopsies and necropsies of 8 ferrets with mastitis caused by hemolytic E. coli (Liberson et al., 1983) revealed extensive edema, hemorrhage, and coagulative and liquefactive necrosis involving the glandular tissue as well as surrounding subcutaneous tissue. Other findings included the presence of a mixed leukocytic infiltrate composed primarily of polymorphonuclear leukocytes; large numbers of bacteria; and thrombosis and necrosis of vessels within and immediately adjacent to areas of inflammation (Liberson et al., 1983). In an outbreak of mastitis in mink due to Staphylococcus aureus and Escherichia coli, histologic examination of affected glands revealed an acute suppurative mastitis with desquamation

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of alveolar epithelium, edema of the connective tissue stroma, alveoli filled with neutrophils and cellular debris, and lactiferous ducts filled with purulent exudate and mats of bacteria within lobules (Trautwein and Helmboldt, 1966). Treatment. Broad-spectrum antibiotic therapy may be instituted pending culture and sensitivity results of the milk. Enrofloxacin (2.5 mg/kg BID PO after a loading dose of 5.0 mg/kg IM) is often effective. Jills may require aggressive care, because acute mastitis may progress rapidly and animals may become septicemic and moribund (Liberson et al., 1983). Oral antibiotic administration to kits nursing affected jills is recommended (Bell, 1997a). Surgical resection and debridement of affected glands and supportive care may be necessary for jills with acute mastitis. Supplementation of kits with milk replacer may also be necessary, because jills with acute mastitis are reluctant to nurse, and jills with the chronic form have diminished lactation as milk-producing tissue is replaced by scar tissue (Bell, 1997a). Maintaining thorough personal hygiene practices when handling affected jills is important in minimizing spread to other lactating jills. Cross-fostering kits may be required; however, kits may spread infection to healthy jills. It is reported that jills with the chronic form of mastitis should be culled (Bell, 1997a).

2.

Viral Infections

a.

Canine Distemper

Etiology. Canine distemper (CD) is caused by a paramyxovirus of genus Morbillivirus that is related to measles and rinderpest (Budd, 1981). There are several strains, including a ferret-adapted strain of canine distemper virus (CDV), that vary in incubation, clinical signs, and duration (Fox et al., 1998b). The virus can be inactivated by heat, light, and various chemicals, including phenol, Roccal, sodium hydroxide, and formalin (Shen and Gorham, 1980; Budd, 1981). Infectious virions have been recovered from fomites after 20 min at room temperature. Canine distemper is the most serious viral infection of ferrets. Mortality approaches 100%, making appropriate husbandry and vaccination imperative. The disease has a catarrhal phase and a neurological, or central nervous system (CNS), phase. The catarrhal phase is 7-10 days postinfection and involves anorexia, pyrexia, photosensitivity, and serous nasal discharge. An erythematous pruritic rash spreads from the chin to the inguinal region. It is suspected that the rash results from cell-mediated immunity to infected endothelial cells, similar to the response seen in humans with measles (Norrby and Oxman, 1990). Hyperkeratosis of footpads, called hard pad, is an inconsistent feature. Secondary bacterial infections result in mucopurulent ocular and nasal discharge and possibly bacterial pneumonia. The CNS phase, with ataxia, tremors, and paralysis, may or may not be preceded by

the catarrhal phase. Death occurs in 12-16 days from ferret strains of CDV and up to 35 days with canine strains. Infection is uniformly fatal. Epizootiology and transmission. Virus is shed from infected hosts from conjunctival, nasal, and oral exudates, urine, feces, and sloughed skin (Gorham and Brandly, 1953). Transplacental infection is not reported in ferrets. Attenuated CDV vaccine strains have not been recovered from the body secretions of ferrets following vaccination (Shen et al., 1981). Unvaccinated dogs and other canids, mustelids, and procyonids may serve as reservoirs of infection. Viremia is detectable 2 days postinfection and persists until the ferret dies or mounts a neutralizing antibody response (Liu and Coffin, 1957). The primary site of replication is the respiratory and lymphatic systems, and CDV has been recovered from the nasal secretions of ferrets 5-13 days postinfection. A decrease in lymphocyte subsets is detectable 5 - 3 0 days postinfection. Clinical signs and necropsy findings. Histologically, intracytoplasmic and intranuclear inclusion bodies may be observed in tracheal, bronchial, epithelia, and bile duct as well as transitional epithelium in the bladder (Liu and Coffin, 1957) (Fig. 4). The eosinophilic (hematoxylin-eosin) inclusions appear orange using Pollack's trichrome stain. Diagnosis and differential diagnoses. Presumptive diagnosis is based on clinical observation, questionable vaccination history, and exposure. A fluorescent antibody test can be used on peripheral blood and conjunctival mononuclear cells to detect infection. Reverse transcriptase-polymerase chain reaction (RT-PCR) has also been used to detect experimental infection (Stephensen et al., 1997). Differential diagnoses should include infection with influenza virus or Bordetella bronchiseptica. Influenza does not rapidly progress to mucopurulent ocular and nasal discharge as CD does. Treatment and control. During an outbreak, clinically affected ferrets should be isolated and the remainder of the colony vaccinated. Distemper infection can be prevented by vaccination with modified live vaccine of chicken embryo tissue culture origin (CETCO) administered subcutaneously or intramuscularly. Kits should be vaccinated every 2 - 3 weeks, starting at age 6 weeks, until 14 weeks and annually thereafter (Fox et al., 1998b). It is important to adhere to the prescribed vaccination protocol, because ferret deaths have been reported following double-dose vaccination (Carpenter et al., 1976). Inactivated distemper vaccines do not elicit consistent, effective immunity and are not recommended. It is important to know the vaccination schedule of your ferret supplier and to vaccinate supplementally as appropriate. New ferrets should be held in quarantine for 2 weeks prior to introduction into the resident colony.

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13. BIOLOGY AND DISEASES OF FERRETS

Fig. 4. Intracytoplasmic(curved arrow) and intranuclear (arrowhead)inclusionbodies of canine distempervirus in the bile duct epitheliumof a ferret.

Ferrets have been experimentally infected with feline panleukopenia, canine parvovirus, canine parainfluenza virus, mink enteritis virus, respiratory syncytial virus, transmissible mink encephalopathy, and pseudorabies, but natural infection with these viruses has not been reported (Fox et al., 1998b). b.

Aleutian Disease

Etiology. Aleutian disease virus (ADV) is a parvovirus with strains of varying virulence and immunogenicity. Mink-derived strains are more virulent to mink than are ferret-derived strains (Fox et al., 1998b). Epizootiology and transmission. Aleutian disease (AD) is a chronic progressive illness that was first described in mink (Oxenham, 1990). It was originally named hypergammaglobulinemia (HGG) because of this remarkable finding. Infection may be subclinical for years. Because the immunomodulation associated with ADV infection is disruptive to biomedical research, it is important to seek sources of ADV-free ferrets (Fox et al., 1998b). Transmission between ferrets may be direct or via aerosol of urine, saliva, blood, feces, and fomites (Kenyon et al., 1963; Gorham et al., 1964). Vertical transmission is established in mink but is unproven in ferrets. Clinical signs. Ferrets infected with ADV as adults develop persistent infection but rarely disease, although chronic progressive weight loss, cachexia, malaise, and melena have been described (Porter et al., 1982). AD may also cause ataxia, paral-

ysis, tremors, and convulsions (Oxenham, 1990; Welchman et al., 1993). The lesions are typically immune-mediated, and there is elevation of the gammaglobulins to generally greater than 20% of the total proteins (Porter et al., 1982; Fig. 5). The precise mechanism of immunomodulation is unknown, but in mink there is depression of B- and T-cell responses. Necropsy. Ferrets may have no lesions upon necropsy, or infrequently they may have hepatosplenomegaly and lymphadenopathy. The most consistent histological finding is periportal lymphocytic infiltrates (Fig. 6). Bile duct hyperplasia and periportal fibrosis have also been reported. Membranous glomerulonephritis has been described (Ohshima et al., 1978). Although lesions are subtle, use of ADV-infected ferrets in biomedical research is contraindicated because histological lesions interfere with the interpretation of study results (Fox et al., 1998b). Diagnosis and differential diagnoses. Presumptive diagnosis is based on HGG and chronic weight loss. Diagnosis is confirmed by immunofluorescent antibody (IFA) or counterimmunoelectrophoresis (CIEP) for antibody to ADV antigen (Palley et al., 1992). PCR-based assays have also been used (Erdman et al., 1996b; Saifuddin and Fox, 1996; Erdman et al., 1997). Differential diagnoses include the neurotropic form of CD, as well as chronic wasting diseases such as neoplasia, malabsorption, maldigestion, and bacterial enteritis (Fox et al., 1998b). Treatment and control. Vaccination against ADV would be contraindicated because of the immune-mediated reaction, and a vaccine is not available. Chemical disinfection may be achieved

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Diagnosis. Diagnosis is based on typical clinical presentation and recovery within 4 days, unlike with CD, which progresses to more severe disease and death. Hemagglutination inhibition antibody titers on acute and convalescent sera are rarely needed. Treatment and control. Antibiotic therapy may be instituted to preclude secondary bacterial infection. Animal technicians and investigators suffering from influenza should avoid contact with ferrets. Ferrets have been used extensively as a model for influenza research because the biological response to infection is similar to that in humans (Fox et al., 1998b). Ferrets have been used in influenza A research to study pathogenesis, to investigate Reye's syndrome, and to evaluate vaccine trials (Deshmukh, 1987; Sweet et al., 1987).

TP - 10.6 g/dl /globulin 9 = 5.75 g%

,

. .

d.

_

Alb Fig. 5. Serum protein electrophoretograms of two ferrets with Aleutian disease-associated syndromes. Note that gammaglobulin concentrations exceed 20% of the total serum protein. (Reprinted from Palley et al., 1992.)

with formalin, sodium hydroxide, and phenolics (Shen et aL, 1981). There is no treatment for AD, and infected ferrets should be culled from the colony.

c.

Influenza

Etiology. Influenza is caused by an orthomyxovirus that is transmissible from humans to ferrets and ferrets to humans (Smith and Stuart-Harris, 1936). Human influenza viruses A and B are pathogenic to ferrets (Fox et al., 1998b). Ferrets are also susceptible to avian, phocine, equine, and swine influenza, although only porcine influenza causes clinical signs. Because the viruses can be readily transmitted from humans to ferrets, handling precautions such as wearing masks and gloves should be in place to minimize transmission. Epizootiology, transmission, and clinical signs. Influenza virus generally remains localized in nasal epithelium in ferrets but may cause pneumonia. Clinical signs appear 48 hr postinfection and include anorexia, fever, sneezing, and serous nasal discharge. Conjunctivitis, photosensitivity, and otitis are also sometimes seen (Fox et al., 1998b). Secondary bacterial infection by Streptococcus sp. and occasionally Bordetella bronchiseptica may prolong recovery. Transmission occurs via aerosol and direct contact.

Rabies

Etiology. Rabies is caused by a rhabdovirus. Rabies infection is infrequently reported in ferrets, and until recently, research on rabies in ferrets was lacking (Fox et al., 1998b). Ferrets in a well-managed facility would have low risk of exposure to rabies virus. Treatment and control. A USDA-approved, killed rabies vaccine given subcutaneously at ages 3 months and 1 year and annually thereafter is recommended to protect ferrets against rabies (Rupprecht et al., 1990). Modified live vaccine (MLV) is not recommended, because there is at least one case of rabies in a ferret that was vaccinated with MLV rabies vaccine (Fox et al., 1998b). There is no treatment for rabies. Clinical signs and pathogenesis. Clinical signs of rabies infection in ferrets may include anxiety, lethargy, and posterior paresis. In one experimental infection, 11 of 40 ferrets died, and Negri bodies were seen in the brain of only 2 of the 11 (Blancou et al., 1982). There is conflicting data on the isolation of rabies virus from the salivary glands following experimental infection. In one study using raccoon variant of rabies for infection, more than half of the ferrets had rabies isolated from the salivary glands (Fox et al., 1998b). Ferrets at risk for exposure to rabies virus that bite or scratch a human should be placed under quarantine for not less than 10 days of observation. Veterinarians and facility managers should seek assistance from state public health officials. Diagnosis and differential diagnoses. Differential diagnosis includes the neurotropic form of CD. Diagnosis is based on direct IFA of brain tissue. Because rabies in ferrets is poorly understood, the head from ferrets that exhibit signs compatible with rabies and that have exposure histories that raise concerns about rabies should be shipped to the state public health authority for confirmation.

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Fig. 6. Lymphocyticinfiltrate of portal triad associated with Aleutian disease virus. e.

Rotavirus

Etiology. Rotaviruses cause diarrhea in young of many species, including humans, calves, pigs, sheep, and rats. Diarrhea in ferret kits is thought to be caused by a poorly characterized, atypical rotavirus that has not been cultivated in vitro (TorresMedina, 1987). Atypical rotaviruses lack the rotavirus common antigen. Epizootiology, transmission and clinical signs. Clinical disease may occur in kits as young as 1 - 4 days old or in older animals up to 6 weeks of age. Diarrhea soils the perineum and possibly the fur and nest material. Mortality rates are agedependent, with high mortality occurring in young kits and lower mortality occurring in kits over 10 days of age (Bell, 1997a; Fox et al., 1998b). Secondary bacterial infection may influence the severity of diarrhea. Necropsy andpathogenesis. Lesions are restricted to the gastrointestinal tract. Yellow-green liquid or mucous feces may be seen in the terminal colon on necropsy. Subtle small-intestinal villous atrophy and epithelial cell vacuolation are detectable histologically. Diagnosis and differential diagnoses. Clinical diagnosis can be confirmed by using clarified and ultracentrifuged fecal pel-

lets for electron microscopy. The ferret rotavirus does not crossreact with commercially available enzyme immunoassays (Torres-Medina, 1987).

Treatment and control. It is desirable to avoid sources that are known to be infected with ferret rotavirus. Affected kits may be supplemented with kitten milk replacer, using a medicine dropper. Mortality is reduced if the kits continue nursing. Treatment of secondary bacterial infections may reduce severity of the diarrhea, and supportive care, including subcutaneous fluid administration for young kits, may be required (Fox et al., 1998b). Jills develop immunity to rotavirus infection, and subsequent litters are protected. f.

Other Viruses

Infectious bovine rhinotracheitis (IBR) was isolated from the liver, spleen, and lung of clinically normal ferrets (Porter et al., 1975). Raw beef was suspected as the source of infection, reinforcing the need to exclude raw meat products from the diet of ferrets used for research. IBR does cause significant respiratory pathology in experimentally infected ferrets (Porter et al., 1975). A transmissible diarrhea, referred to as epizootic catarrhal enteritis, has been observed in adult ferrets several days after direct contact and fomite exposure to affected ferrets (Fox et al.,

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1998b). Clinically the diarrhea is green and bile-tinged, and the ferrets become rapidly dehydrated. Mortality is low. Some ferrets develop elevated liver enzymes. Treatment involves aggressive oral and systemic fluid therapy. A recent study implicates a coronavirus as the cause of this disease (Williams et al., 2000).

3.

Parasitic Infections

a.

Protozoa

i. Enteric coccidiosis Etiology. Three species of the genera Isospora and Eimeria have been reported to infect the ferret: Isospora laidlawi, Eimeria furonis, and E. ictidea (Blankenship-Paris et al., 1993). Epizootiology and transmission. tion of sporulated oocysts.

Treatment and control. Good husbandry practices that include sanitation and frequent disposal of feces reduce the number of oocysts in the environment. Cleaning cages with a strong ammonium hydroxide solution is reported to be effective (Kirkpatrick and Dubey, 1987). Heat treatment of surfaces and utensils may also be effective (Kirkpatrick and Dubey, 1987). Treatment of ferrets with sulfadimethoxine at 50 mg/kg orally once and then 25 mg/kg orally every 24 hr for 9 days is recommended (Rosenthal, 1994). As in dogs and cats, the complete elimination of a coccidial infection requires an immunocompetent host. ii. Cryptosporidiosis Etiology. Cryptosporidiosis is caused by infection with Cryptosporidium spp.

Infection occurs from inges-

Clinical signs. Coccidiosis in ferrets is usually subclinical but has been reported to be associated with diarrhea, lethargy, and dehydration in one ferret (Blankenship-Paris et al., 1993). Clinical signs are often seen in young, newly acquired ferrets and are more common after a stressful event (Rosenthal, 1994). Rectal prolapse can also develop in association with coccidial infection (Rosenthal, 1994). Diagnosis. Diagnosis is generally made by any of the fecal flotation methods commonly used in veterinary practice or by direct wet mount of feces and microscopic examination for sporulated or unsporulated oocysts. Because coccidial oocysts are small, slides should be examined under higher magnification. Necropsy findings. Diagnosis is usually performed antemortem. Pathologic lesions associated with enteric coccidiosis in a laboratory-reared ferret that was euthanized were described in one published report (Blankenship-Paris et al., 1993). Microscopic lesions were confined to the jejunum and ileum and consisted of villous and epithelial thickening. Parasitic cysts and microorganisms within epithelium, and a mild granulomatous inflammation in the villar lamina propria, were also observed. A recent report documents clinical and anatomic pathology associated with biliary coccidiosis in a weanling ferret (Williams et al., 1996). Differential diagnosis. Diarrhea may be observed in ferrets that present with gastroenteritis secondary to gastrointestinal foreign bodies and dietary indiscretion, as well as other nutritional, inflammatory, infectious, or other systemic diseases. Infectious causes such as proliferative colitis, salmonellosis, giardiasis, rotavirus, and campylobacteriosis should be considered. Diarrhea may also be seen in eosinophilic gastroenteritis, an uncommonly reported condition in ferrets.

Epizootiology and transmission. Cryptosporidium is a protozoan in the class Sporozoa, subclass Coccidia, that inhabits the respiratory and intestinal epithelium of birds, reptiles, mammals, and fish (Regh et al., 1988). It is known to cause gastrointestinal tract disease in many species, including rodents, dogs, cats, calves, and people (Hill and Lappin, 1995). It has a life cycle similar to other coccidian parasites and is transmitted by ingestion of sporulated oocysts. Autoinfection is also a characteristic of the life cycle. Transmission may occur through consumption of contaminated food or water. Cattle, dogs, and cats, shedding oocysts, are reported to be potential sources of human infection (Hill and Lappin, 1995; Fox, 1998g). Immunosuppressed people are at greatest risk of developing severe fulminating gastrointestinal disease (Hill and Lappin, 1995). The finding of cryptosporidiosis in two ferrets that died from unrelated causes in one animal facility resulted in a survey of the existing ferret population and new arrivals into the facility to determine the prevalence and incidence of infection (Regh et al., 1988). Findings indicated that 40% of the resident population and 38-100% of new arrivals had oocysts in their feces but showed no clinical signs. Clinical signs. Only subclinical infection has been reported in both immunocompetent and immunosuppressed ferrets (Regh et al., 1988). Diagnosis. Diagnosis is based on the identification of the organism in feces. The oocysts are small when compared with other coccidia and may be overlooked or mistaken for yeasts (Kirkpatrick and Dubey, 1987). Yeasts are oval, whereas cryptosporidium oocysts are spherical or ellipsoidal. Additionally, yeasts will stain with iodine and are not acid-fast, whereas Cryptosporidium has the opposite staining characteristics. The oocyst residuum is seen as a refractive dot under phase-contrast microscopy, a structure lacking in yeast (Kirkpatrick and Dubey, 1987). Sugar-solution centrifugation and fecal sedimentation using formalin-ether or formalin-ethyl acetate are effective di-

13. BIOLOGY AND DISEASES OF FERRETS

agnostic concentration techniques (Hill and Lappin, 1995). Oocysts may then be viewed with phase-contrast or bright-field microscopy of specimens stained with an acid-fast method. A direct fecal smear may be methanol- or heat-fixed and stained with an acid-fast method (Hill and Lappin, 1995).

Necropsy findings. Histologic evaluation reveals the presence of organisl3ns, spherical to ovoid in shape and from 2 to 5 ~tm in diameter, associated with the brush border of the villi. A mild eosinophilic infiltrate was observed in the lamina propria of the small intestine in most animals. The ileum was the most common and heavily infected section of small intestine (Regh et al., 1988). Treatment and control. There is no known definitive treatment for cryptosporidiosis (Fox, 1998g). Supportive and symptomatic care should be provided in clinical cryptosporidiosis. Infections are self-limiting in immunocompetent patients (Fox, 1998g). Control is aimed at eliminating or reducing infective oocysts in the environment and avoidance of contact with known sources. Because of the potential for zoonotic transmission, restricting contact of children and immunosuppressed individuals with infected ferrets and practicing good hygiene may help reduce the potential for infection. Drying, freeze-thawing, and steam cleaning inactivate the organism (Hill and Lappin, 1995). There are few effective commercial disinfectants. b.

Ectoparasites and Mites

i. Sarcoptic mange Etiology. Sarcoptic mange is caused by infection with Sarcoptes scabiei. Epizootiology and transmission. Transmission occurs through direct contact with infected hosts or contact with fomites. This parasitic infection is rare under research conditions. Clinical signs. Infection of ferrets with S. scabiei may occur in a generalized or a pedal form (Bernard et al., 1984). In the generalized form, lesions consist of focal or generalized alopecia with intense pruritus. In the pedal form, lesions are confined to the toes and feet, which become swollen and encrusted with scabs. Nails may be deformed or lost if the condition is left untreated. Diagnosis. Diagnosis is made by finding the mites in skin scrapings or removing crusts, breaking them up, and clearing with 10% KOH for microscopic examination (Phillips et al., 1987). False-negative results are possible; multiple scrapings may be necessary. Differential diagnosis. Differential diagnosis should include other pruritic external parasitic conditions, including flea infes-

503

tation. Demodicosis has been reported to cause mild pruritus and alopecia in ferrets (Noli et al., 1996).

Treatment and control. In the pedal form, treatment consists of trimming the claws and removing the scabs after softening them in warm water (Bernard et al., 1984). Treatments that have been used include ivermectin, 0.2-0.4 mg/kg, administered subcutaneously and repeated every 7-14 days until mites are gone; shampoos or soaks to reduce the pruritus; and topical or systemic antibiotic administration for treatment of secondary bacterial dermatitis (Hillyer and Quesenberry, 1997b). Alternatively, weekly dips in 2% lime sulfur until 2 weeks after clinical cure have been shown to be effective (Fox, 1998a). Treatment of all affected animals as well as contact animals, and decontamination of enclosures and bedding, are recommended. ii. Demodicosis Etiology. Demodicosis is caused by infection by Demodex spp. Epizootiology and transmission. The parasite is found in normal skin of almost all dogs and is not considered contagious. Predisposing factors such as immunologic or genetic conditions have been suggested (Kwochka, 1986). One clinical report describes demodicosis in two adult ferrets that had been treated with an ear ointment containing triamcinolone acetonide for recurrent ear infections daily for 3 periods of 3 months each during the course of a year (Noli et al., 1996). Clinical signs. In the report mentioned above, the ferrets presented with alopecia, pruritus, and orange discoloration of the skin behind the ears and on the ventral surface of the abdomen and an accompanying seborrhea (Noli et al., 1996). Diagnosis. Deep skin scrapings should be performed to demonstrate mites. Finding a large number of live adult mites or immature forms and eggs is necessary to confirm the diagnosis. In very chronic cases, the skin may be so thickened that scrapings may be unrewarding. In these cases, a skin biopsy may be diagnostic (Kwochka, 1986). Necropsy findings. Histologic evaluation of skin biopsies obtained in the case report described above revealed mites with a short, blunted abdomen similar to that of Demodex criceti and located in the infundibulum of hairs. The epidermis was slightly hypertrophic, and there was a mild superficial orthokefatotic hyperkeratosis. A very mild superficial and perivascular mixed cellular infiltrate was also observed in the dermis. Differential diagnosis. Generalized demodicosis should be differentiated from sarcoptic mange and flea infestation. Primary or secondary bacterial dermatitis or pyoderma should also be considered.

504 Treatment and control. The ferrets in the above-mentioned clinical report were treated initially with a suspension of 0.0125% amitraz applied as a dip 3 times at 7-day intervals for 3 treatments. Two drops of the same solution were applied in each ear every other day. After the initial treatment, the ferrets were reexamined, and treatment was continued with the same concentration of solution applied once every 5 days, while the tail was washed with a higher concentration of amitraz (0.025%) once every other day. Thereafter, 3 final treatments with 0.0375% amitraz every 5 days for the body, and every other day for the ears and tail, were administered. The ferrets were evaluated and skin scrapings were performed regularly during treatment and posttreatment to monitor response to therapy. Treatment of any associated pyodermas, systemic illnesses, or management problems should also be included as part of the therapeutic regimen. iii. Ear mites Etiology. The ear mite, Otodectes cynotis, which commonly infects dogs and cats, is also a common clinical problem in ferrets (Fox, 1998g). Epizootiology and transmission. Ear mites are transmitted through direct contact with infested ferrets, dogs, or cats (Fox, 1998g). The entire life cycle is completed in 3 weeks. Clinical signs. Ear mite infestation in the ferret is usually asymptomatic (Orcutt, 1997). However, clinical signs may include head shaking; mild to severe pruritus with inflammation and excoriation; secondary otitis interna with ataxia; circling; torticollis; and Horner's syndrome (Orcutt, 1997; Fox, 1998g). A brownish black waxy discharge is often present. Diagnosis. Diagnosis is based on direct observation of mites via otoscopic examination or microscopic identification of the ear mite or any of the life-cycle stages of the mite in exudate from the ear canal. Treatment and control. Several treatment regimens, including topical and injectable mitocidal treatments, have been recommended (Orcutt, 1997; Fox, 1998g). A recent study using three treatment regimens--two topical and one injectable--revealed that topical treatments were more efficacious than the injectable in reducing or eradicating ear mites (Patterson et al., 1999). Efficacy was evaluated by microscopic evidence of ear mites in debris from aural swabs taken weekly for an 8-week period. Topical 1% ivermectin (Ivomec, Merck AgVet Division, Rahway, New Jersey), diluted 1" 10 in propylene glycol at a dosage of 400 ~tg/kg body weight divided equally between the two ear canals and administered on days 1 and 14 of the study, was the most effective treatment. All susceptible animals in a household should be treated. Ears should be gently cleaned prior to initiating treatment (Orcutt, 1997). High doses of injectable iver-

ROBERT P. MARINIET AL. mectin (0.2 ml of 1% ivermectin) administered to jills at 2 - 4 weeks of gestation resulted in high rates of congenital defects (Orcutt, 1997).

iv. Fleas Etiology. Ctenocephalides species can infest ferrets. Epizootiology and transmission. Transmission requires direct contact with another infested animal or a flea-infested environment. Clinical signs. Flea infestation may be asymptomatic or may cause mild to intense pruritus and alopecia of the dorsal thorax and neck (Timm, 1988). Diagnosis. Diagnosis is based on clinical signs and identification of fleas or flea excrement. Differential diagnosis. Sarcoptic and demodectic mange should be included in the differential diagnosis of pruritic skin disease in the ferret. Close examination of the pelage for fleas or flea excrement should be performed. Skin scrapings may be indicated. Treatment and control. As with flea infestation in dogs and cats, concurrent treatment of the environment, as well as all animals in the household, is essential for effective flea control. Compounds approved for flea control in cats such as rotenone or pyrethrin powders or sprays may be used in ferrets (Hillyer and Quesenberry, 1997a). 4.

Fungal Diseases

Ferrets may develop systemic disease from Blastomyces, Coccidioides, Cryptococcus, and Histoplasma. The reservoir of most of these fungi is the soil, however, making infection unlikely in a research facility. In production facilities, exposure can be minimized through careful selection of source animals, appropriate sanitation, and control of pests, particularly birds.

a.

Pneumocystis carinii

Pneumocystis carinii has been recently reclassified as a fungus. Although P. carinii inhabits the lungs of many different species, recent transmission studies suggest that these fungi are highly species-specific (Gigliotti et al., 1993; Fox et al., 1998b). Clinical disease is evident only in immunocompromised ferrets and can be induced using high doses of exogenous steroids (Stokes et al., 1987). Lesions include interstitial pneumonitis with mononuclear cell infiltrates; cysts and trophozooites are evident with Gomori methanamine-silver nitrate and Giemsa on bronchoalveolar lavage. Treatment with trimethoprim sul-

505

13. BIOLOGY AND DISEASES OF FERRETS famethoxazole probably controls but does not eliminate infection (Fox et al., 1998b).

b.

Mucormycosis

Ferrets are susceptible to secondary fungal infection of the outer ear canal with Absida corymbifera or Malassezia spp. (Dinsdale and Rest, 1995; Fox, 1998d). The fungi are widespread in the environment and will cause a secondary fungal infection in the ears of ferrets infested with Otodectes cynotis. The yeasts can be visualized by impressions of ear exudates. Treatment involves eradication of the underlying mite infestation followed by oral and topical ketoconazole, miconazole, and polymyxin B.

c.

Dermatomycosis

Dermatomycoses in ferrets are caused by Microsporum canis and Trichophyton mentagrophytes. Dermatophytes are transmissible to humans and are a zoonosis; thus affected animals should be quarantined and removed from the facility to minimize risk (Dinsdale and Rest, 1995; Scott et al., 1995; Fox et al., 1998b). Control of infection includes general disinfection and destruction of contaminated bedding. Lesions are circumscribed areas of alopecia and inflammation, which begin as small papules that spread peripherally in a scaly inflamed ring. The yellow-green fluorescence of M. canis under ultraviolet light helps distinguish it from T. mentagrophytes. Skin scrapings digested with 10% potassium hydroxide reveal characteristic arthrospores. Treatment with griseofulvin causes clinical remission but may not clear infection. 5.

Other

Other ectoparasitic infections observed to occur in ferrets include cutaneous myiasis and tick infestation. Granulomatous masses in the cervical region caused by the larval stage of Hypoderma bovis have been reported in ferrets (Fox, 1998g). Cuterebra larvae, although uncommonly observed in ferrets, may cause subdermal cysts found in the subcutis of the neck (Orcutt, 1997). Infestation with the flesh fly has been reported as a problem in commercially reared mink and ferrets housed outdoors (Fox, 1998g). Ticks may be found on ferrets housed outdoors or on those used for hunting rabbits (Fox, 1998g). Ticks should be removed carefully with hemostats or tweezers, ensuring that the entire head and mouthparts are removed from the skin. Appropriate caution should be exercised in tick removal, because ticks are responsible for transmission of various zoonotic pathogens; gloves should be worn.

6.

Nematodes

a.

Heartworm

Etiology. The ferret is susceptible to natural and experimental infection with Dirofilaria immitis. Epizootiology and transmission. Dirofilaria immitis is a filarial parasite that is transmitted by mosquitoes, which serve as the intermediate host and vector. Microfilaria are ingested by mosquitoes and, after two molts, become infective third-stage larvae. Infective larvae are deposited onto the skin when mosquitoes feed, and larvae find their way into the body of the final host through the bite wound and migrate subcutaneously to the thorax and eventually to the heart (Knight, 1987). The primary reservoir of infection is dogs, but heartworm may be found in a variety of mammals, including humans. All other species except wild and domestic canids, domestic felines, ferrets, and the California sea lion are considered aberrant hosts (Knight, 1987). Clinical signs. The following clinical signs have been reported in clinical reports describing cases of D. immitis in the ferret: weakness, lethargy, depression, dyspnea, cyanosis, anorexia, dehydration, cough, and pale mucous membranes (Miller and Merton, 1982; Parrott et al., 1984; Moreland et al., 1986). Moist lung sounds and/or muffled heart sounds were revealed by thoracic auscultation in many of these cases. Pleura/or abdominal effusion may be observed radiologically. The ferrets described in these cases were housed outdoors and either died or were euthanized. Diagnosis. Diagnosis of heartworm is based on clinical signs, radiographic findings, and testing for circulating microfilariae and heartworm antigen. Microfilaremia is not consistently observed in naturally occurring and experimental cases of heartworm infection in ferrets (Fox, 1998g). Testing for heartworm antigen appears to be more diagnostically useful (Stamoulis et al., 1997). In a study to determine the minimum oral dose of ivermectin needed for monthly heartworm prophylaxis in ferrets, the use of an antigen test (Uni-Tec Canine Heartworm test, Pitman-Moore Co., Mundelein, Illinois) detected infection in more untreated control animals than did the modified Knott test for detection of circulating microfilaria in the same ferrets (Supakorndej et al., 1992). Necropsy findings. Cardiomegaly, pleural and/or abdominal fluid, and pulmonary congestion are common findings at necropsy. Grossly, adult worms have been observed in the right atrium, right ventricle, pulmonary artery, and cranial and caudal vena cava. Microscopically, microfilaria may be seen in small and large vessels of the lung. Differential diagnosis. Differential diagnosis should include primary cardiac diseases, such as dilatative cardiomyopathy, and other systemic or pulmonary diseases.

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Treatment and control. Control is best directed at prevention through the administration of heartworm preventative and it is recommended that ferrets in heartworm-endemic areas receive monthly oral ivermectin throughout the year (Stamoulis et al., 1997; Fox, 1998g). The dosage recommended for ferrets by the American Heartworm Society is 0.006 mg/kg body weight monthly (Fox, 1998g). Housing ferrets indoors, particularly during the mosquito season, would help minimize exposure. Successful adulticide treatment in ferrets has been described and includes the administration of thiacetarsemide, with the same precautions used in dogs: antithrombotic therapy, treatment for heart failure, and strict cage confinement (Stamoulis et al., 1997). One should follow up with heartworm antigen tests until negative and resume heartworm prevention 1 month after adulticide treatment (Stamoulis et al., 1997). Ferrets are also susceptible to infection with the following nematodes: Toxascaris leonina; Toxocara cati; Ancylostoma spp.; Dipylidium caninum; Mesocestoides spp.; Atriotaenia procyonis; Trichinella spiralis; Filaroides martis; and Spiroptera nasicola (Rosenthal, 1994; Fox, 1998g).

B.

Metabolic and Nutritional Diseases

1. Pregnancy Toxemia

Pregnancy toxemia in the ferret occurs predominantly in primiparous jills carrying large litters. An inadvertent fast in late gestation is sometimes implicated. At least 75 % of jills carrying more than 8 kits will develop pregnancy toxemia if subjected to 24 hr of food withdrawal in late gestation (Bell, 1997a; Batchelder et al., 1999). Any jill with 15 or more kits may develop pregnancy toxemia because abdominal space is not adequate for both the gravid uterus and the volume of food required to support it. Pregnancy toxemia of the ferret is of the metabolic type and shares features with similar conditions in pregnant sheep, obese cattle, pregnant camelids, obese guinea pigs, and starved pregnant rats, as well as with the condition feline idiopathic hepatic lipidosis. It is characterized by abnormal energy metabolism with consequent hyperlipidemia, hypoglycemia, ketosis, and hepatic lipidosis. In this condition, energy demand exceeds intake, leading to excessive mobilization of free fatty acids and a chain of metabolic events that culminates in a shift from fatty acid metabolism and export to ketosis and hepatic lipidosis. Clinical signs include anorexia, lethargy, melena, dehydration, and easily epilated hair. Differentials include dystocia, metritis, pyometra, septicemia, renal failure, and Helicobacter mustelae-induced gastric ulcer. In a recent study of ferrets with pregnancy toxemia, consistent clinical chemistry abnormalities included azotemia (100%), hypocalcemia (83 %), hypoproteinemia (70%), and elevated liver enzymes (100%) (Batchelder et al., 1999). Anemia was found in 50% of ferrets tested. Necropsy findings include tan or yellow discolored liver, gastric hemorrhage, and gravid uterus. Treatment forjills within

a day of their due date should include cesarean section and intensive postoperative support, including force-feeding a gruel of high-quality cat food and ferret chow, nutritive pastes, intravenous fluids containing glucose, and supplemental heat. Cesarean section should be performed under isoflurane anesthesia because hepatic dysfunction prolongs the metabolism of injectable agents. Agalactia is common after cesarean section, and kits may require hand feeding with kitten or puppy milk replacers, administered per os by fine-tipped syringe 6 times daily for the first 24 hr. Cross fostering is an effective method of enhancing kit survival; hand rearing of kits if the jill fails to nurse within a day postoperatively is energy-consuming and generally unrewarding. For jills that develop pregnancy toxemia before day 40 of gestation, fluids and nutritional support must be provided until viable kits can be delivered by cesarean. Pregnancy toxemia may be avoided by close monitoring of appetite of jills in late gestation, provision of a highly palatable diet with >20% fat and > 35 % crude protein, and avoidance of stress and dietary change. Water should be made available in both bowls and water bottles, and food should be provided ad libitum in several bowls. 2. Hyperestrogenism

Ferrets are induced ovulators and may remain in persistent estrus if they are not bred or if estrus is not terminated chemically or via ovariohysterectomy (Bell, 1997a). Jills that remain in estrus for more than 1 month are at risk for developing estrogeninduced anemia. Hyperestrogenism from persistent estrus causes bone marrow hypoplasia of all cell lines in approximately half of ferrets in prolonged estrus (Ryland et al., 1983). Clinical signs include vulvar enlargement, bilaterally symmetric alopecia of the tail and abdomen, weakness, anorexia, depression, lethargy, weight loss, bacterial infection, and mucopurulent vaginal discharge. Hematology findings may vary from an initial neutrophilia and thrombocytosis early in the disease course to lymphopenia, thrombocytopenia, neutropenia, and anemia. The anemia begins as normocytic normochromic but progresses to macrocytic hypochromic (Sherrill and Gorham, 1985). Coagulopathy associated with hepatic dysfunction and thrombocytopenia combine to produce extensive manifestations of bleeding, pallor, melena, petechiation or ecchymosis, subdural hematoma, and hematomyelia (Hart, 1985; Fox and Bell, 1998). At necropsy, tissue pallor, light tan to pale pink bone marrow, hemorrhage, bronchopneumonia, hydrometra, pyometra, and mucopurulent vaginitis may be seen. Histopathology may reveal cystic endometrial hypoplasia, hemosiderosis, diminished splenic extramedullary hematopoiesis, and mild to moderate hepatic lipidosis (Sherrill and Gorham, 1985; Bell, 1997a). Treatment consists of terminating estrus while supporting the animal with antibiotics, blood transfusion, B vitamins, and nutritional supplementation. Estrus may be terminated by injection with 50-100 IU of human chorionic gonadotropin (hCG) or 20 ~tg of gonadotropin-releasing hormone (GnRH),

13. BIOLOGY AND DISEASES OF FERRETS repeated 1 week after initial injection if required. Ovariohysterectomy may be considered for ferrets that are stable and have adequate numbers of platelets and red cells. Ferrets with a packed cell volume (PCV) of 25% or greater have a good prognosis and require only termination of estrus for resolution of aplastic anemia. Jills with a PCV of 15-25% may require blood transfusions and have a guarded prognosis. Ferrets with a PCV of less than 15% have a poor prognosis and require aggressive therapy with multiple transfusions. The lack of identifiable blood groups in ferrets makes multiple transfusions uncomplicated by potential transfusion reactions (Manning and Bell, 1990b). Estrogen-induced anemia may be avoided by ovariohysterectomy. of nonbreeding females, use of vasectomized hobs, or pharmacologic termination of estrus initiated 10 days after estrus onset. A 40- to 45-day pseudopregnancy then follows, except in the case of ovariohysterectomy. Repeated administration of hCG may result in sensitization and anaphylaxis. After several administrations, hCG is unlikely to be effective in termination of estrus. Anaphylaxis is manifest as incoordination, tremor, vomiting, and diarrhea and may be reversed by prompt administration of diphenhydramine. 3.

Hyperammonemia

Arginine-free diets are unlikely to be fed in the laboratory setting, but administration of such a diet to young ferrets fasted for 16 hr leads to hyperammonemia and encephalopathy within 2 3 hr (Thomas and Desmukh, 1986). Exacerbation of signs may be achieved by challenging young ferrets with influenza virus and aspirin (Desmukh et al., 1985) and constitutes a model of Reye's syndrome in children. Lethargy and aggressiveness yield to prostration, coma, and death in affected ferrets. Hyperammonemia presumably occurs because of the inability of ferrets to produce adequate amounts of ornithine from non-arginine precursors. Detoxification of ammonia is thereby compromised. Ferrets more than 18 months old are unaffected by arginine-free diets. 4.

Zinc Toxicosis

Ferrets of all ages are susceptible to zinc toxicosis, and the condition has been documented in two ferret farms in New Zealand (Straube and Walden, 1981). Leaching of zinc from steam-sterilized galvanized food and water bowls was implicated. Clinical signs included pallor, posterior weakness, and lethargy. Definitive diagnosis requires demonstration of elevated concentrations of zinc in kidney and liver. At necropsy, kidneys are enlarged, pale, and soft; livers are orange, and gastric hemorrhage may be seen. Histopathology reveals glomerular collapse, tubular dilation, tubular proteinaceous debris, focal cortical fibrosis, hepatic periacinar infiltration, and depression of the erythroid series. Avoidance of galvanized materials precludes the development of zinc toxicosis.

507

C.

Traumatic Disorders

Umbilical entanglement may occur in ferrets on the day of parturition and has been associated with fine-particle bedding, large litters, and short kit-birth intervals (Bell, 1997a; Fox et al., 1998a). Jills may neglect to clean placentas from their kits, or kits may be born so rapidly that there is not adequate time for the jill to clean the kits of placental membranes, thereby predisposing to entanglement. Entangled kits may succumb to dehydration, hypothermia, and hypoglycemia because they are unable to nurse and the jill cannot curl around them. Detailed dissection with fine scissors and forceps under a heat lamp or on a heated surface can free the kits. Occasionally, kits may need to be rotated on their umbilical pedicle to achieve adequate clearance to cut the cord; cords should be cut as far from the umbilicus as possible. The use of warm saline or water may help soften the mass. Some kits in the tangle may present with dark, swollen extremities or prolapsed umbilical cords and may require euthanasia. Parturition should be supervised, if possible, to avoid umbilical entanglement. D.

Iatrogenic Diseases

Hydronephrosis may occasionally occur in the ferret and is most commonly associated with inadvertent ligation of the ureter during ovariohysterectomy. Ovarian remnants are another potential sequela to ovariohysterectomy. Ovarian remnants in ferrets may be associated with estrus, vulvar enlargement, and alopecia. Appropriate diagnostic procedures include ultrasonography and plain and contrast radiography for hydronephrosis and ultrasonography and serum hormone concentrations for ovarian remnants. Exploratory celiotomy confirms the diagnosis, and unilateral nephrectomy or ovariectomy is indicated if the remaining kidney is normal and the ferret is otherwise healthy. E.

Neoplastic Diseases

Over the last few decades, increasing numbers of ferrets have been used in research or kept as pets, and as these animals have received veterinary care, it has become evident that ferrets are subject to a wide variety of neoplastic conditions (Li et al., 1998). However, four categories of cancer account for the majority of ferret neoplasms: pancreatic islet cell tumors, adrenocortical cell tumors, lymphoma, and skin cancers. 1. Insulinoma

Functional pancreatic islet cell tumors (insulinomas) are the most common neoplasm diagnosed in ferrets (Li et al., 1998). Disease may be evident in ferrets as young as 2 years old, but later onset (at 4 - 5 years of age) is typical (Caplan et al., 1996; Ehrhart et al., 1996). Nonspecific presenting signs include

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weight loss, vomiting, and ataxia. Weakness is often evident, ranging from lethargy to posterior paresis or outright collapse (Caplan et al., 1996). Hypoglycemia caused by excess production of insulin by neoplastic 13cells may cause tremors, disorientation, or seizures (Fox and Marini, 1998). Excessive salivation (ptyalism) or pawing at the mouth is a frequent finding. Clinical signs are often intermittent or episodic. Other common findings include splenomegaly and lymphocytosis. Presumptive diagnosis is made based on clinical signs in conjunction with the demonstration of hypoglycemia. Blood glucose determinations for the diagnosis of insulinoma are most useful when taken after a 4 hr fasting period. Fasting glucose concentrations below 60 may be diagnostic for the condition (Quesenberry and Rosenthal, 1997), whereas values between 60 and 85 are suspect and the test should be repeated (Fox and Marini, 1998). Other potential causes for hypoglycemia should be ruled out, including anorexia, starvation, hepatic disease, sepsis, and nonpancreatic neoplasia (Antinoff, 1997). Demonstration of concurrent hyperinsulinemia aids the diagnosis (Caplan et al., 1996). Medical management using prednisone and/or diazoxide along with dietary modification such as frequent feeding of high-protein meals can minimize or control clinical signs but will not affect the underlying tumor (Quesenberry and Rosenthai, 1997). Surgical exploration of the pancreas and tumor excision are recommended for animals that are healthy enough to be subjected to anesthesia and surgery. Histological examination of the tissue removed can provide a definitive diagnosis, and although the effect may be transient, clinical signs are often reduced or eliminated after surgical debulking (Figs. 7 and 8) (Ehrhart et al., 1996). Histologically, these tumors reveal ma-

lignant proliferation of pancreatic 13cells, and local recurrence or metastasis to lymph nodes, mesentery, spleen, or liver may occur (Caplan et al., 1996). 2.

Adrenal Tumors

Adrenocortical cell tumor is the second most common type of neoplasia in ferrets (Li et al., 1998) and is generally diagnosed between 3 and 6 years of age. If clinical signs are present, they often include weight loss and a bilateral, symmetric alopecia. Pruritus is a variable finding (Quesenberry and Rosenthal, 1997). Although ferrets with this syndrome have been called "cushingoid," it is rare to diagnose elevated resting levels of glucocorticoids or an abnormal response to adrenocorticotropic hormone (ACTH) stimulation or dexamethasone suppression testing. Elevation of adrenal sex hormones (e.g., androstenedione, 17-hydroxyprogesterone, and/or estradiol) is more likely, and these may lead to characteristic changes such as estruslike vulvar swelling in spayed females and prostatic changes in males (Rosenthal and Peterson, 1996; Coleman et al., 1998). Rule-outs for enlarged vulva include estrus in an intact female or functional ovarian remnants in a spayed female. Abdominal palpation may reveal cranial abdominal masses, and ultrasound may be useful (Barthez et al., 1998). Serum assay for abnormal levels of the sex hormones listed above should be considered (Lipman et al., 1993; Wagner and Dorn, 1994; Rosenthal and Peterson, 1996). In many cases the alopecia begins as a seasonally intermittent partial hair loss that becomes more severe as time goes on (Fig. 9). Even severe manifestations of this endocrine alopecia

Fig. 7. Grossappearance of islet cell tumors in the ferret (arrows). Note the isoflurane-inducedsplenomegaly.

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13. BIOLOGY AND DISEASES OF FERRETS

Fig. 8.

Histologic appearance of an islet cell tumor (arrows) metastatic to lymph node.

Fig. 9.

Adrenal-associated endocrine alopecia in the ferret.

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can spontaneously reverse in the absence of specific therapy, as demonstrated in a group of 5 ferrets referred to our facility for diagnostic workup. In each of these 5 ferrets, near total alopecia resolved within a few months of being housed in a research environment. Despite being asymptomatic at the end of the study, all 5 were shown to have histologic evidence of adrenocortical neoplasia. Although this phenomenon is mediated by hormonal effects, anecdotal reports such as this suggest that the alopecia may be significantly modulated by environmental factors (e.g., photoperiod or diet). Surgical exploration and removal of enlarged adrenals are commonly performed to establish the diagnosis and to remove hyperfunctional tissue. Unilateral adrenalectomy early in the disease may be curative, but because bilateral neoplastic involvement is not uncommon, full or partial removal of both glands may be required. Adrenolytic agents such as mitotane have been used with limited success (Quesenberry and Rosenthai, 1997). Histologically, adrenocortical adenomas are generally 1 cm or less in diameter and are composed of well-differentiated cells with a granular or vacuolated cytoplasm. Adrenal cell carcinomas are less commonly found and are larger, with a more pleomorphic and invasive character (Li et al., 1998). Metastasis to nearby tissues can occur. In our experience, adrenal cortical hyperplasia with or without neoplasia is an extremely common finding in aging ferrets, even in those not showing clinical signs. In one retrospective survey of our necropsy records it was found that more than 90% of ferrets greater than 4 years of age had hyperplastic or neoplastic adrenal changes when examined (data not shown). For this reason, careful considerations of other possible disease processes should be made before attributing clinical signs solely to adrenal enlargement.

3.

Lymphoma

Lymphoma can affect ferrets of almost any age. Ferrets younger than 2 years of age often present with mediastinal lymphoma and/or leukemia, whereas those older than 3 years of age often develop multicentric solid tumors (Erdman et al., 1996a). The early age of onset in some ferrets and reports of case clustering have led to investigation into potential infectious etiologies for lymphoma in the ferret (Erdman et al., 1996b). Earlier reports of feline leukemia virus (FeLV) seroconversion in affected animals have not been substantiated. However, experimental and epidemiological evidence suggests that a retrovirus that is distinct from FeLV may be involved (Erdman et al., 1995). In one study, whole or filtered lymphoma cells from a 3-year-old ferret with spontaneous lymphoma were injected IP into 6 recipient ferrets (Erdman et al., 1995). Two of the 6 ferrets were euthanized after 14 months, but the remaining 4 developed splenomegaly, lymphocytosis, and lymphoma. One

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P. MARINI ET AL.

ferret that received cell-free materials developed multicentric lymphoma with prominent cutaneous lymphoma nodules. Elevated reverse transcriptase activity and retrovirus-like particles evident by electron microscopy were seen in the donor and all of the affected recipient ferrets. Other potential etiologies that have been considered include two infectious agents that are known to cause chronic immune stimulation in affected ferrets, the Aleutian disease virus (ADV) and Helicobacter mustelae. A link with ADV has not been proven, but H. mustelae seems to be responsible for the development of a very specific type of gastric B-cell lymphoma (Erdman et al., 1997). Affected ferrets may exhibit localizing signs (e.g., dyspnea in a ferret with mediastinal involvement or peripheral lymphadenopathy in an animal with a multicentric distribution) but as is the case in many species, lymphoma is a "masquerader," and affected ferrets often present with chronic, nonspecific signs. Weight loss, anorexia, and lethargy are often reported. Splenic and/or hepatic enlargement may be evident. Cutaneous involvement has been documented (Li et al., 1995; Rosenbaum et al., 1996). Although hematological examination typically reveals anemia and lymphopenia, lymphocytosis may be found, especially in younger ferrets. Atypical lymphocytes are identified in the circulation in some cases. Antemortem definitive diagnosis of lymphoma can be made by cytological examination of specimens obtained via fine-needle aspiration or excisional biopsy. Tan-colored masses involving lymph nodes, spleen, liver, or other organs are commonly found at necropsy (Fig. 10). Diffuse involvement may lead to uniform enlargement of these organs or to a thickening of the wall of the stomach or intestines. As in other species, histological evaluation reveals neoplastic lymphocytes in affected tissues, generally evident as a monomorphic population (Fig. 11). Although surgery and radiation therapy may be useful in certain cases, most attempts to treat ferret lymphoma have utilized chemotherapeutic regimens with dosages extrapolated from other domestic animals or humans. Treatment generally results in a remission that may last from 3 months to 5 years (Brown, 1997b; Erdman et al., 1998).

4.

Skin Tumors

Mast cell tumors are among the most commonly reported integumentary tumors in ferrets (Parker and Picut, 1993; Li and Fox, 1998). Cutaneous mastocytomas may occur anywhere on the body and present as firm, nodular skin lesions 2 10 mm in size that are often associated with alopecia or crusty ulceration of the overlying skin. Pruritis is common (Stauber et al., 1990). Histologically, they are composed of well-differentiated mast cells with metachromatic cytoplasmic granules that may be difficult to detect in sections stained with

511

13. B I O L O G Y AND DISEASES OF F E R R E T S

Fig. 10.

Cranial mediastinal mass consistent with lymphoma in a ferret.

hematoxylin-eosin, but are more evident in toluidine bluestained sections. A variety of tumors of epithelial origin occur in ferrets, and they can appear at any site on the body. The most common are the basal cell tumors, which present as firm plaques or pedunculated nodules that are white or pink (Parker and Picut, 1993).

Fig. I1.

They may grow rapidly and become ulcerated. The percentage of basiloid cells present in these tumors, and the degree of associated squamous or sebaceous differentiation can vary, resulting in a spectrum of tumor subtypes and associated histological diagnoses (Orcutt, 1997). However, as is the case with mastocytomas, most are benign and will not recur after

Monomorphic population of lymphocytes in a case of lymphoma in a ferret.

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512

excision. Resected tumors should be examined histologically to rule out less common tumors that might have a more guarded prognosis, such as squamous cell carcinoma or apocrine gland adenocarcinoma. Chordomas are not epithelial tumors, but they often present as readily evident firm masses on the tail that may cause ulceration of the overlying skin. These neoplasms arise along the axial skeleton from notochord remnants and are typically slow-growing (Dunn et al., 1991). Tumors involving the tail generally do not recur after amputation of the affected region, but a wide surgical margin should be maintained by removing several vertebrae proximal to the tumor. The prognosis is guarded for those rare chordomas that arise in the cervical region, and metastasis has been documented (Williams et al., 1993).

F. 1.

Miscellaneous Diseases

Congenital Lesions

Congenital defects identified in ferrets include a variety of neural tube defects, gastroschisis, cleft palate, amelia, corneal dermoids, cataracts, and supernumerary incisors (Willis and Barrow, 1971; Ryland and Gorham, 1978; McLain et al., 1985; Besch-Williford, 1987). Cystic or polycystic kidneys have been observed (Andrews et al., 1979a; Dillberger, 1985). Cystic genitourinary anomalies associated with the prostate, bladder, and/or proximal urethra most likely develop secondary to aberrant hormone secretion by adrenocortical tumors (Li et al., 1996a; Coleman et al., 1998). Newborn ferrets are normally born with a closed orbital fissure and are prone to developing subpalpebral conjunctival abscesses. Treatment involves surgically opening the lids (a minor procedure) to establish drainage and to allow topical antibiotics to be administered (Bell, 1997a). 2.

Aging and Degenerative Disease

Cardiomyopathy is a common cause of disease in aging ferrets. The dilatative form of disease is most commonly diagnosed. Affected animals commonly present with lethargy, weight loss, and anorexia. Physical examination may reveal signs of congestive heart failure such as hypothermia, tachycardia, cyanosis, jugular distension, and respiratory distress (Lipman et al., 1987). Auscultation may reveal a heart murmur and/or muffled cardiac sounds. Hepatomegaly and splenomegaly are often identified. Radiographs may reveal an enlarged cardiac silhouette and evidence of pulmonary edema or pleural effusion (Greenlee and Stephens, 1984). Electrocardiography and echocardiography can help make the definitive diagnosis. Medical therapy (supportive care, diuretics, and inotropic drugs) may relieve clinical signs and improve the qual-

ity of life for a period of months (Stamoulis et al., 1997). The long-term prognosis for survival is guarded to poor. Splenomegaly is a common finding in ferrets. In many cases the enlarged spleen appears to be a secondary manifestation of another disease (e.g., insulinoma, cardiomyopathy, or adrenal tumor) and is of unknown significance (Stamoulis et al., 1997). Histologic examination of affected organs has revealed that the most common cause for splenic enlargement (in the absence of a neoplastic infiltrate) is extramedullary hematopoiesis (EMH) (Erdman et al., 1998). This may be an incidental finding, but it has been suggested that in some cases a pathologically enlarged spleen may play a role in chronic anemia that may respond to splenectomy, a syndrome known as hypersplenism (Ferguson, 1985). Splenomegaly can also be commonly found in conjunction with lymphoma, with or without intrasplenic neoplastic lymphoid accumulations. In anesthetized ferrets, splenomegaly may be caused by splenic sequestration of erythrocytes (Marini et al., 1994, 1997). Because this is a transient effect, the normalization of splenic size upon recovery from anesthesia can help in the differentiation of anesthetic-induced splenomegaly from that due to other causes. Eosinophilic gastroenteritis is an idiopathic disorder characterized by peripheral eosinophilia (10-35% of circulating leukocytes), hypoalbuminemia, and diffuse infiltration of the gastrointestinal tract with eosinophils (Fox et al., 1992a). Presenting signs for this syndrome generally include chronic weight loss, anorexia, diarrhea, and occasionally vomiting. Eosinophilic granulomas have been found in the mesenteric lymph nodes of most affected ferrets, and in some cases other organs (e.g., lung or liver) may be involved. An interesting finding in many ferrets is the presence of Splendore-Hoeppli material in the inflamed lymph nodes, a histological phenomenon that has been associated in other species with helminths, bacteria, fungi, and foreign bodies (Fig. 12). An etiological agent has not been identified; consequently, therapy consists largely of supportive care to treat the chronic enteritis (Fox, 1998b). Based on the biology of eosinophils, however, the use of corticosteroids or ivermectin has been attempted and may be beneficial (Bell, 1997b). Megaesophagus has been diagnosed in ferrets presenting with a variety of signs, including weight loss, anorexia, difficulty in eating, or repeated regurgitation. The cause is generally unknown, and the prognosis is poor, despite efforts at supportive care (Blanco et al., 1994). Gray, yellow, or white small raised lesions may be found on the surface of ferret lungs at gross examination. Histologically, these lesions are composed of a superficial thickening of the lung tissue with mononuclear cell infiltration and varying degrees of fibrosis, with or without cholesterol-like clefts. The etiology of this condition (known as subpleural histiocytosis, pleural lipidosis, or lipid pneumonia) is unknown, and it appears to be an incidental lesion (Fox, 1998f).

513

13. BIOLOGY AND DISEASES OF FERRETS

Fig. 12.

Splendore-Hoeppli phenomenon in the lymph node of a ferret with eosinophilic gastroenteritis (Giemsa stain).

REFERENCES

Allen, R. E, Evans, E. V., and Sibbald, I. R. (1964). Energy: protein relationships in the diets of growing mink. Can. J. Physiol. Pharmacol. 47, 733. Anderson, E. (1989). The phylogeny of mustelids and the systematics of ferrets. In "Conservation Biology and the Black-footed Ferret" (U. S. Seal, E. T. Thorne, M. A. Bogan, and S. H. Anderson, eds.). Yale Univ. Press, New Haven, Connecticut. Andrews, P., Illman, O., and Mellersh, A. (1979a). Some observations of anatomical abnormalities and disease states in a population of 350 ferrets (Mustela furo). Z. Versuchstierkd. 21, 346-353. Andrews, P. L., Bower, A. J., and Illman, O. (1979b). Some aspects of the physiology and anatomy of the cardiovascular system of the ferret, Mustela putorius furo. Lab. Anim. 13, 215-220. Andrews, P. L. R., and Illman, O. (1987). The ferret. In "UFAW Handbook on the Care and Management of Laboratory Animals" (T. Poole, ed.), 6th ed. Longmans, London. Antinoff, N. (1997). Musculoskeletal and neurological diseases. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 126-130. Saunders, Philadelphia. Barthez, P. Y., Nyland, T. G., and Feldman, E. C. (1998). Ultrasonography of the adrenal glands in the dog, cat, and ferret. Vet. Clin. North Am. Small Anim. Pract. 28, 869-885. Batchelder, M. A., Bell, J. A., Erdman, S. E., Marini, R. P., Murphy, J. C., and Fox, J. G. (1999). Pregnancy toxemia in the European ferret (Mustela putorius furo). Lab. Anim. Sci. 49, 372-379.

Baum, M. J. (1998). Use of the ferret in reproductive neuroendocrinology. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), pp. 521-536. Williams and Wilkins, Baltimore. Beck, E, et al. (1976). Comparison of the teratogenic effects of mustine hydrochloride in rats and ferrets. The value of the ferret as an experimental animal in teratology. Teratology 13, 151. Bell, J. (1997a). Periparturient and neonatal diseases. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 58-60. Saunders, Philadelphia. Bell, J. A. (1997b). Helicobacter mustelae gastritis, proliferative bowel disease, and eosinophilic gastroenteritis. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 37-43. Saunders, Philadelphia. Bell, J. A., and Manning, D. D. (1990a). A domestic ferret model of immunity to Campylobacter jejuni-induced enteric disease. Infect. Immun. 58, 1848-1852. Bell, J. A., and Manning, D. D. (1990b). Reproductive failure in mink and ferrets after intravenous or oral inoculation of Campylobacterjejuni. Can. J. Vet. Res. 54, 432-437. Bell, J. A., and Manning, D. D. (1991). Evaluation of Campylobacterjejuni colonization of the domestic ferret intestine as a model of proliferative colitis. Am. J. Vet. Res. 52, 826-832. Bernard, S. L., Leathers, C. W., Brobst, D. E, and Gorham, J. R. (1983). Estrogen-induced bone marrow depression in ferrets. Am. J. Vet. Res. 44, 657-661. Bernard, S. L., Gorham, J. R., and Ryland, L. M. (1984). Biology and diseases of ferrets. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), p. 394. Academic Press, Orlando, Florida.

514 Besch-Williford, C. L. (1987). Biology and medicine of the ferret. Vet. Clin. North Am. Small Anim. Pract. 17, 1155-1183. Blanco, M. C., Fox, J. G., Rosenthal, K., Hillyer, E. V., Quesenberry, K. E., and Murphy, J. C. (1994). Megaesophagus in nine ferrets. J. Am. Vet. Med. Assoc. 205, 444-447. Blancou, J., Aubert, J. E A., and Artois, M. (1982). Rage experimetale du furet (Mustela putorius furo). Rev. Med. Vet. 133, 553. Blankenship-Paris, T. L., Chang, J., and Bagnell, C. R. (1993). Enteric coccidiosis in a ferret. Lab. Anim. Sci. 43, 361-363. Bleavins, M. R., and Aulerich, R. J. (1981). Feed consumption and food passage time in mink (Mustela vison) and European ferrets (Mustela putoriusfuro). Lab. Anim. Sci. 31, 268-269. Brown, S. A. (1997a). Basic anatomy, physiology, and husbandry. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 3-13. Saunders, Philadelphia. Brown, S. A. (1997b). Neoplasia. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 99114. Saunders, Philadelphia. Budd, J. (1981). Distemper. In "Infectious Diseases of Wild Mammals" (J. W. Davis, L. H. Karstad, and D. O. Trainer, eds.). Iowa State Univ. Press, Ames. Caplan, E. R., Peterson, M. E., Mullen, H. S., Quesenberry, K. E., Rosenthal, K. L., Hoefer, H. L., and Moroff, D. S. (1996). Diagnosis and treatment of insulin-secreting pancreatic islet cell tumors in ferrets: 57 cases (19861994). J. Am. Vet. Med. Assoc. 209, 1741-1745. Carpenter, J. W., Appel, M. J. G., and Erickson, R. C. (1976). Fatal vaccineinduced canine distemper virus infection in black-footed ferrets. J. Am. Vet. Med. Assoc. 169, 961-964. Carroll, R. S., Erskine, M. S., Doherty, P. C., Lundell, L. A., and Baum, M. J. (1985). Coital stimuli controlling luteinizing hormone secretion and ovulation in the female ferret. Biol. Reprod. 32, 925-933. Carroll, R. S., Erskine, M. S., and Baum, M. J. (1987). Sex difference in the effect of mating on the pulsatile secretion of luteinizing hormone in a reflex ovulator, the ferret. Endocrinology 121, 1349-1359. Carter, G. R., Chengapa, M. M., and Roberts, A. W. (1995). Campylobacter and Helicobacter. In "Essentials of Veterinary Microbiology," 5th ed., pp. 214215. Williams and Wilkins, Baltimore. Clingerman, K. J., Fox, J. G., and Walke, M. (1991). "Ferrets as Laboratory Animals: A Bibliography." National Agricultural Library, Beltsville, Maryland. Coburn, D. R., and Morris, J. A. (1949). The treatment of Salmonella typhimurium infection in ferrets. Cornell Vet. 39, 183-192. Coleman, G. D., Chavez, M. A., and Williams, B. H. (1998). Cystic prostatic disease associated with adrenocortical lesions in the ferret (Mustela putorius furo). Vet. Pathol. 35, 547-549. Corbet, G. B., and Hill, J. E. (1980). "A World List of Mammalian Species." Cornell Univ. Press, New York. Creed, J. E., and Kainer, R. A. (1981). Surgical extirpation and related anatomy of anal sacs of the ferret. J. Am. Vet. Med. Assoc. 179, 575-577. Desmukh, D. R. (1987). Reye's syndrome. Comp. Pathol. Bull. 19, 2. Desmukh, D. R., Thomas, P. E., and McArthur, M. B. (1985). Serum glutamatedehydrogenase and ornithine carbamyl transferase in Reye's syndrome. Enzyme 33, 171-174. Dillberger, J. E. (1985). Polycystic kidneys in a ferret. J. Am. Vet. Med. Assoc. 186, 74-75. Dinsdale, J. R., and Rest, J. R. (1995). Yeast infection in ferrets [letter]. Vet. Rec. 16, 647. Dunn, D. G., Harris, R. K., Meis, J. M., and Sweet, D. E. (1991). A histomorphologic and immunohistochemical study of chordoma in 20 ferrets (Mustela putorius furo). Vet. Pathol. 28, 467-473. Ehrhart, N., Withrow, S. J., Ehrhart, E. J., and Wimsatt, J. H. (1996). Pancreatic beta cell tumor in ferrets: 20 cases (1986-1994). J. Am. Vet. Med. Assoc. 209, 1737-1740.

ROBERT P. MARINI ETAL.

Erdman, S. E., Reimann, K. A., Moore, E M., Kanki, E J., Yu, Q. C., and Fox, J. G. (1995). Transmission of a chronic lymphoproliferative syndrome in ferrets. Lab. Invest. 72, 539-546. Erdman, S. E., Brown, S. A., Kawasaki, T. A., Moore, E M., Li, X., and Fox, J. G. (1996a). Clinical and pathogic findings in ferrets with lymphoma: 60 cases (1982-1994). J. Am. Vet. Med. Assoc. 208, 1285-1289. Erdman, S. E., Kanki, P. J., Moore, E M., Brown, S. A., Kawasaki, T. A., Mikule, K. W., Travers, K. U., Badylak, S. E, and Fox, J. G. (1996b). Clusters of malignant lymphoma in ferrets. Cancer Invest. 14, 225-230. Erdman, S. E., Correa, P., Li, X., Coleman, L. A., and Fox, J. G. (1997). Helicobacter mustelae-associated gastric mucosa associated lymphoid tissue (MALT) lymphoma in ferrets. Am. J. Pathol. 151, 273-280. Erdman, S. E., Li, X., and Fox, J. G. (1998). Hematopoietic diseases. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 231-246. Williams and Wilkins, Baltimore. Evans, H. E., and An, N. Q. (1998). Anatomy of the ferret. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), pp., 19-69. Williams and Wilkins, Baltimore. Ferguson, D. C. (1985). Idiopathic hypersplenism in a ferret. J. Am. Vet. Med. Assoc. 186, 693-695. Field and Laboratory Service Veterinary Staff. (1984). Diseases of the fitch. Surveillance 11, 18. Forester, N. T., Parton, K., Lumsden, J. S., and O'Toole, P. W. (2000). Isolation of Helicobacter mustelae from ferrets in New Zealand. New Zealand Vet. J. 48, 65-69. Fox, J. G. (1998a). Bacterial and mycoplasma diseases. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 321-354. Williams and Wilkins, Baltimore. Fox, J. G. (1998b). Diseases of the gastrointestinal system. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 273-290. Williams and Wilkins, Baltimore. Fox, J. G. (1998c). Housing and management. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 173-182. Williams and Wilkins, Baltimore. Fox, J. G. (1998d). Mycotic diseases. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 393-404. Williams and Wilkins, Baltimore. Fox, J. G. (1998e). Normal clinical and biologic parameters. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 183-210. Williams and Wilkins, Baltimore. Fox, J. G. (1998f). Other systemic diseases. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 307-320. Williams and Wilkins, Baltimore. Fox, J. G. (1998g). Parasitic diseases. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 375-392. Williams and Wilkins, Baltimore. Fox, J. G., and Bell, J. A. (1998). Growth, reproduction, and breeding. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 211-227. Williams and Wilkins, Baltimore. Fox, J. G., and Marini, R. P. (1998). Diseases of the endocrine system. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 291-305. Williams and Wilkins, Baltimore. Fox, J. G., and McLain, D. E. (1998). Nutrition. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 149-172. Williams and Wilkins, Baltimore. Fox, J. G., Murphy, J. C., Ackerman, J. I., Prostak, K. S., Gallagher, C. A., and Rambow, V. J. (1982). Proliferative colitis in ferrets. Am. J. Vet. Res. 43, 858-864. Fox, J. G., Ackerman, J., and Newcomer, C. E. (1983). Ferret as a potential reservoir for human campylobacteriosis. Am. J. Vet. Res. 44, 1049-1052. Fox, J. G., Edrise, B. M., Cabot, E., Beaucage, C., Murphy, J. C., and Prostak, K. S. (1986a). Campylobacter-like organisms isolated from gastric mucosa of ferrets. Am. J. Vet. Res. 47, 236-239. Fox, J. G., Hotaling, L., Ackerman, J. B., and Hewes, K. (1986b). Serum chem-

13. BIOLOGY AND DISEASES OF FERRETS

istry and hematology reference values in the ferret (Mustela putoriusfuro). Lab. Anim. Sci. 36, 583. Fox, J. G., Ackerman, J. I., Taylor, N., Claps, M., and Murphy, J. C. (1987). Campylobacter jejuni infection in the ferret: An animal model of human campylobacteriosis. Am. J. Vet. Res. 48, 85-90. Fox, J. G., Adldns, J. A., and Maxwell, K. O. (1988a). Zoonoses in ferrets. Lab. Anim. Sci. 38, 500. (Abstract). Fox, J. G., Cabot, E. B., Taylor, N. S., and Laraway, R. (1988b). Gastric colonization of Campylobacterpylori subsp, mustelae in ferrets. Infect. Immun. 56, 2994-2996. Fox, J. G., Chilvers, T., Goodwin, C. S., Taylor, N. S., Edmonds, P., Sly, L. I., and Brenner, D. J. (1989a). Campylobacter mustelae, a new species resulting from the elevation of Campylobacter pylori subsp, mustelae to species status. Int. J. Syst. Bacteriol. 39, 301-303. Fox, J. G., Murphy, J. C., Otto, G., Pecquet-Goad, M. E., Lawson, G. H. K., and Scott, J. A. (1989b). Proliferative colitis in ferrets: epithelial dysplasia and translocation. Vet. Pathol. 26, 515-517. Fox, J. G., Correa, P., Taylor, N. S., Lee, A., Otto, G., Murphy, J. C., and Rose, R. (1990). Helicobacter mustelae-associated gastritis in ferrets: an animal model of Helicobacter pylori gastritis in humans. Gastroenterology 99, 352-361. Fox, J. G., Otto, G., Murphy, J. C., Taylor, N. S., and Lee, A. (1991a). Gastric colonization of the ferret with Helicobacter species: natural and experimental infections. Rev. Infect. Dis. 13, $671-$680. Fox, J. G., Otto, G., Taylor, N. S., Rosenblad, W., and Murphy, J. C. (1991 b). Helicobacter mustelae-induced gastritis and elevated gastric pH in the ferret (Mustela putorius furo). Infect. Immun. 59, 1875-1880. Fox, J. G., Palley, L. S., and Rose, R. (1992a). Eosinophilic gastroenteritis with Splendore-Hoeppli material in the ferret (Mustela putorius furo). Vet. Pathol. 29, 21-26. Fox, J. G., Paster, B. J., Dewhirst, E E., Taylor, N. S., Yan, L.-L., Macuch, P. J., and Chmura, L. M. (1992b). Helicobacter mustelae isolation from feces of ferrets: evidence to support fecal-oral transmission of a gastric Helicobacter. Infect. Immun. 60, 606-611. Fox, J. G., Blanco, M., Yan, L., Shames, B., Polidoro, D., Dewhirst, E E., and Paster, B. J. (1993). Role of gastric pH in isolation of Helicobacter mustelae from the feces of ferrets. Gastroenterology 104, 86-92. Fox, J. G., Andrutis, K., and Yu, J. (1994). Animal models for Helicobacterinduced gastric and hepatic cancer. In "Helicobacter pylori: basic mechanisms to clinical cure" (R. H. Hunt and G. N. J. Tytgat, eds.), pp. 504-522. Kluwer Academic Publ., Dordrecht, Holland. Fox, J. G., Dangler, C. A., Sager, W., Borkowski, R., and Gliatto, J. M. (1997). Helicobacter mustelae-associated gastric adenocarcinoma in ferrets (Mustela putorius furo). Vet. Pathol. 34, 225-229. Fox, J. G., Pearson, R. C., and Bell, J. A. (1998a). Diseases of the genitourinary system. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 247-272. Williams and Wilkins, Baltimore. Fox, J. G., Pearson, R. C., and Gorham, J. R. (1998b). Viral diseases. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 355-374. Williams and Wilkins, Baltimore. Frederick, K. A., and Babish, J. G. (1985). Compendium of recent literature on the ferret. Lab. Anim. Sci. 35, 298-318. Gigliotti, E, Harmsen, A. G., Haidaris, C. G., and Haidaris, P. J. (1993). Pneumocystis carinii is not universally transmissible between mammalian species. Infect. Immun. 61, 2886-2890. Gorham, H. R., and Brandly, C. A. (1953). The transmission of distemper among ferrets and mink. Paper presented at the 90th Meeting of the American Veterinary Medical Association. Gorham, J. R., Gordy, D. R., and Quortrup, E. R. (1949). Salmonella infections in mink and ferrets.Am. J. Vet. Res. 10, 183-192. Gorham, J. R., Leader, R. W., and Henson, K. B. (1964). The experimental transmission of a virus causing hypergammagloblinemia in mink: sources and modes in infection. J. Infect. Dis. 114, 341.

515 Greenlee, E G., and Stephens, E. (1984). Meningeal cryptococcosis and congestive cardiomyopathy in a ferret. J. Am. Vet. Med. Assoc. 184, 840-841. Hahn, E. W., and Wester, R. C. (1969). "The Biomedical Use of Ferrets in Research." Marshall Farms Animals, North Rose, New York. Hammond, J., and Chesterman, F. C. (1972). The ferret. In "The Universities Federation for Animal Welfare Handbook on the Care and Management of Laboratory Animals" (UFAW, ed.), 5th ed. Churchill Livingstone, London. Hart, J. E. (1985). Endocrine factors in hematological changes seen in dogs and ferrets given estrogens. Med. Hypotheses 16, 159-163. Hill, S. L., and Lappin, M. R. (1995). Cryptosporidiosis in the dog and cat. In "Kirk's Current Veterinary Therapy" (J. D. Bonagura and R. W. Kirk, eds.), pp. 728-731. Saunders, Philadelphia. Hillyer, E. V., and Quesenberry, K. E. (1997a). Dematologic diseases. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 119-120. Saunders, Philadelphia. Hillyer, E. V., and Quesenberry, K. E., eds. (1997b). "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery." Saunders, Philadelphia. Jenkins, J. R., and Brown, S. A. (1993). "A Practitioner's Guide to Rabbits and Ferrets." American Animal Hospital Association, Lakewood. Jones, J. W., Pether, J. V. S., Rainey, H. A., and Swinburn, C. R. (1993). Recurrent Mycobacterium bovis infection following a ferret bite [letter]. J. Infect. 26, 225-226. Kauffman, C. A. (1981). Cell mediated immunity in ferrets: delayed dermal hypersensitivity, lymphocyte transformation, and macrophage migration inhibitory factor production. Dev. Comp. Immunol. 5, 125-134. Kenyon, A. J., Helmboldt, C. E, and Nielson, S. W. (1963). Experimental transmission of Aleutian disease with urine. Am. J. Vet. Res. 24, 1066. Kirkpatrick, C. E., and Dubey, J. P. (1987). Enteric coccidial infections. Vet. Clin. North Am. Small Anim. Pract. 17, 1414-1416. Knight, D. (1987). Heartworm infections. Vet. Clin. North Am. Small Anim. Pract. 17, 1463-1518. Krueger, K. L., Murphy, J. C., and Fox, J. G. (1989). Treatment of proliferative colitis in ferrets. J. Am. Vet. Med. Assoc. 194, 1435-1436. Kwochka, K. W. (1986). Canine demodicosis. In "Current Veterinary Therapy 11" (R. Kirk, ed.), pp. 531-537. Saunders, Philadelphia. Lee, E. J., Moore, W. E., Fryer, H. C., and Minocha, H. C. (1982). Haematological and serum chemistry profiles of ferrets (Mustela putorius furo). Lab. Anim. 16, 133-137. Li, X., and Fox, J. G. (1998). Neoplastic diseases. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 405-448. Williams and Wilkins, Baltimore. Li, X., Fox, J. G., Erdman, S. E., and Aspros, D. G. (1995). Cutaneous lymphoma in a ferret (Mustela putoriusfuro). Vet. Pathol. 32, 55-56. Li, X., Fox, J. G., Erdman, S. E., Lipman, N. S., and Murphy, J. C. (1996a). Cystic urogenital anomalies in the ferret (Mustela putorius furo). Vet. Pathol. 33, 150-158. Li, X., Pang, J., and Fox, J. G. (1996b). Coinfection with intracellular Desulfovibrio species and coccidia in ferrets with proliferative bowel disease. Lab. Anim. Sci. 46, 569-571. Li, X., Fox, J. G., and Padrid, P. A. (1998). Neoplastic diseases in ferrets: 574 cases (1968-1997). J. Am. Vet. Med. Assoc. 212, 1402-1406. Liberson, A. J., Newcomer, C. E., Ackerman, J. I., Murphy, J. C., and Fox, J. G. (1983). Mastitis caused by hemolytic Escherichia coli in the ferret. J. Am. Vet. Med. Assoc. 183, 1179-1181. Lipman, N. S., Murphy, J. C., and Fox, J. G. (1987). Clinical, functional, and pathologic changes associated with a case of dilatative cardiomyopathy in a ferret. Lab. Anim. Sci. 37, 210-212. Lipman, N. S., Marini, R. P., Murphy, J. C., Zhibo, Z., and Fox, J. G. (1993). Estradiol-17[3-secreting adrenocortical tumor in a ferret. J. Am. Vet. Med. Assoc. 203, 1552-1555. Liu, C., and Coffin, D. L. (1957). Studies on canine distemper infection by means of fluorescein-labeled antibody. 1. The pathogenesis, pathology, and diagnosis of the disease in experimentally infected ferrets. Virology 3, 115.

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Loeb, W., and Quimby, E, eds. (1999). "The Clinical Chemistry of Laboratory Animals." Taylor and Francis, Philadelphia. Manning, D., and Bell, J. A. (1990a). Derivation of gnotobiotic ferrets: perinatal diet and hand-rearing requirements. Lab. Anim. Sci. 40, 51-55. Manning, D. D., and Bell, J. A. (1990b). Lack of detectable blood groups in domestic ferrets: implications for transfusion. J. Am. Vet. Med. Assoc. 197, 84-86. Marini, R. P., Callahan, R. J., Jackson, L. R., Jyawook, S., Esteves, M. I., Fox, J. G., Wilkinson, R. A., and Strauss, H. W. (1997). Distribution of technetium 99m-labeled red blood cells during isoflurane anesthesia in ferrets. Am. J. Vet. Res. 58, 781-785. Marini, R. P., Fox, J. G., Taylor, N. S., Yan, L., McColm, A., and Williamson, R. (1999). Ranitidine-bismuth citrate and clarithromycin, alone or in combination, for eradication of Helicobacter mustelae infection in ferrets. A. J. Vet. Res. 60, 1280-1286. Marini, R. P., Jackson, L. R., Esteves, M. I., Andrutis, K. A., Goslant, G. M., and Fox, J. G. (1994). The effect of isoflurane on hematologic variables in ferrets. Am. J. Vet. Res. 55, 1479-1483. Marshall, K. R., and Marshall, G. W. (1973). "The Biomedical Use of Ferrets in Research." Marshall Farms Animals, North Rose, New York. McLain, D. E., Harper, S. M., Roe, D. A., Babish, J. G., and Wilkinson, C. (1985). Congenital malformations and variations in reproductive performance in the ferret: effects of maternal age, color, and parity. Lab. Anim. Sci. 35, 251-255. Miller, W. R., and Merton, D. A. (1982). Dirofilariasis in a ferret. J. Am. Vet. Med. Assoc. 180, 1103-1104. Moreland, A. E, Battles, A. H., and Nease, J. H. (1986). Dirofilariasis in a ferret. J. Am. Vet. Med. Assoc. 188, 864. Morgan, J. P., and Travers, K. E. (1998). Use of the ferret in cardiovascular research. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), 2nd ed., pp. 499-510. Williams and Wilkins, Baltimore. Morrow, D. A. (1980). "Current Therapy in Theriogenology." Saunders, Philadelphia. Mullen, H. (1997). Soft tissue surgery. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 131-144. Saunders, Philadelphia. Noli, C., van der Horst, H., and Wjillemse, T. (1996). Demodiciasis in ferrets (Mustela putorius furo). Vet. Q. 18, 28-31. Norrby, E., and Oxman, M. N. (1990). Measles virus. In "Fields Virology" (B. N. Fields, D. M. Knipe, R. M. Chanock, et al., eds.), pp. 1013-1044. Raven Press, New York. Ohshima, K., Shen, D. T., Henson, J. B., and Gorham, J. R. (1978). Comparison of the lesions of Aleutian disease in mink and hypergammaglobulinemia in ferrets. Am. J. Vet. Res. 39, 653-657. Orcutt, C. (1997). Dermatologic diseases. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 115-125. Saunders, Philadelphia. Otto, G., Fox, J. G., Wu, P.-Y., and Taylor, N. S. (1990). Eradication of Helicobacter mustelae from the ferret stomach: an animal model of Helicobacter (Campylobacter) pylori chemotherapy. Antimicrob. Agents Chemother. 34, 1232-1236. Oxenham, M. (1990). Aleutian disease in the ferret. Vet. Rec. 126, 585. Palley, L. S., Corning, B. E, Fox, J. G., Murphy, J. C., and Gould, D. H. (1992). Parvovirus-associated syndrome (Aleutian disease) in two ferrets. J. Am. Vet. Med. Assoc. 201, 100-106. Parker, G. A., and Picut, C. A. (1993). Histopathologic features and post-surgical sequelae of 57 cutaneous neoplasms in ferrets (Mustela putorius furo L.). Vet. Pathol. 30, 499-504. Parrott, T. Y., Greiner, E. C., and Parrott, J. D. (1984). Dirofilaria immitis in three ferrets. J. Am. Vet. Med. Assoc. 184, 582-583. Patterson, M. M., Kirchain, S. M., and Whary, M. T. (1999). Clinical trial to control ear mites in a ferret colony. Lab. Anim. Sci. 49, 437. Peter, A. T., Bell, J. A., Manning, D. D., and Bosu, W. T. (1990). Real-time ul-

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trasonography determination of pregnancy and gestational age in ferrets. Lab. Anim. Sci. 40, 91- 92. Phillips, P. H., O'Callaghan, M. G., Moore, E., and Baird, R. M. (1987). Pedal Sarcoptes scabiei infestation in ferrets (Mustela putorius furo). Aust. Vet. J. 64, 289-290. Porter, D. D., Larsen, A. E., and Cox, N. A. (1975). Isolation of infectious bovine rhinotracheitis virus from Mustelidae. J. CIin. Microbiol. 1, 112113. Porter, H. G., Porter, D. D., and Larsen, A. E. (1982). Aleutian disease in ferrets. Infect. Immun. 36, 379-386. Powell, D. A., Gonzales, C., and Gunnels, R. D. (1991). Use of the ferret as a model for pediatric endotrocheal intubation training. Lab. Anim. Sci. 41, 86-89. Pyle, N. J. (1940). Use of ferrets in laboratory work and research investigators. Am. J. Public Health 30, 787. Quesenberry, K. E., and Rosenthal, K. L. (1997). Endocrine diseases. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 85-98. Saunders, Philadelphia. Regh, J. E., Gigliotti, E, and Stokes, D. (1988). Cryptosporidiosis in ferrets. Lab. Anim. Sci. 38, 155-158. Rosenbaum, M. R., Affolter, V. K., Usborne, A. L., and Beeber, N. L. (1996). Cutaneous epitheliotropic lymphoma in a ferret. J. Am. Vet. Med. Assoc. 209, 1441-1444. Rosenthal, K. (1994). Ferrets. Vet. Clin. North Am. 24, 10. Rosenthal, K. L. (1997). Respiratory diseases. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds.), pp. 77-84. Saunders, Philadelphia. Rosenthal, K. L., and Peterson, M. E. (1996). Evaluation of plasma androgen and estrogen concentrations in ferrets with hyperadrenocorticism. J. Am. Vet. Med. Assoc. 209, 1097-1102. Rupprecht, C. E., Gilbert, J., Pitts, R., Marshall, K. R., and Koprowski, H. (1990). Evaluation of an inactivated rabies virus vaccine in domestic ferrets. J. Am. Vet. Med. Assoc. 196, 1614-1616. Ryan, K. D. (1984). Hormonal correlates of photoperiod-induced puberty in a reflex ovulator, the female ferret. Biol. Reprod. 31, 925. Ryland, L. M., and Gorham, J. R. (1978). The ferret and its diseases. J. Am. Vet. Med. Assoc. 173, 1154-1158. Ryland, L. M., Bernard, S. L., and Gorham, J. R. (1983). A clinical guide to the pet ferret. Compend. Contin. Educ. Pract. Vet. 5, 25. Saifuddin, M., and Fox, J. G. (1996). Identification of a DNA segment in ferret Aleutian disease virus similar to hypervariable capsid region of mink Aleutian disease parvovirus. Arch. Virol. 141, 1329-1336. Schulman, E Y., Montall, R. J., and Hauer, P. J. (1993). Gastroenteritis associated with Clostridium perfringens type A in black-footed ferrets (Mustela nigripes). Vet. Pathol. 30, 308-310. Schultheiss, P. C., and Dolginow, S. Z. (1994). Granulomatous enteritis caused by Mycobacterium avium in a ferret. J. Am. Vet. Med. Assoc. 204, 12171218. Scott, D. W., Miller, W. H., and Griffin, C. E. (1995). "Small Animal Dermatology." Saunders, Philadelphia. Shen, D. T., and Gorham, J. R. (1980). Survival of pathogenic distemper virus at 5~ and 25~ Vet. Med. Small Anim. Clin. 75, 69-72. Shen, D. T., Gorham, J. R., Ryland, L. M., and Strating, A. (1981). Using jet injection to vaccinate mink and ferrets against canine distemper, mink viral enteritis, and botulism, type C. Vet. Med. Small Anim. Clin. 76, 856-859. Sherrill, A., and Gorham, J. R. (1985). Bone marow hypoplasia associated with estrus in ferrets. Lab. Anim. Sci. 35, 280-286. Shump, A. U., and Shump, K. A., Jr. (1978). Growth and development of the European ferret (Mustela putorius). Lab. Anim. Sci. 28, 89-91. Shump, A., et al. (1974). "A Bibliography of Mustelids. Part 1: Ferrets and Polecats." Michigan Agricultural Experiment Station 6977, East Lansing. Sinclair, D. G., Evans, E. V., and Sibbald, I. R. (1962). The influence of apparent digestible energy and apparent digestible nitrogen in the diet on weight

13. BIOLOGY AND DISEASES OF FERRETS gain, feed consumption, and nitrogen retention of growing mink. Can. J. Biochem. Physiol. 40, 1376. Smith, L. (1975). "The Pathogenic Anaerobic Bacteria." Charles C. Thomas, Springfield, Illinois. Smith, W., and Stuart-Harris, C. H. (1936). Influenza infection of man from the ferret. Lancet 2, 21. Stamoulis, M. E., Miller, M. S., and Hillyer, E. V. (1997). Cardiovascular diseases. In "Ferrets, Rabbits, and Rodents: Clinical Medicine and Surgery" (E. V. Hillyer and K. E. Quesenberry, eds., pp. 63-70). Saunders, Philadelphia. Stauber, E., Robinette, J., and Basaraba, R. (1990). Mast cell tumors in three ferrets. J. Am. Vet. Med. Assoc. 196, 766-767. Stephensen, C. B., Welter, J., Thaker, S. R., Taylor, J., Tartaglia, J., and Paoletti, E. (1997). Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection. J. Virol. 71, 15061513. Stokes, D. C., Gigliotti, E, Rehg, J. E., Snellgrove, R. L., and Hughes, W. T. (1987). Experimental Pneumocystis carinii pneumonia in the ferret. Br. J. Exp. Pathol. 68, 267-276. Straube, E. E, and Walden, N. B. (1981). Zinc poisoning in ferrets (Mustela putorius furo). Lab. Anim. 15, 45-47. Supakorndej, E, McCall, J. W., and Lewis, R. E. (1992). "Biology, Diagnosis, and Prevention of Heartworm Infection in Ferrets," pp. 59-69. American Heartworm Society, Batavia, Illinois. Sweet, C., Bird, R. A., Jakeman, K., Coates, D. M., and Smith, H. (1987). Production of passive immunity of neonatal ferrets following maternal vaccination with killed influenza A virus vaccines. Immunology 60, 83-89. Symmers, W., and Thomson, A. (1953). Observations on tuberculosis in the ferret (Mustela furo L.). J. Comp. Pathol. 63, 20-29. Thomas, E E., and Desmukh, D. R. (1986). Effect of arginine-free diet on ammonia metabolism in young and adult ferrets. J. Nutr. 116, 545-551. Thomson, A. (1951). A history of the ferret. J. Hist. Med. Allied Sci. 6, 471. Thornton, E C., Wright, E A., Sacra, E J., and Goodier, T. E. (1979). The ferret, Mustela putoriusfuro, as a new species in toxicology. Lab. Anim. 13, 119-124. Timm, K. I. (1988). Pruritis in rabbits, rodents, and ferrets. Vet. Clin. North Am. 18, 1088-1089. Tompkins, D. S., Watt, J. I., Rathbone, B. J., and West, A. E (1988). The characterization and pathological significance of gastric CampyIobacter-like organisms in the ferret: a model of chronic gastritis? Epidemiol. Infect. 101, 269-278. Torres-Medina, A. (1987). Isolation of atypical rotavirus causing diarrhea in neonatal ferrets. Lab. Anim. Sci. 37, 167. Trautwein, G. W., and Helmboldt, C. E (1966). Mastitis in mink due to Staphylococcus aureus and Escherichia coli. J. Am. Vet. Med. Assoc. 149, 924928. USDHHS. (1996). "Guide for the Care and Use of Laboratory Animals," p. 32. National Academy Press, Washington, D.C.

517 Viallier, J., Viallier, G., and Prave, M. (1983). Place de Mycobacterium avium dans l'epidemiologies mycobacterienne actuelle chez les animaux domestiques et savages. Sci. Vet. Med. Comp. 85, 103-109. Vinegar, A., Sinnett, E. E., Kosch, P. C., and Miller, M. L. (1985). Pulmonary physiology as a model for inhalation toxicology. Lab. Anim. Sci. 35, 246250. Wagner, R. A., and Dorn, D. P. (1994). Evaluation of serum estradiol concentrations in alopecic ferrets with adrenal gland tumors. J. Am. Vet. Med. Assoc. 205, 703-707. Walton, K. C. (1977). The ferret. In "Handbook of British Mammals" (G. Corbet and H. Southern, eds.). Blackwell Scientific, Oxford. Wang, X.-D., Krinksy, N. I., Marini, R. P., Tang, G., Yu, J., Hurley, R., Fox, J. G., and Russell, R. M. (1992). Intestinal uptake and lymphatic absorption of [3-carotene in ferrets: a model for human [3-carotene metabolism. Am. Physiol. Soc. 263, G480-G486. Wang, X.-D., Russel, R. M., Marini, R. P., Tang, G., Dolnikowski, G., Fox, J. G., and Krinsky, N. I. (1993). Intestinal perfusion of [3-carotene in the ferret raised retinoic acid level in portal blood. Biochim. Biophys. Acta 1167, 159-164. Wang, X.-D., Marini, R. P., Hebuterne, X., Fox, J. G., Krinksy, N. I., and Russell, R. M. (1995). Vitamin E enhances the lymphatic transport of [3-carotene and its conversion to vitamin A in the ferret. Gastroenterology 108, 719-726. Welchman, D. d. B., Oxenham, M., and Done, S. H. (1993). Aleutian disease in domestic ferrets: diagnostic findings and survey results. Vet. Rec. 132, 479-484. Wildt, D. E., Bush, M., and Morton, C. (1989). Semen characteristics and testosterone profiles in ferrets kept in a long-day photoperiod, and the influence of hCG timing and sperm dilution medium on pregnancy rate after laporoscopic insemination. J. Reprod. Fertil. 86, 349-358. Williams, B. H., Chimes, M. J., and Gardiner, C. H., (1996). Biliary coccidiosis in a ferret (Mustela putoriusfuro). Vet. Pathol. 33, 437-439. Williams, B. H., Eighmy, J. J., Berbert, M. H., and Dunn, D. G. (1993). Cervical chordoma in two ferrets (Mustela putorius furo). Vet. Pathol. 30, 204206. Williams, B. H., Kiupel, M., West, K. H., Raymond, J. T., Grant, C. K., and Glickman, L. T. (2000). Coronavirus-associated epizootic catarrhal enteritis in ferrets. J. Am. Vet. Med. Assoc. 217, 526-530. Williams, E. S., Thorne, E. T., and Kwiatkowski, D. R. (1992). Comparative vaginal cytology of the estrous cycle of black-footed ferrets (Mustela nigripes), Siberian polecats (M. eversmanni), and domestic ferrets (M. putorius furo). J. Vet. Diagn. Invest. 4, 38-44. Willis, L. S., and Barrow, M. V. (1971). The ferret (Mustela putorius furo) as a laboratory animal. Lab. Anim. Sci. 21, 712-716. Yu, J., Russell, R. M., Salomon, R. N., Murphy, J. C., Palley, L. S., and Fox, J. G. (1995). Effect of Helicobacter mustelae infection on epithelial cell proliferation in ferret gastric tissues. Carcinogenesis 16, 1927-1931.

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Chapter 14 Biology and Diseases of Ruminants: Sheep, Goats, and Cattle Margaret L. Delano, Scott A. Mischler, and Wendy J. Underwood

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Comments about and Examples of Use in Research . . . . . . . . . . . . . . C. Availability and Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Laboratory Management and Husbandry . . . . . . . . . . . . . . . . . . . . . . II. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Unique Physiological Characteristics and Attributes, with Emphasis on Comparative Physiology . . . . . . . . . . . . . . . . . . . . B. Normal Values: Growth, Longevity, Hematology, Clinical Chemistry C. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Genetic, Metabolic, Nutritional, and Management-Related Diseases. C. Traumatic Disorders (Wounds, Bites, and Entrapped Foreign Bodies) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... D. Iatrogenic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Neoplastic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

INTRODUCTION

Since the first edition of this book, the use of r u m i n a n t s as research subjects has c h a n g e d dramatically. Formerly, large animals were p r i m a r i l y used for agricultural research or as m o d e l s of h u m a n diseases. O v e r the past decade, r u m i n a n t s have continued in their traditional agricultural research role but are n o w extensively used for studies in m o l e c u l a r biology, genetic engiLABORATORYANIMALMEDICINE,2ndedition

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neering, and b i o t e c h n o l o g y for basic science, agricultural, and clinical applications. C o n c e r n and interest for the welfare for these species and i m p r o v e d u n d e r s t a n d i n g of their b i o l o g y and b e h a v i o r have c o n t i n u e d during this time, and these are reflected in s o m e respects in the c h a n g i n g h u s b a n d r y and m a n a g e m e n t . This c h a p t e r addresses the basic biology, husbandry, and m o r e c o m m o n and i m p o r t a n t diseases of three r u m i n a n t species m sheep, goats, and c a t t l e - - c o m m o n l y used in the laboratory. O n e chapter is s i m p l y not adequate, however, to address Copyright2002,ElsevierScience(USA).Allfightsreserved. ISBN0-12-263951-0

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the many details and complexities of these species' biology, management, and diseases. References noted in the text offer more information to the interested reader.

A.

Taxonomy

Sheep, goats, and cattle are ungulates, "hooved" animals that are members of the order Artiodactyla (even-toed ungulates, or animals with cloven hooves), suborder Ruminantia (ruminants, or cud-chewing animals) and family Bovidae. Members of the Bovidae group of mammals are distinguished by characteristics such as an even number of toes, a compartmentalized forestomach, and horns. These animals are obligate herbivores and, as adults, derive all their glucose from gluconeogenesis. The subfamily Caprinae includes sheep and goats. The genus and subgenus Ovis includes domestic sheep as well as wild Asian and European sheep species. Domestic sheep are Ovis aries. The subgenus Pachyceros includes the wild North American species as well as snow sheep (O. nivicola) of northeastern Siberia. Capra hircus is the domestic goat that originated from western Asian goats. Capra pyrenaica (Spanish goat), C. ibex (goats of the Red Sea and Caucasus area), and C. falconiere (wild goat of Afghanistan and Pakistan) are other members of the genus. The subfamily Bovinae and genus Bos include all domestic and wild cattle. The subgenus taurus contains all of today's domestic cattle. Common genus and species terminology for modern-day cattle includes Bos taurus and B. indicus. Bos taurus (domestic cattle), originally from the European continent, have no hump over the withers. Bos indicus, also known as Zebu cattle, have a hump over the withers and drooping ears. These cattle include breeds found in the tropics and are extremely heat tolerant, and some breeds are known for parasite resistance. Bos taurus and B. indicus have been crossed, and new breeds have been developed during this century (Briggs and Briggs, 1980; Walker et al., 1983). There are several hundred breeds of sheep worldwide that are distinguished as "meat," "wool" or "hair," or "dual-purpose." Some wool or hair breeds have varying coat colors. Some breeds are raised for milk (cheese) production. Common breeds of European origin that are raised for meat in the United States include the larger breeds such as Dorset, Columbia, Suffolk, and Hampshire. Slightly smaller breeds include Southdown and Border Cheviot. Wool breeds include Merino, Rambouillet, Lincoln, and Romney; wool breeds are subclassified according to the properties of the wool. The Barbados is known as a "hair" breed. Newer breeds that have been developed in the United States include Polypay and Targhee (Briggs and Briggs, 1980). Goat breeds are numerous and are usually classified according to use as dairy, meat, fiber, or skin-type breeds. The major dairy breeds are the Alpine, Nubian, Toggenburg, La Mancha, Saanen, and Oberhaslie; all have origins on the European continent. The Nubian breed was developed from crossbreeding

British stock with Egyptian and Indian goats. This breed is relatively heat tolerant and produces milk with the highest butterfat (about 4-5%). Fiber breeds include the Angora and the Cashmere. The Angora, the source of mohair, originated in Turkey. The Cashmere breed is found primarily in mountainous areas of Central Asia. The La Mancha, a newer breed of dairy goat first registered in the United States in 1958, has rudimentary ears that are a genetically dominant distinguishing characteristic of the breed. The meat breeds include the Boer, Sapel, Ma Tou, Kambling, and Pygmy. The Pygmy goat is small and is sometimes used for both meat and milk. The Mubend of Uganda and the Red Sokoto of West Africa produce quality skins for fine leather (Smith and Sherman, 1994). Most breeds of cattle are classified as "dairy" or "beef"; a few breeds are considered "dual-purpose." Common dairy breeds in the United States include Holstein-Friesian, Brown Swiss, Jersey, Ayrshire, Guernsey, and Milking Shorthorn. Holsteins have the largest body size, whereas Jerseys have the smallest. Of breeds in temperate regions, Jerseys have been considered to be the most heat tolerant, but Holsteins have been found to adapt to warmer climates. There are many beef breeds. The more common in the United States include Angus (also called AberdeenAngus), Hereford (both polled and horned), and Simmental (Briggs and Briggs, 1980; Schmidt et al., 1988). Breeds indigenous to other continents, such as the Cape Buffalo, have been found to have unique innate immune characteristics that protect them from endemic trypanosomiasis (Muranjan et al., 1997). More detailed information regarding these and other ruminant breeds is available in Briggs and Briggs (1980). "Rare" or "minor" breeds of sheep, goats, and cattle are studied for their genetic and production characteristics. Discussions of these and efforts at conservation are described in detail elsewhere (National Research Council, 1993). Several terms are unique to ruminants. In relation to sheep, a ewe is the female, and a ram is the adult intact male. A lamb is the young animal, and ram lamb and ewe lamb are commonly used terms. A wether is a castrated male. The birthing process is referred to as lambing. With respect to goats, a doe or nanny is the female. A buck or billy is the adult intact male. A kid or goatling is a young goat. A young male may be referred to as a buckling, and a young female may be referred to as a doeling. A castrated male in this species is also called a wether. The birthing process is called kidding. With respect to cattle, an adult female is a cow, and an adult male is a bull. A calf is a young animal. A heifer is a female who has not had her first calf. A steer is a castrated male. Calving refers to the act of giving birth.

B.

Comments about and Examples of Use in Research

Ruminants have been used as research models since the inception of the land grant college system, first in production

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

agriculture and now also in basic and applied studies for the anatomic and physiologic sciences and in biomedical research for a variety of purposes. Healthy, normal young ruminants serve as models of cardiac transplantation and as preclinical models for evaluation of cardiac assist or prosthetic devices, such as vascular stents and cardiac valves (Salerno et al., 1998). For many years, ruminants have been useful research subjects for reproductive research, such as research on embryo transfer, artificial insemination, and control of the reproductive cycle (Wall et al., 1997). Several important milestones in gene transfer, cloning, nuclear transfer, and genetic engineering techniques have been developed or demonstrated using these species (Ebert et al., 1994; Schnieke, 1997; Cibelli et al., 1998a,b) (see Fig. 1). One of many proposed uses of genetically engineered ruminants is the production of proteins that will be secreted in the milk and later isolated (Ebert et aL, 1994; Memon and Ebert, 1992). Healthy sheep and goats are also often used for antibody production (Hanly et al., 1995). Genome mapping developed rapidly during the 1990s; extensive information is available and is increasing for sheep and cattle (Broad et al., 1998; Womack, 1998). Sheep are often selected for studying areas such as ruminant physiology and nutrition. These animals provide obvious bene-

Fig. 1.

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fits over the use of cattle in research from the standpoint of size, ease of handling, cost of maintenance, and docile behavior. Sheep are also widely used models for basic and applied fetal and reproductive research (Buttar, 1997; Rees et al., 1998; Ross and Nijland, 1998). The species is used for investigating circadian rhythms related to day length (Lehman et al., 1997), and the interaction between olfactory cues and behavior (Kendrick et al., 1997). The number and diversity of natural- and induceddisease research models in sheep are great and increasing. Natural models include congenital hyperbilirubinemia/hepatic organic anion excretory defect (Dubin-Johnson syndrome) in the Corriedale breed, congenital hyperbilirubinemia/hepatic organic anion uptake defect (Gilbert syndrome) in the Southdown breed, glucose-6-phosphate dehydrogenase deficiency in the Dorset breed, GM~ gangliosidosis in the Suffolk breed, and pulmonary adenomatosis (jaagsiekte) in many breeds (Hegreberg, 198 l a). Induced models include arteriosclerosis, hemorrhagic shock, copper poisoning (Wilson's disease), and metabolic toxocosis (Hegreberg, 198 lb). Goats are used in a wide variety of agricultural and biomedical disciplines such as immunology, mastitis, nutrition, and parasitology research. Vascular researchers select the goat because of the large, readily accessible jugular veins. Goats with

The production of cloned cattle reflects the changing use of ruminants in research.

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SCOTT A. MISCHLER,WENDYJ. UNDERWOOD,AND MARGARETL. DELANO

inherited caprine myotonia congenita ("fainting goats") have been used as a model for human myotonia congenita (Thomsen's disease) (Kuhn, 1993). A line of inbred Nubians serves as models for the genetic disease [3-mannosidosis and prenatal therapeutic cell transplantation strategies (Lovell et al., 1997). (These disorders are discussed in more detail in Section III,B,1.) Goats are used as a model for osteoporosis research (Welch et al., 1996). Cattle are often used as a source of ruminal fluid for research, teaching, or treatment of other cattle, by placing a permanent fistula in the left abdominal wall to allow sampling of ruminal fluid (Dougherty, 1981). Cattle also serve as models of many infectious diseases, including zoonoses, and several inherited metabolic diseases. This species is useful for the basic and comparative research on the pathogenesis and immunology of inherited and infectious diseases. Bovine trichomoniasis, caused by Tritrichomonas (Trichomonas)fetus, has been identified as a useful model for the human infection by Trichomonas vaginalis (Corbeil, 1995). Inherited cardiomyopathies have been found in the Holstein-Friesian, Simmental-Red Holstein, Black Spotted Friesian, and Polled Hereford with woolly coat (Weil et al., 1997). Lipofuscinosis has been identified in Ayrshires and Friesians, and glycogenesis in Shorthorns and Brahmans. Metabolic diseases such as hereditary orotic aciduria and hereditary zinc deficiency have been characterized in HolsteinFriesian or Friesian cattle. Holstein cattle also serve as a model for leukocyte adhesion deficiency syndrome (AFIP, 1995).

C.

Availability and Sources

Common breeds of normal, healthy ruminants are usually readily available, although seasonality may play a role, as noted below. Agricultural sources and reputable farms may be located through land-grant universities or agricultural schools, cooperative extension and 4-H networks, regional ruminant breeders' associations, and farm bureaus. Commercial sources of purposebred animals are found in technical publications and annual listings of research animal vendors. Breeds carrying genetic traits of interest, either as animal models or as valuable production characteristics, may be located through literature or Internet searches, animal science societies, breed or livestock conservation associations, and information resources such as the Armed Forces Institute of Pathology. Organizations such as the Institute for Laboratory Animal Research (ILAR), National Center for Research Resources (NCRR), or the Animal Welfare Information Center (AWIC) may also serve as information sources about the animals needed. Purpose-bred research sheep and goats are available from commercial vendors and are usually maintained in registered facilities under federal standards that are also acceptable to research animal accrediting agencies. These commercial animals are frequently described as specific pathogen-free (SPF) and

housed as biosecure or closed flocks. Animal health programs are in place, and health reports or other quality assurance reports are usually available on request. Agricultural sources of either small ruminant may be acceptable, but specific research needs may not have been addressed or may not be understood. Lambs, kids, and milking goats may be difficult to locate in fall and winter months because most breeds of sheep and goats are seasonal breeders. Management practices exist, however, to extend the breeding and milking seasons. Most cattle used as animal models in research in the United States are from one of the dairy breeds, usually Holstein, because this breed is now the most common. Purpose-bred, specific pathogen-free research cattle are not typically available. Because of selection and the management of dairy production units, calves and young stock are available year-round. Availability of young beef cattle is more seasonal, according to production cycles typically followed by that industry. Auction barns or sales are not appropriate sources for research ruminants. Many of these animals are culls and will be poor-quality research subjects. They may be in poor body condition and stressed, may be sources of disease, and may contaminate other healthy animals, as well as the research facility. Selection of the suppliers should be made only after research needs have been carefully considered. Consistently working with and buying directly from as few sources as possible are best. Certain types of research (i.e., agricultural nutrition studies) may better be served by selecting animals from local agricultural suppliers rather than commercial vendors located in a different geographical area. The selection of sources for research ruminants includes scrutiny of flock or herd record keeping; health monitoring, vaccination, and preventive medicine programs (including hoof care); production standards and management practices consistent with the industry; management of the breeding flock or herd; sanitation and waste handling programs; vermin and insect control measures (especially for flies and other flying insects); rearing programs for and condition of young stock; the location, health, and condition of the other animals on the premises; intensity of housing; and animal housing facilities. Preliminary and periodic visits to the source farms should be conducted. It is important to establish a good relationship with the local attending large-animal veterinarians, who will be valuable resources for current approved therapies and practices. They may need to be oriented on the specific requirements of animal research. Creative ways can be used to initiate and foster a good working relationship between the agricultural supplier and the research facility. Supplying the vaccines or dewormers required for flock health programs, providing services such as quarterly serological testing or fecal examinations for the herd or flock, and paying a premium (rather than market price) for animals that meet the quality criteria established for the research animals are often helpful. A set of testing standards can be developed based on one

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

high-quality supplier, and then flocks or herds can be "qualified" based on those standards. Qualifying entails evaluations utilizing the facility and management aspects mentioned above and testing either a percentage of the herd or flock or the entire herd or flock for a number of infectious agents. The testing regimen itself should be carefully developed and evaluated. Once qualified, each source farm should be reevaluated periodically to maintain its status. Slaughter checks may be appropriate; otherwise necropsy of sentinel animals may be required. Selected animals undergoing screening tests should be quarantined from the rest of flock or herd while awaiting test results. Vaccination and deworming regimens can be instituted during these quarantine periods. A second quarantine should occur when animals arrive at the research facility. The animal screening process also depends on the origin of the animal (state, country) and the scientific program. Federal and state regulations must be followed. Socialization of the animals at the source facility should also be considered in terms of ease of handling and safety for personnel in the confinement of the research lab, barn, or farm. For example, frequently handled calves will be easier to manage, and adult dairy goats that have been acclimated to human contact are preferable. Several texts provide information on industry standards for flock and herd management and preventive medicine strategies that can provide helpful orientation to those unfamiliar with these aspects. These references also provide information regarding vaccination products licensed for use in ruminants and typical herd and flock vaccination parasite control schedules ("Current Veterinary Therapy," 1986, 1993, 1999; "Council report," 1994; "Large Animal Internal Medicine," 1996; Smith and Sherman, 1994) When designing a vaccination program during qualification of a source or at the research facility, it is important to evaluate the local disease incidence and the potential for exposure. Vaccination programs should be conducted with an awareness of duration of passive immunity and stresses in ruminants' lives (e.g., weaning, grouping, management changes, and shipping) that may impair immunity or increase susceptibility to infectious diseases. It is also prudent to evaluate the cost-effectiveness of vaccination; labor and vaccine expenses may be much higher than the potential animal morbidity or mortality for diseases in a particular locality. Not all of the vaccines mentioned subsequently will be necessary in all herds or flocks. Vaccination needs for research animals will also depend on the local disease history, intent of the research, the age of the animals needed for research, and the length of time the animals will be housed. Typical health screening programs for sheep include Q fever (Coxiella burnetii); contagious ecthyma; caseous lymphadenitis (Corynebacterium pseudotuberculosis); Johne's disease (Mycobacterium paratuberculosis); ovine progressive pneumonia; internal parasitism such as nasal bots, lungworms, and intes-

523

tinal worms; and external parasitism such as sheep keds. Each supplier should be queried about vaccination programs for bluetongue, Brucella ovis, Campylobacter spp., Chlamydia (enzootic abortion of ewes), clostridial diseases, pneumonia complex (parainfluenza 3, Pasteurella haemolytica, and P. multocida), ovine ecthyma, rabies, Dichelobacter (Bacteroides) nodosus, Arcanobacterium pseudotuberculosis, Bacillus anthracis, and Fusobacterium necrophorum. Because of the limited number of biologics approved for small ruminants, products licensed for cattle have been used with success in sheep, and some licensed for sheep are used in goats ("Council report," JAVMA, 1994). In some cases, approved feed additives, such as coccidiostats, are fed to sheep. The basic screening profile for goats should include Q fever (Coxiella burnettii), caprine arthritis encephalitis (CAE), brucellosis, tuberculosis, and Johne's disease (Mycobacterium paratuberculosis). Goats may also be tested for caseous lymphadenitis, contagious ecthyma, or Mycoplasma as needed. Herd vaccination programs may include immunizations against tetanus and other clostridial diseases, Chlamydia, Campylobacter, contagious ecthyma, caseous lymphadenitis, Corynebacterium pseudotuberculosis, and Escherichia coli. Cattle herds should be screened for Johne's disease, brucellosis, tuberculosis, respiratory diseases, internal and external parasitism, and foot conditions such as hairy heel warts and foot rot. Determination of the status of the herd with respect to bovine leukemia virus (BLV) may be worthwhile. Herd programs may include essential or highly recommended vaccines against bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (BRSV), parainfluenza 3 (PI-3), Leptospira pomona, Tritrichomonas fetus, rotavirus, coronavirus, Campylobacter (Vibrio), Pasteurella haemolytica and P. multocida, and Brucella abortus. Other vaccination programs, dependent on herd status, endemic diseases, or geographic location, may include immunizations against the Clostridial diseases, Moraxella bovis (pinkeye), Fusobacterium necrophorum (foot rot), Staphylococcus aureus (mastitis), Haemophilus somnus, rabies, tetanus, Bacillus anthracis, enterotoxigenic E. colL Anaplasma, and other Leptospira species. Some products considered to have limited efficacy include vaccines against Salmonella dublin and S. typhimurium. Some autogenous vaccines may be more effective than the commercially available products--for example, the bovine papillomavirus (warts) vaccines. Rearing programs for dairy calves differ from those for the smaller ruminants, including the withdrawal of calves from their dams immediately or by 24 hours after birth. In the cattle industry, antibiotics, ionophores (antibiotics that control selected populations of ruminal organisms), coccidiostats, probiotics, and other approved additives may be part of the milk replacers, grain and concentrate formulations, and/or creep feeding regimens. Use varies by the segment of the industry, and regulations vary by country. Subcutaneous hormonal

524

SCOTT A. MISCHLER, WENDYJ. UNDERWOOD,AND MARGARETL. DELANO

implants (such as estradiol benzoate and progesterone combined, zeranol, or 17[~-estradiol) are administered, especially to beef calves destined for market rather than breeding, to promote growth. Transportation of the animals from the source to the research facility must be carefully planned, and all applicable livestock travel regulations followed. It is best to have the animals transported in vehicles regularly utilized by the source farm. If commercial haulers are used, then disinfecting trucks, trailers, and associated equipment, such as ramps and chutes, beforehand is particularly important. The loading, footing, and distribution of the animals in the trailers and trucks, as well as environmental conditions during shipping, are important to consider to minimize stress and injury to the animals. Sufficient time for acclimation to the facility, pens, handlers, feed, and water must be allowed once at the destination ("Livestock Handling and Transport," 1998).

D. LaboratoryManagement and Husbandry Recent publications address many general considerations as well as specifics about the facilities, husbandry, space requirements, and standard practices for research and production ruminants. Institutions, private entities, researchers, and facility staff must also be aware of the recent adoption by the U.S. Department of Agriculture (USDA) of specific guidelines for regulation of farm animals, such as ruminants, that are used in biomedical and other nonagricultural research. The USDA Animal Care Policy 29 notes that the "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching" and the "Guide for the Care and Use of Laboratory Animals" provide additional information to supplement the existing Animal Welfare Act regulations (CFR, 1985; FASS, 1999; Hays et al., 1998; NRC, 1996a; USDA, 2000). In all cases, stress should be considered and minimized in the husbandry and handling of ruminants. Animals need to be provided adequate time to adapt to new surroundings. Stress decreases feed intake, and the resulting energy, vitamin, and mineral deficiencies will affect the growth and development in younger animals. Reproductive soundness and rumen function are affected by transport and similar stresses. Standard practices such as weaning, castration, dehorning, vaccinations, deworming and treatments for external parasites, shipping and the associated feed and water deprivation, introduction to a new housing environment and new personnel, and intercurrent disease are all stressors (Houpt, 1998). Animals should be acclimated to the use of halters and leads, temporary restraint devices, and other handling equipment associated with the research program. Personnel in the research facility who are unfamiliar with ruminants should be trained in appropriate handling techniques. Ap-

preciation for ruminant behaviors has grown in recent years, and refined ruminant handling techniques have been published (Houpt, 1998; Grandin, 1998). When ruminants are confinement-housed, care should be taken to provide adequate but draft-free ventilation. Ammonia buildup and other waste gases may induce respiratory problems. In cold weather, if the ceiling, walls, or water pipes condense water, then the ventilation should be increased even at the expense of lower temperatures. Even adult goats and younger cattle are quite comfortable in cold, even subfreezing temperatures, if provided with adequate amounts of dry dust-free bedding and draft protection. Sheep, because of their wool, are remarkably tolerant to both hot and cold extremes. Newborn lambs and recently shorn adults are susceptible to hypothermia, hyperthermia, and sunburn. Therefore, in outside housing areas, sheep should be provided with shelters to minimize exposure to sun and inclement weather. Animals housed under intensive confinement should be kept clean, and excreta should be removed from the pens or enclosures daily. Feed and water equipment should be maintained in sound, clean condition and should be constructed to prevent fecal contamination. Waterers should not create a muddy environment in paddocks or pens. There should be sufficient continuous-access waterers placed around the area to prevent competition or fighting. Feeders should be constructed to conform to species size and feeding characteristics and to prevent entrapment of head and limbs. Pens, other enclosures, passageways, chutes, and floors must be very sturdy to withstand such factors as the frequent cleaning; the strength, weight, and curiosity of all ages of animals; and the investigative and climbing behaviors of goats. Chain-link fences are dangerous because goats (as well as some breeds and ages of sheep) are curious and tend to stand on their hind legs against fencing or walls. Forelimbs may be caught easily in the mesh. Floors in any areas where animals will be housed, led, or herded must ensure secure footing at all times to prevent slipping injuries. All ruminants are social and herding animals. Therefore, they should be housed in groups or at least within eyesight and hearing of other animals. Singly housed animals should have regular human contact. Environmental enrichment should be governed by the experimental protocol or standard operating procedures, and durable play objects should be supplied to those animals that are housed in confinement. Calves, in particular, that must be singly housed or that have been recently weaned, need play objects (Morrow-Tesch, 1997). Because sheep and goats are sensitive to changes in light cycle (especially reproductive parameters), photoperiod must be taken into account. Normally, sheep and goats should be maintained on a cycle comparable to natural conditions. Light intensity should be maintained at about 220 lux (ILAR, 1996; FASS, 1999). Light cycles can be manipulated for experimental reasons.

14. BIOLOGYAND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE

lI. A.

BIOLOGY

Unique Physiological Characteristics and Attributes, with Emphasis on Comparative Physiology

The development of the digestive system and the unique function of the rumen are among the most notable comparative anatomic and physiologic characteristics of ruminants. There is a three-compartment forestomach (rumen, reticulum, and omasum) and a true stomach (abomasum). The mature rumen functions as an anaerobic fermentation chamber in which the enzymes, such as cellulase, of the resident bacteria allow the animals to prosper as herbivores. Digestion is also aided by other microorganisms, such as protozoa (105-106/ml) and bacteria (109-101~ that contribute to rumen fermentation. The result is the production of volatile fatty acids (acetic, propionic, and butyric). Unlike in the monogastrics, fermentative digestion and volatile fatty acid absorption also occur in the large intestines. The main sources of energy for ruminants are volatile fatty acids (VFAs) rather than glucose. Glucose is formed from propionic acid (or from amino acids) for metabolism in the central nervous system (CNS), uterus, and mammary glands. Plasma glucose in ruminants is much lower than and is regulated differently from that in nonruminants. The rumen microorganisms also synthesize vitamins, such as B and K, and provide protein that is used by the animals' systems. Large amounts of fermentation gases such as CO2 and methane, and small amounts of nitrogen, are naturally eructed (Hecker, 1983; Schimdt et al., 1988). Intestinal immunoglobulin absorption by pinocytosis in the neonates is crucial to the success of passive transfer. This transfer mechanism is functional for approximately the first 36 hr after birth. Neonatal ruminants are immunocompetent, however, and this condition is used to advantage for vaccinations against some common diseases of the neonatal and later juvenile periods, such as infectious bovine rhinotracheitis (IBR) vaccine (using modified live virus vaccines) to calves when their dams' colostrum is lacking antibody against this virus. Unlike hepatic lipogenesis in humans, lipogenesis in sheep primarily occurs in adipose tissue and the mamrnary gland (Hecker, 1983). In addition to normal lymph node Chains, and as in other ruminants, sheep have small red "nodes" associated with blood vessels. Inadvertently named hemal "lymph nodes," they contain numerous red blood cells. Sheep have a relatively large pituitary gland, and accessory adrenal medullary tissue may be interspersed throughout the abdominal cavity. Three major ovine histocompatability classes have been identified and designated as OVAR (Ovis aries) classes I, II, and III (Franz-Werner et al., 1996). Bovines are recognized as having several unique aspects involving their immune systems. The bovine lymphocyte antigen (BoLA) system ranks after the hu-

525

man (HLA) and murine (H-2) systems in terms of depth of knowledge (Lewin, 1996). Cattle are considered free of autoimmune diseases (Schook and Lamont, 1996). The complexity of the immunobiology of the bovine mammary gland is being studied extensively because mastitis is the most prevalent disease in the dairy industry. Several innate immune mechanisms and cellular defenses, and their variation throughout lactation, have been described (Sordillo et al., 1997).

BO Normal Values: Growth, Longevity,

Hematology, Clinical Chemistry Hematology and clinical reference texts are available for the ruminant species and include overviews of normal values for age, sex, and breed-specific ranges, as well as discussions regarding the influences on the hemogram of many management, nutritional, geographic, metabolic, physiologic (including lactation), medication, and iatrogenic variables (Duncan and Prasse, 1986; Jain, 1986; Kaneko et al., 1997). These references should be consulted when preparing to include blood collection data in research protocols and when reviewing hematologic findings. In addition, most veterinary diagnostic laboratories have also developed databases for normal ranges for hematologic and clinical chemistry values based on subjects from their service areas, and these may be useful as local and breed references. Appropriate control groups must be incorporated into each research plan, however, to establish the normal values (see Table I) for the particular locale, diagnostic facilities, breed, age, sex, and research circumstances. Normal hematologic and clinical biochemistry data are presented in Tables II and III. Some general statements apply to most ruminants. Most ruminants have fewer neutrophils than lymphocytes. The blood urea nitrogen (BUN) values cannot be used as an indicator of renal function because of the metabolism of urea nitrogen by rumen microflora. Because of the large volume of rumen water, ruminants can generally go several days without drinking before significant dehydration occurs. Erythrocytes may become more fragile during rehydration, resulting in some degree of hemolysis and hemoglobinuria. Severe dehydration can occur quickly, however, in animals that are ill. Urine pH is generally alkaline in adult ruminants. Ruminant erythrocytes are smaller than those in other mammals, and hematocrits tend to be overestimated unless blood samples are centrifuged for longer amounts of time for packing of the cell pellet. Increased red-cell fragility is also associated with the smaller erythrocyte. Rouleau formation does not occur in cattle but does to a limited extent in sheep and goats. In addition to fetal hemoglobin, sheep are reported to have at least six different hemoglobins (Hecker, 1983). Blood coagulation in sheep is similar to that in humans.

SCOTT A. MISCHLER, WENDY J. UNDERWOOD, AND MARGARET L. DELANO

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Table 1 Normal Values for Sheep, Goats, and Cattle: Vital Signs, Life Spans, and Weightsa Parameter and age

Sheep

Chromosome number Body temperature(o C) Young Adult Heart rate (beats/min) Young Adult Respiration rate (breaths/min) Young Adult Life span (years) Body weights (lb) Birth 1 month 3 months 6 months 9 months 12 months 18 months 24 months 36 months Deciduous dental formula Permanent dental formula

54

Goats

Cattle

60

60

39.5-40.5 39-40

39-40.5 38.5-39.5

39-40.5 38-39

140 (120-160) 75 (60-120)

140 (120-160) 85 (70-110)

120 (100-140) 60 (40-80)

50 (30-70) 36 (12-72) 10-15

50 (40-65) 28 (15-40) 8-12

48 (30-60) 24 (12-36) 20-25

3-25 110

300 (ram), 200 (ewe) 2(Di 0/3, Dc 0/1, Dp 3/3) = 20 2(10/3, C 0/1, M 3/3) = 32

25 55 85 110 130 155 170 205 2(Di 0/3, Dc 0/1, Dp 3/3) = 20 2(10/3, C 0/1, M 3/3) = 32

400 720 1100 2(Di 0/3, Dc 0/1, Dp 3/3) = 20 2(10/3, C 0/1, M 3/3) = 32

aVital sign data for goats are from "Large Animal Internal Medicine" (1996). Sheep weight data representweights of feeder lamb and adult dry ewe (Federation of Animal Science Societies [FASS], 1998). Goat weight data are for a large-breedmale goat. Cattle weight data represent weights of female Holstein or Guernsey dairy cattle (FASS, 1998). Life span data for sheep and cattle are from Brooks et al. (1984).

Erythrocytes in Pygmy and Toggenburg goats tend to be more fragile than erythrocytes from other goat breeds. Normal caprine erythrocytes lack central pallor because they are fiat and lack biconcavity. Normal caprine erythrocytes may exhibit poikilocytosis. At least five blood groups have been reported in goats: B, C, M, R-O, and X. Because transfusion reaction rates may be as high as 2 - 3 % , cross-matching is advisable although not always practical (Smith and Sherman, 1994). Blood loss of up to 25% of the red cell mass at a single time point can be tolerated by healthy goats. Blood may safely be obtained in volumes of 10 m l / k g body weight and given in volumes Of 1 0 - 2 0 ml/kg. In general, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) are not liverspecific in goats, and alanine aminotransferase (ALT; formally serum glutamic-pyruvic transaminase, or SGPT) cannot be used to evaluate hepatic disease in goats. ~,-Glutamyltransferase (GGT) and alkaline phosphatase (AP) are associated with biliary stasis, and elevations in GGT are generally associated with hepatic damage.

C.

Nutrition

The nutritional needs of ruminants vary considerably according to the species, breed type, different phases of development, the use of the animals, location, and different stresses in their lives. For example, mineral requirements and other nutritional requirements vary even among breeds of cattle. Several references are available that describe the varying requirements and are useful for determining the requirements of ruminants consistent with the parameters noted above and the type of feeds available (Jurgens, 1988; "Large Animal Clinical Nutrition," 1991; NRC, 1981, 1989, 1993, 1996b; "Large Animal Internal Medicine," 1996). Preformulated commercial feeds, concentrates, and supplements are available specifically for the different species of ruminants. Some of these provide complete energy and protein requirements or may be used as supplements for what cannot be provided entirely by pasture, forage, hay, or silage. Concentrate mixtures contain salt, minerals, and other elements. Concentrates should contain a protein source such as soybean meal, cottonseed meal, or linseed meal. Computer programs are also

527

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE Table II

Normal Values for Sheep, Goats, and Cattle: Hematology Parameter (units) Packed cell volume (%) Hemoglobin (g/dl) Red blood cells (RBC) (x 106/~tl) White blood cells (WBC) (x 103) Total protein (g/dl) Mean corpuscular volume (fL) Mean corpuscular hemoglobin (pg) Mean corpuscular hemoglobin concentration (g/dl) Reticulocytes (%) RBC diameter (~tm) RBC life (days) Myeloid: Erythroid ratio Platelets (x 103/~tl) Fibrinogen (mg/dl) WBC differential: absolute count/~tl (% of total) Stabs, bands Segmented neutrophils Lymphocytes Monocytes Eosinophils Basophils Coagulation tests (sec) Prothrombin time Partial thromboplastin time Thrombin time

Sheep 27-45 9-15 9-15 4-12 6.0-7.5 28-40 8-12 31-34 0 3.2-6 140-150 0.77-1.68:10 250-750 100-500 Rare 400-6000 (10-50) 1600-9000 (40-75) 0-750 (0-6) 0-1200 (0-10) 0-350 (0-3) 13.5-15.9 27.9-40.7 4.8-8.0

readily available for those who may need to formulate and balance rations. The palatability of feeds should be taken into account. Mineral deficiencies and supplementation have been shown to influence several physiologic parameters such as immune function. Introduction of young stock should include continuation of the feeding program of the source or gradual transition to appropriate feed for the animals available in the region of the research facility (NRC, 1996). Good-quality pasture can support ruminants under certain circumstances. Lush spring pastures, especially pastures containing alfalfa, can induce bloat, diarrhea, grass tetany, or nitrate poisoning. Ruminants not acclimated to lush pasture should be fed good-quality hay and slowly introduced to pasture environments. When ruminants have access to pasture, it is important to be aware of different eating habits. Sheep and cattle are grazers. Goats are browsers and will readily eat grasses, as well as seeds, nuts, fruit, and woody-stemmed plants. Goats, however, can also be selective eaters and will only eat the leafy, more nutritious parts of the plant. Therefore, goats have a tendency to "waste" hay. Other eating habits should also be considered. Finely ground concentrates are not tolerated well by goats; pelleted concentrates are preferred because the goat will pick out large

Goats

Cattle

22-38 8-12 8-18 4-13 6-7.5 16-25 5.2-8

24-46 8-15 5-10 4-12 7-8.5 40-60 11-17

30-36 0 2.5-3.9 125 0.69:10 300-600 100-400 Rare 1200-6250 (30-48) 2000-9100 (50-70) 0-550 (0-4) 50-1050 (1-8) 0-150 (0-1) 9.0-14.0 20.9-33.4

30-36 0 4.8 160 0.31-1.85:10 100-800 300-700 0-250 (0-2) 600-5400 (15-45) 1800-9000 (45-75) 80-850 (2-7) 80-2400 (2-20) 0-250 (0-2) 6.8-8.4 11.0-17.4 4.3-7.1

particles in mixes. Generally, goats do not prefer "sweet" feeds that contain molasses and do not need supplemental concentrates if a good-quality pasture or hay is fed. When given access to a salt block, goats generally are self-regulating. Grass-fed goats and lactating goats may need supplementation with calcium and phosphorus, whereas alfalfa-fed goats do not (Bretzlaff et al., 1991). Horse and sheep feeds may be fed to goats provided that the feed does not contain much molasses (Bretzlaff et al., 1991). The copper content of horse feed is not excessive for goats, as it is for sheep. Pelleted horse feeds with 2 5 - 2 8 % fiber and 12-14% protein are good goat rations. Goats will consume 5 - 8 % of body weight in dry-matter intake (whereas cattle will usually consume only 4% of body weight). Goats enjoy human contact, and small alfalfa cubes make tasty treats for the goat. Rations that have excessive calcium-phosphorus ratios or elevated magnesium levels may induce urinary calculi in male ruminants. These may also occur when forage grasses are high in silicates and oxalates. To increase ovulation rate in does, some producers "flush" females by feeding 0.5-1 lb concentrate per head per day for several weeks before and after the initiation of the breeding season. Thin pregnant dairy goats should be fed 1 lb concentrate per

SCOTT A. MISCHLER, WENDY J. UNDERWOOD, AND MARGARET L. DELANO

528

Table IIl

Normal Values for Sheep, Goats, and Cattle: Clinical Biochemistry a Parameter (units)

Source

Sheep

Goat

Cattle

Alanine aminotransferase (ALT, SGPT; U/liter) Albumin (g/liter) Alkaline phosphatase (AP; U/liter) Aspartate aminotransferase (AST, SGOT; U/liter) Bicarbonate (HCO3; mmol/liter) Bilirubin Conjugated (mg/dl) Unconjugated (mg/dl) Total (mg/dl) Blood urea nitrogen (BUN; mg/dl) Calcium, total (mg/dl) Carbon dioxide, total (mmol/L) Chloride (C1; mmol/liter) Creatine kinase (CK) U/liter) Creatinine (mg/dl) y-Glutamyltransferase (GGT; U/liter) Globulin (g/liter) Glucose (mg/dl) Lactate dehydrogenase (U/liter) Magnesium (mg/dl) Phosphorus (P; mg/dl) Potassium (K; mmol/L) Sorbitol dehydrogenase (SDH; U/liter) Sodium (Na; mmol/liter) Total protein (TP, g/liter)

s, hp

30 ___4 24-3.0 (27 ___1.9) 68-387 (178 ___102) 60-280 (307 ___43) 20-25

6-19 27.0-39.0 (33.0 _ 3 . 3 ) 93-387 (219 ___76) 167-513

11-40 (27 _ 14) 30.3-35.5 (32.9 _ 1.3) 0-488 (194 ___126) 78-132 (105 +__27) 17-29

s, hp s, p, hp s, p, hp s, hp s, hp s, hp s, hp s, p, hp s, p s s, p, hp s, hp s hp hp hp hp s

0-0.27 (0.12) 0-0.12 0.1-0.5 (0.23 ___0.01) 8-20 11.5-12.8 21-28 (26.2) 95-103 8.1-12.9 (10.3 ___1.6) 1.2-1.9 20-52 (33.5 ___4.3) 35.0-57.0 (44.0 +_ 5 . 3 ) 50-80 (68.4 _ 6.0) 238-440 (352 ___59) 2.2-2.8 5.0-7.3 (6.4 ___0.2) 3.9-5.4 (4.8) 5.8-27.9 (15.7 ___7 . 5 ) 139-152 60.0-79.0 (72.0 + 5 . 2 )

0.01 10-20 (15 __+2.0) 8.9-11.7 25.6-29.6 (27.4 ___1 . 4 ) 99-110.3 (105.1 _+ 2 . 9 ) 0.8-8.9 (4.5 ___2.8) 1.0-1.8 20-56 (38 ___13) 27.0-41.0 (36.0 ___5 . 0 ) 50-75 (62.8 ___7.1) 2.8-3.6 4.2-9.1 (6.5) 3.5-6.7 (4.3 ___0.5) 14.0-23.6 (19.4 __+3 . 6 ) 142-155 (150 ___3.1) 64.0-70.0 (69.0 ___4 . 8 )

0.04-0.44 (0.18) 0.03 0.01-0.5 (0.2) 20-30 9.7-12.4 21.2-32.2 (26.5) 97-111 (104) 4.8-12.1 (7.4 __+2.4) 1.0-2.0 6.1-17.4 (15.7 ___4.0) 30.0-34.8 (32.4 _ 2.4) 45-75 (57.4 _+ 6.8) 692-1445 (1061 ___222) 1.8-2.3 5.6-6.5 3.9-5.8 (4.8) 4.3-15.3 (9.2 ___3.1) 132-152 (142) 67.4-74.6 (71.0 ___1.8)

aData presented as ranges with mean and standard deviation in parentheses, s, Serum; p, plasma; hp, heparinized plasma. Clinical biochemistry data from Kaneko et al. (1997).

day, with the amount increasing to 1.5 lb per head per day during the last 6 weeks of gestation. Forage should be fed a d libit u m during this time. All newborn ruminants must receive passive immunity from colostrum, the first postpartum milk of a dam that contains concentrated protective maternal antibodies (most as IgG1), functional leukocytes, cytokines, vitamins, minerals, and protein. Colostrum also has laxative properties. Trypsin inhibitors in the colostrum allow the passage of intact antibody molecules, by pinocytosis, through the neonate's gut wall and into the bloodstream during the first few days after birth. The quality of the colostrum is directly related to herd or flock management, vaccination programs, and the dam's overall condition and nutrition throughout gestation and at the time of parturition. Ensuring effective colostrum transfer is also dependent on the timing and amount taken by the neonate. Most neonatal ruminants can suckle well within 3 hr of birth. Those that do so have been shown to have significantly less diarrhea (Naylor, 1996). Neonates weakened by dystocia or hypothermia, for example, should be hand-fed or tube-fed colostrum. If necessary, the dam should be hand-milked and the newborn fed colostrum (for example, 2 0 - 4 0 ml for kids) every 2 - 4 hr for the first 1 - 2 days.

In typical m a n a g e m e n t situations, dairy calves either are separated from their dams immediately after birth and bottle-fed colostrum, or they remain with their dams for only about 24 hr and suckle fresh colostrum during this time. Dairy producers then refrigerate and/or freeze the colostrum that cannot be consumed by the calf during that time and then feed this diluted 5 0 : 5 0 with warm water 3 times a day to the calves during the next 2 - 3 days. Extra frozen colostrum for emergencies may be obtained from dairy farmers; it is advantageous to obtain colostrum from well-managed herds and from the multiparous cows in the herd (not heifers) in the same geographic locale. Holstein calves, for example, should receive a m i n i m u m of 3 5 liters within 12 hr of birth and then be fed about 1 0 - 1 5 % of body weight in colostrum by 24 hr of age. After 3 days, calves are then placed on milk replacers. Although young ruminants generally do well receiving their dams' milk, commercially available milk replacers are available and should generally be prepared and fed according to the manufacturer's recommendations. Containers used to prepare and feed these replacers should be sanitized daily. The fat content of both calf and lamb milk replacers is excessive; however, calf milk replacers can be used for kids if care is taken not to overfeed.

14. BIOLOGYAND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE

Young ruminants can be offered good-quality hay (such as second cutting) to nibble on by 1 week of age. Calves may be provided with calf starter, a commercially available concentrate with appropriate levels of energy and protein, fed according to the manufacturer's recommendations at 2 - 3 weeks of age. They can be weaned off milk replacer by 4 - 7 weeks of age. Young ruminants (4-12 months of age) need good-quality forage as well as grain and concentrate supplementation to promote development of the rumen. In farm management situations, forage can be silage, pasture, and hay. In a confinement situation like a research unit, good-quality hay, such as second cutting, is desirable. Animals should not be overfed and should be offered a mineral mix free-choice. In contrast to dairy calves, beef calves remain with their mother cows until weaning at 7 months of age. Calves tend to suckle many times per day. As they mature, calves are creepfed, with the energy and protein content of the ration determined by the milk production of the dams and by the available forage, such as pasture. D.

Reproduction

Several useful references addressing ruminant reproduction in detail are available ("Current Veterinary Therapy: Food Animal Practice," 1986, 1993, 1999; "Large Animal Internal Medicine," 1996; "Current Therapy in Large Animal Theriogenology," 1997; Hafez, 1987). 1.

Reproductive Physiology

Sheep are seasonally polyestrous; most breeds will express estrus in the fall (Northern Hemisphere) and subsequently lamb in the spring. Some breeds of sheep may cycle in both the fall and the spring. Between seasonal periods of receptivity, the females undergo a long period of sexual quiescence called anestrus. In a research environment, ewes can be artificially stimulated to progress from anestrous to estrous cyclicity by maintaining the females in 8 hr of light and 16 hr of dark for 8 10 weeks. Puberty is reached at about 7-8 months (or earlier) in both rams and ewes; rams will typically reach puberty before their female counterparts. Ewes will display signs of estrus for about 2 4 - 3 0 hr and will ovulate spontaneously at the end of estrus. The estrous cycle length is 14-19 days, with an average of about 17 days. Following breeding, the average length of gestation is 147-150 days. Slightly longer gestations are observed in animals carrying single lambs (singlets), in animals carrying rams, and in certain breeds such as those derived from Merinos. Prolificacy, or the number of lambs produced per gestation, tends to be dependent on the maturity of the dam (older dams tend to have multiple lambs) and on breed characteristics (some fine-wool breeds have fewer multiple births). The Finn and Dorset breeds are especially prolific. Lambs vary in size at birth

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from about 3 - 4 lb up to 25 lb. Factors that affect birthweight include parental size, number of lambs in the litter (fewer lambs or singlets tend to be larger), age of the ewe (younger ewes have smaller lambs), lamb gender (males tend to be heavier), nutrition, and season or temperature (spring lambs tend to be larger than fall lambs). Goats are seasonally polyestrous in temperate regions, so that young are born in favorable times of the year. They are shortday breeders, in that estrus (heat) is brought about by the decreasing light of shorter days. In temperate climates of the Northern Hemisphere, goats are normally anestrous during the summer and begin cycling in the fall. The actual length of the sexual cycle depends on day length, breed, and nutrition. Most dairy goats cycle between August and February or March. Nubians often have extended breeding cycles, and the sexual season of some breeds, including the Alpine, can be extended by artificial means. The caprine gestation length averages 150 days with a variation of 145-155 days. Does bear singletons, twins, and triplets, with slightly shorter gestation when the doe is carrying triplets. Cows are polyestrous. Domestication of cattle has included selection against seasonality of the breeding season, particularly in dairy breeds but to some extent also in the beef breeds. In spite of this, cattle have been found to be still sensitive, in varying manifestations, to photoperiodicity. Reproductive physiology in cattle is influenced by many factors. The reproductive programs in source herds and at well-managed facilities will be production-related. Extensive coverage of both physiologic basics and specific industry-related criteriamfor retention of a cow as a breeder, for examplenare addressed in detail in texts and references oriented toward herd and production management ("Current Veterinary Therapy," 1986). Gestation in cattle is approximately 280 days, with a range of 270-292 days. The length of gestation in cattle is influenced by fetal sex; fetal numbers; age and parity of the cow; breed; genotype of cow, bull, or fetus; nutrition; and local environmental factors. As noted, these factors are also important in sheep and goats. Cows usually bear single calves, although twin births do occur. When twins are combinations of male and female calves, the female should be evaluated for freemartinism. 2.

Detection of Estrus and Pregnancy

Ovine estrus detection is usually accomplished by the ram. Nonetheless, because artificial insemination is achievable in ewes, clinical signs of estrus are important. Typically, ewes in heat will show a mild enlargement of the vulva, with slight increases of mucus secretion. Ewes may isolate from the flock and appear anxious. It is often better and clearly more reliable to employ the help of a sterile ram to mark females when they are in standing heat. Two mating systems commonly employed include hand mating and group mating. With hand mating, ewes

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SCOTT A. MISCHLER, WENDYJ. UNDERWOOD,AND MARGARET L. DELANO

are placed either singly or in small groups with the ram of choice. Ewes are removed as serviced. Group mating involves placement of a mature ram with approximately 5 0 - 6 0 ewes for the entire 6-week breeding season. In either mating system, it is best to attach a marking harness to the male so that individual ewes can be identified as serviced. This is important so that parturition dates can be calculated. An easy, natural way to estimate pregnancy is by placing sterile teaser rams with the ewes at the end of the breeding season. Any animal marked by the ram probably has not conceived. Ultrasound scanners are also used for pregnancy detection. The ultrasound transducer is placed against the right abdomen; presence of a fetus is indicated on the machine. Claims of 98% accuracy at 6 weeks postbreeding have been made, although accuracy is generally best beyond 60 days of gestation. Interrectal Doppler ultrasound probes detect fetal pulses. Fetal heart rate is in the range of 130-160 beats per minute, whereas maternal heart rates tend to be 90-110 beats per minute. Accuracy is best beyond 60 days of pregnancy. Rectal-abdominal palpation is an inexpensive alternative. A plastic probe is introduced intrarectally into the ewe, which is restrained on her back in a cradle. The plastic probe is then manipulated toward the abdomen while palpating for the fetus with the opposite hand. The age of the doe when she first expresses heat varies with breed. Some does will express signs of heat between 3 and 4 months old. However, does should be 7-10 months old or at least 8 0 - 9 0 lb in weight before being bred. The caprine estrous cycle lasts 18-24 days. The duration of estrus is 2 4 - 9 6 hr but averages about 40 hr. The estrous cycle can be more erratic in the beginning than in the end of the breeding season (Smith, 1997). "Standing heat" is usually 12-24 hr but can be as short as a few hours. Signs of estrus in goats include uneasiness, tail switching or "flagging," redness and swelling of the vulva, clear vaginal discharge that becomes white by the end of estrus, vocalization such as continuous bleating, and occasionally riding and standing with other does. A doe that is not in heat will not stand to back pressure or for attempts to hold her tail. Does can be induced to show signs of heat by buck exposure and will ovulate within 7-10 days after introduction of the buck. Goats ovulate during the later part of the estrous cycle, most between 2 4 36 hr after the onset of estrus. Nevertheless, goats should be mated once signs of estrus are recognized and every 12 hr until the end of estrus. Most goats kid only once a year, although some goats near the equator may kid twice. Once bred successfully, a goat will only rarely show signs of heat again. In fact, the first sign of pregnancy is usually a failure to return to heat, so animals should be carefully watched. Pregnancy can be affirmed by a variety of means. Goats will generally decrease milk production with pregnancy and should have at least a 6- to 8-week dry period for the udder to fully involute and prepare for the next milking period. In cattle, age of first estrus is dependent on the breed, the

season (with winter delaying), and the level of nutrition (with higher levels hastening puberty). In some cases, the presence of mature cycling cows influences heifer puberty. With adequate nutrition, dairy breeds will reach puberty at 10-12 months and beef breeds at 11-15 months, and estrous cycles will occur regularly after the pubertal (first) estrus, Maturing heifers will often have one or more ovulations before showing overt signs of estrus. Only one follicle usually ovulates per estrous cycle (Hafez, 1987) Estrus, or standing heat, in cattle averages 1216 hr in length, with a range of 6 - 2 4 hr ("Large Animal Internal Medicine," 1996). Detection of standing heat is important because it is closely related to the time of ovulation. Ovulation occurs approximately 25-32 hr after estrus. Detection of estrus is usually accomplished by visual observation of vaginal mucous discharge, mounting behavior by other females (i.e., the cow standing to be mounted is the individual in estrus), and receptivity to a bull (willingness to stand). Successful visual detection of standing heat is dependent on observation skills of handlers, knowledge of the herd, stresses (e.g., detection decreased in Bos taurus during heat stress), barn and yard surfaces (estrus detected better on dirt than on concrete), and maintaining a consistent observation schedule. Teaser animals outfitted with marking devices are also used. Other methods of detecting estrus include monitoring progesterone levels; glass slide and other evaluations of cervical mucus; change in vaginal pH; and body temperature changes (Hafez, 1987). Estrous cycles are usually 21 days in length, with a range of 17-25 days. It is recommended that a heifer deliver her first calf by 2 years of age. After successful conception, progesterone levels in the cow remain elevated for most of the pregnancy, as the result of the 9corpus luteum of pregnancy, and they decline only during the final month. Conceptus implantation occurs beginning at about day 17. If the pregnancy fails before this time, the cow will begin to cycle again between days 18-24, but if the pregnancy ends after day 17, there may be a delayed return to estrus. Realtime ultrasonography can be used to determine pregnancy as early as 9 days after insemination, with embyros seen by days 26-29. Fetal gender can also be determined by experienced personnel by this method by about day 55. Detection of pregnancy can be successful by 2 5 - 4 0 days after conception by observation of failure to return to estrus or by palpation per rectum (detecting fetal membrane slip by days 30-35 and/or amniotic vesicle by days 28-35). Palpation of the fetus is possible by day 65 and placentomes by approximately days 100110. Palpation later in presumed pregnancy will provide information based on differences in size of the two uterine horns, changes in the uterine wall, and fremitus in the miduterine artery. Pregnancy can also be determined with reasonable success rates by determining if progesterone levels are elevated at days 2 0 - 2 4 after insemination. Levels of bovine pregnancy-specific protein B may also be measured; this is produced by trophoblastic cells and is detectable by days 15-24 and elevated throughout pregnancy.

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

531

Placentation in sheep, goats, and cattle is epitheliochorial and 10 ft. Evaluation of a cow's udder prior to breeding and especotyledonary, in contrast to the diffuse or microcotyledonary cially as parturition approaches is important in order to assure placentas of horses and pigs. The placentomes, the infolded adequate nutrition and success of passive transfer by the functional units of the placenta, are formed as the result of fu- neonate. If the udder is edematous or if mastitis is present, for sion of the villi of the fetal cotyledons projecting into the crypts example, an alternate source of colostrum (such as frozen reof the maternal caruncles (specialized projections of uterine " serves) must made be available. Poor udder conformation may mucosa). Caruncles of sheep and goats are concave in shape, also be problematic; contingency plans should be made to enwhereas those of cows are convex. The placentomes are dis- sure adequate support for the young if they cannot suckle from tributed between the pregnant and nonpregant horns of the those udders. Inexperienced heifers may react indifferently or uterus in sheep, and there are 90-100. In cattle, although the aggressively to their offspring and should be monitored more placentomes initially develop around the fetus, they will even- closely than older, multiparous cows with uneventful calving tually be distributed to the limit of the chorioallantoic mem- histories. brane even in the nongravid horn. The placentomes in the nongravid horn will be smaller than in the gravid horn. The total 4. Parturition number will be 70-120. Ewes approaching parturition generally isolate themselves from the flock, become restless, stamp their feet, blat, and peri3. Husbandry Needs odically turn and look at their abdomen. The pelvic region The best birthing preparation for all dams is to ensure a proper will appear relaxed, and milk will be present in the udder. plane of nutrition (not overnutrition) and adequate exercise. If Once hard labor contractions begin, lambs will usually be born possible, the dam should be confined to a birthing pasture or quickly. Animals that do not appear to be progressing correctly sanitized maternity pen a few days prior to parturition. The should be examined for dystocia. Most cases of fetal malprebirthing environment will be very important in the overall sentation or malpositioning can be corrected via vagino-uterine health of the dam and offspring; stress minimization and a clean manipulation. Occasionally cesarean sections will be necessary. environment will benefit the immune health of both in the short Sanitation, cleanliness, and adequate lubrication are of utmost and long term. Outdoor parturition in a small birthing pasture importance when performing obstetrical procedures. has advantages. There is less stress and less intensity of pathoFor about a week before parturition, rectal temperature of the gens. Indoor maternity pens should be clean, dry, warm, well doe will be above normal, or about 103~ depending on envibedded, well ventilated but draft-free, and well lighted. Ade- ronmental temperatures. Approximately 24 hr prior to birth, quate space per pen minimizes losses of neonates from being rectal temperature will fall to slightly below normal. Many stepped and sat on by the dam. Management of these pens, es- large dairy-goat facilities attempt to control the onset of partupecially if concentrated in an area, is important to minimize rition in order to assist birthing. The drug of choice to induce pathogens to which dam and young are exposed. Water troughs parturition in the goat is prostaglandin F2~ (PGF2~) (Ott, 1982). or buckets should be elevated or placed outside the pen, because On day 144 of gestation, goats given PGF2~ (2.5-5 mg) will delambs and kids have a tendency to fall or be pushed into them. liver kids within 28-57 hr. Most goats prefer to kid alone and Soiled bedding should be removed from the birthing pen be- do so unaided. Human interaction can actually interfere with tween dams, the area sanitized and allowed to dry, and fresh normal birthing, especially in young or nervous does. Some bedding installed for the next occupant. Moving the female im- does may reject kids if extensive human interference occurs. mediately before or during parturition may delay the birthing Does nearing parturition have an obviously swollen udder and process. In goats, furthermore, in utero death may occur if par- a red, swollen vulva. Pelvic ligaments at the base of tail relax. turition is unduly delayed. Dams should be monitored closely The doe may circle to make a bed, get up and down, look at her during parturition for dystocias; these may result in loss of tail or sides, push other goats away, and bleat softly. Signs of imyoung or in young severely weakened from the prolonged pending parturition include restlessness; vocalization (bleating birthing process. softly); uneasiness, including getting up and down, pawing, and Prior to parturition, ewes should be sheared or crutched. bedding; and a mucous discharge, leading to a moist tail. Eight Crutching refers to removing wool around the perineal and to 12 hr prior to parturition, the cervix will dilate and the cervimammary areas; this minimizes fetal contamination during the cal mucous plug will be evident as a tan, smeared substance on birth process. Foot trimming can be done at this time as well. the tail and perineum of the dam. Kids should present within 1The tail and perineal area of the doe should be clipped and 6 hr in either anterior or posterior position. A posterior presencleaned to improve postbirth sanitation. In general, the pregnant tation can be recognized by the presence of upward-pointing doe needs a 14 ft 2 (1.2 m X 1.2 m) area for the birthing process, feet. Most does will rest between fetuses and are best left alone. and area needs to be increased after birthing to allow spacing However, if labor is prolonged more than 1 hr, a vaginal exam for kids. Each cow should have a minimum pen area of 10 ft x is indicated.

SCOTT A. MISCHLER, WENDY J. UNDERWOOD, AND MARGARET L. DELANO

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If the pregnant goat is housed with other goats, then herdmates will express great interest in the dam. Unless moved prior to parturition, it is best to leave the dam with the group until after parturition, because removal may delay parturition. Goats are not prone to retained placenta. Normal kids will be quite active and will quickly attempt to stand and nurse. Weak kids should be towel-dried, warmed (via heat lamp, heat pad, or warm water bottle), and assisted to nurse or fed colostrum. The goat is one of the few ungulate species that will exhibit "false pregnancy," or pseudopregnancy. This is a fairly common condition. Does may have characteristically distended abdomens and may develop hydrometra and "deliver" large volumes of cloudy fluid at expected due dates. Subsequent pregnancies can be normal. Goats should be tested for pregnancy by 40 days of age. Veterinary use of prostaglandins has been successful in treating this condition. As in other species, parturition in cattle results from a combination of hormonal changes associated with the maturity of the fetus, notably ACTH (adrenocorticotropic hormone) and subsequent increases in fetal corticosteriods within 2 days of birth. Administration of ACTH to a fetus, or administration to the dam, results in premature birth. Pregnancy is extended if fetal pituitary or adrenal glands are removed surgically. The fetal cortisol probably affects placental steroid production, accounting for sharp increases in the estrogens and estrogen precursors. Coincident with this, maternal progesterone levels fall. The rising levels of estrogen cause release of maternal PGF2~ and induction of oxytocin receptors. Most cows will separate themselves from the rest of the herd. A cow will lift her tail and arch her back when she is within a few hours of delivering the calf, and most cows are recumbent when delivering the calf. Typically, the whole birthing process takes about 100 min. The length of labor of cows carrying larger calves also will be longer. Nervous heifers will take longer to deliver, and if they are disturbed, their labor may cease. All postparturient animals should be monitored for successful passage of these fetal membranes within 12 hr of birth. Veterinary intervention is required if not. Cows occasionally eat placentas, which may subsequently obstruct rumen outflow and require surgical correction. For cattle, it is now recommended practice to remove membranes that have passed, in order to prevent ingestion. 5.

Early Development of the Newborn

Following lambing, it is critical that the newborns be "processed" so that they will have greatest survival chances. In a well-managed flock, many lambs and ewes will not need much assistance. When assistance is given, the newborn lamb's nose and mouth should be wiped free of secretions; gently swinging the lambs, head down, aids in removal of these fluids. The lamb should be dried off and stimulated through rubbing to aid its breathing. The lamb's navel should be dipped in an iodine solution to prevent subsequent navel infections. And the lamb

should be identified by the application of an ear tag or ear notch. It is extremely important that the lamb be supplied with highquality colostrum within the first 12 hr of birth. Lambs that are not nursing on their own should be tube-fed with colostrum that has been collected and saved previously (i.e., frozen in ice cube trays) or collected from the mother after parturition. Passive transfer can be assessed by measuring serum y-glutamyltransferase (GGT) levels (Tessman et al., 1997). After the first few days, colostrum changes over to milk. Nursing lambs will ingest increasing amounts of milk as they grow. If the ewe cannot produce sufficient milk, the lamb should be "grafted" onto another ewe or fed artificially with a baby bottle. Powdered milk replacers are commercially available; the content of ewe milk is much different from that of cow's milk; thus lamb milk replacer should specifically be used. One report notes that 5 0 - 7 0 % of lamb deaths occur during the first week of life and up to 90% occur within the first month. Good management of ewes during gestation, care of the lamb at parturition, application of an appropriate vaccination program, and observation and intervention within the first several weeks of a lamb's life will minimize losses (Ross, 1989). Immediately after birth, the placenta and any birthing materials should be removed from the doe's pen. Kids do not usually need assistance. If kids are to be raised by the dam, they can be left alone; otherwise, kids should be towel-dried and removed from the dam. Kids are cold-sensitive and may require a heat lamp or other source of added warmth in cold weather. Navel cords should be dipped in tincture of iodine, and kids should be dehorned and castrated within the first several days of life. To control caprine arthritis encephalitis (CAE), kids should be immediately removed from the dam and hand-fed heattreated colostrum. Colostrum should be heat-treated for 1 hr at 131 ~E The first feeding can be up to 125 ml of colostrum. Kids should receive a total of 250 ml colostrum within the first 3 6 48 hr of birth. After day 3, kids can be placed on milk replacer. Milk replacers should contain 16-24% fat and 2 0 - 2 8 % milkbased protein. By 14 days of age, kids should be consuming approximately 1.1-1.4 liters of milk per day. Kids should be introduced to forages as soon as possible and may be weaned by 6 - 1 0 weeks or 18-25 lb body weight. Milk that is fed can be reduced by 4 weeks of age by decreasing either the volume fed or the number of feedings. As with other dams, a cow is usually very attentive to her newborn calf, cleaning and softly vocalizing to the neonate. Calves typically are standing by 1 hr after birth and are suckling within 3 hr. As noted previously, dairy calves may be removed from the cow even before suckling, and the colostrum milked from the dam and given to the calf. Assistance may be required for nervous heifers, after dystocias and in extreme circumstances such as severe cold. Cleaning the newborn's nose and mouth, rubbing down the neonate, assuring that the calf does not get chilled, and assuring that it receives adequate colostrum are all important under any of these circumstances. A stressed calf's umbilical

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE may be treated with an iodine or chlorhexidine solution, although some authors note no benefit of navel treatment, specifying that successful transfer of passive immunity and sound sanitary management of birthing area are the most crucial factors in preventing omphalitis (navel ill) (House, 1996; Kersting, 1997; Kasari and Roussel, 1999). Because newborn calves can be deficient in vitamin A and iron, these may be injected to improve disease resistance (Wikse and Baker, 1996). In cases in which the dams' colostrum is known to be deficient in antibodies against common diseases, vaccinations may be administered at 1 day old and followed with boosters at regular intervals. Dehorning is performed when horn buds appear. Castration is performed between 2 and 9 weeks of age or later.

6.

Sexing

Sexing the young in any of the ruminant species is straightforward. The vulva of the female young is located just ventral to the anus. The genitalia of the male include a penis, located along the ventral midline, and a scrotum, located in the inguinal region. The phenomenon of the freemartin, a genetic female born as a twin to a male, is the result of anastomoses between placental circulations of the twin fetuses; the mixing of bloodforming cells and germ cells results in the X X / X Y chimeras. This occurs in 8 5 - 9 0 % of phenotypic bovine females born as co-twins with males. The female will often have abnormal vulva and clitoris, and the vagina will be a blind end because of the lack of a cervix. Sometimes singleton freemartins are born if the male fetus is lost after 30 days' gestation. Multiple births are selected for and are common in sheep; the freemartin phenomenon is regarded as rare. Twinning is common in goats, and freemartinism occurs in about 6% of male-female pairs of twins. Intersexes are seen in some goat breeds and when polled goats are mated. Proof is usually based on evidence of abnormal genital development and reports of abnormal sexual behavior.

7.

Weaning

Prior to weaning, it must be established that lambs can nutritionally survive without mother's milk. Thus, grain, and later roughage, should be offered to lambs well in advance of the day of weaning so that they can adjust to the feedstuff. To prevent the ewes from ingesting the lamb ration, a "creep" should be set up by building an area adjacent to the ewe-lamb pen and devising a slatted entry for the lambs to enter but not the ewes. Therefore, the lambs will be accustomed to the new ration through this creep-feeding process. If lambs and ewes will be pastured later in the spring, it is still beneficial to creep-feed lambs until pasture growth is adequate enough to fulfill the requirements of the growing lambs. Lambs that are consuming 1.5-2 lb of creep feed per day may be weaned. Depending on the individual program, lambs may be

533

weaned as early as 4 weeks of age, although 6 - 8 weeks of age is more common. If ewes are of a breed that will cycle twice a year, and if it is expected that they will be rebred, then the lambs must be weaned as early as possible so that lactational anestrus will resolve and ewes will recycle. Another factor is the cost of lactation rations for the ewes; if lamb grain is more economical than ewe grain, then lambs should be weaned. About 4 - 5 days prior to weaning, feeding of the lactation ration to the ewes should be discontinued, and only roughage fed. At weaning, the lambs should be removed in the creep, and the ewes removed to an area that is not within sight (and preferably sound) of the lambs. The ewes should be monitored for postweaning mastitis and treated as necessary. Ewes that have physical or disease problems or that have not been productive at lambing or feeding their lambs should be culled. The lambs should be monitored to assure that they continue to gain weight and are eating the new ration. Kids should be introduced to forages within the first week of life because the natural curiosity of these animals will cause them to investigate sources of feed. Kids can be weaned by 6 10 weeks or 18-25 lb. Hand-fed milk should be reduced by 4 weeks of age by reducing the volume fed or by decreasing the number of feedings. Dairy calves are now usually removed from their dams immediately after birth. It is less common now to allow the calves to remain with their dams for about 24 hr and suckle fresh colostrum during this time, because their intake will be inadequate. Dairy producers refrigerate and/or freeze the colostrum produced during the first 24 hr and feed this, diluted 50:50 with warm water, twice a day to the calves during the next 2 - 3 days. Holstein calves, for example, should receive a minimum of 3 5 liters within 12 hr of birth and then be fed about 10-15% of body weight in colostrum by 24 hr of age. After 3 days, calves are then placed on milk replacers, preformulated powders reconstituted with water that provide complete nutrition. Milk replacers are commercially available and should be fed according to manufacturer's recommendations Vaccination programs for calves vary with the preventive medicine program for the overall herd. Passive immunity provided by colostrum from cows on sound management programs will last until a calf is about 6 - 7 months old; normally vaccinations are not necessary and are contraindicated during those first 6 months. The duration of passive immunity varies considerably among calves, however; some producers choose to begin vaccinating calves at 1-2 months of age and continue with monthly booster immunizations until the animals are 7 months old, when passive immunity is no longer a possibility. 8.

Artificial Insemination

Artificial insemination (AI) in sheep is more difficult than in cattle because sheep are smaller and cannot be reproductively manipulated via the rectum and because the cervix of sheep is

SCOTT A. MISCHLER, WENDYJ. UNDERWOOD,AND MARGARET L. DELANO

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more difficult to traverse with the insemination pipette. Breeding animals artificially with fresh semen produces pregnancy rates averaging 50% (not unlike that of cattle); artificial insemination with frozen semen is less successful. Several artificial insemination techniques have been used. Laparoscopic AI involves the surgical instillation of semen into the uterus through a small abdominal opening. The procedure is successful but is technically involved and costly. Cervical AI involves the transvaginal introduction of semen into the cervix. A modification of this technique (transcervical AI) allows for penetration through the cervix into the uterus. This method (called the Guelph system for transcervical AI) leads to successful penetration into the uterus in up to 75% of ewes when performed by an experienced inseminator. Artificial insemination is now an integral part of dairy herding; natural insemination as a management practice is relatively rare. Technicians performing the AI technique are available through commercial enterprises. Dairy production employees are also trained. Information regarding the management of the donors and recipients, the storage and handling of the semen, and the skills and record keeping required is covered extensively elsewhere (Nebel, 1997). 9.

Synchronization

Because sheep are hormonally similar to other ruminants, estrous synchronization techniques are comparable. Progesterone suppresses follicle-stimulating hormone (FSH) secretion, preventing animals from developing follicles and exhibiting estrus. Artificial or natural progesterone can be administered in the feed, through parenteral injection, subcuticular implants, and vaginal pessaries. The progesterone is withdrawn in about 1214 days, after which the FSH secretion will initiate the process of follicle development (Trower, 1993). Estrus usually will occur in 3 6 - 6 0 hr (average is 48 hr). A natural method of synchronization, often applied to promote flock breeding within a short period of time (and thus parturition will be within a narrow window as well), is the introduction of sterile rams with the ewes before the beginning of the normal fall mating period. Pheromones released from males naturally stimulate the females to cycle and to synchronize their heats. It should be noted that introduction of a male during late anestrus will often stimulate ovulation in about 6 days; however, this cycle will generally be without clinical signs of estrus (silent heat). Vasectomy of rams is one method of producing sterile "teaser rams." Introduction of the buck to a group of does will induce ovulation and may even synchronize does. Does that are kept separate from the buck will show signs of estrus, will ovulate within 6 10 days, and will have normal pregnancies when introduced to a buck. Bucks with horns and intact scent glands are better able to induce ovulation than dehorned bucks, whose scent glands often been removed. Control of breeding in the goat has been studied mostly in

dairy breeds in order to produce milk throughout the year and to reduce kidding labor. Goats in the luteal phase of the estrous cycle, days 4-16, are sensitive to PGF2~ (2.5-5 mg IM) and will show estrus in 3 6 - 6 0 hr postinjection (Bretzlaff, 1997). Dosing cycling animals twice 11 days apart will synchronize goats, and artificial insemination using this method has resulted in 4 0 - 6 0 % conception rates (Bretzlaff, 1997; Greyling and Van Niekerk, 1986). Programs for timed breeding have been described and involve administering progestogens (Bretzlaff, 1997). Vaginal pessaries of fluorogestone acetate left in place for 21 days in the doe followed by an injection of pregnant mare serum gonadotropin (PMSG) at the time of pessary removal have proven successful. Also, when primed by PGF2~, an 11day regimen of fluorogestone acetate with PMSG given on day 9 has been successful. Synchronization of cattle estrous cycles and superovulation are used as management techniques in certain commercial cattle and dairy production settings where estrus synchronization or embryo transfer is advantageous to production and management. The methodology is also used in the research setting for coordinating donors and recipients of embryos or other genetically manipulated tissues for implantation. The options and dosing regimens are described in detail in veterinary clinical texts (Wenzel, 1997; Vanderboom et al., 1997). In synchronization, the principle is lysis of the existing corpus luteum. The more common practices involve the use of products approved for use in cattle such as PGF2~, one of its analogs, or products containing estradiol valerate. Progestogens are also used in conjunction with estradiol valerate. Other approaches, involving management techniques combined with pharmacologic interventions, are considered less successful. Superovulation regimens involve injections of FSH either alone or with PGF2~ at timed internals. Estrus is expected 48 hr after the final injection, and two inseminations are performed at 12 hr intervals after estrus detection. Preparation of recipients involves injection of PGF2~ or progestogens with gonadotropins such as PMSG. For greatest success as management tools, these must be combined with a consistent program that provides appropriate nutrition for all cattle involved. Synchronization of animals is also influenced by several other factors, however, such as time in the cycle when hormones are administered, response by each individual animal, whether the cow is a dairy or beef animal, parity and maturity of the cows, success of heat detection after the luteolysis, and accurate record keeping. 10.

Embryo Transfer

Embryo transfer involves the removal of multiple embryos from a superovulated embryo donor and transferring them to synchronized recipients. This method maximizes the genetic potential of the donor animal. The donor animal is hormonally superovulated and inseminated. In sheep, about 1 week after breeding, the embryos are surgically removed from the donor's

14. BIOLOGYAND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE uterus. In cattle, the procedure is nonsurgical. About 75% of expected embryos (determined by counting corpora lutea) can be recovered; successful recovery is affected by factors such as age of the donor, reproductive health, and experience of the surgeon or technician. Furthermore, not all collected embryos are of transferable quality. Recipients are hormonally synchronized with the donor animals. On the day of embryo collection, transferable embryos are implanted into the uterus of the recipient; laparoscopy has been used in the past and is now being replaced by nonsurgical methods. Pregnancy rates average about 70%. If recipients are not available, embryos, like sperm, can be frozen and kept for later transfer. Embryo transfer is commonly practiced in cattle as a herd improvement technique and as a research technique for engineered embyros. Disease screening programs for all animals involved are important because several pathogens can be transmitted directly or indirectly, such as bovine viral diarrhea virus, bluetongue virus, infectious bovine rhinotracheitis virus, and mycoplasmal species.

11.

a.

Miscellaneous Management Considerations

Management of Male Animals

In sheep flocks and goat herds, as noted, male young are usually castrated by 1 month of age. The elastrator method is the more popular for animals less than 1 week of age. Other methods include the emasculatome (crushing) and surgical removal ("knife method"). The distress associated with castration and tail docking in lambs is the subject of debate and has been researched recently (Kent et al., 1995). As noted, male calves are usually castrated as early as possible and no later than 3 month of age. In some production situations, however, where maximum hormone responsive muscle development and grouping animals together for procedures dictate scheduling, the procedure may be performed on older males. Open and closed techniques are used, depending on the age of animals and on veterinary or farm practice. Breeding and vasectomized rams and bucks are usually maintained by medium to large production farms. Smaller farms often borrow breeding males. Breeding males are typically selected by production record, pedigree, and/or breed. Vasectomized males are often retired breeders and should be tattooed or identified clearly to avoid any wasted breeding time. The vasectomy technique for both species is comparable (Smith and Sherman, 1994). Rams may be housed together for most of the year, whereas bucks are penned separately. Because ewes will exhibit only a limited number of estrous cycles before becoming reproductively quiescent, it is critical that the male be capable of successfully breeding the female in an expeditious manner. Any defects in the external genitalia, reproductive diseases, or musculoskeletal abnormalities may prevent successful copulatory behaviors. Furthermore, it is impor-

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tant to know the semen quality of the ram as one indicator of fertility. Semen can be collected via electroejaculation or by use of a teaser mount. Once semen is collected, it should be handled carefully and kept warm to prevent sperm death, leading to improper conclusions about the male. Typically, the characteristics usually evaluated as a determinate of sperm quality are volume (normal between 0.7 and 2.0 ml); motility (% of sperm moving in a forward wave; high quality is associated with motility of approximately 90%); concentration (sperm count per unit of volume as measured by a hemocytometer; high-quality semen should contain 1.8 X 109 sperm per ml); morphology (live versus dead cells, as determined by special stains and the percentage of abnormal-appearing sperm; neither the abnormalities nor the dead sperm should exceed 10% in high-quality semen). The extensive use of artificial insemination in the dairy cattle industry has minimized the use of bulls on many farms, although a farm may maintain a few bulls for heat detection and for "cleanup" breeding. Breeding bulls are maintained in beef production establishments. Breeding bulls must be part of the herd vaccination program, with special attention to appropriate timing of immunizations for the commonly transmitted venereal diseases campylobacteriosis and trichomoniasis. b.

Cattle Tail Docking

Tail docking is a relatively recent development in dairy herd management and is practiced in the belief that it will minimize bacterial contamination of the udder and therefore the milk. Tails are typically docked to about 10 inches in length. The practice is more popular in certain regions in the United States. To date, there is no published study indicating that this technique provides any distinctive advantage over keeping the tail switch hair clipped short.

E.

Behavior

Healthy ruminants have good appetites, chew cud, are alert and curious, have healthy intact coats, move without hindrance, and have clear, bright, clean eyes and cool dry noses. Even adult animals, when provided sufficient space, will play. Sheep and goats have tidy "pelleted" dark green feces. Cattle have pasty, moist, dark green-brown feces. Ruminants normally vocalize, and handlers will learn to recognize normal communication among the group or directed at caregivers in contrast to that when animals are stressed. Excessive, strained vocalizations are often a sign of stress in cattle. "Bruxism," or grinding of the teeth by a ruminant, is usually associated with discomfort or pain. Other signs of discomfort, stress, or illness include decreased time spent eating and cud chewing, restlessness, prolonged recumbency with outstretched neck and head, and hunched back when standing. Unhealthy ruminants may be thin,

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may arch their backs or favor a limb, or may have external lumps or swollen joints, an unusual abdominal profile, or rough or dull coats. All ruminants are herd animals to some extent and social individuals; therefore, every effort should be made to allow contact among animals, in terms either of direct contact or of sound, smell, or sight. Human contact and handling should be initiated promptly and maintained regularly and consistently throughout the animal's stay in the research facilities. Animals should be provided sufficient time to acclimate to handlers and research staff. Cattle and sheep can hear at higher frequencies than humans can and may react to sounds not perceived by handlers. Knowledge of the peculiarities of sheep behavior will increase the ease of handling and decrease stress-related effects in research. Generally, fine-wooled breeds, such as Rambouillet, are the most gregarious and are best handled in groups. The meat, or "downs," breeds tend to be less gregarious, and the long-wooled breeds tend to be solitary (Ross, 1989; ASIA, 1996). Nonetheless, movement of animals is simplified by proper facility design. Sheep have a wide-angle visual field and are easily scared by activities that are taking place behind them. Sheep should be moved slowly and gently. To capture individuals within a flock, it is best to confine the flock to a smaller space and use a shepherd's crook or to gently catch the animal in front of the neck/thorax. Grabbing the wool can injure the animals, as well as damage the wool and the underlying tissues. Sheep move best in chutes that have solid walls, and individual animals will generally follow a lead animal. Any escape route will be challenged and, if successfully breached, will disrupt the entire flock movement. Sheep movement is also disrupted by contrasts such as light and shadows that impinge on a chute or corral. Finally, like most animals, sheep have a flight zone (minimum zone of comfort), the penetration of which will result in sheep scattering. This minimal flight distance can be modified by increasing handling of the animals and working at the edge of the zone, but it should always be considered when working with animals in chutes, pens, or other confined areas. Goats exhibit behavioral characteristics that make them quite distinct from other ruminants. Their browsing activity makes them quite orally investigative. Goats will readily nibble or chew just about anything they come in contact with, so researchers should keep all paperwork and equipment out of reach. A herd of goats will readily chew through wood gates and fencing, especially when confined in areas without alternatives for chewing behavior. Goats are also inquisitive, restless, agile jumpers and climbers, and quite mischievous. If maintained in paddocks, strong high fences are essential, as are adequate spaces for exercise or boulders or rock piles for hoof maintenance and recreational climbing. Goats are more tolerant of isolation and are more easily acclimated to human contact than sheep are, but goats will confront unfamiliar intruders and make sneezing noises. Goats with horns will use them to advantage,

and horns may also become entangled in fencing. Although less strongly affected by flock behavior, goats are social animals. Most goats raised in close human contact are personable and cooperative and can easily be taught to stand for various procedures, including blood collection. An understanding of breed behaviors, sources of stress in cattle, play behaviors, calf behaviors, and dominance determinants will contribute to prevention of injuries to handlers and better health and welfare of the animals. Ruminants of all ages, especially cattle of all ages, should be handled with an appreciation of the serious injury to human handlers that may result (Houpt, 1998). Cattle have a wide visual field, as sheep do, and a flight zone that varies in size, according to previous handling experiences (gentle handling and animal tameness make the flight zone smaller) and the circumstances of the moment (Grandin, 1993). Groups of cattle are moved effectively around a facility by utilizing chute systems, with sequences of gates, that minimize chances of animals turning around. Dairy cattle have been bred and selected over centuries for their docile, tractable characters and production characteristics. In contrast, beef breeds have not been selected for docility and are generally more difficult to handle and restrain. Beef breeds, such as Angus, are known for their independent natures and protective maternal instincts. All cattle respond well to feed as a reward for desired behavior. Healthy cattle typically are very curious and watchful and are alert to sounds and smells. When not grazing or eating, they hold their heads up. When sleeping, the head and neck may be tucked back. Because of ruminant digestive and metabolic needs, much of the day is spent eating or cud chewing. Occasionally, adult cows sit upright like dogs. Cattle maintained inside tend to be more docile. In addition to forced isolation from other cattle, sources of stress include rough attitudes of handlers and unfamiliar visual patterns, routines, or environments. These stressors may exacerbate signs of systemic illnesses. Calves are known for non-nutritive suckling, bar licking, and tongue rolling. Non-nutritive suckling behavior is greater in hungry calves and also right after a milk meal. It is best to provide nipples and other clean noninjurious materials for the animals to suck. Non-nutritive suckling can be detrimental in group-housed calves because it can result in disease transmission and hair ball formation. Environmental enrichment devices have been developed to cope with this behavior. The behavior diminishes as the animals are weaned onto solid food (MorrowTesch, 1997). Play activity and vocalizations of calves mimic adult dominance behaviors. Play activity by young adult cattle is more common in males, can be quite rough, and is often triggered by a change in the environment. Dominance behaviors are dependent on direct physical contact among the cattle, and dominance hierarchies are established within a herd. Horns, age, and weight have been reported to be the most important determi-

14. BIOLOGYAND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE nants. Aggressive behaviors in cattle may be triggered by newly introduced animals or unfamiliar visual patterns and by feeding when animals are very hungry. Aggression is more common among intact adult males.

III.

DISEASES

This section focuses primarily on the more common diseases affecting sheep, goats, and cattle in the United States and elsewhere in North America and those that are reportable. For detailed information not included in this limited overview and for diseases of importance internationally, the authors recommend several excellent comprehensive and focused veterinary clinical texts and periodicals that address ruminant diseases, preventive medicine, and individual and flock or herd management. These are listed under "Major References" in the reference list at the end of this chapter. Recommendations for current drug therapies, both approved and off-label use in ruminants, including withholding prior to slaughter, formularies, and related information can be found in the references noted above and in formularies (Hawk and Leary, 1995; Plumb, 1999). In addition, the Food Animal Residue Avoidance Databank (FARAD), accessible on the Internet , should be used as a resource. FARAD is a food safety project of the U.S. Department of Agriculture and is an information resource to prevent drug and pesticide residues in food animals and animal products.

A. 1.

Infectious Diseases

Bacterial, Mycoplasmal, and Rickettsial Diseases

a.

Actinobacillosis ("Wooden Tongue")

Etiology. Actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. In sheep and cattle, A. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. This disease, called wooden tongue, has not been documented in goats. Clinical signs. Skin lesions are common. Tongue lesions are more common in cattle than in sheep. Lip lesions are more common in sheep. Soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. Animals may have difficulty prehending

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food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. Diagnosis is made based on clinical signs and is confirmed by culture.

Epizootiology and transmission. The organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. Rough feed material and foreign bodies may play a role in causing abrasions. Actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. Lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. Necropsy findings. Purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. Exudates will also contain characteristic small white-gray (sulfurlike) granules. The pus is usually nonodorous. Differential diagnosis. Contagious ecthyma and caseous lymphadenitis are the primary differentials. Diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. Treatment. Animals should be fed softer feeds. Antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. Penicillin is not effective. Weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. Surgical excision and drainage are not recommended. Prevention and control. Because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. b. Arcanobacterium Infection (Formerly actinomycosis, or "Lumpy Jaw") Etiology. Arcanobacterium (formerly known as Actinomyces or Corynebacterium) pyogenes and A. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. Arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. This organism has also been associated with pharyngitis and mastitis in cattle. Clinical signs and diagnosis. Arcanobacterium bovis causes mandibular lesions primarily. The mass will be firm, nonpainful, and immovable. Draining tracts may develop over time.

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If teeth roots become involved, painful eating and weight loss are evident. Radiographic studies are helpful for determining fistulas. Diagnosis is based on clinical signs, and culture is required to confirm Arcanobacterium. The prognosis is poor for lumpy jaw.

Epizootiology and transmission.

These organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds.

Necropsy.

Draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed.

Pathogenesis.

Arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis.

Differential diagnosis.

Actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. A major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. There are many differentials for septic joints and polyarthritis: Chlamydia spp., Mycoplasma spp., streptococci, coliforms, Erysipelothrix rhusiopathiae, Fusobacterium necrophorum, and Salmonella spp. Tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. Prevention and control.

Arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. Treatment. Penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. Sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. Extended antibiotic therapy may be necessary. Surgical excision is an option. In addition to medications noted above, isoniazid is somewhat effective for A. bovis infections in nonpregnant cattle. Research complications.

The possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals.

c. Actinomycosis Omphalophlebitis, omphaloarteritis, omphalitis, and navel ill are terms referring to infection of the umbilicus in young animals.

Arcanobacterium pyogenes is the most common organism causing omphalophlebitis, an acute localized inflammation and infection of the external umbilicus. Most cases occur within the first 3 months of age, and animals are presented with a painful enlargement of the umbilicus. Animals may exhibit var-

ious degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, hematuria, and so on. Severe sequelae may include septicemia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, and endocarditis.

Research complications.

Young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. Affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects.

d.

Anthrax

Etiology.

Bacillus anthracis is a nonmotile, capsulated, sporeforming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. Common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. Clinical signs and diagnosis. Anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. Subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. The incubation period is 1 day to 2 weeks. Bloody secretions such as hematuria and bloody diarrhea often occur. Abortion and blood-tinged milk may also be noted. The disease is usually fatal, especially in sheep and goats, after 1-3 days. Death is the result of shock, renal failure, and anoxia. Diagnosis is based on the clinical signs of peracute deaths and hemorrhage. Stained blood smears may show short, single to chained bacilli. Blood may be collected from a superficial vein and submitted for culture. Epizootiology and transmission.

Cattle and sheep tend to be affected more commonly than goats, because of grazing habits. Older animals are more vulnerable than younger, and bulls are more vulnerable than cows. Although the disease occurs worldwide, and even in cold climates, most cases in the United States occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. Spores survive very well in the environment. The anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. Abrasive forages may play a role in infection. Transmission via insect bites or through skin abrasions rarely occurs.

Necropsy.

Necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without

14. BIOLOGYAND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE opening the animals. Incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. Blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. Splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. Pathogenesis. The rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. Encapsulation protects the organisms from phagocytosis. Liberated toxins cause local edema. Differential diagnosis. Although anthrax Should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. Prevention and control. Outbreaks must bereported to state officials. Anthrax is of particular concern as a bioterrorism agent. Any vaccination programs should also be reviewed with regulatory personnel. Herds in endemic areas and along waterways are usually vaccinated routinely with the Sterne-strain spore vaccine (virulent, nonencapsulated, live). Careful hygiene and quarantine practices are crucial during outbreaks. Dead animals and contaminated materials should be incinerated or buried deeply. Biting insects should be controlled. The disease is zoonotic and a serious public health risk. Treatment. Treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. Amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. During epidemics, animals should be vaccinated with the Sterne vaccine. Research complications. Natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. The organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. e.

Brucellosis

Etiology. Brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. Brucella abortus is one of several Brucella species that infects domestic animals but cross-species infections occur rarely. Brucella abortus or B. melitensis may cause brucellosis in sheep, cattle, and goats. Brucella melitensis (biovar 1, 2, or 3) is the primary cause of sheep disease (Garin-Bastuji et al., 1998). Brucella ovis is more

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commonly associated with ovine epididymitis or orchitis than abortion. In the United States, clusters of brucellosis are still found in western areas contiguous to Yellowstone National Park. Bang's disease is the common name given to the disease in ruminants. Clinical signs and diagnosis. Brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about 3 months. However, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. Abortion usually occurs in the third trimester, after which the ewe will appear to recover. It has been reported that up to 20% of infected ewes may abort more than once. Rams will also be infected and may develop orchitis or pneumonia. The disease caused by B. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. Brucella ovis causes decreased fertility. Brucella melitensis is the more common cause of brucellosis in goats. Brucella abortus has been shown to infect goats in natural and experimental infections, and B. ovis has also been shown to infect goats experimentally. Does infected with B. melitensis will also abort during the third trimester. Infections with B. abortus in cattle produce few clinical signs. There may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. In cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. Infection may cause abortions after the fifth month, with resulting retained placentas. Permanent infection of the udder is common and results in shedding of organisms in milk. In bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. Organisms may be in the semen. In infected herds, lameness may also be a clinical sign. Diagnosis of brucellosis can be made by bacterial isolation of the Brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. Many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and others. Test selection is often dependent on state requirements in the United States. Epizootiology and transmission. The primary route of transmission of B. abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. Exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and

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through other mucous membranes such as the conjunctiva. Brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding.

Necropsy findings. A sheep fetus aborted due to Brucella will exhibit generalized edema. The liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. Peritoneal and pleural cavities often contain serofibrinous exudates. The placenta will be leathery. Pathogenesis. Ruminants are considered especially susceptible to Brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. Brucella utilizes erythritol preferentially over glucose as an energy source. Placentas and male genitalia also contain high levels of erythritol. Brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. Abortion is the result of placentitis, typically during the third trimester of gestation. Brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. The organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. Orchitis may be a sequelae of the disease. Epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to B. ovis, and by semen cultures. Differential diagnosis. Differential diagnoses include all other abortion-causing diseases. Many other agents, such as Actinobacillus spp., Arcanobacterium (Actinomyces) pyogenes, Eschericia coli, Pseudomonas spp., Proteus mirabilis, Chlamydia, Mycoplasma, and others may be associated with ovine epididymitis and orchitis. A clinically and pathologically similar agent, Actinobacillus seminis, has been isolated from virgin rams. This organism has morphological and staining characteristics similar to those of B. ovis and complicates the diagnosis (Genetzky, 1995). Prevention and control. The Rev 1 vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the United States. Separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with B. ovis bacterin can prevent the disease. Over the past 20 years, aggressive federal and state regulatory and cattle herd health programs in the United States have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. Most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. Cattle vaccination programs can be

very successful when conducted on a herd basis to reduce likelihood of exposure. Strain 19 and the recently validated attentuated strain RB51 are live vaccines and can be used in healthy heifer calves 4 - 1 2 months old. Vaccination for older animals may be done under certain circumstances. Vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. The strain 19 vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. The vaccine will not, however, protect the animals from becoming infected with B. abortus. Strain 19 vaccine induces an antibody response in cattle. The RB51 vaccine does not result in antibody titers and therefore is advantageous because infection with Brucella can be determined serologically. The RB51 vaccine has been designated as the official calfhood bovine brucellosis vaccine in the United States by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) (Stevens et al., 1997). Brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. The RB51 is regarded as less pathogenic and abortigenic in cattle.

Treatment. Definite confirmation of Brucella infection is important from the standpoint of public and herd health. Culling is considered the treatment of choice in cattle herds. Rams infected with B. ovis should be isolated and treated with tetracyclines. Research complications. Brucellosis represents a research complication as a cause of abortions and of infections in male ruminants. Impairment of the infected host's immune system, especially alteration of phagocytic cells where the bacteria stay in membrane-bound vesicles, should be considered. The potential complications of needle sticks by large-animal veterinarians with the strain 19 vaccine and the public health risks (undulant fever) are well known. Less is known presently regarding the RB51 vaccine effects in humans. Epidemiologic and diagnostic methodologies are being developed to track and monitor these cases. There is also a risk of human infection from handling infected materials during a dystocia or postmortem. Worldwide, B. melitensis is the leading cause of human brucellosis. f.

Campylobacteriosis (Vibriosis)

i. Campylobacter fetus subsp, intestinalis; C. jejuni infection (ovine vibriosis) Etiology. Campylobacter (Vibrio) fetus subsp, intestinalis, a pleomorphic curved to coccoid, motile, non-spore-forming,

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

gram-negative bacterium, causes campylobacteriosis, the most important cause of ovine abortion in the United States. There are few reports of campylobacteriosis in goats in the United States. Vibriosis is derived from the name formerly given to the genus; the term is still frequently used.

Clinical signs and diagnosis. Ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. The organism inhabits the intestines and gallbladder in subclinical carriers. Abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. It is reported that 20-25% of the flock may become infected and up to 5% of the ewes will die (Jensen and Swift, 1982). Some lambs may be born alive but will be weak, and dams will not be able to produce milk. Diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. Tentative identification of the organism can be made by observing curved ("gull-wing") rods in Giemsa-stained or Ziehl-Neelsen-stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. Epizootiology and transmission. Campylobacteriosis occurs worldwide. Campylobacter spp., such as C. jejuni, normally inhabit ovine gastrointestinal tracts. Transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. In abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. Ewes may have active Campylobacter organisms in uterine discharges for several months after abortion. The bacteria will also be shed in feces, and feed and water contamination serve as another source. There is no venereal transmission in the ovine.

Necropsy. Aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. The liver may contain 2 - 3 cm pale foci. Placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. The placental cotyledons may appear gray.

Pathogenesis. The organism enters the bloodstream and causes a short-term bacteremia (1-2 weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus.

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cycline. Aborting ewes should be isolated immediately from the rest of the flock. After an outbreak, ewes will develop immunity lasting 2 - 3 years.

Treatment. Infected animals should be isolated and provided with supportive therapy. Prompt decontamination of the area and disposal of the aborted tissues and discharges are important. Research complications. Losses from abortion may be considerable. Campylobacter ssp. are zoonotic agents, and C. fetus subsp, intestinalis may be the cause of "shepherd's scours." ii. Campylobacter fetus subsp, venerealis infection (bovine vibriosis) Etiology. Campylobacter fetus subsp, venerealis is the main cause of bovine campylobacteriosis abortions. It does not cause disease in other ruminant species.

Clinical signs and diagnosis. Preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. This will be particularly apparent in virgin heifers that may return to estrus by 40 days after breeding. Long interestrous intervals also serve an indication of a problem. Spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. Severe endometritis may lead to salpingitis and permanent infertility. Demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. The vaginal mucous agglutination test is used to survey herds for campylobacteriosis. Serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. Culture from breeding animals may be difficult because Campylobacter will be overgrown by faster-growing species also present in the specimens. Epizootiology and transmission. The bacteria is an obligate, ubiquitous organism of the genital tract. Transmission is from infected bulls to heifers. Older cows develop effective immunity.

Necropsy findings.

Necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. In addition, bronchopneumonia and hepatitis will be seen histologically.

Pathogenesis. Campylobacter organisms grow readily in the Differential diagnosis. Toxoplasma, Chlamydia, and Listeria should be considered in late gestation ovine abortions.

genital tract, and infection is established within days of exposure. The resulting endometritis prevents conception or causes embyronic death.

Prevention and control. A bacterin is available to prevent the disease. Carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral chlortetra-

Differential diagnosis. The primary differential diagnosis for campylobacteriosis is trichomoniasis. Other venereal diseases

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should be considered when infertility problems are noted in a herd. These include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV), and bovine virus diarrhea (BVD). Leptospirosis should also be considered. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered.

Treatment. Severe infections should be treated with antibiotics based on culture and sensitivity. Severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent (Smith and Sherman, 1994).

Prevention and control. Killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. The former is preferred because of duration of immunity but causes granulomas. That vaccine also has specific recommendations regarding administration several months before the breeding season. The latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. In both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. Only one bacterin product is approved for use in bulls. Many combination vaccine products contain only the aluminum hydroxide adsorbed product. Artificial insemination (AI) is particularly useful at controlling the disease, but bulls used for AI must be part of a screening program for this and other venereal diseases such as trichomoniasis.

i. Clostridium perfringens type C infection (enterotoxemia and struck) Etiology. Clostridium perfringens is an anaerobic, grampositive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. The bacteria is categorized by toxin production. Toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. Types of C. perfingens are A, B, C, D, and E. This is a common and economically significant disease of sheep, goats, and cattle.

Treatment. Cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. g.

Caprine Staphylococcal Dermatitis

Etiology. The most common caprine bacterial skin infection is caused by Staphylococcus intermedius or S. aureus and is known as staphylococcal dermatitis (Smith and Sherman, 1994). The Staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. Staphylococcus aureus and S. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. Clinical signs and diagnosis. Small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. Occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. Staphylococcal dermatitis may occur secondary to other skin lesions. Diagnosis is based on lesions. Culture will distinguish

h.

Clostridial Diseases

Clinical signs and diagnosis. The beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first 72 hr of a young ruminant's life. Many animals may be found dead, with no clinical presentation. Affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. Feces may range from loose gray-brown to dark red and malodorous. Morbidity and mortality may be nearly 100%. A similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. Struck is rare in the United States. The disease is also caused by the beta toxin of C. perfringens type C and is often associated with rapid dietary changes or shearing stresses in sheep. Although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. Mortality is usually less than 15%. Diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. Identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. Epizootiology and transmission. Clostridial organisms are ubiquitous in the environment as well as in the gastrointestinal tract and contaminated feeds. Confinement and poor sanitation predisposes to infection with C. perfringens. Transmission is by ingestion of contaminated material.

S. aureus. Pathogenesis. Simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. Differential diagnosis. The presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease.

Necropsy findings. Necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. Petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. Hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. Pulmonary and brain edema may also be

14. BIOLOGYAND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE seen. Histologically, the gram-positive C. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. In cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. In late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces.

Pathogenesis.

Hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of Clostridium perfringens type C. Neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. The trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. The toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. The intestinal injury may result in diarrhea, with small amounts of hemorrhage. Associated electrolyte and water loss result in dehydration, acidosis, and shock.

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Clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. Mature affected sheep may be blind and anorectic and may head-press.

Necropsyfindings. Necropsy findings are similar to those seen with C. perfringens type C. Additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (This phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) Focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. Diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy.

Pathogenesis.

Differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning.

The epsilon toxin causes neuronal death and shock, probably through vascular damage. The noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. The largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. The hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism.

Prevention and control.

Differential diagnosis.

Differential diagnosis.

A commercial toxoid is available and should be administered to the pregnant animals prior to parturition. An alternative includes administration of an antitoxin to the newborn lambs. The disease may become endemic once it is on the premises.

Treatment. Treatment is difficult and usually unsuccessful. Antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak.

Tetanus, enterotoxigenic E. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials.

Prevention and control.

Vaccination prevents the disease. Maternal antibodies last approximately 5 weeks postpartum; thus young animals should be vaccinated at about this time. Feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully.

Research complications.

This disease can be costly in losses of neonates and younger animals.

Treatment. Treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment.

ii. Clostridium perfringens type D infection (pulpy kidney disease) Etiology. Clostridium perfringens type D releases epsilon

iii. Clostridium tetani infection (tetanus, lockjaw) Etiology. Clostridium tetani is a strictly anaerobic, motile,

toxin that is proteolytically activated by trypsin. This disease caused by C. perfringens tends to be associated with sheep and is of less importance in goats and cattle.

Clinical signs.

The peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. Because the disease progresses so rapidly to death (within 1-2 hr), clinical signs are rarely observed. Hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. In acute cases, hyperglycemia and glucosuria are considered almost pathognomonic.

spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. At least 10 serotypes of C. tetani exist.

Clinical signs. Infection by C. tetani is characterized by a sporadic, acute, and fatal neuropathy. After an incubation period of 4 days to 3 weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. Erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. The animal may convulse. Death occurs within 3-10 days, and mortality is nearly 100%, primarily from

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respiratory failure. Diagnosis is based on clinical signs. Musclerelated serum enzymes such as aspartate aminotransferase (AST), creatinine kinase (CK), and lactate dehydrogenase (LDH) might be elevated. (Jensen and Swift, 1982). Serum cortisol may also be elevated, and stress hyperglycemia may be evident. Permanent lameness may result in survivors.

Epizootiology and transmission.

Clostridium tetani is a soil contaminant and is often found as part of the gut microflora of herbivores. The organisms sporulate and persist in the environment. All species of livestock are susceptible, but sheep and goats are more susceptible than cattle. Individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. Mouth wounds may also be sites of entry.

Prevention and control

Like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. The disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. Tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. Tetanus antitoxin can be administered (200 IU in lambs) as a preventive or in the face of disease as an adjunct to therapy. Both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered 4 weeks later. Animals should be vaccinated 2 or 3 times during the first year of life. Does and ewes should receive booster vaccinations within 2 months of parturition to ensure colostral antibodies.

Research complications. Pathogenesis. Tetanus, or lockjaw, is caused by the toxins of C. tetani. All serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. A nonspasmogenic toxin is also produced. Contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. The toxin inhibits the release of glycine and y-aminobutyric acid from Renshaw cells; this resuits in hypertonia and muscular spasms. Proliferation of C. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. The uterus is the most common site of infection in postparturient dairy cattle with retained placentas.

Differential diagnoses.

Early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. Polyarthritis of cattle is a differential for the gait changes in that species.

Necropsy findings.

Findings are nonspecific except for the inflammatory reaction associated with the wound. Because of the low number of organisms necessary to cause neurotoxicosis, isolation of C. tetani from the wound may be difficult.

Treatment. Treatment consists of cleaning the infected wound; administering tetanus antitoxin (e.g., at least 500 IU in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. Supportive fluids and glucose must be administered until the animal is capable of feeding. If the animal survives, revaccination should be done 14 days after the previous dose.

Unprotected, younger ruminants may be affected following routine flock or herd management procedures. Contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites.

iv. Clostridium novyi infection (bighead; black disease; bacillary hemoglobinuria, or red water) and C. chauvoei infection (blackleg) Etiology.

Clostridium novyi, an anaerobic, motile, spore-

forming, gram-positive bacteria, is the agent of bighead and black disease. Clostridium novyi type D (C. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." Clostridium chauvoei is the causative agent of blackleg.

Clinical signs. Bighead is a disease of rams characterized by edema of the head and neck. The edema may migrate to ventral regions such as the throat. Additional clinical signs include swelling of the eyelids and nostrils. Most animals will die within 48-72 hours. Black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with C. novyi. It is more common in cattle and sheep but may be seen in goats. The clinical course is 1-2 days in cattle and slightly shorter in sheep. Otherwise healthy-appearing adult animals are often affected. Clinical signs are rarely seen, because of the peracute nature of the disease. Occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. Bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. Animals that survive a few days will develop icterus. Mortality may be high. Blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema (Jackson et al., 1995). Blackleg is more common in cattle than in sheep. The incubation period is 2 - 5 days and is followed by hyperthermia,

14. BIOLOGYAND DISEASESOF RUMINANTS: SHEEP, GOATS,AND CATTLE muscular stiffness and pain, anorexia, and gangrenous myositis. The clinical course is short, 2 4 - 4 8 hr, and untreated animals invariably die. Blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. Most lesions are associated with muscles of the face, neck, perineum, thigh, and back.

Epizootiology and transmission. Bighead is caused by the toxins of C. novyi, which enters through wounds often associated with horn injuries during fighting. The C. novyi type B organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. Clostridium novyi type D is endemic in the western United States. It is hypothesized that the C. chauvoei organisms enter through the gastrointestinal tract. Black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with Fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. The diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. Birds and wild animals may be vectors of the pathogen. Ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations.

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diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials.

Treatment. For C. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. Treatment for black disease is not rewarding even if the animal is found before death. Carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises.

Prevention and control. Vaccinating animals with multivalent clostridial vaccines can prevent these diseases. Subcutaneous administration of vaccine material is recommended over intramuscular. Vaccinations may be useful in an outbreak. Careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. For bighead, mature rams penned together should be monitored for lesions, especially during breeding season. Control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations.

v. Clostridium septicum infection (malignant edema) Etiology. Clostridium septicum is the species usually associ-

Necropsy.

ated with malignant edema, but mixed infections involving other clostridial species such as C. chauvoei, C. novyi, C. sordellii, and C. perfringens may occur. Clostridium spp. are motile (C. chauvoei, C. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods.

Pathogenesis.

Clinicial signs. Malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by Clostridium spp. The incubation period is approximately 2 - 4 days. The affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. Regional lymphadenopathy and fever may occur. The animal becomes anorexic, severely depressed, and possibly hyperthermic. Edema and crepitation may be noted around the wound; death occurs within 12 hr to 2 days.

Diagnosis of black disease is usually based on postmortem lesions. Subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. Carcasses putrefy quickly. In addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. Blood coagulates slowly in affected animals.

The propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. Clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. Mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. Diagnosis is based on clinical signs. In black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. Associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. The toxins produced by C. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. Clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury.

Differential diagnosis.

Differential diagnoses include other clostridial diseases as well as photosensitization. Hemolytic

Epizootiology and transmission.

The organisms are ubiquitous in the environment and may survive in the soil for years. The disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia.

Necropsy findings.

The tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases.

Pathogenesis.

In most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). Furthermore, the toxins enter the bloodstream and

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central nervous system, resulting in systemic collapse and high mortality. Necropsy. Spreading, crepitant lesions around wounds are suggestive of malignant edema. Affected tissues are inflamed and necrotic. Gas and serosanguineous fluids with foul odors infiltrate the tissue planes. Large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. Intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. Organisms can be cultured from guinea pig tissues. Treatment. Infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. Prevention and control. Proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. Multivalent clostridial vaccines are available. Research complications. Morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. i.

Colibacillosis

Etiology. Escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. Escherichia coli organisms have three areas of surface antigenic complexes (O, somatic; K, envelope or pili; and H, flagellar), which are used to "group" or classify the serotypes. Colibacillosis is the common term for infections in younger animals caused by this bacteria. Clinical signs. Presentation of E. coli infections vary with the animal's age and the type of E. coli involved. Enterotoxigenic E. coli infection causes gastroenteritis and/or septicemia in lambs and calves. Colibacillosis generally develops within the first 72 hr of life when newborn animals are exposed to the organism. The enteric infection causes a semifluid, yellow to gray diarrhea. Occasionally blood streaking of the feces may be observed. The animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." Hyperthermia is rare. Severe acidosis, depression, and recumbancy ensue, and mortality may be as high as 75%. The septicemic form generally occurs between 2 and 6 weeks of age. Animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. Opisthotonos, depression, and death follow. Occasionally, swollen, painful joints may be observed with septicemic colibacillosis. Blood cultures may be helpful in identifying the septicemic form.

In ruminants, E. coli is is a less common cause of cystitis and pyelonephritis. The cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. The infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. In chronic cases, animals will be polyuric and undergo weight loss. Escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. Epizootiology and transmission. Escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. Zeman et al. (1989) classify E. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. Enterotoxigenic E. coli (ETEC) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. Fimbrial (pili) antigens associated with ovine disease include K99 and F41. Enterohemorrhagic E. coli (EHEC) attach and efface the microviUus, produce verotoxins, and occasionally cause disease in humans and animals. Enteropathogenic E. coli (EPEC) colonize and efface the microvillus but do not produce verotoxins. EPEC are associated with disease in humans and rabbits and cause a secretory diarrhea. Enteroinvasive E. coli (EIEC) invade the enterocytes of humans and cause a shigella-like disease. Overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. The organism will be endemic in a contaminated environment and present on dams' udders. The bacteria rapidly proliferate in the neonates' small intestines. The bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. If the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. Diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. Necropsy findings. Swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. Septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. Isolation and serotyping of E. coli confirm the diagnosis. ELISA and latex agglutination tests are available diagnostic tools. Differential diagnosis. Differential diagnoses include the enterotoxemias caused by C. perfringens type A, B, or C; Campylobacter jejuni; Coccidia, rotavirus, coronavirus, Salmonella, and Cryptosporidia. Other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered.

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE

Prevention and control.

The best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. Attention to colostrum feeding techniques and colostral quality are important means of preventing disease. Treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities.

Treatment. Antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. Vaccines are available for prevention of colibacillosis in cattle. Corynebacterium pseudotuberculosis Infection (Caseous Lymphadenitis) Etiology. Corynebacterium pseudotuberculosis (previously C. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. Caseous lymphadenitis (CLA) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. The disease has been reported occasionally in cattle.

Clinical signs and diagnosis. Abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. Radiographs may be helpful in identifying affected central nodes. Peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. The incubation period may be weeks to months. Over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. Fever, increased respiratory rates, and pneumonia may also be common signs. Exotoxin-induced hemolytic crises may occur occasionally. Morbidity up to 15% is common, and morbid animals will often eventually succumb to the disease. Diagnosis is based on clinical lesions; ELISA serological testing is also available. Smears of the exudate or lymph nodes aspirates can be Gram-stained. Lymph node aspirates may also be sent for culturing. Epizootiology and transmission.

The organism can survive for 6 months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. Ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry.

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affected. Cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically.

Pathogenesis.

Corynebacterium pseudotuberculosis produces an exotoxin (phospholipase D) that damages endothelial and blood cell membranes. This process enhances the organisms' ability to withstand phagocytosis. The infection spreads through the lymphatics to local lymph nodes. The necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. Differential diagnosis.

Differentials include pathogens causing lymphadenopathy and abscessation.

Treatment. Antibiotic therapy is not usually helpful. Abscesses can be surgically lanced and flushed with iodinecontaining and/or hydrogen peroxide solutions. Abscessing lymph nodes can be removed entirely from valuable animals. During warmer months, an insect repellent should be applied to and around healing lesions. All materials used to treat animals should be disposed of properly. Because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from CLA-negative animals at least until healed. Commercial vaccines are available (Piontkowski and Shivvers, 1998). Prevention and control.

Minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important.

Research complications.

This pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics.

Corynebacterium renale, C. cystitidis, and C. pilosum Infections (Pyelonephritis; Posthitis and Ulcerative Vulvovaginitis) Etiology. Corynebacterium renale, C. cystitidis, and C. pilosum are sometimes referred to as the C. renale group. These are piliated and nonmotile gram-positive rods and are distinguished biochemically. Corynebacterium renale causes pyelonephritis in cattle, and C. pilosum and C. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. In many references, all these clinical presentations are attributed to

C. renale. Necropsy findings.

Disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. Cut surfaces of the affected lymph nodes may appear lamellated. Lungs, liver, spleen, and kidneys may also be

Clinical signs and diagnosis. Acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. Untreated infections usually become

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chronic, with weight loss, anorexia, and loss of production in dairy animals. Relapses are common, and some infections are severe and fatal. Diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). Urine culturing may not be productive. In chronic cases, E. coli and other gram-negatives may be present. Posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. The area may have a distinct malodor. Necrosis and scarring may be sequelae of more severe infections. Fly-strike may also be a complication. Diagnosis is based on clinical signs and on investigation of feeding regimens.

Epizootiology and transmission.

Ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. The vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. Posthitis occurs in intact and castrated sheep and goats.

Necropsy findings.

Pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy.

Pathogenesis.

Corynebacterium renale is a normal inhabitant of bovine genitourinary tracts. The pilus mediates colonization. Conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. In addition, more basic pH urine levels may block some immune defenses. Infections ascend through the urinary tract. The bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. Corynebacterium cystitidis and C. pilosum are normally found around the prepuce of sheep and goats. High-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. Posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. The ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. Differential diagnosis.

Urolithiasis is a primary consideration for these diseases. Contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. Ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis.

tices must be reconsidered. Clipping long wool and hair also is helpful.

Treatment. Long-term (3 weeks) penicillin treatment is effective for pyelonephritis. Reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. Surgical debridement or correction of scarring may also be indicated in severe cases. l.

Erysipelas

Etiology. Erysipelothrix rhusiopathiae is a nonmotile, nonspore-forming, gram-positive rod that resides in alkaline soils.

Clinical signs. Erysipelothrix causes sporadic but chronic polyarthritis in lambs less than 3 months of age. In older goats, erysipelas has been associated with joint infections. Epizootiology and transmission.

The disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. Following wound contamination and a 1- to 5-day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. Joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged.

Necropsy findings.

Thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. The joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. Diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints.

Differential diagnosis.

Differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. Other bacteria causing septic joints include Areanobacterium pyogenes and Fusobacterium necrophorum. Caprine arthritis encephalitis (CAE) should also be considered.

Prevention and control. Proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. Screening of goat herds for CAE is recommended.

Treatment.

Erysipelas is sensitive to penicillin antibiotic

therapy. m.

Dermatophilosis (Cutaneous Streptothricosis, Lumpy Wool Strawberry Foot Rot)

Etiology. Dermatophilus congolensis is an aerobic, gramPrevention and treatment.

Because high-protein feed is often associated with posthitis and vulvovaginitis, feeding prac-

positive, filamentous bacterium with branching hyphae. Dermatophilosis is a chronic bacterial skin disease characterized by

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

crustiness and exudates accumulating at the base of the hair or wool fibers (Scanlan et al., 1984).

Clinical signs. Animals will be painful but will not be pruritic. Two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. Mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. Strawberry foot rot is rare in the United States but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. Animals will be lame. In goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. Lesions in younger goats are seen along the tips of the ears and under the tail. Diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. Epizootiology and transmission. The disease occurs worldwide, and the Dermatophilus organism is believed to be a saprophyte. Transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. Biting insects may aid in transmission. Necropsy findings. Lymphadenopathy as well as liver and splenic changes may be observed. Histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. Dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. Pathogenesis. Lesions typically begin around the muzzle and hooves and the dorsal midline. Prevention and control. Potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. Minimizing moist conditions is helpful in controlling and preventing the disease. In addition, controlling external parasites or other factors that cause skin lesions is important. Lesions will resolve during dry periods. Treatment. Animals can be treated with antibiotics such as penicillin and oxytetracycline. Treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. n.

Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum Infection (Virulent Foot Rot; Contagious Foot Rot of Sheep and Goats; Foot Scald)

Etiology. Two bacteria, Dichelobacter (Bacteroides) nodosus and Fusobacterium necrophorum, work synergistically in caus-

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ing contagious foot rot in sheep and goats. Other organisms may be involved as secondary invaders. Both Dichelobacter and Fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. Foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues (Bulgin, 1986). It is the leading cause of lameness in sheep. At least 20 serotypes of Dichelobacter are known. Arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. Foot scald, an interdigital dermatitis, is caused primarily by D. nodosus alone.

Clinical signs. Varying degrees of lameness are observed in all ages of animals within 2 - 3 weeks of exposure to the organisms. Severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. The interdigital skin and hooves will be moist, with a distinct necrotic odor. Morbidity may reach 70% in susceptible animals. Diagnosis is based on clinical signs. Smears and cultures confirm the definitive agents. Clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. Epizootiology and transmission. Fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. In contrast, Dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about 2 weeks. Some animals may be chronic carriers. Overcrowded, warm, and moist environments are key elements in transmission. Outbreaks are likely in the spring season. Shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. Pathogenesis. Both organisms are transmitted to the susceptible animal by direct or indirect contact. The organisms enter the hoof through injuries or through sites where Strongyloides papillosus larvae have penetrated. Fusobacterium necrophorum initiates the colonization and is followed by D. nodosus. The latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. The pathogenicity of the serotypes of D. nodosus is correlated with the production of these proteases and numbers of pili. Additionally, F. necrophorum causes a severe, damaging inflammatory reaction. Differential diagnosis. Foot abscesses, tetanus, selenium/ vitamin E deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. Treatment. Affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. Systemic antibiotics

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such as penicillin, oxytetracycline, and erythromycin may be used. Goats have improved dramatically when given a single dose of penicillin (40,000 U/kg) (Smith and Sherman, 1994). Footbaths containing 10% zinc sulfate, 20% copper sulfate, or 10% formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. Affected animals should be separated from the flock. Vaccination has been shown to be effective as part of the treatment regimen. Some breeds of sheep and some breeds and lines of goats are resistant to infection. Individual sheep may recover without treatment or are resistant to infection.

Epizootiology and transmission. Cases may be sporadic, or epizootics may occur. Bos taurus dairy breeds and animals with

Prevention and control.

Pathogenesis.

Prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. Dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. Biannual vaccinination in wet areas may be essential. Some breeds may develop vaccination site lumps. Footbaths of 10% zinc sulfate, 10% formalin (where allowed by state regulations), or 10% copper sulfate are also considered very effective preventive measures. Goats are less sensitive than sheep to the copper in the footbaths.

Research complications.

Treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications.

Fusobacterium necrophorum and Bacteroides melaninogenicus Infection (Foot Rot of Cattle, Interdigital Necrobacillosis of Cattle) Etiology.

Interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by Fusobacterium necrophorum and Bacteroides melaninogenicus. Like F. necrophorum, B. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. Dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. This is a common cause of lameness in cattle.

Clinical signs. Clinical signs include mild to moderate lameness of sudden onset. Hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. The interdigital space will be swollen, as will be the coronet and bulb areas. Characteristic malodors will be noted, but there will be little purulent discharge. In more severe cases, animals will have elevated body temperature and loss of appetite. The les~ons progress to fissures with necrosis until healing occurs. The diagnosis is by the odor and appearance. Anaerobic culturing confirms the organisms involved.

wide interdigital spaces are more commonly affected. The factors here are comparable to those present in foot rot of smaller ruminants.

Necropsy findings.

Findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. Although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. The bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. Colonization leads to cellulitis. In addition, F. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by B. melaninogenicus. Zinc deficiency may play a role in the pathogenesis in some situations.

Differential diagnoses.

The most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. Bluetongue virus should also be considered. Grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. The exotic footand-mouth disease virus would be considered in areas where that pathogen is found.

Prevention and control.

As with foot rot in smaller ruminants, management of the area and herd are important. Paddocks and pens should be kept dry, well drained, and free of material that will damage feet. Footbaths and chlortetracycline in the feed have been shown to control incidence. Affected animals should be segregated during treatment. Chronically affected or severely lame animals should be culled. New cattle should be quarantined and evaluated.

Treatment. Successful treatment regimens that result in healing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as 10% zinc sulfate, 2.5% formalin, or 5% copper sulfate) twice a day. In severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. Animals can recover without treatment but will be lame for several weeks. Acquired immunity is reported to be poor. Research complications.

Research complications are comparable to those noted for foot rot in smaller ruminants.

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

p.

Fusobacterium necrophorum infection (Foot Abscesses)

Fusobacterium necrophorum is also associated with foot abscesses, the infection of the deeper structures of the foot, in sheep and goats. Only one claw of the affected hoof may be involved. The animals will be three-legged lame, and the affected hoof will be hot. Pockets of purulent material may be in the heel or toe. Heel Warts (Bovine Digital Dermatitis, Interdigital Papillomatosis, Papillomatous Digital Dermatitis, Foot Warts, Heel Warts, Hairy Foot Warts, Mortellaro's Disease) Etiology.

Bacteria such as Fusobacterium spp., Bacteroides spp., and Dichelobacter nodosus have been isolated from bovine heel lesions. Spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (PDD), in the United States and Europe; these have culturing requirements similar to those of Treponema species.

Clinical signs. All lesions occur on the haired, digital skin. One or all feet may be affected. Most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. Progression of lesions, typically over 2 - 3 weeks, includes erect hairs, loss of hair, and thickening skin. Moist plaques begin as red and remain red or turn gray or black. Exudate or blood may be present on the plaque. Plaques enlarge and "hairs" protrude from the roughened surface. Lesioned areas are painful when touched. The lesions may or may not be malodorous. Epizootiology and transmission.

Facility conditions and herd management are considered contributing factors. The following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. These interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. The disease is seen only in cattle.

Pathogenesis. The organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. Differential diagnosis.

Differentials for lameness will include sole abscesses, laminitis, and trauma.

Prevention and control.

Each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. All equipment used for hoof trimming must be cleaned and disinfected after every use. Trucks and trailers should also be sanitized between groups of animals.

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Treatment. Antibiotic and antiseptic regimens have been used successfully for this problem. Antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. Antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. The footbaths must be well maintained, minimizing contamination by feces and other materials. Tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. Other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. Research complications.

Infectious, contagious PPD is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. The outbreaks are generally worse in younger animals in chronically infected herds. The immune response is not well understood, and it may be temporary in older animals.

Haemophilus somnus infection (Thromboembolic Menin goencephalitis) Etiology.

Haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. Diseases caused by this organism include thromboembolic meningoencephalitis (TEME), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. Haemophilus somnus is a also major contributor to the bovine respiratory disease complex. Haemophilus spp. have been associated with respiratory disease in sheep and goats. Clinical signs.

The neurologic presentation may be preceded by 1-2 weeks of dry, harsh coughing. Neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. High fever, extreme morbidity, and death within 36 hr may occur. Respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza 3, Mycoplasma, and Pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (BRDC). In acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. Abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. In all cases, asymptomatic infections may also occur. Diagnosis based on culture findings is difficult because H. somnus is part of the normal nasopharyngeal flora. Paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. In cases of abortion, other causes should be eliminated from consideration.

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Epizootiology and transmission. Because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. Outbreaks are associated with younger cattle in feedlots in western United States, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. Adult cattle have also been affected. Vaccination for viral respiratory pathogens may increase susceptibility. Transmission is by respiratory and genital tract secretions. The organism does not persist in the environment. Necropsy findings. Pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. Many thrombi with bacterial colonies will be seen in these affected areas. Ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. Usually animals with neurological disease will not have respiratory tract lesions. The respiratory tract lesions include bronchopneumonia and suppurative pleuritis. When combined with Pasteurella infection, the pathology becomes more severe. Aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. Pure cultures of H. somnus may be possible from these tissues. Pathogenesis. Inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. The anatomical location of bacterial residence within the carriers has not been identified. After gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. The bacteria are phagocytosed by neutrophils but are not killed. The thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. Antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. Differential diagnosis. Differentials in all ruminants include other pathogens associated with neurological disease and respiratory disease such as Pasteurella hemolytica, P. multocida, and P. aeruginosa. In smaller ruminants, Corynebacterium pseudotuberculosis should be considered. Prevention and control. Stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). The late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. Planning vaccination programs carefully will decrease chances of outbreaks. For example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during

times of stress to the cattle is worthwhile. Killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. Control of other clinical aspects of the H. somnus disease by these bacterins has not been well described.

Treatment. Rapid treatment at the first signs of neurologic disease is important in an outbreak. Haemophilus somnus is susceptible to several antibiotics, such as oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. s.

Leptospirosis

Etiology. Seven different species of the spirochete genus Leptospira are now recognized, and pathogenic serovars exist within each species; previously pathogenic leptospires were all classified as members of the species L. interrogans. The serovars pomona, icterohaemorrhagiae, grippotyphosa, interrogans, and hardjo are recognized pathogens. Leptospira hardjo and L. pomona are the serovars most commonly diagnosed in cattle, with L. hardjo causing endemic infection. Leptospira hardjo is also the major sheep serovar. Goats are susceptible to several serovars. Clinical signs. Leptospirosis is a contagious but uncommon disease in sheep and goats. The disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. After a 4- to 10-day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. Leptospiremia may last up to 7 days. Immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; crossserovar protection does not occur. During active bacteremia, hemolysis may result in hemoglobin levels of 50% below normal. Hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. Abortion usually occurs only once. Mortality rates of above 50% have been reported in infected ewes and lambs (Jensen and Swift, 1982). Subclinical infection is more common in nonpregnant and nonlactating animals. Sheep infected with leptospirosis may display a hemolytic crisis associated with IgM acting as a cold-reacting hemagglutinin. Acute and chronic infections in cattle are more common than infections in sheep and goats. Acute forms in cattle display signs similar to those in sheep. Acute infection in calves may progress to meningitis and death. Lactating cows will have severe drops in production. Chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. Infertility may also be a sequela. Epizootiology and transmission. Leptospires are a large genus, and leptospirosis is a complicated disease to prevent,

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE treat, and control. The organism survives well in the environment, especially in moist, warm, stagnant water. Cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. Wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. Organisms are shed in urine, in uterine discharges, and through milk. Animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-hostadapted serovar (Heath and Johnson, 1994). Infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. Placental or venereal transmission may occur. As the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). Chronically infected animals may shed the organism in the urine for 60 days or longer.

Necropsy.

Diagnosis is confirmed by identification of leptospires in fetal tissues. The leptospires are visible in silver- or fluorescent antibody-stained sections of liver or kidney. Leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. Fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars.

Differential diagnosis.

More than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. Because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases.

Prevention and control.

Polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. Immunity is serovar specific. Because serological titers tend to diminish rapidly (40-50 days in sheep [Jensen and Swift, 1982]), frequent vaccination may be necessary. Other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted.

Treatment. Antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. Treatment methods for acute leptospirosis include oxytetracycline for 3 - 6 days. Addition of oxytetracycline or chlortetracycline to the feed for 1 week may be helpful. These antibiotics are considered best for removal of the carrier state of some serovars. Vaccination and antibiotic therapy can be combined in an outbreak. Research complications.

Leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans.

t.

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Listeria (Circling Disease, Silage Disease)

Etiology.

Listeria monocytogenes is a pleomorphic, motile, non-spore-forming, [3-hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. Of the 16 known serovars, several produce clinical signs in ruminants. Listeria ivanovii (associated with abortions in sheep) is serovar 5.

Clinical signs. Listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. The overall case rate is low. The disease may present as an isolated case or with multiple animals affected. Three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. The encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs (Scarratt, 1987). Clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of 2 3 weeks. As the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. Facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves V-XII. The neck will by flexed away from the affected side. Facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. The hypersalivation may lead to metabolic acidosis in advanced cases in cattle. Anorexia, prostration, coma, and death follow. The placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. The affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. Abortion usually occurs within 2 weeks of Listeria infection. In cattle, abortion occurs during the last 2 months of gestation and has been induced experimentally 6 - 8 days after exposure. Cows present with the range of clinical signs seen in smaller-ruminant dams. There is no long-term effect on the fertility of affected dams. Epizootiology and transmission.

The organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. By the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. When severe outbreaks occur, feedstuffs should be assessed for spoilage. Listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing.

Diagnosis and necropsy findings.

Diagnosis is usually made from clinical signs. Culture confirms the diagnosis (cold enrichment at 20~ is preferable but not essential for isolation). Impression smears will show the pleomorphic gram-positive

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characterisitics of the pathogen. Tissue fluorescent antibody techniques may also be utilized. Gross lesions are not observed with the encephalitic form. Microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. The pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. Microabscesses of the midbrain are characteristic of Listeria encephalitis in sheep. Aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. Necrotic lesions of the fetal spleen and lungs may also be seen. In goats, Listeria-induced neurological lesions occur only in the brain stem. Placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms.

Pathogenesis.

With the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. The Listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). In the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. After infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). A toxin produced by Listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated.

successful, and mortality is especially high in sheep. Recovered animals tend to resist reinfection.

Research complications.

In addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. Listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. Listeriosis in humans is a reportable disease.

Lyme Disease (Borrelia burgdorferi Infection, Borreliosis) Etiology. Lyme disease is caused by the spirochete Borrelia burgdorferi. Clinical signs and diagnosis. Reports in ruminants indicate seroconversion to B. burgdorferi, but there are few definitive correlations to the arthritis that is present. Diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. Epizootiology and transmission.

The organism is present throughout much of the Northern Hemisphere and has been reported in many mammals and also in birds. Ticks of the Ixodes ricinus complex are the major vectors of the spirochete and must be attached for 24 hr for successful transmission.

Differential diagnoses.

Rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. In sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to Clostridium perfringens type D. In goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. In both species, scrapie is a differential. In cattle, aberrant parasite migration or Hemophilus somnus infection must also be considered.

Pathogenesis.

The Ixodes ticks have three life stages: larval, nymphal, and adult. Feeding occurs once during each stage, and wild animals are the source of blood meals. The larval stages feed from rodents, such as the white-footed deer mouse, Peromyscus leucopus, from which they acquire the spirochete. The nymphal stage is that which usually infects other animals. The adult ticks are usually found on deer.

Differential diagnosis.

Prevention and control.

Affected dams should be segregated and treated. Other animals in the group may be treated with oxytetracycline as needed. Aborted tissues should be removed immediately. Proper storage of fermented feeds minimizes this source of contamination. When silage spoils, the pH increases, producing a suitable growth environment for the organism. Commercial vaccines are not available in the United States.

Treatment.

Affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. Exceptionally high levels of penicillin are required for treating affected cattle. Severely affected animals should receive appropriate fluid support and other nursing care. Treatment is less

Seroconversion to B. burgdorferi does not necessarily confirm the cause of arthritis. Other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, Mycoplasma spp., Chlamydia psittaci, Erysipelothrix spp., Arcanobacterium pyogenes, Brucella spp., and rickets.

Prevention and control.

Control of the tick vector is the most important factor in preventing the possibility of exposure or disease.

Treatment.

Antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected Lyme arthritis.

14. BIOLOGYAND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE

Research complications. Lyme disease is zoonotic, and the lxodes ticks transmit the disease to humans. v.

Mastitis

i. Ovine mastitis Mastitis in ewes may be acute, subclinical, or chronic. Acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. Pasteurella haemolytica is the most common cause of acute mastitis. Additional isolates may include, in order of prevalence, Staphylococcus aureus, Actinomyces (Corynebacterium) spp., and Histophilus ovis. Escherichia coli and Pseudomonas aeruginosa have also been found to cause acute mastitis. As many as six serotypes of Pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. Furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes (Watkins.and Jones, 1992). Subclinical mastitis is detected only indirectly, by counting somatic cells. The most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. Other isolates include Actinomyces bovis, Streptococcus uberis, S. dysgalactiae, Micrococcus spp., Bacillus spp., and fecal streptococci. Most of these organisms are commonly found in the environment. Diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. Both glands are usually affected, but no inflammation is present. Serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (OPP or maedi/visna virus). Other bacterial agents or Mycoplasma have not usually been isolated from udders with this type of mastitis. Acute mastitis occurs in approximately 5% of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are 3 - 4 months old (Lasgard and Vaabenoe, 1993). Subclinical mastitis occurs in 4 - 5 0 % of lactating ewes (Kirk and Glenn, 1996). Subclinical mastitis is more common in ewes from high-milk-producing breeds. Skin or teat lesions and dermatitis increase the prevalence of disease. Acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. Subclinical mastitis is often suggested by somatic cell counts elevated above 1 x 106 cells/ml. When high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. The California mastitis test may also be helpful as an indicator of mastitis. Manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. Treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. It is may be helpful to milk out the infected ud-

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der frequently; oxytocin injections preceding milking will improve gland evacuation. Because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. There is currently no treatment available for diffuse chronic mastitis.

ii. Caprine mastitis Lactating goats are subject to inflammation of mammary gland, or mastitis. The primary causative organisms are Staphylococcus epidermidis and other coagulasenegative Staphylococcus spp. Clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. Large abscesses may be present in the affected gland. Staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. This organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. Caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. Diagnosis is based on careful culture of mastitic milk. Treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. Oxytocin (5-10 U) may help milk letdown for frequent strippings. Bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. In severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. Other less common causes of mastitis in goats include Streptococcus spp. (S. agalactiae, S. dysgalactiae, S. uberis, and zooepidemicus). Gram-negative causes of caprine mastitis include Escherichia coli, Klebsiella pneumoniae, Pasteurella spp., Pseudomonas, and Proteus mirabilis. Corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas Mycoplasma mycoides may cause agalactia and systemic disease. "Hard udder" can be caused by caprine arthritis encephalitis virus (CAEV). Brucellosis and listeriosis can cause a subclinical interstitial mastitis (Smith and Sherman, 1994). iii. Bovine mastitis Mastitis is the disease of greatest economic importance for the dairy cattle industry. The majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. The most common bovine mastitis pathogens include Staphylococcus aureus and Streptococcus agalactiae, S. dysgalactiae, and S. uberis; coliform agents such as Escherichia coli, Enterobacter aerogenes, Serratia marcescens, and Klebsiella pneumoniae; mycoplasmal species such as Mycoplasma bovis, M. bovigenitalium, M. californicum, M. canadensis, and M. alkalescens; and Salmonella spp. such as S. typhimurium, S. newport, S. enteritidis, S. dublin, and S. muenster. Many of these agents such as Staphylococcus

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spp., Salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. These must be regarded as herd and environmental pathogens in terms of treatment and prevention. The pathogenesis of staphylococcal infections is comparable to that in goats. Staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. In contrast, S. uberis and S. dysgalactiae are environmental organisms and can be highly resistant to pencillin. Mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. Transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. Treatments for mycoplasmal mastitis are not successful; culling is recommended. There are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. Management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. Culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. Herd management will diminish teat lesions. Bacterin vaccines are available for preventing and controlling coliform mastitis and S. aureus mastitis. At the time of dry-off, all cows must be treated by intramammary route. Some infections can be successfully cleared during this time. Younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. Milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. Teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre- and postmilking dipping. Milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. Facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. In that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust.

W.

Moraxella bovis Infection (Infectious Bovine Keratoconjunctivitis, Pinkeye)

Etiology.

Moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (IBK) in cattle. This organism is not a cause of keratoconjunctivitis in sheep and goats. The disease includes conjunctivitis and ulcerative keratitis. The pathogenic M. bovis strain is piliated, and at least seven serotypes exist.

Clinical signs. Lacrimation, photophobia, and blepharospasm are seen initially. Conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. Anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. Corneal vascularization begins by 10 days after onset. Reepithelialization of the corneal ulcers occurs by 2 - 3 weeks after onset. Diagnosis is usually based on clinical signs, but culturing is helpful and fluoroscein staining is useful for demonstrating corneal ulceration. Epizootiology and transmission.

The disease is more severe in younger cattle. The clinical signs of IBK tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (IBR) virus or those that have been vaccinated recently with modified live IBR vaccine. The bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. Transmission is by fomites, flies, aerosols, and direct contact. Incidence in winter months is very low. Nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics.

Necropsy findings.

Necropsy is not typically performed on these cases. Corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection.

Pathogenesis.

The pili ofM. bovis bind to receptors of corneal epithelium. The virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. Other factors contributing to infection include ultraviolet light and trauma from dust and plant materials.

Differential diagnoses.

Infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of IBK is not seen with M. bovis infections. Mycoplasma, Listeria, Branhamella (Neisseria), and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals.

Prevention and control.

Cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during IBK outbreaks; this will likely exacerbate the infection. New animals should be quarantined and treated prophylactically before introduction to herds. The available vaccines, containing. M. bovis pili or killed M. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the M. bovis strain may not be homologous to that used for the vaccine preparation. Other preventive measures include 10% permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as 5% coumaphos) to control flies throughout the season and premises; mowing of high pasture

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14. BIOLOGYAND DISEASESOF RUMINANTS: SHEEP, GOATS,AND CATTLE grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age.

Treatment. Cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. Antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. Several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. These should be administered twice per day. Subconjunctival injections of antibiotics, such as penicillin G, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. Intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and 2 doses 72 hr apart eliminate carriers. Third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. Research complications. This pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. The overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. x.

Mycobacterial Diseases

Mycobacterium bovis Infection (Tuberculosis) Etiology. Mycobacteria are aerobic, nonmotile, non-sporeforming, acid-fast pleomorphic bacteria. Most cases of tuberculosis in sheep are related to Mycobacterium bovis or M. avium. Cases in goats have been attributed to M. bovis, M. avium, or M. tuberculosis. Mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. Other agents of mammalian tuberculosis include M. microti and M. africanum. Clinical signs. Tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. The infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. The respiratory system (M. bovis) or the digestive system (M. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. Locations of the characteristic tubercles will determine whether clinical signs are seen. Respiratory signs may include dyspnea, coughing, and pneumonia. Digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. Lymphadenopathy occurs in advanced cases. Fever and generalized disease may be seen after calving. Infected goats lose weight and develop a persistent cough.

Epizootiology and transmission. Although M. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. Animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. Clinically normal animals may serve as carriers. The bacilli stimulate an initial neutrophilic tissue response. Neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called Langhans' giant cells. An outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. Vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. Necropsy findings. Yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. Caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. Prevention and control. Significant progress has been made in eradication programs in the United States during the past several decades, but during the 1990s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. The intradermal tuberculin test, using purified protein derivative (PPD), is usually used as a diagnostic indicator in live animals. This test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. Notification to state officials is required following identification of intradermal-positive animals. Great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. Treatment. No treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. Slaughter is preferred, to prevent potential transmission to humans. Research complications.

The pathogen is zoonotic.

Paratuberculosis, or Johne 's disease (Mycobacterium paratube rculo sis) Etiology. Mycobacterium paratuberculosis, the causative agent of Johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. The organism is actually a subspecies of M. avium, but M. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of M. avium. Clinical signs and diagnosis. Johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is

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characterized by unthriftiness, weight loss, and intermittent diarrhea. In sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. In cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. Usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. Although clinical signs are nonspecific, Johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. The disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than 50% sensitive. In addition, the sensitivity of the serological tests differs between species. The standard is the fecal culture that takes 8 12 weeks. Theenzyme-linked immunosorbent assay (ELISA) is now considered the most reliable serological test, but false negatives do occur. Other serological tests such as agar gel immunodiffusion (AGID) and complement fixation are useful. Herd screening may be done using the AGID or ELISA serological tests. Identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. Some animals serologically negative for Johne's disease, however, have been found to be positive on fecal culture. Commercial AGID tests approved for use in cattle may be useful in diagnosing Johne's disease in sheep (Dubash et al., 1996). Serological tests cross-react with other species of Mycobacterium, especially M. avium.

Epizootiology and transmission.

The organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. Although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. Chronic carriers may intermittently shed the organisms.

Pathogenesis.

Mycobacteriumparatuberculosis is an obligate

parasite that grows only in macrophages of infected animals. Nursing infected dams are a primary source of infection of neonates. If the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. The organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. After an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. Gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats.

Necropsy and diagnosis.

The ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. Ileal and

ileocecal lymph nodes provide the best samples for histology and acid-fast staining.

Differential diagnosis.

Diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. These include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. Deer can be infected, and the lesions can be confused with those of tuberculosis.

Treatment.

Treatment is not worthwhile.

Prevention and control.

Prevention is the most effective method to manage this pathogen. Efforts should be focused on eliminating the disease through test and slaughter. Neonates should not be reared by infected dams. Some states have Johne's disease eradication programs. Facilities and pastures where animals testing positive for Johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling.

Other considerations. Mycobacterium paratuberculosis is being investigated as a factor in the development of Crohn's disease in humans. Navel Ill (Omphalitis, Omphalophlebitis, Omphaloarteritis, Joint Ill) Etiology.

The most common organism causing infection of the umbilicus is Arcanobacterium (formerly Actinomyces, Corynebacterium) pyogenes; other bacteria may be present. Arcanobacterium spp. are anaerobic, nonmotile, non-sporeforming, gram-positive, pleomorphic rods to coccobacilli. Other environmental contaminants are also associated with this disease, such as Escherichia coli, Enterococcus spp., Proteus, Streptococcus spp., and Staplylococcus spp.

Clinical signs and diagnosis. Navel ill is an acute localized inflammation and infection of the external umbilicus. Animals present with fever and painful enlargement of the umbilicus. Animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. Involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. Other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. Epizootiology and transmission.

Many cases occur in neonates, and most cases occur within the first 3 months of age. Cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. Dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. Navel ill is diagnosed by typical clinical signs. The presence

14. BIOLOGYAND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. Assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. Navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. Arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection.

Differential diagnosis. The major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. Mycoplasmal arthritis is a differential in kids. In the past, Erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. Treatment. Omphalitis can be treated with a 10 to 14 day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. If an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. Surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. The prognosis for recovery is good if systemic involvement has not occurred. Prevention and control. The disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (Lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. Research complications. The disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. Pasteurellosis (Shipping Fever, Hemorrhagic Septicemia, Enzootic Pneumonia) Etiology. Pasteurella hemolytica and P. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. Biotype A serotypes are associated with pneumonia and septicemia in all ruminants (Ellis, 1984). Serotype 1 of P. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. Clinical signs. Pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. The organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. The lungs and liver are frequent areas of formation of microabscesses. Acute rhinitis or pharyngitis often precedes the respiratory form. The organism also

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may invade the bloodstream, causing disseminated septicemia. Clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. With the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. The disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. In cultures, P. hemolytica is distinguished from P. multocida by hemolysis on blood agar; only P. multocida produces indole.

Epizootiology and transmission. The organism is ubiquitous in the environment and in the respiratory tracts of these animals. Younger ruminants, between 2 and 12 months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. The pneumonic form appears as a complex associated with concurrent infections such as parainfluenza 3, adenovirus type 6, respiratory syncytial virus, mycoplasmas, chlamydia, Pasteurella multocida and Bordetella parapertussis (Martin, 1996; Brogden et al., 1998). The organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. Necropsy findings. Necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal 1 mm lesions distributed on the surfaces of the lungs and liver. With the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. Fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident.. Pathogenesis. A leukotoxin is considered to be a key factor in the pathogenesis of the P. hemolytica infection. Macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. Treatment. Treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. Newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. In outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. Prevention and control. The incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. Early Pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. Pasteurella

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multocida bacterins and live streptomycin-dependent mutant vaccines are available. In young animals, passive immunity is protective. Preventive measures also include maintaining good ventilation in enclosures and barns. New animals to the flock or herds should be quarantined for at least 2 weeks before introduction. aa.

Salmonellosis

Etiology. Salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. It is a common inhabitant of the gastrointestinal tract of ruminants. Current nomenclature categorizes S. typhimurium as a serovar within the species S. enteritidis (the other two species are S. typhi and S. choleraesuis). Salmonella typhimurium, S. dublin, and S. newport are the common species seen in bovine cases. Salmonella typhimurium, S. dublin, S. anatum, and S. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. Ovine abortions due to various Salmonella species are not reported in the United States but are enzootic in other countries. Salmonella serotypes have been associated with aborted fetuses in all ruminant species. Clinical signs and diagnosis. Salmonellosis causes acute gastroenteritis, dysentery, and septicemia (Anderson and Blanchard, 1989). Clinically, the animals become anorexic and hyperthermic. Diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. Animals become severely depressed and weak, losing a high percentage of their body weight. Animals may die in 1-5 days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. Morbidity may be 25%, and mortality may be high. Septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. Chronically infected animals may have intermittent diarrhea. In goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. Adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. Morbidity and mortality will be highest in neonates, and some may simply be found dead. The older animals generally tend to fare better during the disease. Abdominal distension with profuse yellow feces is common. Kids become severely depressed, anorexic, febrile (with temperatures as high as 106~176 dehydrated, acidotic, recumbent, and comatose. Salmonella abortions may occur throughout gestation. There may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. Hemorrhage, placental necrosis, and edema will be present. Metritis and placental retention may occur. Some mortality of dams may occur. Diagnosis is based on clinical signs and can be confirmed by

culturing fresh feces or at necropsy. Because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. Leukopenia and a degenerative shift to the left are not uncommon hematological findings.

Epizootiology and transmission. Stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. Birds and rodents may be natural reservoirs of Salmonella in external housing environments. Transmission is fecal-oral. After ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the Peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. Animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally (Arora, 1983). Fecal-oral transmission is also associated with Salmonella abortion; veneral transmission has not been reported. Necropsy findings and diagnosis. Animals will have noticeable perineal staining. Intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. Petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. The wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. This membrane is not normally seen in the goat (Smith and Sherman, 1994). Splenomegaly may be present. Aborted fetuses will often be autolysed. Placentitis, placental necrosis, and hemorrhage are commonly seen. Serologic evidence of recent infection can be demonstrated in the dam. Salmonella can be isolated from the aborted tissues. Pathogenesis. After ingestion, the organism proliferates in the intestine. Damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. Although the Salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. Septicemia is a common sequela, with the bacteria localizing throughout the body. In latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. Younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal pH. Carriers may develop clinical disease when stressed. Differential diagnoses. In young animals, differentials include other enteropathogens: Escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. These pathogens may also be present in the affected animals. Differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants.

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

Prevention and control. Affected animals should be isolated during herd outbreaks. Samples for culture should include herdmates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. Repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. The bacteria survive for about a week in moist cow manure. Vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the J-5 bacterin is now considered better.

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negative, rod-shaped bacterium. Type A is more virulent than type B.

Clinical Signs. Although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. The disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. Infected animals move stiffly, are depressed, and are hyperthermic. Anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. Mortality may reach 40%. Animals that recover will have immunity of long duration.

Treatment. Nursing care includes rehydration and correction of acid-base abnormalities. Antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. Gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful.

Epizootiology and transmission. The disease is most commonly transmitted by ticks or biting flies. The wood tick, Dermacentor andersoni, is an important vector in transmitting the disease in the western United States, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen.

Research complications. Salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. Attempts should be made to identify and cull carrier animals.

Pathogenesis. The organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. The organisms can also be transmitted orally through contaminated water.

bb.

Spirochete-Associated Abortion in Cattle (Epizootic Foothill Abortion)

Etiology. Spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. The disease has been reported only in the foothills bordering the central valley of California. Clinical signs. Cows that become infected with the causative agent before 6 months of gestation abort or give birth to weak calves without any clinical sign of infection. Cows infected after 6 months of gestation give birth to normal calves. Affected cows rarely abort in subsequent pregnancies. Epizootiology and transmission. dorus coriaceus.

The tick vector is Ornitho-

Necropsy. Fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. The calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. Histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. Treatment. Chlortetracycline treatment has been effective in controlling this disease. cc.

Tularemia

Etiology. Tularemia is caused by Pasteurella (Francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-

Necropsy findings. Ticks may also be present on the carcasses. Suppurative, necrotic lymph nodes are typical. Lungs will be congested and edematous. Diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; P. tularensis does not survive for long periods in carcasses. Serological findings may also be helpful. Treatment. Infected animals can be treated with oxytetracycline, aminoglycosides, or cephalosporins. Differential diagnosis. When tick infestations are heavy, P. tularensis should be suspected. Pasteurella haemolytica (sheep), Haemophilus somnus (cattle), and Mycoplasma mycoides (goats), and anthrax (all ruminant species) should be considered as differentials. Control and prevention. Eliminating the tick vectors can prevent tularemia. Animals should be provided with fresh water frequently. The organism can survive in freezing conditions and in water and mud for long periods of time. Caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. Research complications. The disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. Although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the United States should be aware of it. The organism is antigenically related to Brucella spp.

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Yersinia

Etiology. Yersiniosis is caused by infections with Yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family Enterobacteriaceae. There are 50 serotypes reported for Y. enterocolitica. Yersinia pseudotuberculosis infections have also been seen in ruminants. Enteric infections predominate in the diseases caused by these bacteria.

Research complications.

Yersinia is zoonotic. The risk of severe enteric disease is considered particularly great for immunocompromised persons. ee.

Mycoplasmal Diseases

i. Mycoplasma bovigenitalium and M. bovis infections Etiology.

Clinical signs and diagnosis.

Clinical disease may be seen rarely in many groups of ruminants. Goats of 1-6 months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting 1 or more days. Spontaneous abortions and weak neonates are also clinical manifestations of infection. Lactating does may have mastitis that becomes chronically hemorrhagic. Bacteremia results in internal abscesses, abortion, and acute deaths. Yersinia pseudotuberculosis has been associated with laboratory goat epizootics (Obwolo, 1976). Diarrhea in pastured sheep, stressed by other factors, has also been reported. Diagnosis is based on culture and serology.

Mycoplasma bovigenitalium and M. bovis are associated sporadically with bovine infertility and abortions. This pathogen has also been reported associated with similar clinical signs in sheep and goats. Clinical signs and diagnosis. Infertility is more commonly caused by M. bovigenitalium infections, and granular vulvo-

The bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water.

vaginitis and endometritis will be present. Granular vulvovaginitis is characterized by raised papules on the mucous membranes and mucopurulent exudate. Abortions and mastitis are associated with M. bovis infections. Calves that are born may be weak. It is rare to have a definitive diagnosis of an abortion due to Mycoplasma. After consideration of other causes of abortion and evaluation of tissues for placentitis or fetal inflammation, diagnosis is confirmed by isolation of Mycoplasma from the genital tract or aborted tissues.

Necropsy findings.

Epidemiology and transmission.

Epizootiology and transmission.

Edema of mesenteric lymph nodes is the most common postmortem finding. Liver abscesses, microabsecesses in the intestines, and granuloma formation have also been reported. Placentas are white, with opaque white foci found on cotyledons. Histologically, suppurative placentitis and suppurative pneumonia are found in the fetal tissue.

Mycoplasmal species are considered ubiquitous, are carried in the genital tracts of males and females, and are transmitted during natural breeding or through contaminated insemination materials. Aerosols also serve as a means of transmission. In addition, transmission occurs by passage through the birth canal, by direct contact, and by contamination from urine of infected animals.

Pathogenesis.

After ingestion, the bacteria cause an enteric infection, and bacteremia follows.

Pathophysiology.

Experimental infections of M. bovis have resulted in placentitis and fetal pneumonia.

Differential diagnoses.

Other causes of abortions, including abortion storms, acute deaths, enteritis, neonatal deaths, and white foci on cotyledons, should be considered. In young animals, differentials include coccidiosis and nematode parasitism. Corynebacterium pseudotuberculosis and tuberculosis are differentials for the internal abscesses.

Prevention and control.

Control measure are not well defined, because the epidemiology of the disease is poorly understood (Smith and Sherman, 1994). Tissues from affected goats must be handled and disposed of properly. Areas housing affected goats must be thoroughly sanitized.

Treatment. In case of an abortion storm, treatment of goats with tetracycline has been useful. Other broad-spectrum antibiotics may also be useful.

Differential diagnoses. Acholeplasma, Ureaplasma, and Haemophilus somnus are differentials for granular vulvovaginitis. Treatment. Fluoroquinolone antibiotics may be useful for treating Mycoplasma-induced reproductive diseases. ii. Mycoplasma ovipneumoniae (ovine mycoplasmal pneumonia) Etiology. Mycoplasma ovipneumoniae causes acute

or

chronic pneumonia in lambs.

Clinical signs. Mycoplasmas induce serious diseases in sheep, causing pneumonia, conjunctivitis, and genitourinary disease. The disease may be coincidental with pasteurellosis. Respiratory distress, coughing, and nasal discharge are ob-

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

served in infected animals. Bronchoalveolar lavage followed by culture is the best method for diagnosis (mycoplasmas are fastidious organisms requiring special handling techniques). Mycoplasmas are isolated from the genitourinary tract of sheep. Vulvovaginitis and reproductive problems are associated conditions.

Treatment. Tylosin, quinolones, oxytetracycline, and gentamicin are good choices for therapy. Prevention.

No vaccine is available.

iii. Mycoplasma mycoides biotype F38 (contagious caprine pleuropneumonia, caprine pneumonia, pleuritis, and pleuropneumonia) Etiology. Mycoplasma mycoides biotype F38 is the agent of contagious caprine pleuropneumonia and is found worldwide. In the United States, caprine pneumonia is also caused by M. ovipneumoniae, M. mycoides subsp, capri, and M. mycoides subsp, mycoides (large colony type).

Clinical signs. Contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever (McMartin et al., 1980). Infections with other Mycoplasma species also have similar clinical signs. Septicemia without respiratory involvement may also be a presentation. Epizootiology and transmission. This disease is highly contagious, with high morbidity and mortality. Transmission is by aerosols. Mycoplasma mycoides subsp, mycoides has become a serious cause of morbidity and mortality of goat kids in the United States.

Necropsy. Large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. Some lung consolidation may be present. Meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. Diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs.

Differential dagnosis. In the United States, the principal differential for M. mycoides subsp, mycoides is caprine arthritis encephalitis.

Treatment. Tylosin and oxytetracycline are effective. Some infections are slow to resolve.

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bidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. Considerable attention is presently given to this genus as a source of morbidity and mortality in goats.

iv. Mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) Etiology. Mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. Mycoplasma mycoides subsp, mycoides, M. agalactiae, M. arginini, and Acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. Respiratory disease and other infections, such as mastitis, may also be observed.

Clinical signs and diagnosis. All ages of animals may be affected. Initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. Sometimes uveitis is evident. Although the presentation is usually unilateral, bilateral involvement is possible. Recurring infections are common. Culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. Epizootiology and transmission. The infection is passed easily between animals by direct contact. Animals can become reinfected, and carrier animals may be a factor in outbreaks.

Necropsy.

It is unlikely that animals would die or be euthanized and undergo necropsy for this problem. Conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. Epithelial cell cytoplasm should be examined for organisms.

Differential diagnosis. The primary differential in sheep and goats is Chlamydia, as well as Branhamella, Rickettsia (Colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. It is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual.

Treatment. Animals do recover spontaneously within about 10 weeks. Tetracycline ointments and powders are also used. Third-eyelid flaps may be necessary if corneal ulceration develops.

Prevention and control. New animals should be quarantined Prevention and control. Vaccines are available in some areas. Infected herds are quarantined. New goats should be quarantined before introduction to the herd.

Research complications. The worldwide distribution of the F38 biotype, as well as the aerosol transmission and high mor-

and, if necessary treated, before introduction to the flock or herd.

ff.

Rickettsial Diseases

i. Eperythrozoonosis (Eperythrozoon, Haemobartonella) Etiology. Eperythrozoonosis is a rare, sporadic, noncontagious,

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blood-borne disease in ruminants worldwide caused by the rickettsial agent Eperythrozoon. Host-specific species of importance are E. ovis, the causative species in sheep and goats, and E. wenyoni, E. tegnodes, and E. tuomii, the causative agents in cattle. Although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. Haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases.

Clinical signs and diagnosis. The disease is more severe in sheep. Following an incubation period of 1-3 weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. Losses may be greater in younger lambs. Cattle are usually latently infected but may have swollen and tender teats and legs. Fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. Diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. Epizootiology and transmission. The rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. Necropsyfindings. Necropsy findings include splenic enlargement and tissue icterus. Pathogenesis. The organism invades and destroys red blood cells. It is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. As with other red blood cell parasites, splenectomy aggravates the disease. Differential diagnosis. Clontridium novyi type D, babesiosis, and leptospirosis are the primary differentials. Prevention and control. Following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. Treatment. Treatment is not usually recommended, but oxytetracycline has been used. Sheep will develop immunity if supported nutritionally during the disease. Research complications. Splenectomized animals are the experimental models used to study these diseases. ii. Q fever, or query fever (Coxiella burnetii) Etiology. Coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. Clinical signs. Infection of ruminants with C. burnetii is usually asymptomatic. Experimental inoculation in other mammals

has resulted in transient hyperthermia, mild respiratory disease, and mastitis. Abortions, stillbirths, and births of weak lambs are also seen.

Epizootiology and transmission. Coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. The organism is associated with either a free-living or an arthropod-borne cycle. Coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. Once introduced into a mammal, Coxiella may be maintained without a tick intermediate. The organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. Additionally, the organism is shed in milk, urine, feces, and oronasal secretions. Necropsy findings. No specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. The placenta will contain white chalky plaques and a red-brown exudate. The disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. The organism has been found in the placentas of clinically normal animals. The organism stains red with modified Ziehl-Neelsen and Macchiavello stains and purple with Giemsa stain. Differential diagnosis. Because of the organisms' similarity to Chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, ELISA, and complement fixation tests. Treatment. Coxiella can be treated with oxytetracyclines. A vaccine is not commercially available. Prevention and control. Any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. Serologic screening of ruminant sources should be performed routinely. Barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. All placentas and all aborted tissues should be handled and disposed of carefully. Q fever has been reported in many mammalian species, including cats. Research complications. Coxiella burnetii-free animals are particularly important in studies involving fetuses and placentation. Because of its zoonotic potential, C. burnetii presents a unique problem in the animal research facility environment. A single organism has been shown to cause disease. Some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example.

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE 2.

Viral Diseases

a. Adenovirus Infections Etiology.

The ruminant adenoviruses are DNA viruses that cause respiratory and reproductive tract diseases. Nine antigenic types of the bovine adenovirus have been identified, with type 3 associated with respiratory disease. Two of the ovine and two of the caprine antigenic types have been identified.

Clinical Signs. Signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. Respiratory tract and intestinal tract diseases may be concurrent. Infections caused by this virus are often found associated with other viral and bacterial infections. Epizootiology and transmission.

The virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. Transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes.

Necropsy findings.

Lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs.

b.

Bluetongue Virus Infection (Reoviridae)

Etiology. The bluetongue virus is an RNA virus in the Orbivirus genus and Reoviridae family. Five serotypes (2, 10, 11, 13, and 17) have been identified in the United States, where it is seen mostly in western states. Bluetongue is an acute arthropodborne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities (Bulgin, 1986).

Clinical signs and diagnosis. Sheep are the most likely to show clinical signs. Clinical disease is less common in goats and cattle. Early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. The virus name, bluetongue, is associated with the typical cyanotic membranes. The fever may subside, but tissue lesions erode, causing ulcers. Increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. Chorioretinitis and conjunctivitis are also common signs in cattle and sheep. Lameness may be observed associated with coronitis and is evident in the rear legs. Skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. Secondary bacterial pneumonia may also occur. Animals may also develop severe diarrhea and become recumbent. Sudden deaths due to cardiomyopathy may occur at any time during the disease. Hematologically, animals will be leukopenic. The course of the disease is about 2 weeks, and mortality may reach 80%.

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If animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. Abortions may occur at any stage of gestation in cattle. Prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. Cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. Diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. Virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. Fluorescent antibody tests, ELISA, virus neutralization tests, PCR, and agar gel immunodiffusion (AGID) tests are also used to confirm the diagnosis.

Epizootiology and transmission.

Severe outbreaks have occurred in other countries during this century. Screening for this disease has limited the strains present in the United States. The disease is most common in outdoor-housed animals primarily in the western United States. The virus is primarily transmitted by biting midges, Culicoides. Culicoides variipennis is the most common vector in the United States. A combination of factors associated with viral strain, available and susceptible hosts, environmental conditions (such as damp areas where flies breed), and vector presence are factors in the severity of outbreaks. The disease is rarely transmitted by animal-to-animal contact or by infected animal products. Virus-contaminated semen, transplacental transfer, and carriage on transferred embyros are other possible means of transmission.

Necropsy findings.

At necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. Ulceration or hyperemia of the coronary bands may also be seen. Many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery.

Pathogenesis.

The virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. After entering the host, the virus causes prolonged viremia. The incubation period is 6 - 1 4 days. The virus migrates to and attacks the vascular endothelium. The resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. Ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. The effects on fetuses appear to be due to generalized infections of developing organs.

Differential diagnosis.

Differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. Rinderpest is a differential in countries

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where it is endemic. Photosensitization should be considered. Foot rot is a differential for the lameness and coronitis. Differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as Charolais cattle and Merino sheep.

Prevention and control.

Cellular and humoral immunity are necessary for protection from infection. The bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. In addition, there is little to no cross protection between strains. Modified live vaccines are available in some parts of the United States but should not be used in pregnant animals. Vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating lategestation ewes may cause birth defects or abortions. Congenital defects are more common from vaccine use than from naturally occurring infection. Minimizing exposure to the vector in endemic areas will decrease the incidence of the disease.

Treatment. Supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. Nonsteroidal anti-inflammatory drugs are often administered. For the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. Research complications.

This is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases.

Bovine Lymphosarcoma (Bovine Leukemia Virus Infection, Bovine Leukosis) Etiology.

Bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (BLV) infection, and those in older cattle that are associated with B LV. B LV is a B lymphocyte-associated retrovirus (Johnson and Kaneene, 1993a,b,c). Clinical signs. Forms of bovine lymphosarcoma that are not associated with BLV infection are calf, or juvenile; thymic, or adolescent (animals 6 months to 2 years old); and cutaneous (any age). The calf form is rare and characterized by generalized lymphadenopathy. Onset may be sudden, and the disease is usually fatal within a few weeks. Signs include lymphadenopathy, anemia, weight loss, and weakness. Some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. The adolescent form is also rare, the course rapid, and the prognosis poor. The disease is seen most often in beef breeds such as Hereford cattle and is characterized by space-occupying masses in the neck or thorax. These masses are also often present in the brisket. Secondary

effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. The cutaneous presentation has a longer course and may wax and wane. The masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. The animals are anemic, and neoplastic involvement may affect cardiac function. Generalized or limited lymphadenopathy may be apparent. Only the adult, or enzootic, form of bovine lymphosarcoma is associated with BLV infection. Many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. Some cows manifest disease only during the periparturient period. Malignant lymphoma is the more common, whereas leukosis, due to B-lymphocyte proliferation, is rare. Clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. Tumors are associated with lymphoid tissues. Common sites also include the abomasum, spinal canal, and uterus. Cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. The common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. Many internal organs may be involved, and tumors may be palpable per rectum. Secondary infections will be due to immunosuppression and the weakened state of the animal. Sheep have acquired BLV infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. Goats seroconvert but do not develop the clinical syndromes. Diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. Kits are available for running AGID, for which the BLV antigens gp-51 and gp-24 are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. ELISA and PCR diagnostic aids will also be helpful.

Epizootiology and transmission.

This disease is present worldwide. It is estimated that at least 50% of the cattle in the United States are infected with BLV. As few as 1% of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the United States. Larger herds tend to have higher rates. Genetic predisposition may be involved; in addition to the presence of BLV, the type of bovine lymphocyte antigen (BoLA) may be correlated to resistance or susceptibility and to the course of the disease. Transmission is believed to be by inhalation of BLV in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). Natural-service bulls may transmit the infection to cows. Cows infected with BLV may transmit the infection to

14. BIOLOGYAND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE their calves in utero. Tabanid and other flies also serve as vectors, but these represent a minor means of transmission.

Necropsy findings. Neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. Tumors may be local or widely distributed in the enzootic form. Definitive diagnosis of neoplastic tissue specimens is by histology. Pathogenesis. As with other retroviruses, the BLV integrates viral DNA into host target cell DNA by means of the reverse transcriptase enzyme, creating a provirus. Prevention and control. There is no vaccine for this disease. Development and maintenance of a BLV-free herd, or controlling infection within a herd, requires financial and programmatic commitments: BLV-positive and BLV-negative animals maintained separately; serologic testing (such as at least every 6 months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile singleuse products), and equipment for ear tagging and dehorning and other such equipment between animals. A fresh rectal exam sleeve and lubricant should be used for each animal examined. Otherwise serologically positive cows may have undetectable antibodies during the periparturient period. Embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. Calves should be fed colostrum from serologically negative cows. Treatment. Treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. In cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. Research complications. The United States and several countries, some in Europe, have official programs for eradication of enzootic bovine leukosis. Bovine Herpes Mammillitis (Bovine Herpesvirus 2 Bovine Ulcerative Mammillitis) Etiology. Bovine herpesvirus 2 causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. Clinical signs and diagnosis. Lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. The udder lesions may extend to the perineum. The lesions progress to vesicles, then to ulcers; these may take 10 weeks to heal. Lesions rarely may also develop focally around the mouth and generally on the skin of the udder. Secondary mastitis may occur, because of bacteria associated with the scabs. Diagnosis is by clinical signs and serologically.

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Epizootiology and transmission. The virus is reported to be widespread. Occurrence is often seasonal, and biting insects may be vectors. Transmission with successful infection requires deep penetration of the skin. Transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. Differential diagnosis. Differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. Other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. Prevention and control. Established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. Treatment. There is no treatment, and affected animals should be separated from the herd and milked last. Lesions can be cleaned and treated with topical antibacterials. e.

Bovine Viral Diarrhea Virus

Etiology. The bovine viral diarrhea virus (BVDV) is a pestivirus of the Flaviviridae family. The Flaviviridae include hog cholera virus and border disease virus of sheep. The virus contains a single strand of positive-sense RNA. A broad range of disease and immune effects is produced by B VDV only in cattle. In addition, this virus is important in the etiology of bovine pneumonias. Bovine viral diarrhea/mucosal disease (BVD/MD) is one of the most important viral diseases and one of the most complex diseases of cattle. Strains of BVDV are characterized as cytopathic (CP) and noncytopathic (NCP), based on cell-culture growth characteristics. The virus has also been categorized as type 1 and type 2 isolates. Heterologous strains exist that may confound even sound vaccination programs. Clinical signs and diagnosis. Signs of BVDV infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (PI) with gradual debilitation, and acute and fatal disease. The presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. An acute form of the disease, caused by type 2 BVDV, occurs in cattle without sufficient immunity. After an incubation period of 5 - 7 days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. The disease course may be shorter with hemorrhagic syndrome and death within 2 days. Clinical signs of B VDV in calves also include severe enteritis and pneumonia.

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When susceptible cows are infected in utero from gestational days 50-100, or gestational cows are vaccinated with a modifled live vaccine, abortion or stillbirth result. Congenital defects caused by BVDV during gestational days 90-170 include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. Typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. Some severely affected calves will not be able to stand. Ophthalmic effects include retinal degeneration and microphthalmia. Fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (PI). The term mucosal disease is commonly associated with this form of the infection. Many PI animals do not survive to maturity, however, and many have weakened immune systems. The PI animals are important because they shed virus and will probably show the clinical signs of mucosal disease (MD) caused by a CP B VDV strain derived from an NCP B VDV strain. These MD clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. Many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. Secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. Animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. Diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to B VDV in precolostral serum. Viral culturing from blood may be useful. In older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. Leukopenia, and especially lymphopenia, are seen. Serology must be interpreted with the awareness of the possibility of PI immunotolerant animals. Vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against CP or NCP strains. The condition of the animals is also a variable.

Epizootiology and transmission.

BVDV is present throughout the world. Transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. PI females transmit the virus to their fetuses. Semen also is a source of virus.

Necropsy findings.

In affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. Later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. Older calves may have areas of intestinal necrosis. In cases where oral erosions occur, erosions will

be found extending throughout the gastrointestinal tract to the cecum. The respiratory tract lesions will often be complicated by secondary bacterial pneumonia. When the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present.

Pathogenesis.

The CP and NCP strains are thought to be related mutations of the BVDV; the CP short-lived isolates are believed to arise from the NCP strains. The NCP strains are those present in the PI animals, and the strains are maintained in cattle populations. CP and NCP isolates vary in virulence, and classification of these types is based on viral surface proteins. Considerable antigenic variation also exists between strains and types. Other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. The pathology caused by B VDV is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. In type 2 BVDV hemorrhagic syndrome, death results from viral-induced thrombocytopenia. In fetuses, the virus infects developing germinal cells of the cerebellum. The Purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. The immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. All of these predispose the affected animals to bacterial infections with Pasteurella haemolytica. B VDV damages dividing cells in fetal organ systems, resulting in abortions and congenital effects.

Differential diagnosis.

Many differentials must be considered for the clinical manifestations of B VDV infections. Differentials for enteritis of calves include viral infections, Cryptosporidia, Escherichia coli, Salmonella, and Coccidia. Salmonella, winter dysentery, Johne's disease, intestinal parasites, malignant catarrhal fever (MCF), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. Respiratory tract pathogens such as bovine respiratory syncytial virus, Pasteurella, Haemophilus, and Mycoplasma must be considered for the respiratory tract manifestations. Oral lesions are also produced by MCF, vesicular stomatitis, bluetongue, and papular stomatitis. Infectious bovine herpesvirus 1, leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion.

Prevention and control.

Combined with sound management in a typical cattle herd, vaccination is the best way to prevent B VDV and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. Vaccine preparations for B VDV are modified live virus (MLV) or killed virus. Each has advantages and disadvantages. The former induces rapid immunity (within 1 week) after a single dose, provides longer duration of immunity against sev-

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

eral strains, and induces serum neutralizing antibodies. MLV vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. The immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. The MLV strains may cross the placenta, resulting in fetal infections. The killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given 2 3 times per year, and do not induce cell-mediated immunity. Passive immunity may protect most calves for up to 6 8 months of age. Subsequent vaccination with MLV may provide lifelong immunity, but this is not guaranteed. Annual boosters are recommended to protect against vaccine breaks. The virus persists in the environment for 2 weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. Maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. Isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. Other management strategies may require a program for testing and culling PI cattle. This can be expensive but may be a worthwhile investment to remove the virus shedders from a herd.

Treatment. No specific treatment is available. Supportive care and treatment with antibiotics to prevent secondary infection are recommended. Animals that survive the infection should be evaluated a month after recovery to determine their status as PI or virus-free. f.

Cache Valley Virus

Etiology.

Cache Valley virus (CVV), of the arbovirus genus of the Bunyaviridae family, is a cause of congenital defects in lambs.

Clinical signs and diagnosis. Teratogenic effects of in utero CVV infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. Stillbirths and mummified fetuses are seen. Lambs will be born weak and will act abnormally. Diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. Epizootiology and transmission.

The virus is present in the western United States, although it has been isolated in a few Midwestern states. Although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants

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and antibodies found in white-tailed deer. Transmission is by arthropods during the first trimester of pregnancy.

g.

Caprine Arthritis Encephalitis Virus

Etiology.

Caprine arthritis encephalitis virus (CAEV) occurs worldwide, with a high prevalence in the United States. Caprine arthritis encephalitis (CAE) is considered the most important viral disease of goats. The CAEV is in the Lentivirus genus of the Retroviridae family. It causes chronic arthritis in adults and encephalitis in young. CAEV is in the same viral genus as the ovine progressive pneumonia virus (OPPV).

Clinical signs and diagnosis. The most common presentation in goats is an insidious, progressive arthritis in animals 6 months of age and older. Animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. Carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." In dairy goats, milk production decreases, and udders may become firmer. This retrovirus also causes neurological clinical signs in young kids 2 - 6 months old. Kids may be bright and alert, afebrile, and able to eat normally even when recumbent. Some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. Mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. Clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. Older animals in the group may experience interstitial pneumonia or chronic arthritis. The pneumonia is similar to the pneumonia in sheep caused by OPPV; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. Some animals in a herd may not develop any clinical signs. Diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to CAEV. An agar gel immunodiffusion (AGID) test identifies antibodies to the virus and is used for diagnosis. Kids acquire an anti-CAEV antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. The antibody does not prevent viral transmission. Ep&ootiology and transmission.

The virus is prevalent in most industrialized countries. The common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-CAEV antibody in the colostrum. Transmission may occur among adult goats by contact. Intrauterine transmission is believed to be rare. Transmission to sheep has occurred only experimentally; there is no documented case of natural transmission.

Necropsy findings.

Necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of

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lymphocytes, macrophages, and plasma cells. Other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. In severe cases of mastitis, the udder may appear to be composed of lymphoid tissue.

Pathogenesis.

The virus infects cells of the mononuclear system, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. Immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. Most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. The infection is lifelong.

Differential diagnosis.

The differential diagnosis for the neurologic form of CAEV should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. The differential diagnosis for CAEV arthritis should include chlamydia and mycoplasma.

Prevention and control.

Herds can be screened for CAE by testing serologically, using an AGID or an enzyme-linked immunosorbent assay (ELISA) test. The ELISA is purported to be more sensitive, whereas the AGID is more specific. Individual animals show great variation in development of antibody. Because CAE is highly prevalent in the United States, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. Recently, an immunoprecipitation test for CAE has been developed that has high sensitivity and specificity. Control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. Parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum (56~ for 1 hr). CAEV-negative goats should be separated from CAEVpositive goats.

Treatment.

Clinical signs and diagnosis. Diseases caused by the virus include conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. The respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. The mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. Fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. Open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. Neonatal calves may develop respiratory as well as general systemic disease. In these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. Young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. This form is usually fatal within 5 days. Abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as 3 months after the respiratory tract disease signs. Infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. Otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. The cow will often carry her tail elevated away from these lesions. These soon coalesce, and a fibrous membrane covers the ulcerated area. If uncomplicated, the infection lasts about 4 - 5 days, and lesions heal in 2 weeks. Younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows.

There is no treatment for CAEV.

Epizootiology and transmission. h.

Infectious Bovine Rhinotracheitis Virus (Infectious Bovine Rhinotracheitis-Infectious Pustular Vulvovaginitis)

Etiology.

The infectious bovine rhinotracheitis virus (IBRV) is also referred to as bovine herpesvirus 1 (BHV-1) and is an alphaherpesvirus. IBRV causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. It is one of the primary pathogens in the bovine respiratory disease complex. Strains include BHV-I.1 (associated with respiratory disease), BHV 1.2 (associated with respiratory and genital diseases), and BHV 1.4 (associated with neurological diseases), which has been reclassified as bovine herpesvirus 5.

IBRV is widely distributed throughout the world, and adult animals are the reservoirs of infection. The disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. Transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. Modified live vaccines are capable of causing latent infections.

Necropsy findings.

Fibrinonecrotic rhinotracheitis is considered pathognomic for IBRV respiratory tract infections. There will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. When there are secondary bacterial infections, such as Pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic

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hemorrhages in that tissue. Lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). Intranuclear inclusion bodies are not a common finding with this herpesvirus.

Pathogenesis.

In the encephalitic form, the virus first grows in nasal mucosa and produces plaques. These resolve within 11 days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. Latent infections are also established in neural tissue.

Differential diagnosis.

The severe oral erosions seen with BVDV infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV). The conjunctivitis of IBR may initially be mistaken for that of a Moraxella bovis (pinkeye) infection; the IBR will be peripheral, and there will not be corneal ulceration. Bovine viral diarrhea virus and IBRV are the most common viral causes of bovine abortion. Differentials for balanoposthitis include trauma from service.

Prevention and control.

Vaccination options include inactivated, attenuated, modified live, and genetically altered preparations. Some are in combination with parainfluenza 3 (PI-3) virus. The MLV preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. Some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. Bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity.

Treatment. Uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. Antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. The encephalitic animals may need to be treated with anticonvulsants. i.

Parainfluenza 3 (PI-3)

Etiology.

Parainfluenza 3, an RNA virus of the family Paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. The viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. Viral strains are reported to vary in virulence. Serotypes seen in the smaller ruminants are distinct from those isolated from cattle.

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Clinical signs and diagnosis. Infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. Clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. In pregnant animals, exposure to PI-3 can result in abortions. Clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as Pasteurella haemolytica infection, is involved. Greater morbidity and mortality will be sequelae of the bacterial infections. Viral isolation or direct immunofluorescence antibody (IFA) from nasal swabs can be used for definitive diagnosis. Epizootiology and transmission.

The virus is considered ubiquitous in cattle and is a common infection in sheep. Presently it is assumed that the virus is widespread in goats, but firm evidence is lacking.

Necropsy findings.

For an infection of PI-3 only, findings will be negligible. Some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. Intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. Findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. Immunohistochemistry may also be used.

Pathogenesis.

PI-3 infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated.

Differential diagnosis.

Differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: IBRV, BVDV, bovine respiratory syncytial virus, and type 3 bovine adenovirus.

Prevention and control.

Immunization, management, and nutrition are important for this respiratory pathogen, as for others. In cattle, modified live vaccines for intramuscular (IM), subcutaneous (SC), or intranasal (IN) administration are available. The IM and SC routes provide immune protection within 1 week after administration but will not provide protection in the presence of passively acquired antibodies. It is contraindicated for pregnant animals because it will cause abortion. The IN route immunizes in the presence of passively acquired antibodies, provides immunity within 3 days of administration, and stimulates the production of interferon. Other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered IM, and followup immunizations are needed

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within 4 weeks. Booster vaccinations are recommended for all preparations within 2 - 6 months after the initial immunization. All presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. The humoral immunity protects against PI-3 abortions. There is no approved PI-3 vaccine for sheep and goats. The use of the cattle formulation in these smaller ruminants is not recommended. Sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control.

Treatment. j.

Uncomplicated disease is not treated.

Respiratory Syncytial Viruses of Ruminants

Etiology.

The respiratory syncytial viruses are pneumoviruses of the Paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. Two serotypes of the bovine respiratory syncytial virus (BRSV) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats.

Clinicalfindings and diagnosis.

Infections may be subclinical or develop into severe illness. Clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. Interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. Development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. Secondary bacterial pneumonia, especially with Pasteurella haemolytica, with morbidity and mortality, is also a common sequela. Abortions have been assciated with BRSV outbreaks. Diagnosis is based on virus isolation and serology (acute and convalescent). Nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease.

Differential diagnosis.

Differentials should include other ruminant respiratory tract viruses.

Prevention and control.

Vaccination should be part of the standard health program, and all animals should be vaccinated regularly. Vaccinations should be administered within 1-2 months of stressful events, such as weaning, shipping, and introduction to new surroundings. Currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. Passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. But this immunity suppresses the mucosal IgA response and serum antibody responses. The basis for successful immune protection is the mucosal memory IgA, but this is difficult to achieve with present vaccine formulations. The virus is easily inactivated in the environment. Preventive measures in preweaning animals should include preconditioning to minimize weaning stress.

Treatment. Recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. In severe cases, antihistamines and corticosteriods may also be necessary. Use of vaccine during natural infection is not productive and may result in severe disease. Ulcerative Dermatosis (Ovine Venereal Disease, Balanoposthitis ) Etiology.

Ulcerative dermatosis is a contagious disease of sheep only. It is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ("Current Veterinary Therapy," 1993).

Gross lesions include consolidation of anteroventral lung lobes. Edema and emphysema are present. As the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. Necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically.

Clinical signs and diagnosis. Lesions include ulcers and crusts associated with the skin and mucous membranes of the genitalia, face, and feet (Bulgin, 1986). Genital lesions are much more common than the facial or coronal lesions. Discomfort may be associated with the lesions. Paraphimosis occasionally occurs. These lesions are painful; during breeding season, animals will avoid coitus. Morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. Diagnosis is based on clinical signs.

Pathogenesis.

Epizootiology and transmission.

Epizootiology and transmission.

These viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols.

Necropsy findings.

The severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. Virulence may vary greatly among viral strains.

Endemic to the western United States, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet.

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Necropsy findings.

Necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. Findings will be similar to those described for contagious ecthyma.

when no contact with cattle has occurred. Persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives.

Pathogenesis.

Necropsy findings.

Following an incubation period of 2 - 5 days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. Pustules rapidly break, forming weeping ulcers. The ulcers scab over and eventually form a fibrotic scar. The disease usually resolves in 2 - 6 weeks. Rarely, the disease will persist for many months to more than a year.

Differential diagnosis.

The main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. This is also a feature of ulcerative dermatosis.

Prevention and control.

No vaccine is available. Affected animals, especially males, should not be used for breeding.

Treatment. Affected animals should be separated from the rest of the flock. Treatment is supportive, including antiseptic ointments and astringents. Research complications.

Breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak.

I. Border Disease Etiology.

Border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern United States), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (BVDV), a pestivirus of the Togaviridae family. Goats are also affected. The virus causes few pathogenic effects in cattle.

Clinical signs and diagnosis. Border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. Lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. Later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. The virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. Diagnosis includes the typical signs described above, as well as serological evidence of viral infection. Virus isolation confirms the diagnosis. Epizootiology and transmission.

The virus is present worldwide, and reports of disease are sporadic. Disease has occurred

Lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. Histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures.

Pathogenesis.

The virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. Infection during the first 45 days of gestation causes embryonic death. In lambs infected between 45 and 80 days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. Infection after 80 days of gestation results in lambs that are born persistently infected. Infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus.

Prevention and control.

Border disease can be prevented by vaccinating breeding ewes with killed-BVDV vaccine. Congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. New animals to the flock should be screened serologically. If cattle are housed nearby, vaccination programs for BVDV should be maintained.

Treatment.

There is no treatment other than supportive care for affected animals. m.

Contagious Ecthyma (Contagious Pustular Dermatitis, Sore Mouth, Orf)

Etiology.

Contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or off, is an acute dermatitis of sheep and goats caused by a parapoxvirus. This disease occurs worldwide and is zoonotic. Naturally occurring disease has also been reported in other species such as musk ox and reindeer. Other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox.

Clinical signs and diagnosis. The disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. Lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. Off is usually found in young animals less than 1 year of age. Younger lambs and kids will have difficulty nursing and become weak. Lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. The scabs may appear nodular and raised above the surface of the surrounding

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skin. Morbidity in a susceptible group of animals may exceed 90%. Mortality is low, but the course of the disease may last up to 6 weeks. Diagnosis is based on characteristic lesions. Biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. Electron microscopy will reveal the virus itself. Disease is confirmed by virus isolation.

Epizootiology and transmission.

All ages of sheep and goats are susceptible. Seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. Older animals develop immunity that usually prevents reinfection for at least 1 or more years. Resistant animals may be present in some flocks or herds. The virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. Transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries.

Necropsy findings.

Necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. Secondary bacterial infection may also be evident.

Pathogenesis.

The virus is typical of the Poxviridae, resembling sheep poxvirus (not found in the United States) and vaccinia virus and replicating in the cytoplasm of epithelial cells. Following an incubation period of 2-14 days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. The vesicles form pustules that rupture and finally scab over.

Differential diagnosis.

Ulcerative dermatosis and bluetongue virus should be considered in both sheep and goats. An important differential in goats is staphylococcal dermatitis.

Prevention and control.

Individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. Clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. Colostral antibodies may not be protective. Vaccinating lambs and kids with commercial vaccine best prevents the disease. Dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. Animals newly introduced to infected premises should be vaccinated upon arrival. Precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate

animals in flocks already free of the disease. Affected dairy goats should be milked last, using disposable towels for cleaning teat ends.

Treatment. Affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. Treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. Treatment for myiasis may also be necessary. The viral infection is self-limiting, with recovery in about 4 weeks. Research complications.

Carrier animals may be a factor in flock or herd outbreaks. Contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. The virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as 6 weeks. Lesions heal without scarring.

n.

Foot-and-Mouth Disease

Etiology.

Foot-and-mouth disease (FMD) is caused by the foot-and-mouth disease virus, a picornavirus in the Aphthovirus genus. The disease is also referred to as aftosa or aphthous fever. Seven immunologically distinct types of the virus have been identified, with 60 subtypes within those 7. Epidemics of the disease have occurred worldwide. North and Central America have been free of the virus since the mid-1950s. This is a reportable disease in the United States; clinical signs are very similar to other vesicular diseases. Cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats.

Clinical signs and diagnosis. In addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. Vesicles may be as large as 10 cm, rupture after 2 days, and subsequently erode. Secondary bacterial infections often occur at the erosions. Anorexia is likely due to the pain associated with the oral lesions. High morbidity and low mortality, except for the high mortality in young cattle, are typical. Diagnosis must be based on ELISA, virus neutralization, fluorescent antibody tests, and complement fixation. Epizootiology and transmission.

Domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. Asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. Greater mortality occurs in younger animals. The United States, Great Britain, Canada, Japan, New Zealand, and Australia are FMD-free, whereas the disease is endemic in most of South

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE America, parts of Europe, and throughout Asia and Africa. The virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. Transmission may also occur by fomites, such as shoes, clothing, and equipment. Human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus.

Necropsy findings.

Vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. Myocardial and skeletal muscle degeneration (Zenker's) is most common (and accounts for the greater mortality) in younger animals. Histological findings include lack of inclusion bodies. Vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium.

Pathogenesis.

The incubation period is 2 - 8 days. The virus replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. Virus shedding begins about 24 hr before clinical signs are apparent. Vesicles result from the separation of the superficial epithelium from the basal epithelium. Fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. Persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known.

Differential diagnosis.

Vesicular stomatitis is the principal differential. Other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection.

Prevention and control.

Movement of animals and animal products from endemic areas is regulated. Quarantine and slaughter are practiced in outbreaks in endemic areas. Quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated 2 or 3 times per year to be effective and will provide only partial protection. Autogenous vaccines are best in an outbreak. Passive immunity protects calves for up to 5 months after birth. The virus is inactivated by extremes of pH, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid.

Treatment. Nursing care and antibiotic therapy to minimize secondary reactions help with recovery. Humoral immunity is considered the more important immune mechanism, with cellmediated immunity of less importance. Research complications.

Rare cases in humans have been reported. Importation into the United States of animal products from endemic areas is prohibited.

o.

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Malignant Catarrhal Fever

Etiology.

Malignant catarrhal fever (MCF) is a severe disease primarily of cattle. The agents of MCF are viruses of the Gammaherpesvirinae subfamily. Alcelaphine herpesvirus 1 and 2 and ovine herpesvirus 2 are known strains. The alcelaphine strains are seen in Africa. The ovine strain is seen in North America. The alcelaphine and ovine strains differ in incubation times and duration of illness. Disease may occur sporadically or as outbreaks.

Clinical signs and diagnosis. Signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. Sudden death may also occur in cattle. Presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. Corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. Other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. As the disease progresses, cattle may shed horns and hooves. In North America, cattle will also have severe diarrhea. The course of the disease may extend to 1 week. Recovery is usually prolonged, and some permanent debilitation may occur. The disease is fatal in severely affected individuals. History of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. Serology, PCR-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. Because of the susceptibility of rabbits, inoculation of this species may be used. In less severe outbreaks or individual animal disease, definitive diagnosis may never be made. Epizootiology and transmission.

Most ruminant species are susceptible to MCE Sheep are sources of infection for cattle, which are dead-end hosts. Other ruminants, including goats, may harbor the virus. Both the African and North American strains are transmissible to rabbits; these animals develop a fatal lymphoproliferative disease. The virus is shed from the nasopharynx. Infection of lambs is horizontal from direct contact. Other sources of the virus include water troughs, placental tissues, contaminated fomites, aerosols, birds, and caretakers.

Necropsy.

Gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. Lymph nodes should be submitted for histological examination. Histological findings include nonsuppurative vasculitis and encephalitis;

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large numbers of lymphocytes and lymphoblasts will be present without evidence of virus.

Pathogenesis. The incubation period may be up to 3 months. Vascular endothelium and all epithelial surfaces will be affected. The virus is believed to cause proliferation of cytotoxic T lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. Differential diagnoses. The differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. Causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. In Africa, rinderpest is also a differential. Other differentials are arsenic toxicity and chlorinated naphthalene toxicity. Prevention and control. No vaccine is available at this time. In North America, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. If there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. Lambs can be free of the infection if removed from the flock at weaning. The virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. Treatment. Affected and any exposed animals should be isolated from healthy animals. There is no specific treatment for MCF; supportive treatment may improve recovery rates. Corticosteroids may be useful.

p.

Ovine Progressive Pneumonia (Maedi/Visna)

Etiology. An RNA virus in the lentivirus group of the Retroviridae family causes ovine progressive pneumonia (OPP), or maedi/visna. Maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in Iceland. Visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. Clinical signs and diagnosis. OPP is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia (Pepin et al., 1998; de la Concha Bermejillo, 1997). Clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to 2 years. Respiratory rate and dyspnea gradually increase as the disease progresses. The animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. Additionally, mastitis is a common clinical feature. Thoracic auscultation reveals consolidation of ventral lung

lobes; and hematological findings indicate anemia and leukocytosis. The rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. Death occurs in weeks to months. The disease can be serologically diagnosed with agar gel immunodiffusion (AGID) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (ELISA) tests.

Epizootiology and transmission. Sixty-eight percent of sheep in some states have been infected with the virus (Radostits et al., 1994). It is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. In addition, transmission through the milk or colostrum is considered common (Knowles, 1997). Necropsy findings. Lesions are observed in lungs, mammary glands, joints, and the brain. Pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. Meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (CNS). Histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. Meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the CNS. Pathogenesis. The virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. After initial infection, the virus integrates into the DNA of mature monocytes and persists as a provirus. Later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. The virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. Pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. Secondary bacterial pneumonia may contribute to the animal's death. Differential diagnosis. ential diagnosis.

Pulmonary adenomatosis is the differ-

Prevention and control. Isolating or removing infected animals can prevent the disease. Facilities and equipment should also be disinfected. Treatment. q.

Treatment is unsuccessful.

Poxviruses of Ruminants

i. Ovine viral dermatosis. Ovine viral dermatosis is a venereal disease of sheep caused by a parapoxvirus distinct from

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14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE contagious ecthyma. The disease resolves within 2 weeks in healthy animals, but lesions are painful and resemble those of Corynebacterium renale posthitis/vulvovaginitis. Symptomatic treatment may be necessary in some cases. There is no vaccine. Animals should not be used for breeding while clinical signs are present.

ii. Proliferative stomatitis (bovine papular stomatitis) Etiology. A parapoxvirus is the causative agent of bovine papular stomatitis. This virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. It is also a zoonotic disease. The disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. In addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. The disease occurs worldwide.

Clinical signs and diagnosis.

Raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. In some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. Lesions persist or may come and go over a span of several months. Morbidity among herds may be 100%. Mortalities are rare. Bovine papular stomatitis is associated with "rat tail" in feedlot cattle. Animals continue to eat and usually do not show a fever. No lesion is seen on the feet. The infection may also be asymptomatic. Diagnosis is based on clinical signs, histological findings, and viral isolation.

Epizootiology and transmission.

Cattle less than 1 year of age are most commonly affected, and disease is rare in older cattle. Transmission is by animal-to-animal contact.

Necropsy findings.

Raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. Histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. Eosinophilic inclusions will be in the cytoplasm of infected epithelial cells.

Pathogenesis.

Following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. These become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. Some infections may recur or persist, with animals showing lesions intermittently or continuously over several months.

Differential diagnosis.

Pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. The differential for the "rat tail" clinical sign is Sarcocystis infection.

Prevention and control.

There is no vaccine available for bovine papular stomatitis. Because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. Vaccination using the local strain, and the skin scarification technique for off, have been protective. Handlers should wear gloves and protective clothing.

Treatment. Cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. Research complications.

Handlers may develop lesions on their hands at sites of contact with lesions of cattle.

iii. Pseudocowpox Etiology. Pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see Sections III,A,2,m and III,A,2,q,ii). Lesions are confined to the teats. This is also a zoonotic disease.

Clinical signs and diagnosis. Minor lesions are usually confined to the teats. These are distinctive because of the ring- or horseshoe-shaped scab that develops after 10 days. Additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. The teat lesions may predispose to mastitis. Pathogenesis.

The virus is spread by contaminated hands, equipment, and fomites.

Differential diagnosis.

Differentials include bovine herpes mammillitis and papillomatosis.

Prevention and control.

Milking hygiene is helpful in control.

Treatment. Lesions should be treated symptomatically, and affected animals milked last. Research complications.

Like other related poxviruses, this virus causes nodular lesions on humans.

r. Pulmonary Adenomatosis (Jaagsiekte) Etiology.

Pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. Pulmonary adenomatosis is caused by a type D retrovirus antigenically related to the Mason-Pfizer monkey virus. Jaagsiekte was the designation when the disease was described originally in South Africa.

Clinical signs and diagnosis. Typical clinical signs include progressive respiratory signs such as dyspnea, rapid respiration,

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and wasting. The disease is diagnosed by these chronic clinical signs and histology.

Epizootiology and transmission.

The disease is transmitted by aerosols. Body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus.

Necropsy.

The adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. The adenocarcinomas metastasize to regional lymph nodes.

Pathogenesis.

As with ovine progressive pneumonia (OPP), the incubation period is up to 2 years long. Adenocarcinomatous lesions arising from type II alveolar epithelial cells may be discrete or confluent and involve all lung lobes.

Differential diagnosis.

This disease occurs coincidentally with or is a differential diagnosis for OPP.

Treatment. s.

No treatment is effective.

Papillomatosis (Warts, Verrucae)

Etiology.

Cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. The disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. These tumors are caused by a papillomavirus (DNA virus) of the Papovaviridae family, and the viruses are host-specific and often body site-specific. Most are benign, although some forms in cattle and one form in goats can become malignant. In cattle, the site specificity of the papillomavirus strains are particularly well recognized. Designations of the currently recognized bovine papillomavirus (BPV) types are BPV-1 through BPV-5.

Clinical signs and diagnosis.

The papillomas may last up to 12 months and are seen more frequently in younger animals. Lesions have typical wart appearances and may be single or multiple, small (1 mm) or very large (500 mm). The infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. In addition, when infections are severe, weight loss may occur. When warts occur on teats, secondary mastitis may develop. In cattle, BPV-1 and BPV-2 cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. BPV-3 causes flat warts that occur in all body locations, B PV-4 causes warts in the gastrointestinal tract, and B PV-5 causes small white warts (called rice-grain warts) on teats. Warts caused by BPV-3 and BPV-5 do not regress spontaneously. Prognosis in cattle is poor only when papillomatosis involves more than 20% of the body surface. In sheep, warts are the verrucous type. The disease is of little consequence unless the warts develop in an area that causes dis-

comfort or incapacitation such as between the digits, on the lips, or over the joints. In adult sheep, warts may transform to squamous cell carcinoma. In goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. Warts on goat udders tend to be persistent. Diagnosis is made by observing the typical proliferative lesions.

Epizootiology and transmission.

Older animals are less sensitive to papillomatosis than young animals, although immunosupressed animals of any age may develop warts as the result of harbored latent infections. The virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos.

Pathogenesis.

The incubation period ranges from 1 to 6 months. The virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. The disease is generally self-limiting.

Differential diagnosis.

In sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox.

Prevention and control.

Commercial vaccines (available only for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. Autogenous vaccines are generally considered more effective. Some vaccine preparations are effective at prevention but not treatment of outbreaks. Viricidal products are recommended for disinfection of contaminated environments. Minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. Halters, brushes, and other items may also be sources of virus.

Treatment. Warts will often spontaneously resolve as immunity develops. In severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. Warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. Cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. Topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. t.

Pseudorabies (Mad Itch, Aujeszky's Disease)

Etiology.

Pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus 1. One serotype is recognized, but strain differences exist. The disease has worldwide distribution. It is a primarily a clinical dis-

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14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE ease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats.

Clinical signs and diagnosis. A range of clinical signs is seen during the rapid course of this usually fatal disease. At the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. Pruritus will not be asymmetric. Animals will also become hyperthermic and will vocalize frantically. Other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. Nystagmus and strabismus are also seen. Animals will be fearful or depressed, and aggression is sometimes seen. Recumbency and coma precede death. Diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. Serology of affected animals is not productive, because of the rapid course. If swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals.

Differential diagnoses.

Differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. Those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis.

Prevention and control.

Pseudorabies is a reportable disease in the United States, where a nationwide eradication program exists; states are rated regarding status. Effective disinfectants include sodium hypochlorite (10% solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. Five minutes of contact time is required, and then surfaces must be rinsed. Other disinfectant methods for viral killing include 6 hr of formaldehyde fumigation, or 360 min of ultraviolet light. Transport vehicles should be cleaned and disinfected between species. Serological screening for pseudorabies of swine housed near ruminants is essential.

Treatment.

There is no treatment, and most affected ani-

mals die.

Research complications. Epizootiology and transmission.

Swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. The infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. Other animals are dead-end hosts. The unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. Transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. When the virus is inhaled, the clinical signs of pruritus are less likely to be seen. Transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. Spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. Transport vehicles used for swine may also be sources of the virus. Raccoons are believed to be vectors of the virus. Horses are resistant to infection.

Necropsy findings.

There is no pathognomonic gross lesion. Definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. Eosinophilic intranuclear inclusion bodies (Cowdry type A) may be present in some affected neurons. Methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus 1.

Pathogenesis.

The incubation period is 90-156 hr and duration of the illness is 8 - 7 2 hr. The longest duration is seen in animals with pruritus around the head.

Swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. Humans have been reported to seroconvert. The porcine herpesvirus 1 shares antigens with the infectious bovine rhinotracheitis virus.

u.

Rabies (Hydrophobia)

Etiology.

Rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. The rabies virus is a neurotropic RNA virus of the Lyssavirus genus and the Rhabdoviridae family. Sheep, goats, and cattle are susceptible. The zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. Rabies is endemic in many areas of the world and within areas of the Unites States. This is a reportable disease in North America.

Clinical findings and diagnosis. Animals generally progress through three phases: prodromal, excitatory, and paralytic. Many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. During the short prodromal phase, animals are hyperthermic and apprehensive. Animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. Repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. The final paralytic stage, with recumbency and death, occurs over several hours to days. This paralytic stage is common in cattle, and animals may simply be found dead. The clinical course is usually 1 - 4 days.

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Diagnosis is based on clinical signs, with a progressive and fatal course. Confirmation presently is made with the fluorescent antibody technique on brain tissue.

Epizootiology and transmission.

The rabies virus is transmitted via a bite wound inflicted by a rabid animal. Cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in North America. Virus is also transmitted in milk and aerosols.

Necropsy findings.

Few lesions are seen at necropsy. Many secondary lesions from manic behaviors during the course of disease may be evident. Histological findings will include nonsuppurative encephalitis. Negri bodies in the cytoplasm of neurons of the hippocampus and in Purkinje's cells are pathognomonic histologic findings.

Pathogenesis.

After exposure, the incubation period is variable, from 2 weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. The rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). The fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary.

Differential diagnosis.

Rabies should be included on the differential list when clinical signs of neurologic disease are evident. Other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis.

Prevention and control.

Vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the United States for goats. Ruminants in endemic areas, such as the East Coast of the United States, should be routinely vaccinated. Any animals housed outside that may be exposed to rabid animals should be vaccinated. Vaccination programs generally begin at 3 months of age, with a booster at 1 year of age and then annual or triennial boosters. Awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. Monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. The virus is fragile and unstable outside of a host animal.

Research complications.

Aerosolized virus is infective. Personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs.

v.

Transmissible Spongiform Encephalopathies

i. Bovine spongiform encephalopathy (mad cow disease). Bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (TSE), is not known to occur in the United States, where since 1989 it has been listed as a reportable disease. The profound impact of this disease on the cattle industry in Great Britain during the past two decades is well known. The disease may be caused by a scrapielike (prion) agent. It is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. The incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other TSE diseases such as scrapie, which affects sheep and goats. In addition, the infectious agent is extremely resistant to dessication and disinfectants. Confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. Molecular biology techniques, such as Western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. Differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. Metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. There is no vaccine or treatment. Prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent USDA regulations prohibit feeding any mammalian proteins to ruminants. ii. Scrapie Scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. Scrapie is a reportable disease. It is much more common in sheep than in goats. The disease is similar to transmissible mink encephalopathy, kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (mad cow disease). Prions are nonantigenic, replicating protein agents.

Etiology.

Clinical signs and diagnosis. During early clinical stages, animals are excitable and hard to control. Tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. In advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. Blindness and abortion may also be seen. Morbidity may reach 50% within a flock. Most animals invariably die within 4 - 6 weeks; some animals may survive 6 months. In goats, the disease is also fatal. Pruritus is generally less severe but may be localized. A wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and

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erect tail and ears. As with sheep, the disease gradually progresses to anorexia and debilitation. Diagnosis can be made by clinical signs and histopathological lesions. A newer diagnostic test in live animals is based on sampling from the third eyelid. Tests for genetic resistance or susceptibility require a tube of EDTA blood and are reasonably priced.

Epizootiology and transmission.

The Suffolk breed of sheep tends to be especially susceptible. Scrapie has also been reported in several other breeds, including Cheviot, Dorset, Hampshire, Corriedale, Shropshire, Merino, and Rambouillet. It is believed that there is hereditary susceptibility in these breeds. Targhees tend to be resistant. Genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons 171 (Q, R, or H genes can be present) and 136 (A or V genes can be present). Of the five genes, R genes appear to confer immunity to clinical scrapie in Suffolks in the United States. Affected Suffolks in the United States that have been tested have been AA QQ. The disease is also enzootic is many other countries. The disease tends to affect newborns and young animals; however, because the incubation period tends to range from 2 to 5 years, adult animals display signs of the disease. Scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. Vertical transmission is questioned, and transplacental transmission is considered unlikely.

Necropsy findings.

At necropsy, no gross lesion is observed. Histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. Inflammatory lesions are not seen.

Pathogenesis.

Replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue.

Differential diagnosis.

In sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization.

Prevention and control.

If the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. The U.S. Department of Agriculture has approved the use of 2% sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. Prions are highly resistant to physicochemical means of disinfection. Artificial insemination or embryo transfer has been shown to decrease the spread of scrapie (Linnabary et al., 1991).

Treatment.

No vaccine or treatment is available.

Research complications.

As noted, this is a reportable disease. Stringent regulations exist in the United States regarding importation of small ruminants from scrapie-infected countries.

w.

Vesicular Stomatitis Virus

Etiology.

Vesicular stomatitis (VS) is caused by the vesicular stomatitis virus (VSV), a member of the Rhabdoviridae. Three serotypes are recognized: New Jersey, Indiana, and Isfahan. The New Jersey and Indiana strains cause sporadic disease in cattle in the United States. The disease is rare in sheep.

Clinical signs and diagnosis. Adult cattle are most likely to develop VS. Fever and development of vesicles on the oral mucous membranes are the initial clinical signs. Lesions on the teats and interdigital spaces also develop. The vesicles progress quickly to ulcers and erosions. The animal's tongue may be severely involved. Anorexia and salivation are common. Weight loss and decreased milk production are noticeable. Morbidity will be high in an outbreak, but mortality will be low to nonexistent. Diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. Diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. Virus isolation, enzyme-linked immunosorbent assay (ELISA), competitive ELISA (CELISA), complement fixation, and serum neutralization are used for diagnosis. Epizootiology and transmission.

This disease occurs in several other mammalian species, including swine, horses, and wild ruminants. VSV is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of pH, and low temperatures. Outbreaks of VS occur sporadically in the United States, but it is not understood how or in what species the virus survives between these outbreaks. Incidence of disease decreases during colder seasons. Equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. Transmission may also be from contaminated water and feed. Transmission is also believed to occur by insects (blackflies, sand flies, and Culicoides) that may simply be mechanical vectors. It is believed that carrier animals do not occur in this disease.

Necropsy.

It is rare for animals to be necropsied as the result of this disease. Typical vesicular lesion histology is seen, with ballooning degeneration and edema. There is no inclusion body formation.

Pathogenesis.

Lesions often begin within 24 hr after exposure. The virus invades oral epithelium. Injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. Animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus.

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Differential diagnosis. Foot-and-mouth disease lesions are identical to VS lesions. Other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. Prevention and control. Quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. Vaccines are available for use in outbreaks and have decreased the severity of lesions. Phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. Treatment. Affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. These animals should be cared for after unaffected animals. Any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. Topical or systemic antibiotics control secondary bacterial infections. Cases of mastitis secondary to teat lesions must be treated as necessary. Any abrasive materials that could cause further trauma to the animals should be removed. Research complications. Animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. Personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. VSV causes a flulike illness in humans. x.

Viral Diarrhea Diseases

i. Ovine. Rotavirus, of the family Reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. Four antigenic groups (A-D) have been identified by differences in capsid antigens VP3 and VP7. Primarily group A, but also groups B and C, have been isolated from sheep. The disease is characterized by yellow, semifluid to watery diarrhea occurring 1-4 days after infection. The disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. The virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. The villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. Virus may remain in the environment for several months. The disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal ELISA tests (marketed tests generally detect group A rotavirus), and fecal latex agglutination tests. Rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. A rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ("Current Veterinary Therapy," 1993).

Coronavirus, of the family Coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. Clinical signs are similar to above, although the incubation period tends to be shorter (20-36 hr), and animals exhibit less anorexia than those with rotavirus. Additionally, mild respiratory disease may be noted (Janke, 1989). Like rotavirus, coronavirus also destroys enterocytes of the villus tips. The virus can be visualized with electron microscopy. Treatment is supportive; close consideration of hydration and acid-base status is essential. Bovine vaccines are available.

ii. Caprine. Rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. It appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. Bovine. Rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (BVDV) are associated with diarrheal disease in calves. Each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. These viral infections may be complicated by parasitic infections (e.g., Cryptosporidium, Eimeria) or bacterial infections (e.g., Escherichia coli, Salmonella, Campylobacter). Treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. Diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. Prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in Section II,B,5. Combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic E. coli. Additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. Rotaviruses of serogroup A are the most common type in neonatal calves; 4- to 14-day old calves are typically affected, but younger and older animals may also be affected. The small intestine is the site of infection. Antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. Transmission is likely from other affected calves and asymptomatic adult carriers. The diarrhea is typically a distinctive yellow. Colitis with tenesmus, mucus, and blood may be seen. This virus may be zoonotic. Coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small- and large-intestinal epithelial cells. The virus infection may extend to mild pneumonia. Transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at

14. BIOLOGYAND DISEASESOF RUMINANTS:SHEEP, GOATS,AND CATTLE the time of parturition. Calves that appear to have recovered continue to shed virus for several weeks. Parvovirus infections are usually associated with neonatal calves. B VDV infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in Section III,A,2,e.

iv. Winter Dysentery.

Winter dysentery is an acute, winterseasonal, epizootic diarrheal disease of adult cattle, although it has been reported in 4-month-old calves. The etiology has not yet been defined, but a viral pathogen is suspected. Coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. Outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. Individual cows are ill for only a few days. The incubation period is estimated at 2 - 8 days. The outbreaks of disease are often seen in herds throughout the local area. Clinical signs include explosive diarrhea, anorexia, depression, and decreased production. The diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. Animals will become dehydrated quickly but are thirsty. Respiratory symptoms such as nasolacrimal discharges and coughing may develop. Recovery is generally spontaneous. Mortalities are rare. Diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as Salmonella or Mycobacterium paratuberculosis, and viruses such as B VDV. Pathology is present in the colonic mucosa, and necrosis is present in the crypts. 3.

Chlamydial Diseases

a.

Enzootic Abortion of Ewes (Chlamydial Abortion)

Etiology. Chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. Clinical signs.

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antigen is common among all Chlamydia; the specific antigen is common to related subgroups. Two subgroups are recognized, one that causes EAE and one that causes polyarthritis and conjunctivitis. The disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water.

Necropsy.

Placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. Histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. Fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. Diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. Impression smears in placental tissues stained with Giemsa, Gimenez, or modified Ziehl-Neelsen can provide preliminary indications of the causative agent. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) methods also aid in diagnosis.

Differential diagnosis.

Q fever will be the major differential for late-term abortion and necrotizing placentitis. Campylobacter and Toxoplasma should also be considered for late-term abortion.

Treatment. Animals may respond to treatment with oxytetracycline. Abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. This is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. Research complications.

In addition to losses or compromise of research animals, pregnant women should not handle aborted tissues.

b.

Chlamydial Polyarthritis of Sheep

Enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids (Rodolakis et al., 1998). The only presenting clinical sign may be serosanguineous vulvar discharges. Other animals may present with arthritis or pneumonia. Infection of animals prior to about 120 days of gestation results in abortion, stillbirths, or birth of weak lambs. Infection after 120 days results in potentially normal births, but the dams or offspring may be latently infected. Latently infected animals that were infected during their dry period may abort during the next pregnancy. Ewes or does generally only abort once, and thus recovered animals will be immune to future infections.

Clinical signs. Clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. Lambs may be anorexic and febrile. Animals frequently also exhibit concurrent conjunctivitis. The disease usually resolves in approximately 4 weeks. Joint inflammation usually resolves without causing chronic articular changes.

Epizootiology and transmission.

Epizootiology and transmission.

Chlamydia possess group and specific antigens associated with the cell surface. The group

Etiology.

Chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. Chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness (Bulgin, 1986), and conjunctivitis (see Section III,A,3,c) in growing and nursing lambs.

The disease is transmitted to susceptible animals by direct contact as well as by contaminated

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feed and water. The organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. The organism causes acute inflammation and associated fibrinopurulent exudates.

The chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. Diagnosis is suggested by the clinical signs. Cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis.

Necropsy findings.

Lesions are found in joints, tendon sheaths, conjunctiva, and lungs. Pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. Lesions will be infiltrated with mononuclear cells. Lung lesions include atelectasis and alveolar inspissation. Diagnosis is based on clinical signs. Synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies.

Treatment. Animals respond to treatment with parenteral oxytetracycline.

Differential diagnosis.

Nonchlamydial keratoconjunctivitis also occurs in sheep and goats. The primary agents involved include Mycoplasma conjunctiva, M. agalactiae in goats, and Branhamella (Neisseria) ovis. A less common differential for sheep and cattle is Listeria monocytogenes. Other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents.

Prevention and control. Chlamydial Conjunctivitis (Infectious Keratoconjunctivitis, Pinkeye) Etiology. Chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. Chlamydia and Mycoplasma are considered to be the most common causes of this disease in goats. Chlamydial conjunctivitis is not a disease of cattle.

Clinical signs. Infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. Perforation may result from the ulceration. Animals will be photophobic. In less severe cases, corneal healing associated with fibrosis and neovascularization occurs in 3 - 4 days. Lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. Morbidity may reach 80-90%. Bilateral and symmetrical infections characterize most outbreaks. Relapses may occur. Other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. Epizootiology and transmission.

Direct contact, and mechanical vectors such as flies easily spread the organism.

Necropsy.

If the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. Conjunctival smears are also useful.

Pathogenesis.

The pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. The infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate.

Source of mechanical irritation should be minimized whenever possible. Quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. Shade should be provided for all animals.

Treatment. The infections can be self-limiting in 2 - 3 weeks without treatment. Treatment consists of topical application of tetracycline ophthalmic ointments. Systemic or oral oxytetracycline treatments have been used with the topical treatment. Atropine may be added to the treatment regimen when uveitis is present. Shade should be provided. 4.

Parasitic Diseases

a.

Protozoa

i. Anaplasmosis Etiology. Anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan

Anaplasma marginale. Anaplasma is a member of the Anaplasmatacae family within the order Rickettsiales. In sheep and goats, the disease is caused by A. ovis and is an uncommon cause of hemolytic disease. Anaplasmosis has not been reported in goats in the United States. Some controversy exists regarding the classification. Most recently it is classified as a protozoal disease because of similarities to babesiosis. It has also been classified as a rickettsial pathogen. This summary addresses the disease in cattle with limited reference to A. ovis infections, but there are many similarities to the disease in cattle.

Clinical signs and diagnosis. Acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. Weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. Four disease stagesnincubation, developmental, convalescent, and carriermare recognized. The incubation stage may be long, 3 - 8 weeks, and is characterized by a rise in body temperature as the infection moves to the next

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE stage. Most clinical signs occur during the 4- to 9-day developmental stage, with hemolytic anemia being common. Death is most likely to occur at this stage or at the beginning of the convalescent stage. Death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. Reticulocytosis characterizes the convalescent stage, which may continue for many weeks. Morbidity is high, and mortality is low. The carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and Anaplasma organisms and any parasitemia are not discernible. Common serologic tests are the complement fixation test and the rapid card test. These become positive after the incubation phase and do not distinguish between the later three stages of disease. Definitive diagnosis is made by clinical and necropsy findings. Staining of thin blood smears with Wright's or Giemsa stain allows detection of basophilic, spherical A. marginale bodies near the red blood cell peripheries. Evidence will most likely be found before a hemolytic episode. A negative finding should not eliminate the pathogen from consideration.

Epizootiology and transmission.

The disease is common in cattle in the southern and western United States. Anaplasma organisms are spread biologically or mechanically. Mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. Sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. Biological transmission occurs when the tick stage of the organism is passed by Dermacentor andersoni and D. occidentalis ticks. The carrier stage covers the time when discernible Anaplasma organisms can be found on host blood smears. Recovered animals serve as immune carriers and disease reservoirs.

Necropsy.

Pale tissues and watery, thin blood are typical findings. Splenomegaly, hepatomegaly, and gallbladder distension are common findings.

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whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. Neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. Vaccinated animals can still become infected and become carriers. The cattle vaccine has shown no efficacy in smaller ruminants, and there is no A. ovis vaccine. Identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. Removing carriers to a separate herd is also an approach. Interstate movement of infected animals is regulated.

Treatment. Oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. Other tetracycline treatment programs have been described to help control carriers. ii. Babesiosis (red water, Texas cattle fever, cattle tick fever) Etiology. Babesia bovis and Ba. bigemina are protozoa that cause subclinical infections or disease in cattle. These are intraerythrocytic parasites. Babesia bovis is regarded as the more virulent of the two organisms. This disease is not seen in the smaller ruminants in the United States.

Clinical signs and diagnosis. The more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. Hemoglobinuria indicates a poor prognosis. Acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. The encephalitic form generally also has a poor prognosis. Sudden death may occur. Thin blood smears stained with Giemsa will show Babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. The trophozoites occur in a variety of shapes, such as piriform, round, or rod. Complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. Epizootiology and transmission.

The clinical disease closely resembles the protozoal disease babesiosis.

Babesiosis is present on several continents, including the Americas. In addition to domestic cattle, some wild ruminants, such as white-tailed deer and American buffalo, are also susceptible. Bos indicus breeds have resistance to the disease and the tick vectors. Innate resistance factors have been found in all calves. If infected, these animals will not show many signs of disease during the first year of life and will become carriers. Stress can cause disease development.

Prevention and control.

Necropsy findings.

Pathogenesis.

The parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. The four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages.

Differential diagnosis.

Offspring of immune carriers resist infection up to 6 months of age because of passive immunity. Vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. There is no entirely effective means, however, to prevent and control the disease. Vaccination (killed

Signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. Animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver.

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Pathogenesis. The protozoon is transmitted by the cattle fever ticks Boophilus annulatus, B. microplus, and B. decoloratus; these one-host ticks acquire the protozoon from infected animals. It is passed transovarially, and both nymph and adult ticks may transmit to other cattle. Only B. ovis is transmitted by the larval stage. Clinical signs develop about 2 weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. Hemolysis is due to intracellular reproduction of the parasites and occurs intra- and extravascularly. In addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. The development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. Differential diagnosis. In addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. Several differentials in the United States for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. Prevention and control. Control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in Section III,A,3,a,i, are important preventive measures. Some vaccination approaches have been effective, but a commercial product is not available. Treatment. Supportive care is indicated, including blood transfusions, fluids, and antibiotics. Medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. Treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. Research complications. United States.

This is a reportable disease in the

iii. Coccidiosis Etiology. Coccidiosis is an important acute and chronic protozoal disease of ruminants. In young ruminants, it is characterized primarily by hemorrhagic diarrhea. Adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. Intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. Coccidia are protozoal organisms of the phylum Apicomplexa, members of which are obligatory intracellular parasites. There are at least 11 reported species of coccidia in sheep, of which several are considered pathogenic: Eimeria ashata, E. crandallis, and E. ovinoidalis (Schillhorn van Veen, 1986). At

least 9 species of Eimeria have been recognized in the goat (Foreyt, 1990). Eimeria ninakohlyakimovae, E. arloingi, and E. christenseni are regarded as the most pathogenic. Eimeria bovis and E. zuernii (highly pathogenic), and E. auburnensis and E. alabamensis (moderately pathogenic), are among the 13 species known to infect cattle. Eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis."

Clinical signs and diagnosis. Hemorrhagic diarrhea develops 10 days to 3 weeks after infection. Fecal staining of the tail and perineum will be present. Animals will frequently display tenesmus; rectal prolapses may also develop. Anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. The diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. The intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. Depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. Concurrent disease with other enteropathogens may also be part of the clinical picture. In sheep, secondary bacterial infection with organisms such as Fusobacterium necrophorum may ensue. Young goats may die peracutely or suffer severe anemia from blood loss into the bowel. Older goats may lose the pelleted form of feces. Cattle may have explosive diarrhea and develop anal paralysis. The disease is usually diagnosed by history and clinical signs. Numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. Laboratory results are usually reported as number of oocysts per gram of feces. Coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. Epkzootiology and transmission. As noted, coccidiosis is a common disease in young ruminants. In goats, young animals aged 3 weeks to 5 months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. Coccidia are host-specific and also host cell-specific. The disease is transmitted via ingestion of sporulated oocysts. Coccidial oocysts remain viable for long periods of time when in moist, shady conditions. Necropsy. Necropsies provide information on specific locations and severity of lesions that correlate with the species involved. Ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. Mucosal scrapings will frequently yield oocysts. Various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. Fibrin and cellular infiltrates will be found in the lamina propria.

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Pathogenesis.

This parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes (Speer, 1996). The severity of the disease is correlated primarily with the number of ingested oocysts. Specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. In most cases, the disease is well established by the time clinical signs are seen. Oocysts must undergo sporulation over a 3- to 10-day period in the environment. After ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. Schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. The organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage.

Differential diagnosis.

Differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, Yersinia, viruses, and other intestinal parasites such as helminths. In cattle, for example, bovine viral diarrhea virus and helminthiasis caused by Ostergia must be considered. Management factors, such as dietary-induced diarrheas, are also differentials. In older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms.

Prevention and control. Good management practices will help prevent the disease. Oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. Proper sanitation of animal housing and minimizing overcrowding are essential. Coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure.

Treatment. Affected animals should be isolated. On an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. Coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. Although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. Other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. Animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. Pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. Mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). Immunity may result when low numbers are ingested and there is only mild disease. Immunity also may develop after more severe infections.

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iv. Cryptosporidiosis Etiology. Cryptosporidium organisms are a very common cause of diarrhea in young ruminants. Four Cryptosporidium species have been described in vertebrates: C. baileyi and C. meleagridis in birds and C. parvum and C. muris in mammals. Cryptosporidium parvum is the species affecting sheep (Rings and Rings, 1996). Debate continues regarding whether there are definite host-specific variants.

Clinical signs and diagnosis. Cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. The diarrhea may last only 6 - 1 0 days or may be persistent and fatal. The diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. Infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. In relapsing cases, animals become cachectic. Overall, morbidity will be high, and mortality variable. Mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. The disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-Schiff stain or methenamine silver. Cryptosporidium also stains red on acid-fast stains such as Kinyoun or ZiehlNeelsen. Fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of 1.27 (Foryet, 1990). Fecal immunofluorescent antibody (IFA) techniques have also been described. Epizootiology and transmission.

Younger ruminants are commonly affected: lambs, kids (especially kids between the ages of 5 and 10 days old), and calves less than 30 days old. Like other coccidians, Cryptosporidium is transmitted via the fecal-oral route. In addition to local contamination, water supplies have also been sources of the infecting oocysts. The oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months.

Necropsy findings.

The lesions caused by Cryptosporidium are nonspecific. Animals will be emaciated. Moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. Cecal and colonic mucosae may sometimes be involved. Gastrointestinal smears may be made at necropsy and stained as described above.

Pathogenesis.

Although Cryptosporidium infections are clinically similar to Eimeria infections (Moore, 1989), Cryptosporidium, in contrast to Eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. There is no intermediate host. The oocysts are half the size of Eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. Within 2 - 7 days of exposure, diarrhea and oocyst shedding occur. The diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. Autoinfection within the lumen of the intestines may also occur and result

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in persistent infections. In addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. Environmental stressors such as cold weather increase mortality. Intensive housing arrangements increase morbidity and mortality.

Differential diagnosis.

Other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic Escherichia coli; Clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). In addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. Eimeria is more likely to cause diarrhea in calves and lambs at 3 - 4 weeks of age. Giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease.

Prevention and control.

Precautions should be taken when handling infected animals. Affected animals must be removed and isolated as soon as possible. Animal housing areas should be disinfected with undiluted commercial bleach or 5% ammonia. Formalin (10%) fumigation has proven successful (Foryet, 1990). After being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. Because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined.

Treatment. No known drug treatment is available. The disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. Supplementation with vitamin A may be helpful. Age resistance begins to develop when the animals are about 1 month old. Research complications.

Cryptosporidiosis is a zoonotic disease. It is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. Adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. The disease can be life-threatening in immunocompromised individuals.

v. Giardiasis Etiology. Giardia lamblia (also called G. intestinalis and G. duodenalis) is a flagellate protozoon. Giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds (Kirkpatrick, 1989), but it not considered to be a significant pathogen in ruminants.

Clinical signs and diagnosis.

Diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain

blood. Animals exhibit fever, dehydration, and depression. Chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. Giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. Oval cysts can be floated with zinc sulfate solution (33%). Standard solutions tend to be too hyperosmotic and to distort the cysts. Newer enzyme-linked immunosorbent assay (ELISA) and IFA tests are sensitive and specific.

Epizootiology and transmission.

Giardia infection may occur at any age, but young animals are predisposed. Chronic oocyst shedding is common. Transmission of the cyst stage is fecaloral. Wild animals may serve as reservoirs.

Necropsy findings.

Gross lesions may not be evident. Villous atrophy and cuboidal enterocytes may be evident histologically.

Pathogenesis.

Following ingestion, each Giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. The organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. Disturbances in intestinal motility may also occur (Rings and Rings, 1996).

Prevention and control.

Intensive housing and warm environments should be minimized. Cysts can survive in the environment for long periods of time but are susceptible to desiccation. Effective disinfectants include quaternary ammonium compounds, bleach-water solution (1:16 or 1:32), steam, or boiling water. After cleaning, areas should be left empty and allowed to dry completely.

Treatment. Giardia has been successfully treated with oral metronidazole. Benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. Research complications.

Giardia is zoonotic. Precautions should be taken when handling infected animals.

vi. Neosporosis Etiology. Neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species Neospora caninum. Abortions have also been reported in sheep and goats. Neonatal disease is seen in lambs, kids, and calves. Until 1988, these infections were misdiagnosed as caused by Toxoplasma gondii. Some similarities exist between the life cycles and pathogeneses of both organisms.

Clinical signs and diagnosis. Abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. Bovine abortions occur between the third

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and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. Although cows that abort tend to be culled after the first or second abortion, repeated N. caninum-caused abortions will occur progressively later in gestation (up to about 6 months) and within a shorter time frame in the same cow (Thurmond and Hietala, 1997). Although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. Many Neospora-infected calves will be born asymptomatic. Weakness will be evident in some infected calves, but this resolves. Rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. Some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. Immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. Serology (IFA and ELISA) is useful, including precolostral levels in weak neonates, but this indicates only exposure. Titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. Earlier and rapid infections are less likely to yield antibodies against Neospora. None of the currently available tests is predictive of disease.

Epizootiology and transmission. The parasite is now acknowledged to be widespread in dairy and cattle herds. The life cycle of N. caninum is complex, and many aspects remain to be clarified. The definitive host is the dog (McAllister et al., 1998). Placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. The many intermediate hosts include ruminants, deer, and horses. Transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. A less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. Reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. Seropositive immunity does not protect a cow from future abortions. Many seropositive cows and calves will never abort or show clinical signs, respectively. Some immunological cross-reactivity may exist among Neospora, Cryptosporidia, and Coccidium. Necropsy findings. Aborted fetuses will usually be autolysed. In those from which tissue can be recovered, tissue cysts are most commonly found in the brain. Spinal cord is also useful. Histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. Pathogenesis. As with Toxoplasma, cell death is the result of intracellular multiplication of Neospora tachyzoites. Neospora

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undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. The intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. Infections of this type are latent and lifelong. Except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. Fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. It usually takes 2 - 4 weeks for a fetus to die and to be expelled. Many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of Neospora abortions remain to be elucidated.

Differential diagnosis. Even when there is a herd history of confirmed Neospora abortions, leptospirosis, bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), salmonellosis, and campylobacteriosis should be considered. BVDV in particular should be considered for abortion storms. Differentials for weak calves are B VDV, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, Toxoplasma, exposure to teratogens, or congenital defects. Prevention and control. The primary preventive measure is preventing contact with contaminated feces. Oocysts will not survive dry environments or extremes of temperature. Dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. Dogs should also be restricted from feed bunks and other feed storage areas. Preventive culling is not economically practical for most producers. A vaccine recently became available. If embryo transfer is practiced, recipients should be screened serologically before use. Treatment.

There is no known treatment or immunoprophy-

laxis.

vii. Sarcocystosis Etiology. Sarcocystosis is the disease caused by the cystforming sporozoon Sarcocystis. Sarcocystis capricanus, S. ovicanus, and S. tenella are the species that infect sheep and goats. Sarcocystis cruzi, S. hirsuta, and S. hominis are the species that infect cattle. Definitive hosts are carnivores, and all ruminant species are intermediate hosts. Clinical signs and diagnosis. Clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. Often the infections are asymptomatic. Fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. Abortions in cattle occur during the second

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trimester and in smaller ruminants 28 days after ingestion of the sporulated oocysts. Definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. Clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. Sarcocystis-specific IgG will increase dramatically by 5 - 6 weeks after infection. There is no cross-reaction between Sarcocystis and Toxoplasma.

Epizootiology and transmission. Infection rates among cattle in the United States are estimated to be very high. Transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. Dogs are the definitive hosts for the species that infect the smaller ruminants. Cats, dogs, and primates (including humans when S. hominis is involved) are the definitive hosts for the species that infect cattle.

Necropsy. Aborted fetuses may be autolysed. Lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. Lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. Cysts in cardiac and skeletal muscles are common incidental findings during necropsies.

Pathogenesis. Ingestion of muscle flesh from an infected ruminant results in Sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. The sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. Merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts.

Clinical signs and diagnosis. Clinical signs depend on the organ or tissue parasitized. Toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young (Underwood and Rook, 1992; Buxton, 1998). It has also been shown to cause pneumonia and nonsuppurative encephalitis. The enteritis at the early stage of infection may be fatal in some hosts. Hydrocephalus does not occur in animals as it does in human fetal Toxoplasma infections. Rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. Infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. Congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. In sheep, weak young will develop normally if they survive the first week after birth. Infected adult sheep show no systemic illness. Infected adult goats, however, may die. Diagnosis may be difficult, and biological, serological, and histological methods are helpful. Serological tests are the most readily available. Complement fixation and the Sabin-Feldman antibody test may assist in diagnosis. Antibodies found in fetuses are indicative of congenital infection and are typically detectable 35 days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. Biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. Epizootiology and transmission. This protozoon is considered ubiquitous. Fifty percent (50%) of adult western sheep and 20% of feedlot lambs have positive hemagglutination titers (1:64 or higher) (Jensen and Swift, 1982). Transmission among the definitive host is by ingestion of tissue cysts.

Necropsy findings. Prevention and control. Feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. These animals should be controlled and must also not have access to carcasses. In larger production situations, monensin may be fed as a prophylactic measure.

Treatment. Monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. viii. Toxoplasmosis Etiology. Toxoplasmosis is caused by the obligate intracellular protozoon Toxoplasma gondii, a coccidial parasite of the family Eimeridae. Cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. The disease is a major cause of abortion in sheep and goats and less common in cattle.

At necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. Fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. Histologically, granulomas with Toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. Impression smears of these tissues, stained appropriately (e.g., with Giemsa), provide a rapid means of diagnosis. Identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. Toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages.

Pathogenesis. The protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. The definitive hosts, felids, become infected by ingesting cyst

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stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. Ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. Cats rarely show clinical signs of infection. One cat can shed millions of oocysts in 1 gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. Oocysts sporulate in cat feces after 1 day. Ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. As in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. The organism migrates to tissues such as the brain, liver, muscles, and placenta. Placental infection develops about 14 days after ingestion of the oocysts. The damage caused by an infection is due to multiplication within cells. Toxoplasma does not produce any toxin.

Differential diagnosis. Differentials for abortion include Campylobacter, Chlamydia, and Q fever. Prevention and control.

Feline populations on source farms should be controlled. Eliminating contamination of feed and water with cat feces is the best preventive measure. Sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. Vaccines for abortion prevention in sheep are available in New Zealand and Europe.

Treatment. Toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful (Underwood and Rook, 1992). (Monensin is not approved for this use in the Unites States.) Weak lambs that survive the first week after birth will mature normally and will not deliver Toxoplasmainfected young. Research complications.

Because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. Infections in immunocompromised humans have been fatal.

ix. Trichomoniasis Etiology. Trichomoniasis is an insidious venereal disease of cattle caused by Tritrichomonas (also referred to as Trichomonas) fetus, a large, pear-shaped, flagellated protozoon. The organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. In the United States, it is now primarily a disease seen in western beef herds. There are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems.

Clinical signs and diagnosis. Clinical signs include infertility manifested by high nonpregnancy rates as well as periodic py-

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ometras and abortions during the first half of gestation. Often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. The abortion rate varies from 5% to 30%, and placentas will be expelled or retained. Tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. Other than these manifestations, infection with T. fetus causes no systemic signs. Diagnosis is based on patterns of infertility and pyometras. For example, pyometras in postcoital heifers or cows are suggestive of this pathogen. Diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. Other nonpathogenic protozoa from fecal contamination may be present in the sample. The trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. Culturing must be done on specific media, such as Diamond's or modified Pastridge.

Epizootiology and transmission.

All transmission is by venereal exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment.

Necropsy findings.

Nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. Histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing.

Pathogenesis.

Tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. Tritrichomonas fetus does not interfere with conception. Embryonic death occurs within the first 2 months of infection. Affected cows will clear the infection over a span of months and maintain immunity for about 6 months. Infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. Older bulls become chronic carriers, probably because of the ability of T.fetus to colonize deeper epithelial crypts of the prepuce and penis. Differential diagnosis.

Campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. Other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. In addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered.

Prevention and control.

A bacterin vaccine is available. Heifers, cows, and breeding bulls are vaccinated subcutaneously

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twice at 2 to 4 week intervals, with the booster dose administered 4 weeks before breeding season starts. Similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. Bulls used for artificial insemination (AI) are screened routinely for T.fetus (and Campylobacter). AI reduces but does not eliminate the disease. The use of younger, vaccinated bulls is recommcmded in all circumstances. New animals should be tested before introduction to the herd. Control measures also include culling affected cows or else removing them from the breeding herd for 3 months to rest and clear the infection. Culling chronically infected bulls is strongly recommended.

Treatment. Imidazole compounds have been effective, but the use of these is not permitted in food animals in the United States. Therapeutic immunizations are worthwhile when a positive diagnosis has been made. These will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. Research complications.

Trichomoniasis should be considered whenever natural service is used and fertility problems are encountered.

b.

Nematodes

Nematodes are important ruminant pathogens that cause acute, chronic, subclinical, and clinical disease in adults and adolescents. The major helminths may cause gastroenteritis associated with intestinal hemorrhage and malnutrition. Nematodiasis is associated with grazing exposure to infective larvae; animals procured for research may have had exposure to these helminths. Mixed infections of these parasites are common. Generally, older animals develop resistance to some of the species; thus, animals between about 2 months and 2 years of age are most susceptible to infection. Because of the parasites' effects on the animals' physiology, infection in these younger animals is a major contributor to a cycle of poor nutrition and digestion, compromised immune responses, and impaired growth and development. Diagnosis is primarily based on fecal flotation techniques; however, because many of these nematodes have similar-appearing ova, hatching the ova and identifying the larvae are often required (Baermann technique). A number of anthelmintics can be used to interrupt nematode life cycles. See Zajac and Moore (1993) and Pugh et al. (1998) for comprehensive reviews of treatment and control of nematodiasis.

younger cattle and are similar to the disease in sheep. Haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. Severe anemia may lead to death. Weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. Diarrhea is not seen in all cases but may sometimes be severe or chronic. The life cycle is direct. Under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in 3 weeks. Embryonated eggs may develop into infective larvae within a week. Hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. Periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). Resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of 2 dosings given 2 - 3 weeks apart. Levamisole is also used. In severe cases, animals may benefit from blood transfusions and iron supplementation. Because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. Haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions.

ii. Ostertagia (Teladorsagia) circumcincta (medium stomach worm). Ostertagia circumcincta is also highly pathogenic for sheep and goats and, like Haemonchus, attaches to the abomasal mucosa and ingests blood. The life cycle is comparable to that of Haemonchus, including the phenomenon of hypobiosis. Larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. Larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric pH from about 2.0 to near 7.0, leading to decreased digestive enzyme activity and malnutrition. Clinical syndromes are categorized as type 1 or type 2. The former type is associated with infections acquired in fall or spring and is seen in younger animals. The latter type is associated with emergence of the arrested larvae during spring or fall. Clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. Diarrhea is usually seen in type 1 only; the symptoms of type 2 are comparable to those of Haemonchus infections. Anthelmintic drug therapy is comparable to that for Haemonchus, and drug resistance is also a problem with Ostertagia.

i. Haemonchus contortus, H. placei (barber's pole worm, large stomach worm). Haemonchus contortus is the most im-

iii. Ostertagia ostertagi (cattle stomach worm). Ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. Ostertagia leptospicularis and O. bisonis also cause

portant internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. Haemonchus contortus and H. placei infections do occur in

disease. The life cycle is direct, and egg shedding by the cattle may occur within 3 - 4 weeks of ingestion of infective larvae. Hypobiosis is also a characteristic of O. ostertagi. In the initial

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. Moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. Cycles of infection and morbidity depend on geographic location, climate, and production cycles. Type 1 cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than 2 years old, and causes diarrhea and anorexia. Type 2 ostertagiasis occurs in cattle 2 - 4 years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type 1, hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. Treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. Ostertagia is susceptible to desiccation but is resistant to freezing.

iv. Trichostrongylus vitrinus, T. axei, T. colubriformis (hair worms). Trichostrongylus species favor cooler conditions, and some larvae may overwinter. Although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. Trichostrongylus vitrinus and T. colubriformis infect the small intestine of sheep and goats. Trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal pH similar to those seen with Ostertagia. Mucosal hyperplasia is not seen. The prepatent period is about 3 weeks. Affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. These worms show intermediate resistance to freezing temperatures and dry conditions.

v. Nematodirus spathiger, N. battus (thread-necked worms). Nematodirus has lower pathogenicity compared with other gastrointestinal nematodes. The larvae cause small-intestinal necrosis and inflammation. The larvae are especially resistant to desiccation and freezing. Clinical signs include depression, weight loss, anorexia, and diarrhea.

vi. Cooperia (small intestinal worms). Cooperia primarily affects younger animals less than 1 year of age. Cooperia curticei infects the small intestine of sheep and goats; C. punctata and C. oncophora infect the small intestines of cattle, sheep, and goats. Cooperia pectinata infects the stomach of cattle. Large numbers lead to clinical infection, and the prepatent period is about 3 weeks. Cooperia and Osteragia infections, like infections of some other nematode species, may act synergistically. Because these nematodes suck blood, clinical signs include anemia, gastrointestinal hemorrhage, and malnutrition. Animals exhibit weight loss, diarrhea, and depression. Cooperia species are intermediate to resistant to the effects of cold temperatures.

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vii. Strongyloides papillosus. Strongyloides papillosus is a small-intestinal parasite of sheep and cattle. Strongyloides has a different life cycle from that of many nematodes. The eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. The parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. Penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. Adult females may reproduce in the small intestines by parthenogenesis. Clinical signs associated with Strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. The current broad-spectrum anthelmintics are effective against Strongyloides. viii. Bunostomum trigonocephalum (hookworm). Bunostomum trigonocephalum is a hookworm that occasionally infects sheep in locales in the southwestern United States. Like

Strongyloides, Bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). The larvae mature in the small intestines and suck blood. Larvae are susceptible to desiccation and freezing. Heavy infection with Bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss.

ix. Oesophagostomum columbianum, O. venulosum (nodule worms). Oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. Oesophagostomum columbianum and O. venulosum infect sheep and cattle. These nematodes may affect sheep from 3 months to 2 years of age, and the prepatent period is about 6 weeks. Larvae are highly sensitive to freezing and desiccation and rarely overwinter. Larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. The resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. Intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. Clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. Nodular lesions are typical at necropsy.

x. Chabertia ovis (large-mouth bowel worm). Chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. Signs of infection are not usually seen in cattle. Prepatent periods are up to 50 days. Heavy infection, which may result from as few as 100 worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia.

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xi. Trichuris (whipworms). Trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. Trichuris has a rather long prepatent period, extending from 1 to 3 months. The oval eggs are double-operculated and survive well in pasture environmental extremes. The adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. The nematodes cause a minor cecitis and will feed on blood. Clinical infection is rare and results in diarrhea with mucus and blood. Treatment and prevention methods are similar to those for other nematodes. xii. Dictyocaulus (lungworms). Dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. In sheep, Dictyocaulus filaria, Protostrongylus rufescens, and Muellerius capillaris cause disease; Dictyocaulus is the most pathogenic. Goats are infected by the same species as sheep, but infections are uncommon. Dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." Infections with these parasites in the United States tend to be associated with cooler, moister climates. Lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately 2 and 18 months of age. Sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. Coughing and dyspnea are symptoms in goats. Diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. The Baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. Dictyocaulus has a direct life cycle. The adult worms reside in the large bronchi. Dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. The expelled larvae are infectious in about 7-10 days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about 5 weeks. Dictyocaulus filaria causes an especially severe bronchitis in sheep. Protostrongylus inhabits smaller bronchioles. Muellerius is of minor pathogenicity. Protostrongylus and Muellerius require the snail or slug as an intermediate host. Infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of Dictyocaulus larvae. Immunity wanes over a year. Viral and bacterial respiratory tract infections may be associated with the parasitic infection. Dictyocaulus viviparus causes the obvious signs in cattle. More severe illness is seen after infections with Cooperia and Ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. Hypobiosis (arrested development of immature worms in lung tissue) is associated with Dictyocaulus infections; cattle will

be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. Pastures can be heavily contaminated during the next grazing season. Necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. Nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. Prevention and control of the disease involve appropriate pasture management. Elimination of intermediate hosts is important in sheep and goat pastures. In a laboratory setting, animals may be procured that are already harboring the disease. Infected animals can be treated with anthelmintics such as ivermectin or levamisole. Muellerius tends to be resistant to levamisole. There is no anthelmintic currently approved for goats, but fenbendazole, administered 2 weeks apart, has been effective for all three nematodes. Treating D. viviparus depends on the type and stage of life of the cattle; label directions must be followed. There is no vaccine for D. viviparus in the United States. Even if infections are not severe and do resolve with treatment, permanent lesions may be inflicted on the lung tissue.

c.

Cestodes (Tapeworms)

i. Moniezia expansa and Thysanosoma actinoides infections. Tapeworms are rarely of clinical or economic importance. In younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. Moniezia expansa, and less commonly Moniezia benedini, inhabit the small intestines of grazing ruminants. Moniezia expansa has the widest distribution of the tapeworm species in North America. Soil mites (Galumna spp. and Oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to 16 weeks. Cysticercoids released from the mites are grazed, pass into the small intestines, and mature. No clinical or pathological sign is usually observed with Moniezia infection; diagnosis is made by observing the characteristic triangularshaped eggs in fecal flotation examinations. Infection is treated with cestocides. Thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the Mississippi River in the United States. Thysanosoma is of the family Anoplocephalidae. The life cycle is indirect, and the intermediate host is the psocid louse. Larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. Typically, no clinical signs are observed with Thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. Necropsy lesions include bile and/or ductal hyperplasia and fibrosis. Thysanosoma is diagnosed premortem by identifying the gravid segments in the feces.

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE

ii. Abdominal or visceral cysticercosis. Abdominal or visceral cysticercosis is an occasional finding at slaughter. The socalled bladder worms typically affect the liver or peritoneal cavity and are the larval form of Taenia hydatigena, the common tapeworm of the dog family. Taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. After ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. The larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next 1-9 months into small fluid-filled bladders. The life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. Although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. At necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. Migration tracts may result in fibrosis and inflammation. Diagnosis is usually made at necropsy. Because of the migration through the liver, Fasciola hepatica is a differential diagnosis. Minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. Research animals may have been exposed prior to purchase. iii. Echinococcosis

(hydatidosis,

hydatid cyst disease).

Echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. The hydatid cyst is the larval intermediate of the adult tapeworm Echinococcus granulosus, which resides in the small intestines of dogs and wild canids. Embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. The eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. The cystic structure develops and potentially ruptures, forming new cystic structures. Clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. Cysts are typically observed at necropsy. Prevention should be aimed at decreasing fecal contamination of feed and water by canids. Additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. Treatment of infected ruminants is uncommon.

iv. Gid. Coenuris cerebralis, the larval form of the canid tapeworm Taenia (Multiceps) multiceps, is the causative agent of the rare condition called gid. The disease occurs in ruminants as well as many other mammalian species. The larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. The resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia,

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and convulsions are observed. Diagnosis is usually made at necropsy. Eliminating transfer from the canid hosts prevents the disease.

d.

Trematodes

i. Fascioliasis (liver fluke disease). Liver flukes are an important cause of acute and chronic disease in grazing sheep and cattle. There are three common species of flukes in ruminants of the continental United States: Fasciola hepatica, Fascioloides magna, and Dicrocoelium dendriticum. Fasciola hepatica infections are primarily seen in Gulf Coast and western states. Fascioloides magna infections are typically seen in Gulf, Great Lake, and northwestern states, where ruminants share pasture with deer, elk, and moose. Dicrocoelium dendriticum infections occur only in New York State. Liver fluke eggs are passed in the bile and feces and hatch in 2 - 3 weeks to form the freeswimming miracidia. It is important to note that each fluke egg represents the source of eventually thousands of cercariae or metacercariae. The miracidia penetrate the body of an intermediate host (usually freshwater snails) and develop through sporocyst and redia stages, finally forming cercariae. (Dicrocoelium is unique because it utilizes a land snail that expels slime balls, each containing several hundred cercariae. These are eaten by a second intermediate host, the ant Formica fusca.) The cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. The metacercariae may remain in a dormant stage on the grass for 6 months or longer until ingested by a ruminant. The ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. There they locate in a bile duct, mature, and remain for up to 4 years. Acute liver fluke disease is related to the damage caused by the migration of immature flukes. Migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. Fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. In cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. Liver fluke damage may predispose to invasion by anaerobic Clostridium species such as C. novyi that could lead to fatal black disease or bacillary hemoglobinuria. Chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. Blood loss into the bile may lead to anemia and hypoproteinemia. Liver damage also is evidenced by increases in liver enzymes such as y-glutamyl transpeptidase (GGT). Persistent eosinophilia is also seen with liver fluke disease. Other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. At necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. Bile ducts will be enlarged, and areas of fibrosis will be evident. Diagnosis can be made from clinical signs and postmortem

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analyses. Blood chemistries suggestive of liver disease and eosinophilia support the diagnosis. Liver fluke control involves removal of the intermediate hosts. In a laboratory setting, liver fluke infection is unlikely. Nonetheless, incoming animals from pasture environments may be infected. Liver flukes can be treated by using the anthelmintic albendazole.

ii. Rumen fluke infections (paramphistomosis). Paramphistomosis is an uncommon disease found in sheep and cattle in southern states. Paramphistomum microbothrioides and P. cervi inhabit the duodenum and rumen of affected sheep. Eggs are passed in the feces and hatch in approximately 1 month, and the miracidia penetrate the intermediate snail hosts. Cercariae develop in the snail over the next month, emerge, and encyst on grasses as metacercariae. When eaten, the metacercariae develop into adult flukes and attach to the mucosal lining. The life cycle is complete in approximately 100 days. The flukes cause localized injury to the mucosa and, by interfering with digestive processes, cause diarrhea and protein loss. Clinically, animals may experience anorexia, dehydration, weight loss, and diarrhea with or without blood. Mortality may reach 25%. Diagnosis is based on clinical findings as well as the identification of flukes or eggs in the feces. Animals can be treated with flukicides. Eliminating the intermediate host prevents the disease.

e.

Mites (Mange)

Mites cause a chronic dermatitis. The principal symptom of these infections is intense pruritus. In addition, papules, crusts, alopecia, and secondary dermatitis are seen. Anemia, disruption of reproductive cycles, and increased susceptibility to other diseases may also occur. Mites are rare in ruminants in the United States, but infections of Sarcoptes and Psorergates mange must be reported to animal health officials. Ruminants in poorly managed facilities are generally the most susceptible to infection, and infections are more frequent during winter months. Diagnosis is based on signs, examination of skin scrapings, and response to therapy. No effective treatment for demodectic mange in large animals has been found. The differential for mite infestations is pediculosis. Several genera of mites may affect sheep. These have been eradicated from flocks in the United States or are very rare and include Psoroptes ovis (common scabies), Sarcoptes scabiei (head scabies, barn itch), Psorergates ovis (sheep itch mite), Chorioptes ovis (foot scabies, tail mange), and Demodex ovis (follicular mange). Goats can also be infected by sarcoptic, chorioptic, and psoroptic mange. The scabies mite Sarcoptes rupicaprae invades epidermal tissue and causes focal pruritic areas around the head and neck. The chorioptic mite, either Chorioptes bovis or C. caprae, does not invade epidermal tissue but rather feeds on dead skin tissue. The chorioptic mite prefers distal limbs, the udder, and the scrotum and can be a significant cause of pruri-

tus. The psoroptic mite Psoroptes cuniculi commonly occurs in the ear canal and causes head shaking and scratching. Repeated treatments of lime sulfur, amitraz, or ivermectin may be effective (Smith and Sherman, 1994). Goats are also susceptible to demodectic mange caused by Demodex caprae. Adult mites invade hair follicles and sebaceous glands. Pustules may develop with secondary bacterial infection. Psoroptes bovis continues to be present in cattle in the United States, although it has been eradicated from sheep. Chorioptes bovis typically infects lower hindlimbs, perineum, tail, and scrotum but can become generalized. The sarcoptic mange mite S. scabei can survive off the host, so fomite transmission is a factor. The mange usually begins around the head but then spreads. This parasite can be transmitted to humans. Demodex bovis infects cattle; nodules on the face and neck are typical. Demodex bovis infections may resolve without treatment. Lindane, coumaphos, malathion, and lime sulfur are used to treat Psoroptes and Psorergates. Ivermectin is effective against Sarcoptes and is approved for use in cattle.

f.

Lice (Pediculosis)

Lice that infect ruminants are of the orders Mallophaga, biting or chewing lice, and Anoplura, sucking lice. These are wingless insects. Members of the Mallophaga are colored yellow to red; members of the Anoplura are blue gray. Lice produce a seasonal (winter-to-spring), chronic dermatitis. In sheep, biting lice include Damalinia (Bovicola) ovis (sheep body louse). Sucking lice that infect sheep include Linognathus ovillus (blue body louse) and L. pedalis (sheep foot louse). In goats, biting lice infection are caused by D. caprae (goat biting louse), D. limbatus (Angora goat biting louse), and D. crassipes. Suckir/g louse infections in goats are caused by L. stenopis and L. africanus. Damalinia bovis is the cattle biting louse. Sucking lice include L. vituli, Solenopotes capillatus, Haematopinus eurysternus, and H. quadripertusus. Pruritus is the most common sign and often results in alopecia and excoriation. The host's rubbing and grooming may not correlate with the extent of infestation. Hairballs can result from overgrooming in cattle. In severe cases, the organisms can lead to anemia, weight loss, and damaged wool in sheep and damaged pelts in other ruminants. Young animals with severe infestations of sucking lice may become anemic or even die. Pregnant animals with heavy infestations may abort. In sheep infected with the foot louse, lameness may result. Lice are generally species-specific. Those infecting ruminants are usually smaller than 5 mm. Goats may serve as a source of infection for sheep by harboring Damalinia ovis. Transmission is primarily by direct contact between animals. Transmission can also occur by attachment to flies or by fomites. Some animals are identified as carriers and seem to be particularly susceptible to infestations. Biting or chewing lice inhabit the host's face, lower legs, and

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flanks and feed on epidermal debris and sebaceous secretions. Sucking lice inhabit the host's neck, back, and body region and feed on blood. Lice eggs or nits are attached to hairs near the skin. Three nymphal stages, or instars, occur between egg and adult, and the growth cycle takes about 1 month for all species. Lice cannot survive for more than a few days off the host. All ruminant mite infestations are differentials for the clinical signs seen with pediculosis. Animals that are carriers should be culled, because these individuals may perpetuate the infection in the group. Lice are effectively treated with a variety of insecticides, including coumaphos, dichlorvos, crotoxyphos, avermectin, and pyrethroids. Label directions should be read and adhered to, including withdrawal times. Products should not be used on female dairy animals. Treatments must be repeated at least twice at intervals appropriate for nit hatches (about every 16 days) because nits will not be killed. Fall treatments are useful in managing the infections. Systemic treatments in cattle are contraindicated when there may be concurrent larvae of cattle grubs (Hypoderma lineatum and H. bovis). Back rubbers with insecticides, capitalizing on self-treatment, are useful for cattle. Sustained-release insecticide-containing ear tags are approved for use in cattle.

g.

Ticks

Etiology.

Ruminants are susceptible to many species of Ixodidae (hard-shell ticks) and Argasidae (softshell ticks). Many diseases, including anaplasmosis, babesiosis, and Q fever are transmitted by ticks.

Clinical signs and diagnosis. Tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. Feeding sites on the host vary with the tick species. Ticks are associated with an acute paralytic syndrome called tick paralysis. This disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. Diagnosis is based on identification of the species. Epizootiology and transmission.

Ticks are not as host-specific as lice. Ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt.

Pathogenesis of tick infestations.

Patterns of feeding on the host differ between Argasidae and Ixodidae. The former feed repeatedly, whereas the latter feed once during each life stage.

Pathogenesis of tick paralysis.

Following a tick-feeding period of 4 - 6 days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve

transmission. Removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. In these cases, death due to respiratory failure occurs.

Treatment.

Ticks can be treated using systemic or topical

insecticides.

h.

Other Parasites

i. Nasal bots (nasal myiasis, head grubs). Nasal myiasis causes a chronic rhinitis and sinusitis. The disease is caused by the larval forms of the botfly Oestrus ovis. The botfly deposits eggs around the nostrils of sheep. The ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. In 2-10 months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. The mature larvae penetrate the soil and pupate for 1-1.5 months and emerge as botflies. Clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. Hypersensitivity to the larvae occurs (Dorchies et al., 1998). Later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. At necropsy, larvae will be observed in the nasal cavity or sinuses. Mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. The disease is diagnosed by observing the behaviors or identifying organisms at necropsy. Up to 80% of a flock will potentially be infected; treatment should be employed on the rest of the flock. Ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. Fly repellents may be helpful at preventing additional infections. ii. Screwworm flies. Cochliomyia hominivorax (Callitroga americana) is the the screwworm that causes occasional disease in the southwestern United States along the Mexico border. Eradication programs have been pursued, and the disease is reportable. Large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. Eggs hatch within 24 hr. Larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. Larvae eventually drop off, pupate best in hot climates, and hatch in 3 weeks. Large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. Single animals or entire herds may be affected. Treatment is intensive, with dressings and larvicidal applications. If there is no intervention, the host succumbs to secondary infections and fluid loss. Effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies.

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iii. Sheep keds ("sheep ticks"). In sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. The disease is caused by Melophagus ovinus, which is a fiat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. The adult fly lives entirely on the skin of sheep. Females mate and produce 10-15 larvae following a gestation of about 10-12 days. The larvae attach to the wool or hair and then pupate for about 3 weeks. The adult female feeds on blood and lives for 4 - 5 months; the life cycle is completed in about 5 - 6 weeks. Infection is highest in fall and winter. Pruritus develops around the neck, sides, abdomen, and rump. In severe cases, anemia may occur. Keds can transmit bluetongue virus. Keds are diagnosed by gross or microscopic identification. Ivermectin or other insecticides are useful treatment agents.

portant, other immune mechanisms are not well understood. Immunity may not be of long duration. Recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. A number of topical treatments, such as 2 - 5 % lime-sulfur solution, 3% captan, iodophors, thiabendazole, and 0.5% sodium hypochlorite, can be used. In severe cases, systemic therapy with griseofulvin may be successful.

Prevention and control. The animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. Pens should be thoroughly cleaned and disinfected. Research complications.

5. Fungal Disease: Dermatophytes (Ringworm) Etiology. Dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera Trichophyton and Microsporum. The primary causes in sheep are T. mentagrophytes and T. verrucosum. In goats, the agents are T. mentagrophytes, M. canis, M. gypseum, T. verrucosum, T. schoenleinii, and Epidermophyton floccosum. In cattle, T. verrucosum is the primary causative agent. Dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. Clinical signs and diagnosis. Multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. Lesions will vary in size. In all ruminants, lesions will be around the head, neck, and ears. In goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. Cattle lesions are unique in the marked crustiness, which progressively appears wartlike. Hair shafts become brittle and break off. Intense pruritus is often associated with the alopecic lesions. The disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and 20% potassium hydroxide digestion. Dermatophyte test media (DTM) cultures are the most reliable means to diagnose the fungus. Broken hairs from the periphery of the lesion are the best sources of the fungus. Epizootiology and transmission. Younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. Transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. Pathogenesis. Incubation can be as long as 6 weeks. The organisms invade and multiply in hair shafts. Treatment. Spontaneous recovery occurs in all species in 14 months. Although cell-mediated immunity is considered im-

Ringworm is a zoonotic disease.

B. Genetic, Metabolic, Nutritional, and Management-Related Diseases 1. Genetic Diseases a.

Entropion

Inverted eyelids are a common inherited disorder of lambs and kids of most breeds. Generally, the lower eyelid is affected and turns inward, causing various degrees of trauma to the conjunctiva and cornea. Young animals will display tearing, blepharospasm, and photophobia initially. If the disorder is left uncorrected, corneal ulcers, perforating ulcers, uveitis, and blindness may occur. Placing a suture or a surgical staple in the lower eyelid and the cheek, effectively anchoring the lid in an everted position, successfully treats the condition. The procedure likely results in the formation of some degree of scar tissue within the lower lid, because when the suture eventually is removed, the condition rarely returns. Other treatments include the injection of a "bleb" of penicillin in the lid, regular manual correction over a 2-day period early in the animal's life, and application of ophthalmic ointments, powders, and solutions. Boric acid or 10% Argyrol solutions have been used as treatments. Because of the genetic predisposition, prevention of the condition requires removal of maternal or paternal carriers.

b.

~-Mannosidosis of Goats

[3-Mannosidosis is an autosomal recessive lysosomal storage disease of goats. The disease affects kids of the Nubian breed and is identified by intention tremors and difficulty or inability of newborns to stand. Cells of affected animals are vacuolated because of a lack of lysosomal hydroxylase, which results in accumulation of oligosaccharides. Newborn kids are unable to rise, and they have characteristic flexion of the carpal joint and hyperextension of the pastern joint. Kids are born deaf and with musculoskeletal deformities such as domed skull, small narrow muzzle, small palpebral fissures, enophthalmos, and depressed

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

nasal bridge (Smith and Sherman, 1994). Carrier adults can be identified by plasma measurements of [3-mannosidase activity.

c.

Congenital Myotonia of Goats

Caprine congenital myotonia is an inherited autosomal dominant disease that affects voluntary striated skeletal muscles. Goats with this disease are commonly known as fainting goats. "Fainting" is actually transient spasms of skeletal musculature brought about by visual, tactile, or auditory stimuli (Smith and Sherman, 1994). Muscle fiber membranes appear to have fewer chloride channels than normal, resulting in decreased chloride conduction across the membrane, with subsequent increased membrane excitability and repetitive firing (Smith and Sherman, 1994). Contractions of skeletal muscle are sustained for up to 1 min. Kids exhibit the condition by 6 weeks of age, and males appear to exhibit more severe clinical signs than females (Smith and Sherman, 1994). Electromyographic studies produce an audible "dive-bomber" sound characteristic of hyperexcitable cell membranes (Smith and Sherman, 1994).

d.

Inherited Conditions of Cattle

i. Congenital erythropoietic porphyria. Congenital erythropoietic porphyria (CEP) is an autosomal recessive disease of cattle seen primarily in Holsteins, Herefords, and Shorthorns. The disease also occurs in Limousin cattle, humans, and some other species. In the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. A reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. Bones are more fragile compared with bones of normal animals. A regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. The genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen III synthase, in the porphyrin-heme synthesis pathway in erythrocytic tissue. The ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. Porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a Wood's lamp. Diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. Heterozygotes may have milder symptoms. Many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ("Large Animal Internal Medicine," 1996). In many cases, the genetic basis has been clarified, and associated defects also noted. Many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. The bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming (Womack, 1998).

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Some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of Holstein cattle. Some of the more commonly reported defects include syndactyly in Holsteins and other breeds and polydactyly in Simmentals; lysosomal storage diseases such as a-mannosidosis in some beef breeds; enzyme deficiencies such as citrullinemia in Holsteins; and progressive degenerative myeloencephalopathy ("weaver") in Brown Swiss.

ii. Goiter of sheep. A defect in the synthesis of thyroid hormone has been identified in Merino sheep (Radostits et al., 1994). Lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. Edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. Immaturity of the lungs at birth causes neonatal respiratory distress and resuits in dyspnea and respiratory failure. iii. Spider lamb syndrome (hereditary chondrodysplasia). Spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in Suffolk and Hampshire breeds. Severely affected lambs die shortly after birth. Animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. With time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. Radiologically, secondary ossification centers--especially the physis, subchondral areas, and cuboidal bonesmare affected. Abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. Lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. Muscle atrophy is common. Diagnosis can be based on typical clinical signs, which are similar to those seen with Marfan syndrome in humans (Rook et al., 1986). Long-term survival is rare; treatment is unsuccessful. 2.

Metabolic Diseases

a.

Abomasal Disorders

i. Abomasal and duodenal ulcers. Abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. Like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. Concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. Copper deficiency, dietary changes, mycotic infections, Clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. In older adult cattle, abomasal lymphosarcoma may be the underlying condition. Gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. Deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. Chronic hemorrhage

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may lead to anemia. Although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. Dark feces or melena and abdominal pain may be observed. Arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. Fecal occult blood is as an easy diagnostic test. Treatment includes gastrointestinal protectants and histamine antagonists. Anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. Preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus.

ii. Abomasal emptying defect. Abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. Suffolks tend to be especially predisposed, although the disease has been diagnosed in Hampshires, Columbias, and Corriedales. The mechanism of the disease is unknown. Affected animals will exhibit a gradual weight loss with a history of normal appetites. Feces will continue to be normal. Ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. Diagnosis is primarily based on history and clinical signs. Elevations in rumen chloride concentrations (> 15 mEq/liter) are commonly found. Radiography or ultrasonography may be helpful at identifying the distended abomasum. Abomasal emptying defect is usually eventually fatal. Medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. Abomasal displacement. Displaced abomasum (DA) is a sporadic disorder usually associated with multiparous 4- to 7year-old dairy cows in early lactation, but the condition can occur even in young calves. Displacement to the right (RDA) may be further complicated by torsion (RTA), a surgical emergency. Left displacement (LDA) is more common than RDA. Clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. Diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. Cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. DA occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. No exact cause of DA has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. Factors such as body size and conformation indicate the possibility of genetic predisposition. Treatments include surgical and nonsurgical techniques for LDA; the former has a better chance of per-

manent correction. Emergency surgery is necessary for RTA; the disorder is fatal within 72 hr. Recurrence is rare after surgical correction. Electrolyte and acid-base imbalances are likely in severe cases and especially with RTA. Prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above (Rohrbach et al., 1999).

b.

Fat Cow Syndrome, Hepatic Lipidosis

Fat cow syndrome is seen in peri- or postparturient overconditioned or obese multiparous dairy cows. Factors in the development of the condition include negative energy balance related to the normal decreased dry matter intake as parturition approaches; hormonal changes associated with parturition; and concurrent diseases of parturition that decrease feed intake and increase energy needs. The possible concurrent diseases include metritis, retained fetal membranes, mastitis, parturient paresis, and displaced abomasum. Signs are nonspecific and include depression, anorexia, and weakness. Prognosis is usually guarded. Diagnosis is based on herd management, the animal's condition, ketonuria, and clinical signs. In prepartum cattle and in lactating cows, blood levels of nonesterified fatty acids (NEFA) greater than 1000 ~tEq/liter and 325-400 ~tEq/liter, respectively, are abnormal (Gerloff and Herdt, 1999). Triglyceride analysis of liver biposy specimens are useful. In affected cows, body fat is mobilized, in the form of NEFA in response to the energy demands. Hepatic lipidosis occurs rapidly as the NEFA are converted into hepatic triglycerides. The ability of the liver to extract the albumin-bound NEFA from the blood is better than that of other tissues that need and can also use NEFA as an energy source. Treatment for any concurrent diseases must be pursued aggressively, as well as measures to increase and stabilize blood glucose, decrease NEFA production, and increase forestomach digestion to improve production of normally metabolized volatile fatty acids. Therapeutic measures include intravenous glucose drips, insulin (NPH or Lente) injections every 12 hr, and transfaunation of ruminal fluid from a normal cow. Prevention includes minimizing stress to lategestation cows. Dry and lactating cows should be maintained separately; their energy, protein, and dry matter requirements are very different. Cows with prolonged lactation or delayed breeding should be managed to prevent weight gain.

c.

Rumen and Reticulum Disorders

i. Bloat. Bloat or tympanites refers to an excessive accumulation of gas in the rumen. The condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. Bloat is classified into two broad categories: frothy bloat and free-gas bloat. Frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. Fermentation gases such as CO2, CH4, and minor gases such as N2, 02, H2, and H2S incorporate into the froth, overdistend the rumen, and eventu-

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14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE

ally compromise respiration by limiting diaphragm movement. The froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. Typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). Free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. Some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. Clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. Additional signs may include colic-like pain of the abdomen and dyspnea. Passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. Once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. Frothy bloat is more difficult to treat, because the foam blocks the stomach tube. Addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. In acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. Limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. Additionally, poloxalene or monensin will decrease the incidence of frothy bloat.

ii. Lactic acidosis. Lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. The disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. Diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. Biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive Streptococcus and Lactobacillus species. The gram-positive organisms efficiently convert the starches to lactic acid. The lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. Lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. Clinically, animals

will become anorexic, depressed, and weak within 1-3 days after the initial insult. Incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted 9Rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. Diagnosis is based on history and clinical signs. Blood, urine, or milk ketones can be detected (Moore and Ishler, 1997). Additionally, rumen pH, which is normally above 6.0, will drop to less than 5.0 and in severe cases may achieve levels as low as 3.8. Similarly, urine pH will become acidic, blood pH will drop below 7.4, and hematocrit will appear to increase due to the relative hemoconcentration. Necropsy findings will be determined by secondary conditions. The primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. Treatment must be applied early in the syndrome. In early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. The patient should be given mineral oil and antlfermentatlves to prevent the continued conversion of starches to acids and the absorption of metabolic products. Bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen pH. Furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. 9

t 9

iii. Rumen parakeratosis. Parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium 9 Excessive and continuous feeding of diets low in roughage causes the mucosal changes 9 Generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. Clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. At necropsy, papillae will be thickened and rough. They will frequently be dark in color, and multiple papillae will clump together. Abscessation may be observed. Histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. Chronic laminitis may be observed. However, diagnosis of parakeratosis is generally made at necropsy. Feeding adequate roughage, such as stemmy hay, will prevent the disease. Antibiotics may be administered to prevent secondary liver abscess formation. iv. Rumenitis. Rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis 9 In addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder 9 Rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. Because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. The inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. Endogenous rumen bacteria such as Fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. Clinically, the animals will appear depressed and

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anorexic. Rumen motility will be decreased, and animals will lose weight. The disease may resolve in a week to 10 days; mortality may reach 20%. Necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. Granulation tissue and scarring may be observed following healing. Rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. The disease can be prevented by minimizing the incidence of lactic acidosis.

Traumatic Reticulitis-Reticuloperitonitis (Hardware Disease) Etiology.

Traumatic reticulitis-reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. The disease is rarely seen in smaller ruminants.

Clinical signs. Clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. Many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. The prognosis is poor when peritonitis becomes diffuse. Sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. Epizootiology and transmission.

This is a noncontagious disease. The occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow.

Necropsy findings.

In severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. Cardiac puncture will be present in those animals succumbing to sudden death.

Pathogenesis.

Consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. This sets off a localized inflammation or a localized or more generalized peritonitis. The inflammation may also temporarily or permanently affect innervation of local tissues and organs. Further damage may result from migration and penetration of the diaphragm, pericardium, and heart. Diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. Radiographs and abdominocentesis may be useful.

Differential diagnosis.

Differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. Infectious diseases that are differentials include systemic leptospirosis and internal parasitism. Diseases causing sudden death may need to be considered.

Prevention and control.

This problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. Adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a bailing gun in young stock at 6 - 8 months of age) are also significant prevention measures.

Treatment. Provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. In severe cases, rumenotomy may be considered. Pregnancy Toxemia (Ketosis), Protein Energy Malnutrition Etiology.

Pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. Beef heifers are susceptible to protein energy malnutrition (PEM) syndrome, which is also referred to as pregnancy toxemia.

Clinical signs. In sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. Hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. They become anorexic and act uncoordinated, frequently leaning against objects. Advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. Body temperature, heart rate, respiratory rate, and rumen motility continue normally. Up to 80% of infected ewes may die from the disease. The course of the disease may last up to a week. In goats, the disease usually occurs in the last 6 weeks of gestation, especially in does carrying triplets. Pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. The doe may separate herself from the herd, stagger, or circle and may appear blind. Appetite is poor, and tremors may be evident. A rapid metabolic acidosis results in subsequent recumbency. Urinalysis will readily reveal ketonuria. If fetal death occurs, acute toxemia and death of the doe may result. In beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. Some cows develop diarrhea. Because the catabolic state is often so advanced, most affected heifers die even if treated. Pregnancy toxemia is diagnosed by evidence of typical clinical signs. Sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. Blood glucose levels found to be below 25 mg/dl and

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE ketonuria are good diagnostic indicators. In cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present.

Epizootiology.

Pregnancy toxemia occurs primarily in ewes that are obese or bearing twins or triplets. The disease develops during the last 6 weeks of pregnancy. PEM most frequently occurs in heifers during the final trimester of pregnancy.

Necropsy findings.

At necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. The liver will be enlarged, yellow, and friable, with fatty degeneration. The adrenal gland may also be enlarged. In cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present.

Pathogenesis.

Rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. The ewe develops a severe hypoglycemia in early stages of the disease. The ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. Propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. When the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. Glycerol is converted to glucose while the fatty acids are metabolized for energy. The oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal Krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and [3-hydroxybutyric acid), thus causing the condition ketoacidosis. Heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. Additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. Marginal diets and poor-quality forage will place the cows in a negative energy balance.

Differential diagnosis.

Hypocalcemia is a common differential diagnosis. In cattle, differentials include chronic or untreated diseases such as Johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases.

Prevention and control.

Pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. In late pregnancy, the dietary energy and protein should be increased 1.5-2 times the maintenance level. PEM can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester.

Treatment. In sheep, because the morbidity may be as high as 20%, treatment should be directed at the flock rather than the in-

603

dividual. Treating the individual is usually unsuccessful. Oral administration of 200 ml of propylene glycol or 50% glucose twice a day, anabolic steroids, and high doses of adrenocorticosteroids may be helpful. If ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. In goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. Parturition can be induced in does by either dexamethasone (10 mg) or PGF2a (10 ~tg). In addition, goats may be treated with 10% dextrose (100 to 200 ml iv) or propylene glycol (60 ml per os 2 or 3 times a day). Adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin B 12and transfaunation. Heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on IV 50% glucose drips, and treated for concurrent disease.

Research complications.

In research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time.

f

Hypocalcemia (Parturient Paresis, Milk Fever)

Etiology.

Hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows.

Clinical signs and diagnosis. In sheep, the disease is seen in ewes during the last 6 weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. As calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. Later, muscular tremors, muscular weakness, and recumbency will ensue. Animals will frequently be found breathing rapidly despite a normal body temperature. Morbidity may approach 30%, and mortality may reach as high as 90% in untreated animals. Affected does become bloated, weak, unsteady, and eventually recumbent. Cows are affected within 2 4 - 4 8 hr before or after parturition. Cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. Tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. The terminal stage of disease is a rapid progression from coma to death. Heart rates will be high, but pulse may not be detectable. Hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. It is later confirmed by laboratory findings of low serum calcium. With hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of 8-12 mg/dl to values of 3 - 6 mg/dl. In cattle, plasma

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levels below 7.5 mg/dl are hypocalcemic; at the terminal stages levels may be 2 mg/dl.

animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease.

Epizootiology.

Hypocalcemia occurs primarily in overweight ewes during the last 6 weeks of pregnancy or during the first few weeks of lactation. The disease is not as common in the dairy goat as in the dairy cow. High-producing, older, multiparous dairy cows are the most susceptible, and the Jersey breed is considered susceptible. Cows that have survived one episode are prone to recurrence. In addition, dry cows must be managed carefully regarding limiting dietary calcium. The disease is not common in beef cattle unless there is an overall poor nutrition program.

Necropsy findings.

There is no pathognomonic or typical find-

ing at necropsy.

Pathogenesis.

During the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. Additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. After parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. Recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation (Rings et al., 1997). Thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal.

Prevention and control.

Maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. In cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful.

Treatment. Hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. Twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. Response will often be rapid, with the resolution of the animal's dull mentation. Less severely affected animals will often try to stand in a short time. Relapses are common, however, in sheep and cattle. Hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. These imbalances should be considered when animals appear to be unresponsive to treatment. Hypocalcemia in the goat can be treated with 50-100 ml of calcium borogluconate. Heart rate should be monitored closely throughout calcium administration. If an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. Calcium gels and boluses are also available for treatment (Rings et aL, 1997). Prognosis is generally good if the

g.

Urinary Calculi (Obstructive Urolithiasis, Water Belly)

Etiology.

Urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (Murray, 1985). The disease occurs rarely in female ruminants.

Clinical signs and diagnosis. Affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. These postures may be mistaken for tenesmus. Male cattle may develop swelling along the ventral perineal area. Affected animals will not stay with the herd or flock. Small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. Additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. The pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. As the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. The abdomen will swell as the bladder enlarges, and rupture can occur within 36 hr after development of clinical signs. Bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. The disease may progress over a period of 1-2 weeks, and the mortality is high unless the blockages are reversed. Diagnosis is made by the typical clinical signs. Abdominal taps may yield urine. Calculi are usually composed of calcium phosphate or ammonium phosphate matrices. Epizootiology and transmission.

Clinical disease is usually seen in growing intact or castrated males. The disease may be sporadic or there may be clusters of cases in the flock or herd.

Necropsy findings.

Necropsy findings include urine in the abdomen with or without bladder or urethral rupture. Renal hydronephrosis may be evident. Calculi or struvite crystal sediment will be observed in the bladder and urethra. Histologically, trauma to the urethra and ureters will be present.

Pathogenesis.

Urolithiasis is multifactorial and involves dietary, anatomical, hormonal, and environmental factors. Male sheep and goats have a urethral process that predisposes them to entrapment of calculi. In cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. Additionally, the removal of testosterone by early castration is thought to

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE result in hypoplasia of the urethra and penis. This physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. Grains fed to growing animals tend to be high in phosphorus and magnesium content. These calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. Other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses).

Differential diagnosis.

Grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. Trauma to the urethral process should be considered. Urinary tract infections are uncommon in ruminants.

Prevention and control.

One case often is indicative of a potential problem in the group. Urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. The normal ratio should be 2:1. Additionally, increasing the amount of dietary roughage will help balance the mineral intake. Increasing the amount of salt (sodium chloride, 2 - 4 % ) in the diet to increase water consumption, or adding ammonium chloride to the diet, at 10 gm/head/day or 2% of the ration, to acidify the urine, will aid in the prevention of this disease. Palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment.

Treatment.

Treatment is primarily surgical (Van Metre et al. 1996). Initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. As the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. In more advanced stages, perineal urethrostomy may yield good results. The prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required.

Research complications.

Young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder.

h. Rickets Rickets is a disease of young, growing animals but rarely occurs in goats. It is a metabolic disease characterized by a failure of bone matrix mineralization at the epiphysis of long bones due to lack of phosphorus. The condition can occur as an absolute deficiency in vitamin D2, an inadequate dietary supply of phosphorus, or a long-term dietary imbalance of calcium and phosphorus. The syndrome must be differentiated from epiphisitis (unequal growth of the epiphyses of long bones in young, rapidly growing kids fed diets with excess calcium). Clinical signs include poor growth, enlarged costochondral

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junctions, narrow chests, painful joints, and reluctance to move. Spontaneous fractures of long bones may occur. Animals will recover when dietary phosphorus is provided and if joint damage is not severe.

3.

Nutritional Diseases

a.

Copper Deficiency (Enzootic Ataxia, Swayback)

Etiology.

Chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. In goats, this deficiency also causes swayback in the fetuses.

Clinical signs and diagnosis. This disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about 3 months of age. Additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. Affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. When the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. Kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. Diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. Diagnosis is based on clinical signs, feed analysis, and pathological findings. Epizootiology and transmission.

Enzootic ataxia is rarely seen in western states; most North American diets have sufficient copper levels to prevent this disease. Copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies.

Pathogenesis.

The maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. Copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth (Smith and Sherman, 1994).

Differential diagnosis.

The differential diagnosis for newborns includes [3-mannosidosis, hypoglycemia, and hypothermia. For older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis.

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Prevention and control.

Copper deficiency can be prevented by providing balanced nutrition for pregnant animals.

Necropsy findings.

Gross encephalomalacia has been noted. Histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. Extensive nerve demyelination and necrosis are evident. Postmortem lesions include extensive demyelination and neuronal degeneration.

Treatment. Because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them.

Necropsy findings.

Common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. Hemoglobin-stained urine will be visible in the bladder. Copper accumulations in the liver reaching 1000-3000 ppm are toxic.

Pathogenesis.

Hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. Stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis.

Differential diagnosis. b.

Copper Toxicosis

Etiology:

Acute or chronic copper ingestion or liver injury often causes a severe, acute hemolytic anemia in weanling to adult sheep and in calves and adult dairy cattle. Growing lambs may be the most susceptible. Copper toxicosis is rare in goats.

Clinical signs and diagnosis.

The clinical course in sheep can be as short as 1 - 4 days, and mortality may reach 75%. Hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. Some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. Weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. Hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (AST) and y-glutamyltransferase (GGT) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage.

Epizootiology and transmission.

A single toxic dose for sheep and goats is the range of 20-100 mg/kg, and for cattle it is 220-880 mg/kg. Chronic poisoning in sheep may occur when 3.5 mg/kg is ingested. Copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. Phytogenous sources include certain pastures such as subterranean clover. Feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. A common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. Chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. Copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. Pregnant dairy cattle may be more susceptible to copper toxicity. Rare sources of copper ingestion may include copper sulfate footbaths.

Other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. Arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. Urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain.

Control and prevention. The disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. Sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. Molybdenum may be administered to animals considered at high risk. Molybdenum-deficient pastures may be treated with molybdenum superphosphate. Herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. Treatment. Oral treatment for sheep consists of ammonium or sodium molybdenate (50-100 mg/day), and sodium thiosulfate (0.5-1.0 mg/day) for 3 weeks aids in excretion of copper. Oral D-penicillamine daily for 6 days (50 mg/kg) has also been shown to increase copper excretion in sheep. Ammonium molybdenate has been administered intravenously to goats at 1.7 mg/kg for 3 treatments on alternate days. Cattle have been treated orally with sodium molybdenate (3 gm/day) or sodium thiosulfate (5 gm/day). Treatment for anemia and nephrosis may be necessary in severe cases. Research complications.

Some breeds of sheep, such as Merino crosses and the British breeds, may be more susceptible to copper toxicosis caused by phytogenous sources.

Selenium~Vitamin E Deficiency (Nutritional Muscular Dystrophy, Nutritional Myodegeneration, White Muscle Disease, Stiff Lamb Disease) Etiology. White muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin E.

14. BIOLOGYAND DISEASESOF RUMINANTS: SHEEP, GOATS,AND CATTLE

Clinical signs and diagnosis. Clinically two forms of the disease have been identified: cardiac and skeletal. The cardiac form occurs most commonly in neonates. In these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. Young will be able to nurse when assisted. In slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. Clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. Mortality may reach 70% (Jensen and Swift, 1982). Paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. With the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. Young will be reluctant to get up but will readily nurse when assisted. Peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. Serum selenium levels are usually below 50 ppb (normal is 158-160 ppb) (Nelson, 1983). Diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin E. Glutathione peroxidase levels in red blood cells can be measured as an indirect test. Clinical biochemistry findings of significant elevations of aspartate aminotransferase (AST) in creatinine kinase (CK) are also supportive of the diagnosis. Epizootiology and transmission.

Selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. Rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease.

Necropsy findings.

Necropsy lesions include petechial hemorrhages and muscle edema. Hallmarks are pale white streaking of affected skeletal and cardiac muscle. These are due to coagulation necrosis. Pale striated muscles of the limb, diaphragm, and tongue are also seen.

Pathogenesis.

Selenium and vitamin E function together as antioxidants that protect lipid membranes from oxidative destruction. Selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. Lack of one or both results in loss of membrane integrity.

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quality roughage, vitamin E and selenium supplementation, and parenteral injections prior to parturition and weaning.

Treatment. Affected animals may be treated by administering vitamin E or selenium injections. Administering vitamin E or selenium to ewes in late pregnancy can prevent white muscle disease (Kott et al., 1998). The label dose for selenium is 2.53 mg/45 kg of body weight. Combination products are available and can be used in goats at the sheep dose (Smith and Sherman, 1994). Proper mineral balance in the diet is critical. d.

Selenium Toxicity

Selenium toxicity occurs most frequently as the result of excessive dosing to prevent or correct selenium deficiency or as the result of ingestion of selenium-converting plants. The main preventive measure for the former is the use of the appropriate product for the species. Secondarily, the concentration of the available product should be double-checked. In the United States, ruminants in the Midwest and western areas may be subject to selenium toxicity when pastured in areas containing selenium-converting plants. Signs of overdosing include weakness, dyspnea, bloating, and diarrhea. Shock, paresis, and death may occur. Initial clinical signs of excessive selenium intake from plants are observed in the distal limb, with cracked hoof walls and subsequent infection and irregular hoof growth.

e.

Thiamin Deficiency (Polioencephalomalacia)

Etiology.

Polioencephalomalacia (PEM) is a noninfectious, noncontagious disease characterized by neurological signs. Growing and adult ruminants on high-concentrate diets are typically affected. Animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk.

Clinical signs and diagnosis. An early sign may be mild diarrhea. Acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. In subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. Hypersalivation may be present, but body temperatures and ocular reflexes are normal. Morbidity and mortality may be high, especially in younger animals. Diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride.

Differential diagnosis.

In neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. Differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration.

Prevention and control.

Awareness of regional selenium deficiencies is important. Control involves providing good-

Epizootiology and transmission.

PEM is caused by a thiamin deficiency. The disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. Pastured animals are also vulnerable if grain is feed. Thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. Recent studies have also indicated that high

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levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. Medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses.

Sherman, 1994). Vitamin A deficiencies associated with hyperkeratosis have been reported, as well as vitamin E-responsive and selenium-responsive dermatitis.

Necropsy signs. Cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. Microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. Gliosis and cerebral capillary proliferation may be observed.

4. Management-Related Diseases

Pathogenesis. A lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. Differential diagnosis. Several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin A deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type D clostridial enterotoxemia. Prevention and control. The disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of B vitamins. If excess sulfur is the primary factor, immediate removal of the source is critical. Treatment. Early aggressive treatment is essential to save animals. The disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. Dexamethasone, B vitamins, and diazepam may also be required. Treatment is less successful when sulfur plays a prominent role in the etiology. Research complications. This disease is preventable. Although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. f

Vitamin D Toxicity

Vitamin D toxicity can result either from iatrogenic overadministration or ingestion of the plant Trisetum flavescens. Serum calcium levels may be high enough that blood in EDTA tubes will clot.

g.

Nutritional Deficiencies

In goats, nutritional deficiencies often manifest as a generalized poor coat that is dry, scaly, thin, and erectile. Zincresponsive dermatitis has been reported in goats (Smith and

a.

Failure of Passive Transfer

Neonatal ruminants are born without immunoglobulins and must receive colostrum by 24 hr after birth. The morbidity and mortality associated with failure of or inadequate passive transfer, such as enteric and respiratory illnesses, can be severe. Measures to assure passive immunity for neonatal ruminants are covered in Section II,B,5, and clinical signs of illness associated with lack of immunity are addressed in the discussions of bacterial diseases (e.g., Escherichia coli infections) and, of viral diseases (e.g., diarrheas) in Section III,A,1 and III,A,2. Generally, transfer of less than 600 mg/dl of immunoglobulins in the serum is classified as failure of transfer, 600-1600 mg/dl is partial, and above 1600 mg/dl is complete transfer. Methods to determine success of transfer should be performed within a week of birth and include single radial immunodiffusion (quantitates immunogloblin classes); zinc sulfate turbidity (semiquantitative); sodium sulfite precipitation (semiquantitative); glutaraldehyde coagulation (coagulates above specific level); and, y-glutamyltransferase (assays enzyme in high concentration in colostrum and absorbed simultaneously with colostrum).

b.

Laminitis

Laminitis is common in ruminants and can be caused by sudden changes in diet, excess dietary energy, and grain overload (or overeating). Laminitis is also associated with mastitis and metritis. Facility conditions, such as concrete flooring, poor manure management, and inadequate resting areas may also contribute to the pathogenesis of the disease. The complete pathogenesis of laminitis is poorly understood; however, it is thought that changes in the diet cause changes in rumen microbial populations, resulting in acidosis and endotoxemia. Dramatic changes in the vascular endothelium result in chronic inflammation of the sensitive laminae of the hoof, separation of corium and hoof wall, and rotation of the third phalanx. Affected animals may be reluctant to get up or walk, will shift their weight frequently, and will grind teeth or walk on carpi. Chronically, the hoof wall takes on a "slipper" appearance. Treatment consists of identifying the underlying cause, administering antiinflammatories (phenylbutazone, flunixin meglumin), feeding good-quality forages only, and regular foot trimming.

c.

Nutritional Diarrhea

Otherwise normal, well-managed lambs, kids, and calves can develop loose, pasty feces due to a nutritional imbalance caused by overfeeding and/or improper mixing of milk replacers. Only

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS,AND CATTLE milk replacer formulated for the particular species should be used. Once nutritional imbalances are corrected, the feces readily return to normal. Sudden changes in diet can also result in loose feces.

d.

Photosensitization(Bighead)

Photosensitization is an acute dermatitis associated with an interaction between photosensitive chemicals and sunlight. The photosensitive chemicals are usually ingested, but in some cases exposure may be by contact. Animals with a lack of pigment are more susceptible to the disease. Three types of photosensitization occur: primary; secondary, or hepatogenous; and aberrant. Primary photosensitization is related to uncommon plant pigments or to drugs such as phenothiazine, sulfonamides, or tetracyclines. Secondary photosensitization is more common in large animals and is specifically related to the plant pigment phylloerythrin. Phylloerythrin, a porphyrin compound, is a degradation product of chlorophyll released by rumen microbial digestion. Liver disease or injury, which prevents normal conjugation of phylloerythrin and excretion through the biliary system, predisposes to photosensitization. The only example of aberrant photosensitization is congenital porphyria of cattle (see Section III,B,1). Pathologically, the photosensitive chemical is deposited in the skin and is activated by absorbed sunlight. The activated pigments transfer their energy to local proteins and amino acids, which, in the presence of oxygen, are converted to vasoactive substances. The vasoactive substances increase the permeability of capillaries, leading to fluid and plasma protein losses and eventually to local tissue necrosis. Photosensitization can occur within hours to days after sun exposure and produces lesions of the face, vulva, and coronary bands; lesions are most likely to occur on white-haired areas. Initially, edema of the lips, corneas, eyelids, nasal planum, face, vulva, or coronary bands occurs. The facial edema, nostril constriction, and swollen lips potentially lead to difficulty in breathing. With secondary photosensitization, icterus is also common. Necrosis and gangrene may occur. Diagnosis is based on clinical lesions and exposure to the photosensitive chemi-cals and sunlight. Treatment is symptomatic. The prognosis for hepatogenous type may be guarded if hepatic disease is severe.

e. ReproductiveProlapses (Vaginal Uterine) Vaginal and uterine prolapses occur in ewes, does, and cows. The conditions are not common in does. Vaginal prolapses usually occur during late gestation and may be related to relaxation of the pelvic ligaments in response to hormone levels. In sheep, these are most common in overconditioned ewes that are also carrying twins or triplets. Overconsumption of roughages, which distends the rumen, and lack of exercise leading to intraabdominal fat may predispose an animal to vaginal prolapse by increasing intra-abdominal pressure. The condition may result

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from excessive straining associated with dysuria from the pressure of the fetuses and/or abdominal contents on the bladder. If the prolapse obstructs subsequent urination, rupture of the bladder may occur. The vaginal prolapse can be reduced and repaired if discovered early, and techniques in small and large ruminants are comparable. The animal should be restrained, and the prolapsed tissue should be cleansed with disinfectants. Best done under epidural anesthesia, the vagina is replaced into the pelvic canal and the vulvar or vestibular opening is sutured closed (Buhner suture). Alternatively, a commercial device called a bearing retainer (or truss) can be placed into the reduced vagina and tied to the wool, thereby holding the vagina in proper orientation without interfering with subsequent lambing. Vaginal prolapses may have a hereditary basis in ewes and cows and may prolapse the following year. These animals should be culled. Vaginal prolapses may occur in nonpregnant animals that graze estrogenic plants or as a sequela to docking the tail too close to the body (Ross, 1989). Uterine prolapses occur sporadically in postpartum ewes and cattle. The gravid horn invaginates after delivery and protrudes from the vulva. The cause is unknown, but excessive traction utilized to correct dystocia or retained placenta, uterine atony, hypocalcemia, and overconditioning or lack of exercise have been implicated. In cattle, the uterine prolapses usually develop within 1 week of calving, are more common in dairy cows than in beef cows, and are often associated with dystocia or hypocalcemia. Cows may also have concurrent parturient paresis. Initially, the tissue will appear normal, but edema and environmental contamination or injuries of the tissue develop quickly. Clinical signs will include increased pulse and respiratory rates, straining, restlessness, and anorexia. If identified early, the uterus can be replaced as for vaginal prolapses. Electrolyte imbalances should be corrected if present. Additional supportive therapy, including the use of antibiotics should always be considered. Tetanus prophylaxis should be included. Oxytocin should be administered to induce uterine reduction. Vaginal closures are less successful at retaining uterine prolapses. Preventive and control measures include regular exercise for breeding animals, and management of prepartum nutrition and body condition.

f

Rectal Prolapse

Rectal prolapse is common in growing, weaned lambs and in cattle from 6 months to 2 years old. The physical eversion of the rectum through the anal sphincter is usually secondary to other diseases or management-related circumstances. Rectal prolapses may occur secondary to gastrointestinal infection or inflammation, especially when the colon is involved. Diseases that cause tenesmus, such as coccidiosis, salmonellosis, and intestinal worms, may result in prolapse. Urolithiasis may result in prolapses as the animal strains to urinate. Any form of cystitis or urethritis, vaginal irritation, or vaginal prolapse and some

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forms of hepatic disease may lead to rectal prolapse. Abdominal enlargement related to advanced stages of pregnancy, excessive rumen filling or bloat, and overconditioning may cause prolapse. Finally, excessive coughing during respiratory tract infections, improper tail docking (too short), growth implants, prolonged recumbency, or overcrowded housing with animal piling may lead to prolapses. Diagnosis is based on clinical signs. Early prolapses may be corrected by holding the animal with the head down, while a colleague places a pursestring suture around the anus. The mucosa and underlying tissue of prolapses that have been present for longer periods of time will often become necrotic, dry, friable, and devitalized and will require surgical amputation or the placement of prolapse rings to remove the tissue. Rectal prolapse may also be accompanied by intestinal intussusceptions that will further complicate the treatment and increase mortality. Occasionally, acute rectal prolapse with evisceration will result in shock and prompt death of the animal. Prognosis depends on the cause and extent of the prolapse as well as the timeliness of intervention. In all cases of treatment, determination and elimination of the underlying cause are essential.

g.

Trichobezoars

Gastrointestinal accumulations or obstructions of hair (and/ or sometimes very coarse roughage, forming bezoars) occur in cattle and sheep. Cattle that are maintained on a low-roughage diet, that lick their coats frequently, that have long hair coats from outdoor housing, or that have heavy lice or mite infestations and associated pruritus will often develop bezoars. In addition, younger calves with abomasal ulcers have been found to be more likely to have abomasal tric.hobezoars as well. Clinical signs may be mild or severe according to size, number, and location. Ruminal trichobezoars rarely result in clinical signs. Obstruction will be accompanied by signs of pain, development of bloat, and decreased fecal production. Serum profiles will show hypochloridemia; other imbalances depend on the duration of the problem. Diagnosis is also based on abdominal auscultation, rectal palpation, and ultrasound (useful in calves and smaller ruminants). Treatment is surgical, such as paracostal laparotomy (for abomasal), paralumbar celiotomy with manual breakdown, or enterotomy. Supportive care should be administered as necessary to correct electrolyte imbalances and to prevent inflammation and sepsis. Prognosis is generally good if the condition is diagnosed and treated before dehydration and imbalances become severe and peritonitis develops. Prevention includes providing good-quality roughage and treating lice and mange infestations. Co Traumatic Disorders (Wounds, Bites,

and Entrapped Foreign Bodies) Wounds may be sustained from poorly constructed pens or fences, or from skirmishes among animals. Predators will usu-

ally be sources of bite wounds. Standard veterinary wound assessment and care are essential for wounds or bites. Tetanus antitoxin may be indicated. Use of approved antibiotics may be appropriate. The lesion should be cleaned with disinfectants and repaired with primary closure if it is clean and uncontaminated. Thorough cleaning, regular monitoring, and healing by second intention are recommended for older wounds. Abscesses may also occur in the soft tissues of the hooves (sole abscesses; see Section III,C,3) because of entrapped foreign bodies or hoof cracks that fill with dirt. Preventive measures include improvement of housing facilities, pens, and pastures; monitoring hierarchies among animals penned together; and implementing predator control measures, such as sound fencing, flock guard dogs, or donkeys, in pasture situations.

D.

Iatrogenic Diseases

1. Anaphylactic Reactions Acute anaphylatic reactions in sheep, goats, and cattle are often clinically referable to the respiratory system. Anaphylactic vaccine reactions cause acute lung edema; lungs are the primary site of lesions if collapse and death are sequelae. The animals will also be anxious and shivering and will become hyperthermic. Salivation, diarrhea, and bloat also occur. Immediate therapy must include epinephrine by intravenous infusion at (1 ml of 1:1000 per 50 kg of body weight for goats and 1:10,000 (0.1 mg/ml) or 0.01 mg/kg (about 5 ml) for adult cows.) Furosemide (5 mg/kg) may be beneficial to reduce edema. Prognosis is usually guarded. Recovery can occur within 2 hr.

2. Catheter Sites and Experimental Surgeries In a research environment, catheter sites or experimental surgeries may be sources of iatrogenic infection. Traumatic injuries to peripheral nerves can cause acute lameness. Improper administration of therapeutics can easily cause this type of lameness. Injections given in gluteals or between the semimembranosus and semitendinosus can cause irritation to the sciatic nerve and subsequent lameness. Contraction of the quadriceps results in the limb being pulled forward. Injections in the caudal thigh can damage the peroneal nerve and cause knuckling at the fetlock. Traumatic injury to the radial nerve can result in a "dropped elbow" (Nelson, 1983). Husbandry procedures such as tail docking, castration, dehorning, dosing with a bailing gun, and shearing may result in superficial lesions, dermal infections, or cases of tetanus. Bailing-gun injuries to the pharynx may lead to cellulitis with coughing, decreased appetite, and sensitivity to palpation. Standard veterinary assessment and care are essential for these cases. Local and systemic antibiotics with supportive care may be indicated. Swelling around peripheral nerves caused by inoculations may be reduced by diuretics and anti-inflammato-

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ries. Mild cases of peripheral nerve damage may recover in 714 days. Personnel training, including review of relevant anatomy, preprocedure preparation, appropriate technique, careful surgical site preparation, rigorous instrument sanitation, and sterile technique will minimize the incidence of potential complications from surgical procedures.

albumin values and foaming urine. The proteinuria also distinguishes amyloidosis (and glomerulonephritis) from other causes of weight loss and diarrhea in cattle such as Johne's disease, parasitism, copper deficiency, salmonellosis, and bovine viral diarrhea virus infection. Prognosis is poor, and no treatment is reported. 2. Dental Wear

E.

Neoplastic Diseases

Neoplasia and tumors are relatively rare in ruminants. Lymphosarcoma/leukemia in sheep has been shown to result from infection by a virus related (or identical) to the bovine leukemia virus. Pulmonary carcinoma (pulmonary adenomatosis) and hepatic tumors are found in sheep. Virus-induced papillomatosis (warts), discussed in Section III,A,2,s, and squamous cell carcinomas have also been reported in sheep. In goats, thymoma is one of the two most common neoplasias reported, although no distinct clinical syndrome has been described. Cutaneous papillomas are the most common skin and udder tumor of goats, and although outbreaks involve multiple animals, no wart virus has been identified. Persistent udder papillomas may progress to squamous cell carcinoma. Lymphosarcoma is reported rarely in goats. Although adrenocortical adenomas have been reported frequently and almost exclusively in older wethers, no clinical condition has been described. Lymphosarcoma of various organ systems and "cancer eye" (bovine ocular squamous cell carcinoma, or OSCC) are the most commonly reported cancers in cattle. Lymphosarcoma is described in Section III,A,2,c. Lack of periocular pigmentation and the amount and intensity of exposure to solar ultraviolet light are considered important factors in OSCC. Genetic factors may also play a role. Many cases occur in Herefords. This is a disease of older cattle; no case has been reported in animals less than 4 years of age. The cancer metastasizes through the lymph system to major organs. Treatment in either lymphosarcoma or OSCC is recommended only as a palliative measure. The extent of ocular neoplastic involvement is a significant criterion for carcass condemnation. Papillomatosis (warts) are common in cattle (see Section III,A,2,s).

F. I.

Miscellaneous

Amyloidosis

Amyloidosis in adult cattle is due to accumulations of amyloid protein in the kidney, liver, adrenal glands, and gastrointestinal tract. The disease has been classified as AA type, or associated with chronic inflammatory disease, although other unknown factors are believed to be involved in some cases. Clinical signs include chronic diarrhea, weight loss, decreased production, nonpainful renomegaly, and generalized edema. The loss of protein in the urine contributes to abnormal plasma

Dental wear is seen most commonly in sheep. As sheep age, excessive dental wear may lead to an inability to properly masticate feed, manifesting as weight loss and unthriftiness. Several factors predisposing to dental wear should be considered. The diet should be properly balanced for minerals, especially calcium and phosphorus, because primary or secondary calcium deficiency during teeth development results in softening of the enamel and dentin. Dietary contamination with silica (i.e., hays and grains harvested in sandy regions) will lead to mechanical wear on the teeth. Likewise, animals grazing or being fed in sandy environments will have excessive tooth wear. Sheep older than about 5 years of age are especially prone to tooth wear and should be checked frequently, especially if signs of weight loss or malnutrition are evident. Managing the content and consistency of the diets can best prevent the disease. 3. Sole abscesses Of the ruminants, cows are the most frequently affected by subsolar absesses. Dirt becomes packed into cracks in the horny layer of the sole of the hoof, and contamination eventually extends into the sensitive areas of the hoof, with lameness and infection resulting. Animals maintained in very soiled or muddy conditions, combined with poor hoof care, are more likely affected. Fusobacterium necrophorum is often the pathogen involved. Separation of the animal, supportive care, surgical drainage, and antibiotic treatment are indicated.

REFERENCES

Major References Cited Addressing Husbandry, Management, and Clinical Medicine Advances in sheep and goat medicine (1990). In "VCNA Food Animal Practice" (M. C. Smith, ed.). Saunders, Philadelphia. American Sheep Industry Association (ASIA) (1996). "The Sheep Production Handbook." C&M Press, Denver. Code of Federal Regulations (CFR) (1985). Title 9, Animals and Animal Products, Subchapter A, Animal Welfare. "Current Therapy in Large Animal Theriogenology" (1997). Edited by R. S. Youngquist. Saunders, Philadelphia. "Current Veterinary Therapy: Food Animal Practice" (1986). Vol. 2 (J. L. Howard, ed.). Saunders, Philadelphia. "Current Veterinary Therapy: Food Animal Practice" (1993). Vol. 3 (J. L. Howard, ed.). Saunders, Philadelphia.

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"Current Veterinary Therapy: Food Animal Practice" (1999). Vol. 4 (J. L. Howard and R. L. Smith, eds.). Saunders, Philadelphia. Federation of Animal Science Societies (FASS) (1999). "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching." FASS, Savoy, Illinois. Hawk, C. T., and Leary, S. L. (1995). "Formulary for Laboratory Animals." Iowa State Univ. Press, Ames. Houpt, K. A. (1998). "Domestic Animal Behavior for Veterinarians and Animal Scientists," 3rd ed. Iowa State Univ. Press, Ames. Jain, N. C. (1986). "Schlam's Veterinary Hematology," 4th ed. Lea and Febiger, Philadelphia. Jensen, R., and Swift, B. L. (1982). "Diseases of Sheep." Lea and Febiger, Philadelphia. Jurgens, M. H. (1988). "Animal Feeding and Nutrition," 6th ed. Kendall/Hunt Publ. Co., Dubuque, Iowa. Kaneko, J. J., Harvey, J. W., and Bruss, M. L. (1997). "Clinical Biochemistry of Domestic Animals." Academic Press, San Diego. "Large Animal Clinical Nutrition" (1991). Edited by J. M. Naylor and S. L. Ralston. Mosby, St. Louis. "Large Animal Internal Medicine" (1996). Edited by B. L. Smith. Saunders, Philadelphia. "Livestock Handling and Transport" (1998). Edited by T. Grandin. CAB International, United Kingdom. National Research Council (1981). "Nutrient Requirements of Goats," 7th rev. ed. National Academy Press, Washington, D.C. National Research Council (1985). "Nutrient Requirements of Sheep," 6th rev. ed. National Academy Press, Washington, D.C. National Research Council (1989). "Nutrient Requirements of Dairy Cattle," 6th rev. ed., update. National Academy Press, Washington, D.C. National Research Council (1993). "Managing Global Livestock Genetic Resources." National Academy Press, Washington, D.C. National Research Council (1996a). "Guide for the Care and Use of Laboratory Animals." National Academy Press, Washington, D.C. National Research Council (1996b). "Nutrient Requirements of Beef Cattle," 7th rev. ed. National Academy Press, Washington, D.C. Plumb, D. C. (1999). "Veterinary Drug Handbook." Iowa State Univ. Press, Ames. Radostits, O. M., Blood, D. C., and Gay, C. C. (1994). "Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats, and Horses." Bailliere Tindall, Philadelphia. Ross, C. V. (1989). "Sheep Production and Management." Prentice-Hall, Englewood Cliffs, New Jersey Smith, M. C., and Sherman, D. M. (1994). "Goat Medicine." Lea and Febiger, Philadelphia. U.S. Department of Agriculture (USDA) (2000). Animal and Plant Health Inspection Service (APHIS), Policy #29, Farm Animals Used for Nonagricultural Purposes, February 11. . Williams, C. S. E (1995). Goats. In "The Experimental Animal in Biomedical Research" (B. E. Rollin, and M. L. Kesel, eds.), Vol. 2. CRC Press, Boca Raton, Florida.

Additional References Cited or Used in the Preparation of This Chapter Anderson, M., and Blanchard, E (1989.) The clinical syndromes caused by Salmonella infection. Vet. Med. 84(8), 816-819. Armed Forces Institute of Pathology (AFIP) (1995). "Animal Models of Human Disease." Registry of Comparative Pathology, Washington, D. C. Arora, A. K. (1983). The effect of stress on the carrier state of Salmonella typhimurium in goats. Vet. Arhiv. 53, 181-187.

Auza, N. J., Olson, W. G., Murphy, M. J., and Linn, J. G. (1999). Diagnosis and treatment of copper toxicosis in ruminants. J. Am. Vet. Med. Assoc. 214, 1624-1628. "Bibliography of Induced Models of Human Disease" (1981b). Edited by G. Hegreberg, and C. Leathers, Washington State Univ., Pullman. Students Book Corp., N.E. 700 Thatuna St., Pullman WA 99163. "Bibliography of Naturally Occurring Models of Human Disease" (1981a). Edited by G. Hegreberg, and C. Leathers. Washington State Univ., Pullman. Students Book Corp., N.E. 700 Thatuna St., Pullman WA 99163. Blackwell, T. E., and Butler, D. G. (1992). Clinical signs, treatment, and postmortem lesions in dairy goats with enterotoxemia: 13 cases. J. Am. Vet. Med. Assoc. 200, 214-217. Bretzlaff, K. (1997). Control of the estrous cycle. In "Current Therapy in Large Animal Theri0genology" (R. S., Youngquist, ed.), pp. 510-514. Saunders, Philadelphia. Bretzlaff, K., Haenlein, G., and Huston E. (1991). The goat industry: feeding for optimal production. In "Large Animal Clinical Nutrition" (J. M. Naylor and S. L. Ralston, eds.), pp. 339-350. Mosby, St. Louis. Brewer, B. D. (1983). Neurologic disease in sheep and goats. Vet. Clin. North Am. Large Anim. Pract. 6, 677-700. Briggs, H. M., and Briggs, D. M. (1980). "Modern Breeds of Livestock," 4th ed. Macmillan, New York. Broad, T. E., Hill, D. E, Maddox, J. E, Montgomery, G. W., and Nicholas, E W. (1998). The sheep gene map. ILAR J. 39, 160-170. Brogden, K. A., Lekmkuhl, H. D., and Cutlip, R. C. (1998). Pasteurella haemolytica complicated respiratory infections in sheep and goats. Vet. Res. 29, 233-254. (Abstract). Brooks, D. L., Tillman, P. C., and Niemi, S. M. (1987). Ungulates as laboratory animals. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.). Academic Press, Orlando. Bulgin, M. S. (1986). Diagnosis of lameness in sheep. Compend. Contin. Educ. Pract Vet. 8, F122-F128. Buttar, H. S. (1997). An overview of the influence of ACE inhibitors on fetalplacental circulation and perinatal development. Mol. Cell. Biochem. 176(1-2), 61-71. Buxton, D. (1998). Protozoan infections (Toxoplasma gondii, Neospora caninum, and Sarcocystis spp.) in sheep and goats: recent advances. Vet. Res. 29, 289-310. (Abstract). Cibelli, J. B., Stice, S. L., Golueke, E J., Kane, J. J., Jerry, J., Blackwell, C., Ponce de Leon, E A., and Robl, J. M. (1998a). Cloned transgenic calves produced from nonquiescent fibroblasts. Science 280, 1256-1258. Cibelli, J. B., Stice, S. L., Golueke, E J., Kane, J. J., Jerry, J., Blackwell, C., Ponce de Leon, E A., and Robl, J. M. (1998b). Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells. Nat. Biotechnol. 16, 642-646. Corbeil, L. B. (1995). Use of an animal model of trichomoniasis as a basis for understanding this disease in women. Clin. Infect. Dis. 21(Suppl. 2), S 158S161. Council report: vaccination guidelines for small ruminants (sheep, goats, llamas, domestic deer, and wapiti) (1994). J. Am. Vet. Med. Assoc. 205,15391544. de la Concha Bermeiillo, A. (1997). Maedi-visna and ovine progressive pneumonia. Vet. Clin. North Am. Food Anim. Pract. 13, 13-33. (Abstract). Dorchies, P., Curanton, C., and Jacquiet, P. (1998). Pathophysiology of Oestrus ovis infection in sheep and goats: A review. Vet. Rec. 142, 487-489. Dougherty, R. W. (1981). "Experimental Surgery in Farm Animals." Iowa State Univ. Press, Ames. Dubash, K., Shulaw, W. P., Bech-Nielsen, S., Stills, H. E, Jr., and Slemons, R. D. (1996.) Evaluation of an agar gel immunodiffusion test kit for detection of antibodies to Mycobacterium paratuberculosis in sheep. J. Am. Vet. Med. Assoc. 208, 401-403. (Abstract). Duncan, J. R., and Prasse, K. W. (1986). "Veterinary Laboratory Medicine." Iowa State Univ. Press, Ames. Ebert, K. M., DiTullio, P., Barry, C. A., Schindler, J. E., Ayres, S. L., Smith,

14. BIOLOGY AND DISEASES OF RUMINANTS: SHEEP, GOATS, AND CATTLE T. E., Pellerin, L. E., Meade, H. M., Denman, J., and Roberts, B. (1994). Induction of human tissue plasminogen activator in the mammary gland of transgenic goats. Bio/Technology 12, 699-702. Ellis, J. A. (1984). Pasteurella haemolytica infections in sheep. Compend. Contin. Educ. Pract. Vet. 6, $360-$366. Foreyt, W. (1990). Coccidiosis and cryptosporidiosis in sheep and goats. Vet. Clin. North Am. Food Anim. Pract. 6(3), 655-670. Franz-Werner, S., Maddox, J., Ballingall, K., Buitkamp, J., Crawford, A. M., Dutia, B. M., Epplnr, J. T., Ferguson, E. D., Groth, D., Hopkins, J., Rhind, S. M., Sargan, D., Wetherall, J., and Wright, H. (1996). The ovine major histocompatibility complex. In "The Major Histocompatibility Complex Region of Domestic Animal Species" (L. B. Schook and S. J. Lamont, eds.), pp. 121-176. CRC Press, Boca Raton, Florida. Garin-Bastuji, B., Blasco, J. M., Grayon, M., and Verger, J. M. (1998). Brucella melitensis infection in sheep: present and future. Vet. Res. 29, 255-274. (Abstract). Garrick, M. D. (1983). Hemoglobin switching. In "Red Blood Cells in Domestic Animals" (N. S. Agar and P. G. Board, eds.), pp. 209-226. Elsevier, Amsterdam. Genetzky, R. M. (1995). Epididymitis in rams. Compend. Contin. Educ. Pract. Vet. 17, 447. Gerloff, B. J., and Herdt, T. H. (1999). Fatty liver in dairy cattle. In "Current Veterinary Therapy: Food Animal Practice" (J. L. Howard and R. L. Smith, eds.). Saunders, Philadelphia. Grandin, T. (1988). Livestock Handling Guide: Management Practices that Reduce Livestock Bruises and Injuries, and Improve Handling Efficiency.

Livestock Conservation Institute, Madison, WI. Greyling, J. P. C., and Van Niekerk, C. H. (1986). Synchronization of oestrus in the Boer goat doe: Dose effect of prostaglandin in the double injection scheme. South Afr. J. Anim. Sci. 16, 146. Habel, R. E. (1975). "Guide to the Dissection of Domestic Ruminants." Published by the author, Ithaca, New York. Hafez, E. S. E. (1987). "Reproduction in Farm Animals," 6th ed. Williams and Wilkins, Baltimore. Hanly, W. C., Artwohl, J. E., and Bennett, B. T. (1995). Review of polyclonal antibody production procedures in mammal and poultry. In "Adjuvants and Antibody Production." ILAR J. 37, 93-118. Hays, J. T., Suckow, M. A., Jackson, G. E., and Douglas, E E. (1998). Considerations in the design and construction of facilities for farm species. Lab. Anita. 27(6), 22-25. Heath, S. E., and Johnson, R. (1994). Clinical update: leptospirosis. J. Am. Vet. Med. Assoc. 205, 1518-1523. Hecker, J. F. (1983). The Sheep as an Experimental Animal. Academic Press, London. Hegreberg, G., and Leathers, C. (1980a). Bibliography oflnduced Animal Models of Human Disease. Pullman. Hegreberg, G., and Leathers, C. (1980b). Bibliography of Naturally Occurring Models of Human Disease. Pullman. House, J. K. (1996). Postpartum assessment and care of the newborn ruminant. In "Large Animal Internal Medicine" (B. L. Smith, ed.), pp. 321-322. Saunders, Philadelphia. Hutt, E B. 1964. "Animal Genetics." Ronald Press Co., New York. ILAR, NRC. 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. Jackson, C. A., Rebhun, W. C., and McDonough, P. L. (1995). Blackleg: A new perspective on an old disease. Compend. Contin. Educ. Pract. Vet. 17, 1299. Janke, B. H. (1989). Protecting calves from viral diarrhea. Vet. Med. 84(8), 803-810. Johnson, R., and Kaneene, J. B. (1993a). Bovine leukemia virus. Part 1: Descriptive epidemiology, clinical manifestations, and diagnostic tests. In "Infectious Disease in Food Animal Practice," pp. 122-129. Compendium Collection, Veterinary Learning Systems, Trenton. Johnson, R., and Kaneene, J. B. (1993b). Bovine leukemia virus. Part 2: Risk

613

factors of transmission. In "Infectious Disease in Food Animal Practice," pp. 131-140. Compendium Collection, Veterinary Learning Systems, Trenton. Johnson, R., and Kaneene, J. B. (1993c). Bovine leukemia virus. Part 3: Zoonotic potential, molecular epidemiology, and an animal model. In "Infectious Disease in Food Animal Practice," pp. 141-148. Compendium Collection, Veterinary Learning Systems, Trenton. Johnson, R., and Kaneene, J. B. (1993d). Bovine Leukemia Virus. Part 4: Economic Impact and Control Measures. In "Infectious Disease in Food Animal Practice," pp. 149-157. Compendium Collection, Veterinary Learning Systems, Trenton. Kahler, S. C. (1998). Brucella abortus strain RB51 vaccine: its advantages and risks. J. Am. Vet. Med. Assoc. 213, 21-22. Kasari, T. R., and Roussel, A. J. 1999. Omphalitis and its sequelae in ruminants. In "Current Veterinary Therapy: Food Animal Practice" (J. L. Howard and R. L. Smith, eds.), Vol. 4, pp. 62-65. Saunders, Philadelphia. Kendrick, K. M., Da Costa, A. P. C., Broad, K. D., Ohkura, S., Guevara, R., Levy, E, and Keverne, E. B. (1997). Neural control of maternal behavior and olfactory recognition of offspring. Brain Res. Bull. 44, 383-395. Kent, J. E., Molony, V., and I. S. Robertson. (1995). Comparison of the Burdizzo and rubber ring methods for castrating and tail docking lambs. Vet. Rec. 136, 192-196. Kersting, K. (1997). Postpartum care of the cow and calf. In "Current Therapy in Large Animal Theriogenology" (R. S. Youngquist, ed.). Saunders, Philadelphia. Kimberling, C. V., and Ellis, R. P. (1990). Advances in the control of foot rot in sheep. Vet. Clin. North Am. Food Anim. Pract. 6(3), 671-681. Kirk, J. H., and Glenn, J. S. (1996). Mastitis in ewes. Compend. Contin. Educ. Pract. Vet. 18, 582. Kirkpatrick, C. E. (1989). Giardiasis in large animals. Compend. Contin. Educ. Pract. Vet. 11, 80-84. Knowles, D. P., Jr., (1997). Laboratory diagnostic tests for retrovirus infections of small ruminants. Vet. Clin. North Am. Food Anim. Pract. 13, 1-11. (Abstract). Kott, R. W., Thomas, V. M., Hatfield, P. G., Evans, T., and Davis, K. C. (1998). Effects of dietary vitamin E supplementation during late pregnancy on lamb mortality and ewe productivity. J. Am. Vet. Med. Assoc. 212, 9971000. (Abstract). Kuhn, E. (1993). [Myotonia congenita (Thomsen) and recessive myotonia (Becker)]. Nervenarzt 64, 766-769. Lasgard, A. G., and Vaabenoe, A. (1993.) Genetic and environmental causes of variation in mastitis in sheep. Small Ruminant Res. 12(3), 339-347. Lehman, M. N., Goodman, R. L., Karsch, E J., Jackson, G. L., Berriman, S. J., and Jansen, H. T. (1997). The GnRH system of seasonal breeders: anatomy and plasticity. Brain Res. Bull. 44, 445-457. Lewin, H. A. (1996). Genetic organization, polymorphism, and function of the bovine major histocampaticulity complex. In "The Major Histocompatibility Complex Region of Domestic Animal Species" (L. B. Schook and S. J. Lamont, eds.). CRC Press, New Yqrk. Linnabary, R. D., Hall, R. E, Wilson, R. B., and Knowles, A. M. (1991). Scrapie in sheep. Compend. Contin. Educ. Pract. Vet. 13, 511-516. Lovell, K. L., Matsuura, E, Patterson, J., Baeverfjord, G., Ames, N. K., and Jones, M. Z. (1997). Biochemical and morphological expression of early prenatal caprine beta-mannosidosis. Prenatal Diagn. 17, 551-557. Martin, W. B., (1996). Respiratory infections of sheep. Comp. Immunol. Microbiol. Infect. Dis. 19, 171-179. (Abstract). McAllister, M. M., Dubey, J. P., and Lindsay, D. S. (1998). Dogs are the definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473-1478. McMartin, D. A., MacOwan, K. J., and Swift, L. L. (1980). A century of classical contagious caprine pleuropneumonia from original description to aetiology. Br. Vet. J. 136, 507-515. Melling, M., and Alder, M. (1998). "Sheep and Goat Practice 2." Saunders, Philadelphia. Memon, M. A., and Ebert, K. M. (1992). Gene manipulation in goats through

614

SCOTT A. MISCHLER, WENDY J. UNDERWOOD, AND MARGARET L. DELANO

biotechnology. In "Pre-Conference Proceedings of Veterinary International Conference on Goats" (R. R. Lokeshwat, ed.). IGA, New Delhi, India. Moore, D. A. (1989). Minimizing morbidity and mortality from cryptosporidiosis. Vet. Med., 811-815. Moore, D. A., and Ishler, V. (1997). Managing dairy cows during the transition period: focus on ketosis. Vet. Med. 92(12), 1061-1072 Morrow-Tesch, J. (1997). Environmental enrichment for dairy calves and pigs. AWIC Newsl. 7, 3-8. Muranjan, M., Wang, Q., Li, Y., Hamilton, E., Otieno-Omondi, E P., Wang, J., Van Praagh, A., Grootenhuis, J. G., and Black, S. J. 1997. The trypanocidal Cape buffalo serum protein is xanthine oxidase. Infect. Immun. 65, 38063814. Nappert, G., Zello, G. A., and Naylor, J. M. (1997). Oral rehydration therapy for diarrheic calves. Compend. Contin. Educ. Pract. Vet. 19(8), S181-S189. Naylor, J. M. (1996). Neonatal ruminant diarrhea. In "Large Animal Internal Medicine" (B. L. Smith, ed.), pp. 396-417. Saunders, Philadelphia. Nebel, R. L. (1997). Techniques for artificial insemination of cattle with frozenthawed semen. In "Current Therapy in Large Animal Theriogenology" (R. S. Youngquist, ed.). Saunders, Philadelphia. Nelson, D. (1983). Noninfectious causes of lameness. Vet. Clin. North Am. Large Animal Pract. 5(3), 491-497. Neosporosis: its prevalence and economic impact. (1998). Compend. Contin. Educ. Pract. Vet. Suppl. 20, 11 (D). Obwolo, M. J. (1976). A review of yersinosis (Yersinia pseudotuberculosis infection). Vet. Bull. 46, 167-171. Ott, R. S. (1982). Dairy goat reproduction. Compend. Contin. Educ. Pract. Vet. 4, S 164-S 172. Pepin, M., Vitu, C., Russo, P., Mornex, J. E, and Peterhans, E. (1998). Maedivisna virus in sheep: a review. Vet. Res. 29, 341-367. (Abstract). Piontkowski, M. D., and Shivvers, D. W. (1998). Evaluation of a commercially available vaccine against Corynebacterium pseudotuberculosis for use in sheep. J. Am. Vet. Med. Assoc. 212, 1765-1768. (Abstract). Pugh, D. G., Hilton, C. D., and Mobini, S. M. (1998). Ruminant production management: control programs for gastrointestinal nematodes in sheep and goats. Compend. Contin. Educ. Pract. Vet. 20, S112-S123. Rees, S., Mallard, C., Breen, S., Stringer, M., Cock, M., and Harding R. (1998). Fetal brain injury following prolonged hypoxemia and placental insufficiency: a review. Comp. Biochem. Physiol. AmMol. Integrative Physiol. 119, 653-660. Rings, D. M., and Rings, M. B. (1996). Managing Cryptosporidium and Giardia infections in domestic ruminants. Vet. Med. 91(12), 1125-1131. Rings, M. B., Rings, D. M., and Welker, B. (1997). Milk fever: seeking new solutions to an old problem. Compend. Contin. Educ. Pract. Vet. 19(8), S175-S180. Rodolakis, A., Salinas, J., and Papp, J. (1998). Recent advances on ovine chlamydial abortion. Vet. Res. 29, 275-288. (Abstract). Rohrbach, B. W., Cannedy, A. L., Freeman, K., and Slenning, B. D. (1999). Risk factors for abomasal displacement in dairy cows. J. Am. Vet. Med. Assoc. 214, 1660-1663. Rook, J. S., Kopcha, M., Spaulding, K., Coe, P., Benson, M., Krehbiel, J., and Trapp, A. L. (1986). The spider syndrome: a report on one purebred flock. Compend. Contin. Educ. Pract. Vet. 8, $402-$405. Ross, M. G., and Nijland, M. J. (1998). Development of ingestive behavior. Am. J. Physiol. 274 (4, Part 2), R879-R893. Salerno, C. T., Droel, J., and Bianco, R. W. (1998). Current state of in vivo preclinical heart valve evaluation. J. Heart Dis. 7, 158-162. Scanlan, C. M., Garrett, P. D., and Geiger, D. B. (1984). Dermatophilus congolensis infections in cattle and sheep. Compend. Contin. Educ. Pract. Vet. 8, F52-F58. Scarratt, W. K. (1987). Ovine listeric encephalitis. Compend. Contin. Educ. Pract. Vet. 9, F28-F32. Schillhorn van Veen, T. W. (1986). Coccidiosis in ruminants. Compend. Contin. Educ. Pract. Vet. 8, F52-F58.

Schmidt, G. H., Van Vleck, L. D., and Hutjens, M. E (1988). "Principles of Dairy Science," 2nd ed. Prentice-Hall, Englewood Cliffs, New Jersey. Schnieke, A. K., Kind, A. J., Ritchie, W. A., Mycock, K., Scott, A. R., Ritchie, M., Wilmut, I., Colman, A., and Campbell, K. H. S. (1997). Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278(5346), 2130-2136. Schook, L. B., and Lamont, S. J. (1996). Future perspectives. In "The Major Histocompatibility Complex Region of Domestic Animal Species" (L. B. Schook and S. J. Lamont, eds.), p. 29. CRC Press. New York. Smith, M. C. (1997). Clinical reproductive anatomy and physiology of the doe. In "Current Therapy in Large Animal Theriogenology" (R. S. Youngquist, ed.), pp. 505-507. Saunders, Philadelphia. Sordillo, L. M., Sharer-Weaver, K., and DeRosa, D. (1997). Immunobiology of the mammary gland. J. Dairy Sci. 80, 1851-1865. Speer, C. A. (1996). Coccidiosis. In "Large Animal Internal Medicine" (B. L. Smith, ed.). Saunders, Philadelphia. Stevens, M. G., Olsen, S. C., Palmer, M. V. (1997) Brucella abortus strain RB51: a new brucellosis vaccine for cattle. Compend. Contin. Educ. Pract. Vet. 19, 766-774. Tessman, R. K., Tyler, J. W., Parish, S. M., Johnson, D. L., Gant, R. G., and Grasseschi, H. A. (1997). Use of age and serum gamma-glutamyltransferase activity to assess passive transfer status in lambs. J. Am. Vet. Med. Assoc. 211, 1163-1164. (Abstract). Thurmond, M. C., and Hietala, S. K. (1997). Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle. Am. J. Vet. Res. 58, 1381-1385. Trower, C. J. (1993). Artificial control of breeding in ewes. Compend. Contin. Educ. Pract. Vet. 15, 642-645. Underwood, W. J., and Rook, J. S. (1992). Toxoplasmosis infection in sheep. Compend. Contin. Educ. Pract. Vet. 14, 1543-1548. Vanderboom, R. J., McCauley, T. C., Tappan, R., and Ax, R. L. 1997. Bovine reproductive biotechnology. In "Current Therapy in Large Animal Theriogenology" (R. S. Youngquist, ed.), pp. 457-473. Saunders, Philadelphia. Walker, E. P., Nowak, R. M., and Paradiso, J. L. (1983). "Mammals of the World." Johns Hopkins Univ. Press, Baltimore. Wall, R. J., Kerr, D. E., and Bondoli, K. R. (1997). Transgenic dairy cattle. Genetic engineering on a large scale. J. Dairy Sci. 80, 2213-2224. Watkins, G. H., and Jones, J. E. T. (1992). The effect of intra-mammary inoculation of lactating ewes with Pasteurella haemolytica isolates from different sources. J. Comp. Pathol., 455-564. Weaver, A. D. (1986). "Bovine Surgery and Lameness." Blackwell Scientific, Boston. Weil, J., Eschenhagen, T., Magnussen, O., Mittman, C., Orthey, E., Scholz, H., Scafrer, H., and Scholtysik, G. (1997). Reduction of myocardial myoglobin in bovine dilated cardiomyopathy. J. Mol. Cell. Cardiol. 29, 743-751. Welch, R. D., Ashman, R. B., Baker, K. J., and Browne, R. H. (1996). Intraosseous infusion of prostaglandin E2 prevents disuse-induced bone loss in the tibia. J. Orthop. Res. 14, 303-310. Wenzel, J. G. W. (1997). Estrous cycle synchronization. In "Current Therapy in Large Animal Theriogenology" (R. S. Youngquist, ed.), pp. 290-294. Saunders, Philadelphia. Wikse, S. E., and Baker, J. C. (1996). The bronchopneumonias (respiratory disease complex of cattle, sheep, and goats). In "Large Animal Internal Medicine" (B. L. Smith, ed.), pp. 632-654. Saunders, Philadelphia. Womack, J. E. (1998). The cattle gene map. In "Comparative Gene Mapping." Inst. Lab. Anim. Res. J. 39, 153-159. Zajac, A. M., and Moore, G. A. (1993). Treatment and control of gastrointestinal nematodes in sheep. Compend. Contin. Educ. Pract. Vet. 15, 999-1009. Zeman, D. H., Thomson, J. U., and Francis, D. H. (1989). Diagnosis, treatment, and management of enteric colibacillosis. Vet. Med. 84(8), 794-802.

Chapter 15 Biology and Diseases of Swine Kathy E. Laber, Mark T. Whary, Sarah A. Bingel, James A. Goodrich, Alison C. Smith, and M. Michael Swindle

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Introduction .................................................

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L a b o r a t o r y M a n a g e m e n t and H u s b a n d r y

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U s e in R e s e a r c h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Biology

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Nutrition

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INTRODUCTION

Swine have been increasingly used in biomedical research both as general large-animal biological models in teaching and research, and for the study of specific disease conditions due to their anatomic and physiologic characteristics (Swindle, 1998). Textbooks specific to the use of swine as laboratory animals are available (Swindle, 1998, 1983; Stanton and Mersmann, 1986; Bollen, Hansen, and Rasmussen, 2000). Proceedings books from symposia on the use of swine in research are also available (Swindle, 1992; Tumbleson, 1986; Tumbleson and Schook, 1996). LABORATORY ANIMAL MEDICINE, 2nd edition

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Taxonomy

Order: Artiodactyla (even-toed ungulates) Family: Suidae Species: Sus scrofa domestica

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Availability and Sources

Commercial breeds of domestic swine raised for meat production are readily available worldwide. There is a wide variability in the health status of the various herds. In the United Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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States the designation SPF (specific pathogen-free) has a proprietary connotation. It is a program based on management procedures that reduce or eliminate diseases that stunt growth. Pigs designated SPF are a good source for biomedical research; however, it must be remembered that the designation does not mean that the animals are completely free of diseases that may interfere with research. It is best to buy animals from a herd in which the institutional veterinarian has screened for complicating diseases. Commercial breeds have limited availability from commercial suppliers of laboratory animals (Saffron and Gonder, 1997; Swindle et al., 1994). When using domestic breeds of swine, the growth factors are a major consideration. Swine reach sexual maturity and a commercial slaughter weight of approximately 250-280 kg at 5 - 6 months of age. At birth, swine weigh approximately 3 kg (average); consequently, there is an exponential growth phase during the adolescent period. Most swine used in research programs are 15-30 kg and are 8-12 weeks of age. Weight gain during this period may be 2 - 5 kg per week. When selecting a model, the age and maturity factors must also be considered. Consequently, domestic swine are rarely used for long-term projects unless the study includes the effect of growth and maturity factors or the animals are involved in agricultural research. Generally, most projects involving a length of > 3 weeks would best be performed in miniature swine (Swindle, 1998; Swindle et al., 1994; Fisher, 1993). Miniature swine are available from commercial breeders of laboratory animals. Commonly used breeds include Yucatan, Hanford, Sinclair, Hormel, and Gottingen. Other breeds of miniature pigs are available in limited quantities from some market areas and include the Panepinto, Vietnamese potbellied, Ohmini, Pitman-Moore, and Chinese dwarf. Generally, the health status of these animals is higher than that of SPF animals, and they are suitable for most biomedical research projects. These animals range from 30 to 50 kg in body weight at sexual maturity and, consequently, are more amenable than larger commercial breeds to long-term projects (Swindle, 1998; Fisher, 1993; Panepinto, 1986).

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Laboratory Management and Husbandry

Individual shipments of swine are best separated by time and distance and in particular, mixing animals from multiple vendors is poor practice. Swine should be purchased from vendor herds that are validated brucellosis-free and qualified pseudorabies-negative by the U.S. Department of Agriculture (USDA). Commercial sources typically implement a vaccination and parasite-control program beginning at weaning age and dependent on the intended experimental use of the animal, such efforts may or may not need additional attention at the research facility. Quality source herds will worm piglets at 4- to 6-week intervals and administer preventive treatments for ectoparasites.

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Weanling animals are commonly vaccinated against erysipelas and leptospirosis, and breeding-herd animals should be vaccinated in addition against porcine parvovirus, Bordetella bronchiseptica, Pasteurella multocida, and Escherichia coli. Newly received animals should be given a minimum of 72 hr to adjust to the new environment during which time physical exams and screening tests for parasites can be performed. Diet changes should be gradual over several days, with fiber increased if stress-induced diarrhea develops. Adult swine that are housed long-term should have, at a minimum, periodic physical exams that include weight and parasite checks. Vaccination programs for adult swine should be implemented based on risk assessment that considers how the animal will be used in research, what the housing conditions are, and how close the research herd is to new animals of uncertain health status. Ideally, pigs should be purchased from one source of established health status to take advantage of natural herd immunity. The value of good herd health management is illustrated by the observation that swine herds that maintain specific pathogen-free status have an odds ratio of 0.2 relative to that of conventional herds for the development of diarrhea (Moiler et al., 1998). Swine are best housed in pens rather than cages. Pens may be constructed of either chain-link fencing or stainless steel or aluminum bars. Wood is best avoided because of pigs' ability to chew it and the difficulty of sanitation. The chosen material should be of sturdy construction because swine can be very destructive. It is best to provide them with indestructible toys or balls to preoccupy them and to satisfy their rooting instincts (Swindle, 1998). Flooring for swine deserves special consideration. Smooth flooring, such as seamless epoxy, is best avoided. Swine have difficulty with firm footing on these floors, especially when the floors are wet. If contact flooring is used, it should either have a rough surface to provide traction and provide wear on the hooves or it should be covered with deep wood-chip bedding. Wood-chip bedding keeps swine clean and satisfies their rooting instinct. However, wood-chip bedding is eaten by swine, especially when they are fasted. Raised flooring has been found to be satisfactory in many laboratory situations. Plastic-coated metal grids are sturdy and easy to sanitize. However, if a cut becomes apparent in the plastic, swine will strip the flooring and eat the plastic. Slatted fiberglass floors with grit to provide hoof wear are generally ideal in most situations. They are lightweight and easy to remove from pens for sanitation (Swindle, 1998). Swine readily use automatic watering systems. The system should be checked daily to ensure that the water supply is functional because swine are susceptible to "salt poisoning," which results in a neurologic syndrome when they are deprived of water. Food dishes should be secured to the cage or flooring. Swine will tip movable dishes and lose their feed, especially on raised flooring. They will also chew their feeders, which are best made of an indestructible material such as stainless steel (Swindle, 1998).

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Swine prefer to have contact with other members of their species. They may be housed together in groups, but dominance fighting will occur unless animals are socialized. This social instinct may also be satisfied by providing cage walls that allow visual and snout contact between animals (Swindle, 1998; Fisher, 1993; Panepinto, 1986). Swine can be restrained in slings, such as the Panepinto sling (Panepinto et al., 1983). This method of humane restraint is preferable to agricultural methods such as snout tying. Small swine can also be restrained manually in a manner similar to that of dogs. Swine may be trained to walk on a leash and can also be restrained against the side of the cage with movable handheld panels (Swindle, 1998). Intramuscular injections may be administered in the neck or hindlimb. Venous access sites include the following veins: auricular, cephalic, external and internal jugular, precava, lateral saphenous, cranial abdominal, and femoral (Figs. 1-9). Most of the peripheral vessels are deep and not visible; consequently, a knowledge of their anatomic location is essential. Most of the vessels can be accessed with standard-sized needles and a

Fig. 2.

Venipunctureof the digital vein.

20-gauge 1.5-inch needle is the largest size that will be required in swine up to 50 kg (Swindle, 1998; Bobbie and Swindle, 1986). Surgical procedures, anesthesia, and anatomy, including surgical approaches for vascular access and fistulation procedures, are described in detail in other references (Swindle, 1998).

D.

Fig. 1.

Venipunctureof the cephalic vein.

U s e in R e s e a r c h

Swine have been used mainly for research involving the cardiovascular system because of their unique anatomy and physiology, which makes them similar to humans (Swindle, 1998; Stanton and Mersmann, 1986). Cardiovascular diseases of interest include atherosclerosis, coronary arterial stenosis and infarction, congenital heart disease, volume- and pressureoverload heart failure, electrophysiology, and testing of grafts, stents, and interventional devices. Swine are uniquely susceptible to atherosclerosis, which may be induced by feeding of cholesterol and fat-enhanced diets. A more rapid form of atherosclerosis may be induced by damaging the endothelium with a

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organs, the surgical anatomy, and response to immunosuppressive therapy make them ideal for many of these studies. Swine are being developed as models and donors for xenotransplantation, which has included the development of transgenic strains (Swindle, 1998a). The anatomic and physiologic characteristics of the skin have made swine a definitive plastic surgical model. Swine have also been developed as models in a wide variety of other surgical procedures, including fetal surgery and procedures in the musculoskeletal, central nervous, gastrointestinal, urogenital, and cardiopulmonary systems. Many other biological models have been developed in swine, including the areas of systemic and dermal toxicology, septic and hemorrhagic shock, immunology, diabetes, malignant melanoma, malignant hyperthermia, and gastric ulceration. An exhaustive list of all the developed and potential models in swine is beyond the scope of this chapter. Extensive reviews of that information may be found in general reference and proceedings books.

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Fig. 3.

Venipuncture of the tail vein.

balloon catheter. The induced form has the advantage of producing a lesion in a specific anatomic region. Genetic models of high membranous ventricular septal defect (VSD) and von Willebrand's disease are also available (Swindle et al., 1996). Nutritional and gastrointestinal models in swine are studied because of the physiology of their digestion, which is similar to that of humans, and their omnivorous diet. Areas studied include nutrient absorption and growth, gastrointestinal transport, hepatic metabolism, total parenteral nutrition, and necrotizing enterocolitis. Renal diseases are another area of interest in research. Swine have been used in studies of renal hypertension, vesicoureteral reflux, intrarenal reflux, and urinary obstruction. Swine have been increasingly used in research and teaching studies that involve surgery, both as a substitute for dogs and as a model based on physiologic characteristics (Swindle, 1986). Swine are the model of choice for most of the laparoscopic and endoscopic procedures because of their size and anatomy. Catheter delivery of interventional devices has also been studied extensively in swine. Transplantation research has been performed on heart, lung, liver, kidney, and viscera. The size of the

BIOLOGY

Unique Physiologic Characteristics and Attributes

References with complete descriptions of swine anatomy and physiology are available (Swindle, 1998). However, some of the unique characteristics of swine will be covered in this section. The cardiovascular system is similar to that of humans, especially the coronary anatomy (Swindle, 1998). The blood supply from the coronary artery is right-side dominant and does not have preexisting collateral circulation. This makes the coronary blood flow situation similar to 90% of that of the human population, unlike that in other species such as the dog. The electrophysiologic system is more neurogenic than myogenic, and there are prominent Purkinje fibers. The left azygous (hemiazygous) vein drains the intercostal vessels into the coronary sinus unlike in most other species. This vessel may be ligated or blocked with a balloon catheter to provide total coronary venous drainage into the coronary sinus. The aorta has a true vaso vasorum like that of humans. Normal values for hematology and serum chemistry for Hanford miniature swine are listed in Table I, and Table II contains data on serum chemistry and urine physiology for Yucatan micropigs and domestic swine. The gastrointestinal tract has unique anatomic characteristics (Swindle, 1998). The stomach has a muscular outpouching, the torus pyloricus, near the pylorus. The bile duct and pancreatic duct enter the duodenum separately in the proximal portion. The anatomic divisions between the duodenum, ileum, and jejunum are indistinct. The mesentery is thin and friable. The mesenteric branches form their vascular arcades in the subserosa rather than in the mesentery as in other species. The

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15. B I O L O G Y AND DISEASES OF SWINE

Fig. 4.

Venipuncture of the mammary vein.

majority of the large intestine is arranged in a spiral colon in the left upper quadrant of the abdomen. This series of centrifugal and centripetal coils includes the cecum and ascending, transverse, and majority of the descending colon. Tenia and haustra are present on the cecum and large intestine. In spite of the

Fig. 5.

anatomic differences from humans, the physiology of digestion and intestinal transport are very similar. Other unique anatomic features need to be considered (Swindle, 1998). The lymph nodes are inverted with the germinal centers being located in the internal portion of the node. The

Venipuncture of the anterior vena cava.

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Fig. 6.

Venipunctureof the externaljugular vein.

thymus is located on the ventral midline of the trachea near the thoracic inlet rather than proximal to the larynx. A major portion of the thymus is located in the neck, and the single pair of parathyroid glands is located in the medial aspect of the proximal portion of this gland. The penis is fibromuscular with a corkscrew-shaped tip located in a preputial diverticulum near the umbilicus. The penis has a sigmoid flexure. The male accessory glands include the ductus deferens, prostate, vesicular gland, and bulbourethral gland. The female reproductive system is bicornuate with lengthy tortorus fallopian tubes. The pancreas is bilobed and surrounds and encompasses the superior mesenteric vein. The liver is divided into lobules by microscopic fibrous septae. The cytochrome P450 system is similar to that in humans.

B.

Nutrition

A comprehensive text on swine nutrition has been published (Lewis and Southern, 2000). There is considerable variation of the genetic capacity for accretion of lean body mass among the various breeds of swine utilized in biomedical research. The "farm swine" include breeds developed for meat production and at 6 months of age may have a lean body weight 5- to 6-fold greater than that of a micropig breed. The published research on swine nutrition is focused on farm swine and maximization of lean growth (Table III). The majority of mini- and microswine nutritional research is proprietary and is reflected in the com-

mercially available formulations offered by feed companies. In general, the nutrient requirements of these breeds are similar; however, the small breeds often require fixed-quantity feeding to control obesity, especially for long-term research studies. This in turn necessitates a higher margin of safety for many nutrient concentrations to prevent deficiencies, since most commercially available diets are designed for free-choice feeding. Diets formulated for the mini- and microbreeds generally have lower-energy and higher-fiber concentrations. The daily energy (Fig. 10) and quantity of feed (Fig. 11) required by farm pigs will predispose the mini- and microbreeds to obesity. One nutrient requirement that is particularly important for newborn piglets is iron. Nursing piglets require 21 mg of iron for each kilogram of growth (National Research Council [NRC], 1998), and sow's milk contains approximately 1 mg of iron per liter (Brady et al., 1978). Therefore, a microcytic, hypochromic anemia can develop. Nursing piglets can obtain some additional iron if allowed access to the feces of the sow; however, deficiency is still a common clinical problem. Consequently, it is routine practice in most swine herds to give 100200 mg of iron dextran IM within 48 hr of farrowing to prevent iron deficiency anemia. Swine, unlike ruminants, do not require elemental sulfur in their diets when adequate sulfur-containing amino acids (methionine and cystine) are available. Sulfur is essential for synthesis of various body compounds such as taurocholic acid, chondroitin sulfate, glutathione, and lipoic acid. Methionine alone can meet the total sulfur-containing amino acid re-

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15. BIOLOGY AND DISEASES OF SWINE

days. Estrus typically lasts 48 hr (range 1-3 days). Prior to the onset of estrus, sows will exhibit signs of vulvar reddening and swelling, mucous discharge, nervousness, and increased activity. During estrus, sows will stand immobile when pressure is applied to the rump (Braun, 1993). Silent estrus is common in swine, but the presence of a boar can facilitate estrus detection (Dial et al., 1992). Optimal fertilization rates occur when insemination takes place 12 hr prior to ovulation. However, the variability in the interval between onset of estrus and ovulation makes it difficult to determine when females ovulate. As a result, commercial producers usually breed sows twice during estrus to maximize conception rates. Litter size also tends to increase with multiple matings per estrus. In pen mating, the sow and boar are left together during estrus. Hand mating involves placing the sow and boar in the same pen at 12-24 hr intervals during estrus until the female is no longer receptive (Dial et al., 1992). Swine may also be bred by artificial insemination; however, conception rates are typically 10-15% lower than when natural service is used. Satisfactory results are obtained if sows are inseminated 10-30 hr after the beginning of estrus (Einarsson, 1980). 2.

Fig. 7. Dilationof the lateral auricularvein, using a tourniquet.

quirement in swine because cystine can be synthesized from methionine. The amino acid requirements (Table III) refer to the L-isomer, which is the most biologically active form in swine and most common form found in plants and animals (NRC, 1988, 1998).

C. 1.

Reproduction

Reproductive Physiology

Swine reach sexual maturity at 3 - 7 months of age, with most miniature breeds becoming sexually mature at 4 - 6 months of age. Litter size varies among breeds, with domestic swine usually having an average of 8-12 pigs per litter and miniature breeds, 4 - 6 pigs. Litter size also varies with parity, being smallest at the first parity, increasing to a maximum between the third and seventh paritities, and then remaining stable or decreasing (Dial et al., 1992). The average estrous cycle is 21 days with a range of 17-25

Pregnancy

Failure to return to estrus 18-24 days following mating is the first sign of pregnancy. Nonestrous sows are most easily detected by daily exposure to a boar during this time. Behavioral changes are seen in only 50% of sows in the absence of a boar. In the absence of a boar, determination of pregnancy can be based on whether or not the physical and behavioral changes of estrus are observed (Braun, 1993). Estrus detection has been reported to be 98% accurate and can be used to determine pregnancy status soon after failure of conception or death of a litter (Dial et al., 1992). Other pregnancy-detection procedures include the use of ultrasound and hormone assays. Ultrasound is < 90% accurate and cannot be performed prior to the fourth week of gestation Amplitude-depth ultrasound units can be used to detect pregnancy reliably between 30 and 90 days and as early as 18 days with some equipment. They are handheld devices that detect interfaces between fluid and tissues, which is the reason why they lose sensitivity at either early or late gestation. Doppler ultrasonography can be used from 4 weeks until farrowing and can also be used to determine litter size as well as fetal viability in late gestation (Dial et al., 1992; Braun, 1993). Activity of the corpora lutea can be measured by progesterone assays. Progesterone concentrations of < 1 ng/ml on days 1719 of the estrous cycle are typical of nonpregnant females. An elevated progesterone concentration on day 18 after breeding is indicative of pregnancy. Estrone sulfate assays are more accurate for determining pregnancy status than progesterone assays. Estrone sulfate, produced by the fetus, reaches peak blood levels at 2 3 - 3 0 days gestation (Braun, 1993).

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Fig. 8.

3.

Venipunctureof the medial saphenousvein.

Parturition and Neonatal Care

The gestation period of miniature pigs and commercial pigs is typically 114-115 days. Signs of impending parturition are usually evident during the last week of gestation. The vulva becomes swollen and more reddened during the last 3 - 4 days. Development and distension of individual mammary glands occur during the last 2 - 3 days of gestation, and drops of clear or straw-colored fluid can be expressed. This is followed by the initiation of milk secretion. Characteristically, abundant milk can be expressed at the onset of farrowing. The interval between the initiation of milk flow to parturition is typically 6 - 1 2 hr and provides a somewhat reliable sign of farrowing. Respiratory rate is most reliable. Behavioral changes occur during the 24 hr preceding farrowing and include restlessness and nesting. Frequent urination, defecation, and chewing or biting on surrounding objects may also be noted. However, just prior to birth, this activity diminishes and the sow becomes recumbent (Day, 1980; Braun, 1993). Use of a farrowing crate is seldom necessary. The week prior to the anticipated farrowing date, sows should be placed in a quiet room in a stall with abundant bedding material for nest building. Wood chips are ideal for farrowing stalls since they allow the sow to engage in nesting behavior. They also help maintain the neonates' body temperature since newborn piglets lack the ability to effectively thermoregulate. Environmental temperature should be 85~176 with a supplemental heat source in the stall that results in a temperature of approximately 90~ at

pig level (Fisher, 1993). Hanging heat lamps are commonly used and should be positioned to be effective without causing burns. The sow's comfort level is approximately 68~176 which is the reason for having a supplemental heat source just for the neonates. Newborns should not be exposed to drafts or moisture. The duration of farrowing ranges from less than 1 up to 8 hr, but typically lasts 3 - 4 hr. Larger litters may have a longer farrowing duration. The sow displays little physical exertion during the birth process. Sows generally remain laterally recumbent while giving birth but will occasionally change to a standing or ventrally recumbent position. The interval between the birth of piglets is typically 15 min. Assistance should be provided if more than 3 0 - 6 0 min elapse between the delivery of piglets (Day, 1980; Braun, 1993). The most important factors that contribute to neonatal survival are the ability of the piglets to receive colostrum within the first 12 hr of birth, adequate nutrition, and appropriate environmental conditions (Reeves, 1993). Competition is normal among littermates during nursing and can result in inadequate colostrum and milk intake in less dominant animals. Neonates will compete for, and establish, teat order on their day of birth. This hierarchy remains until weaning (Sawatsky, 1993). If necessary, the technique of split suckling can be used to ensure that all animals can nurse. This involves removing half of the litter comprising the largest piglets 3 - 4 times a day to allow the smaller animals to nurse adequately (Reeves, 1993; Dial et al., 1992). The sow's milk supply should be checked daily to

623

15. BIOLOGY AND DISEASES OF SWINE

rooms with temperatures at 75~176 Swine are generally weaned at 3 - 5 weeks by allowing them access to a solid ration.

D.

Fig. 9.

Venipuncture of the femoral vein.

prevent piglet deaths from dysgalactia. Commercial pig milk replacers are available and should be provided to piglets by bottle or pan feeding if the sow is unable to produce an adequate milk supply. Preweaning mortality is endemic in most herds, but mortality varies depending on the prevalence of the various causes, which include poor viability at birth, chilling, starvation, trauma, diarrhea, and other diseases (Dial et al., 1992). Trauma includes incidences of piglets that are stepped on, suffocated when lain on, and savaged by the female. Savaging is a behavior observed occasionally in individual animals, resulting in injury to and/or death of the piglets. The only recourse is to remove the piglets from the sow and to cull her from the breeding herd. Day 1 care for piglets includes disinfection of the navel, clipping of the canine or "needle" teeth, injection of an iron supplement, identification of individual animals, weighing, and clinical exam (Reeves, 1993; Fisher, 1993). The environmental temperature should remain at 85~176 for animals up to 3 4 weeks of age. Animals 4 to 8 weeks old can be housed in

Behavior

Swine are highly social and intelligent animals. They have a highly developed sense of smell, but poor eyesight. Grouphoused swine are frequently observed vocalizing to each other. Pigs have an innate need to root, which can become destructive if they are not provided with an adequate outlet for expression. Housing strategies should accommodate swine behavioral needs as much as possible within the constraints of experimental design. Group housing or housing 2 animals per cage can be used to allow social interactions among animals. If individual housing is necessary, cages should be close together, and their design should include openings at the bottom to facilitate contact. Providing bedding material such as wood shavings is an excellent way to satisfy pigs' rooting behavior. Bedding material has the additional advantage of absorbing excreta but can be more labor-intensive for the husbandry staff than slatted or mesh flooring. Alternatively, a variety of toys, such as balls, chains, or hoses, can be supplied to help provide cage enrichment (Sawatsky, 1993; Fisher, 1993). Swine are readily trained and respond well to positive reinforcement in contrast to conventional agricultural handling practices. This characteristic can be used to advantage in the research setting when animals must be handled or restrained for research manipulations. Acclimating and training swine to tolerate research equipment that will be used on them should be a standard procedure and can include the use of various types of food rewards given for reinforcing wanted behaviors. Gentle handling and the use of a humane restraint sling is warranted whenever swine need to be transported from their home cages or when restraint is necessary during noninvasive procedures. Many pigs respond to gentle rubbing of the ventral abdomen by rolling over onto their sides, enabling caregivers to perform such minor procedures as wound cleansing or suture removal without restraining the animals. This type of handling is very effective for positively reinforcing contact between pigs and their caretakers and has a calming effect on most animals.

E.

Immunology and Use of Swine in Xenotransplantation

1. Immunology Swine immunology is of increasing interest because of the potential for using pigs in xenotransplantation. The comparable anatomy and physiology of the pig and the human, the availability of inbred lines with a defined health status, and the emerging abilities to genetically modify the pig to lessen the risk of rejection make it an attractive source of much-needed

624

KATHY E. LABER ET AL. Table I

Hematology and Serum Chemistry for Hanford Miniature Swine a Females

Males Parameter Hematology WBC (x 1000/~tl) RBC (X 106/~tl) Hemoglobin (gm/dl) Hematocrit (%) MCV (fl) MCH (pg) MCHC (gm/dl) SEG N (x 1000//~tl) Lymphocytes (x o) Monocytes (x o) Basophils (• o) Eosinophils (• ~ NRBC/100WBC Chemistry Glucose (mg/dl) BUN (mg/dl) Creatinine (mg/dl) Phosphorus Calcium (mg/dl) Total Protein Albumin (gm/dl) Globulin (gm/dl) A/G Sodium (mEq/liter) Chloride (mEq/liter) Potassium (mEq/liter) CO2 AGAP Total bilirubin Direct bilirubin Indirect bilirubin Alkaline Phosphatase (U/liter) GGT (IU/liter) AST (U/liter) LDH (U/liter) CK (U/liter) Na/K a

Mean

SD

Range

Quantity

Mean

SD

Range

Quantity

25.1 7.4 12.2 38.0 57.1 16.6 32.0 16.8 6.6 1.5 0.1 0.1 0.1

3.4 0.8 0.5 1.6 5.0 1.6 0.9 5.2 3.4 0.8 0.2 0.2 0.3

21.3-32.4 6.6-9.3 11.4-12.8 35-40 48.1-63.1 13.7-18.6 30.8-33.7 10.8-24.6 3.4-12.7 0.7-3.2 0.0-0.7 0.0-0.5 0.0-1.0

9 9 9 9 8 9 9 9 9 9 9 9 9

20.8 7.0 12.1 37.2 59.8 17.4 32.5 13.3 6.4 1.0 0.0 0.1 0.1

3.0 0.7 0.8 2.3 4.0 1.1 0.5 4.1 3.2 0.8 0.1 0.1 0.3

16.8-26.7 6.4-8.3 11.4-13.5 35-41 54.1-63.9 15.9-18.8 31.7-33.1 7.6-19.5 3.2-13.0 0.2-2.9 0.0'0.3 0.0-0.2 0.0-1.0

10 10 10 10 10 10 10 10 10 10 10 10 10

104.6 14.7 0.8 6.3 11.2 6.5 3.6 2.9 1.3 142.2 100.4 5.7 26.6 15.2 0.2 0.0 0.2 370.8 47.4 56.5 580.0 456.8 25.3

9.3 1.6 0.2 0.5 0.4 0.3 0.3 0.4 0.3 1.9 0.8 0.7 1.6 1.5 0.1 0.0 0.1 112.0 14.1 14.3 75.0 185.7 3.1

94-118 12-17 0.5-1.1 5.9-7.3 10.4-11.8 6.1-7.0 3.3-4.0 2.4-3.7 0.9-1.7 140-146 99-102 4.7-6.8 24-28 12-17 0.11-0.41 0.0-0.02 0.09-0.41 166 -484 31-75 42-90 510 -758 270-735 21- 30

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 8 10 10 10 10

108.7 13.2 0.7 9.4 10.8 6.2 3.7 2.6 1.5 142.5 100.1 5.6 25.9 16.5 0.1 0.0 0.1 413.5 37.7 42.2 548.3 358.9 26.1

11.0 1.7 0.1 0.9 0.5 0.3 0.3 0.4 0.3 1.7 1.4 0.6 3.1 2.2 0.0 0.0 0.0 106.1 6.6 8.5 40.0 144.2 2.6

91-123 10-16 0.6-0.8 7.7-10.7 10.0-11.4 5.8-6.6 3.1-4.3 2.1-3.5 0.9-1.9 139-144 98-102 4.6-6.3 20-29 14-20 0.09-0.16 0.01-0.02 0.07-0.15 206 -576 29-49 33-59 490 -593 221-628 23 - 31

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Samples taken from unanesthetized 3-month-old animals. Unpublished data courtesy of Charles River Laboratories, Inc.

transplant organs. C o m p r e h e n s i v e reviews of swine i m m u n o l ogy (Pescovitz, 1998) and xenotransplantation (Gianello et al., 1998) have been published. In c o m p a r i s o n to those of m o s t other m a m m a l s , cortical and m e d u l l a r y areas of l y m p h nodes have an inverted relationship, a trait shared with the elephant, rhino, dolphin, hippo, and warthog (Pescovitz, 1998). Afferent l y m p h percolates from the central cortex to the outer paracortex (equivalent of medulla), where cells migrate through high endothelial venules and back into the blood; thus, efferent l y m p h is relatively acellular. M e d u l l a r y areas of the porcine l y m p h node are denser in cell

n u m b e r s than those of m o s t other species, being rich in m a c r o p h a g e s , p l a s m a cells, and eosinophils. The pig also has p r o m i n e n t intraluminal m a c r o p h a g e s within respiratory airways. Similar to ruminants, pigs have two types of Peyer's patches present in the small bowel. A l t h o u g h there is no clearly established functional distinction, the multiple (20+__) discrete Peyer's patches found in the j e j u n u m contain B and T cells, while the singular long ileal Peyer's patch contains B cells almost exclusively ( B a r m a n et al., 1997). D u e to a relative lack of specific antibodies, identification of cluster of differentiation (CD) markers to p h e n o t y p e l y m p h o c y t e subsets has lagged such

625

15. BIOLOGY AND DISEASES OF SWINE Table II

Clinical Chemistry Reference Ranges for Swinea Yucatan Micropigs (Mature) Parameter Serum analyte Glucose (mg/dl) BUN (mg/dl) Creatinine (mg/dl) Sodium (mEq/liter) Potassium (mEq/liter) Chloride (mEq/liter) Bicarbonate Calcium (mg/dl) Phosphorus (mg/dl) Iron (~tg/dl) GGT (IU/liter) AST (U/liter) Alkaline Phosphatase LDH CK (U/liter) Total Protein (gm/dl) Albumin (gm/dl) Cholesterol (mg/dl) Triglycerides (mg/dl) Total bilirubin (mg/dl) Urine analyte Urine flow (ml/min/kg) Osmolality (mOsm/kg H20) Sodium (~tmol/min/kg) Potassium (~tmol/min/kg) Glomerular filtration rate (ml/min/kg) Renal plasma flow filtration rate (ml/min/kg)

Female 68.5 _ 10.1 18.9 _ 3.0 0.9 _ 0.13 139.8 _ 2.90 5.46 -+ 0.84 103.6 +_ 6.9 10.9 _ 0.64 6.66 _+ 0.74 60 -+ 9.6 41.4 _ 27.6 63.0 _ 28.8 55.2 _ 19.8 704.4 _ 1158 74.9 -+ 4.8 82.0 __+15.1 31.9 _+ 9.7 0.338 _+ 0.146

Yorkshire/ Duroc (Immature) Male 65.2_ 19.4 _ 0.85 _ 139.8 _ 5.50 _ 101.8 _

Female/Male 12.1 3.4 0.19 2.80 0.84 3.6

10.9 _ 0.72 6.63 _ 0.74 169 _ 25 60.0 _ 12.0 40.2 _ 22.2 787.8 + 178.8 759.6 _ 180.2 717.0 _ 995 72.86 _ 4.4 47_+3 73.1 _+ 12.8 27.5 _+ 8.9 0.330 _+ 0.288

82.9 _+ 11.9 9.0 _ 3.2 1.01 _+ 0.22 138.0 _ 3.49 4.40 _+ 0.37 106.0 _ 7.80 29.0 -+ 2.2 9.6 _ 0.58

0.1-0.16 115-546 0.25-8.13 0.09-3.31 1.0-4.5 7.7-32.2

aSummarized from Loeb and Quimby (1999).

studies of the mouse and the human. Two International Swine Cluster of Differentiation Workshops have been held for comparison of novel antibodies to swine CD antigens and crossreactive antibodies available from mouse and h u m a n studies (Saalmuller et al., 1998). M a n y homologous CD markers have now been identified, and a limited number are available commercially from the American Type Culture Collection (Manassas, Virginia) and Pharmingen, Inc. (San Diego, California). A monoclonal antiporcine CD3 antibody has been identified that is capable of activating or depleting T cells in vitro and inducing an immunosuppressive state in vivo, which will greatly facilitate studies of the swine i m m u n e system, in particular, induction of tolerance in xenotransplantation research (Huang et al., 1999). Bone marrow of swine is more similar to that of humans than of rodents in toxicity response to lethal irradiation, allowing studies that have demonstrated the benefit of T-cell de-

pletion of donor tissues in preventing graft-versus-host disease. Normative data for the swine i m m u n e system, such as lymphoid tissue weights and percentages of cell subsets represented in different tissues, are influenced by the animal health status, as data derived from animals of conventional health status (i.e., farm environments) differ significantly from data derived from those housed under specific p a t h o g e n - f l e e , gnotobiotic, or axenic conditions. The pig has a large population of what were initially considered as "null" cells, lacking expression of CD2, CD4, or CD8, but now known to express CD3, classifying them as T cells. This lymphoid population is largely y5 T cells and is found in large numbers in various tissues, especially mucosal sites, as in ruminants (Davis et al., 1998). Expression of CD4 (T helper) and CD8 (T cytotoxic or T suppressor) is mutually exclusive in most species, but swine have a unique lymphocyte subset that expresses both CD4 and CD8 (Thome et al., 1994).

626

KATHY E. LABER E T AL. Table III

Daily Nutrient Requirements of Growing Swinea Body weight (kg) Parameters

10-20

20-50

5 0 - 80

Average weight in range (kg) Digestible energy of diet (kcal/kg) Estimated digestible energy intake (kcal/day) Metabolizable energy of diet (kcal/kg) b Estimated metabolizable energy intake (kcal/day)b Estimated feed intake (gm/day) Crude protein (%) Water (liters) (2.5 liters per kg feed consumed) Fatty acid requirements Linoleic acid (gm) Amino acid requirements (gm/day) (Total basis) Arginine Histidine Isoleucine Leucine Lysine Methionine Methionine + cystine Phenylalanine Phenylalanine + tyrosine Threonine Tryptophan Valine Mineral elements Calcium (gm) Phosphorus, total (gm) Phophorus, available (gm) Sodium (gm) Chlorine (gm) Magnesium (gm) Potassium (gm) Copper (mg) Iodine (mg) Iron (mg) Manganese (mg) Selenium (mg) Zinc (mg) Vitamins Vitamin A (IU) Vitamin D 3 (IU) Vitamin E (IU) Vitamin K (menadione) (mg) Biotin (mg) Choline (gm) Folacin (mg) Niacin, available (mg) Pantothenic acid (mg) Riboflavin (mg) Thiamin (mg) Vitamin B 6 (mg) Vitamin B12 (lxg)

15 3400 3400 3265 3265 1000 20.9 2.5

35 3400 6305 3265 6050 1855 18.0 4.6

65 3400 8760 3265 8410 2575 15.5 6.4

1.00

1.86

2.58

4.6 3.7 6.3 11.2 11.5 3.0 6.5 6.8 10.6 7.4 2.1 7.9

6.8 5.6 9.5 16.8 17.5 4.6 9.9 10.2 16.1 11.3 3.2 11.9

7.1 6.3 10.7 18.4 19.7 5.1 11.3 11.3 18.0 13.0 3.6 13.3

7.00 6.00 3.20 1.50 1.50 0.40 2.60 5.00 0.14 80.00 3.00 0.25 80.00

11.13 9.28 4.27 1.86 1.48 0.74 4.27 7.42 0.26 111.30 3.71 0.28 111.30

12.88 11.59 4.89 2.58 2.06 1.03 4.89 9.01 0.36 129.75 5.15 0.39 129.75

1750 200 11 0.50 0.05 0.40 0.30 12.50 9.00 3.00 1.00 1.50 15.00

2412 278 20 0.93 0.09 0.56 0.56 18.55 14.84 4.64 1.86 1.86 18.55

3348 386 28 1.29 0.13 0.77 0.77 18.03 18.03 5.15 2.58 2.58 12.88

The values in this table are minimum reqirements only and are not recommended allowances. A margin of safety should be added for most of the nutrients with the exception of selenium. The National Research Council's (NRC) (1998) "Nutrient Requirements of Swine," 10th rev. ed., should be consulted by researchers developing their own diet formulations. Adapted from NRC (1998), with permission. aAssumes allowed feed ad libitum that is 90% dry matter and corn-soybean based. bAssumes that metabolizable energy is 96% of digestible energy.

627

15. B I O L O G Y AND D I S E A S E S O F S W I N E 14000 12000 10000 8000 6000 4000 2000 t

5

i

i

!

i

t

1

i

!

i

i

t

i

i

i

i

i

i

i

!

i

i

I

i

i

15 25 35 45 55 65 75 85 95 105 115 125 Body weight in kilograms

Fig. 10. Expected daily digestible energy intake for growing farm swine. (Adapted from computer model program, NRC, 1998, with permission.)

This subset may represent a type of memory cell or a lineage that differentiates into CD4§ -, since CD8 expression is low and the CD8 dimer, normally a13 in chain structure, is a s in these cells. Immunoglobulins (Ig) of the pig are the most studied of those in farm species (reviewed in Ober et al., 1998). There is no transplacental transfer of maternal immunity; thus, neonates are colostrum-dependent. Access to IgG-rich colostrum within the first 6 hr postpartum is most critical for 3-week survival rate and weight gain. Colostral leukocytes, largely neutrophils and T cells, are also absorbed by intercellular migration. Intestinal closure for absorption of colostrum is complete by 24 to 48 hr of age. In contrast to most other species, the pig lacks the gene for IgD, which is a precursor immunoglobulin in the differentiation pathway to IgM. The pig does have a large number of IgG subclasses: IgG1, IgG2a, IgG2b, IgG3, and IgG4. Immunoglobulin A circulates as a dimer in blood and tissues and as a monomer in mucosal secretions; IgE is found in serum and mucosal tissues. High endothelial venules of transplanted swine tissues express adhesion molecules, but information on the relative homology of these "addressins" is limited in scope due to lack of reagents. Cytokines and lymphokines in the pig have been studied in models associated with inflammation, such as sepsis, atrophic Digestible Energy concentrationof 3, 330 kcal / kg food 4 '~ 3.5

3 2.5

rhinitis, erysipelas, arthritis, and viral infections (Murtaugh, 1994; Ober et al., 1998). Reports on swine cytokine regulation and function suggest that the biology is similar to that of humans and mice and that there is some limited homology; swine lymphocytes will respond to recombinant human interleukin (IL)-2 in vitro (Whary et al., 1995). Located on chromosome 7, swine leukocyte antigens (SLA), the equivalent of the mouse major histocompatibilite complex (MHC), have been cloned and sequenced. As they are in other species, SLA class I genes are universal in tissue expression and function to restrict T-cell activation, particularly antiviral immune responses, and are pivotal for inducing tolerance for "self." The SLA class II genes have been cloned and are restricted in expression to B cells, activated macrophages, a subset of CD8 § T cells, and vascular endothelium. The number of SLA class III genes that have been cloned is lower than that found in other species. Member genes of the SLA class III complex function in the complement system, which in the pig is closely aligned with the human systems of classical and alternative pathways of complement activation. One difference is that elimination of antigen-antibody immune complexes occurs through the lung in the pig, in contrast to the target organs of liver and spleen in humans (Davies et al., 1995). Red blood cell antigen classification is very complex in the pig, with 16 genetic systems having been developed that consist of 78 blood factors, which are either antigens of the red blood cell itself or become cell-associated from other tissues when serum concentrations are high (Pescovitz, 1998). Knowledge of red cell surface expression of carbohydrate antigens is important to xenotransplantation because of their role in complement activation, which mediates hyperacute rejection of transplanted tissues. There are no primary immunodeficiencies that have been identified in the pig, but acquired immunodeficient states can be surgically induced (thymectomy, splenectomy). Spontaneous cases of immunodeficiency have been attributed to inadequate colostrum, stress, or poor nutrition (Pescovitz, 1998). Autoimmune disease in swine is largely undocumented except for hemolytic disease in neonates related to postnatal absorption of maternal immunoglobulins (erythroblastosis fetalis) and two forms of glomerulonephritis. One form appears to be inherited in Norwegian Yorkshire swine, and a second involves spontaneous IgA nephropathy reported in Japanese slaughter pigs (both reviewed in Ober et al., 1998). Arthritis in pigs attributable to infection with Mycoplasma hyorhinis or Erysipelothrix rhusiopathiae has similar pathogenesis to human rheumatoid arthritis.

1.5

2.

0.5 0 5

Fig. 11.

15 25 35 45 55 65 75 85 95 105 115 125 Body weight in kilograms

Expected daily food intake for growing farm swine. (Adapted from

computer model program, NRC, 1998, with permission.)

Use of Swine in Xenotransplantation

Experimental use of swine organs or tissues for humans faces significant scientific challenges of overcoming hyperacute and chronic rejection by the host as well as societal issues involving ethics, cost, and the risk of zoonotic diseases. Nonetheless, the

628

pig is considered to be the prime candidate for systematic production of suitable organs because of its many advantages. Miniature swine are readily available and have body weight, organ size, and physiology similar to those of humans. Additionally, their transplant potential is supported by emerging transgenic technology to minimize rejection and by a large database of technical information generated by their use in other areas of biomedical research (Swindle, 1992). Disease resistance of swine organs has also been promoted as a strategy to circumvent failure of transplanted organs resulting from infectious agents specific to the human, such as hepatitis B virus (Mueller et al., 1999). Three lines of miniature swine homozygous for a different set of SLA alleles have been developed by David Sachs of the Massachusetts General Hospital (Boston) (Lunney et al., 1986). Designated as S L A aa, SLA cc, and SLA aa, these lines are heterozygous at minor histocompatibility and other loci and thus are useful genetic models for studying rejection. Pairing donor and recipient within a line is used to model for transplants between identical human siblings, between lines as a model for nonidentical siblings, and between F1 hydrids for parentoffspring transplants. Intra-SLA recombinant strains have permitted study of SLA class I and II differences and demonstrated the effect of SLA mismatches on graft survival of various tissues. Matching SLA skin grafts typically survive for 7 to 12 days, but vascular grafts and liver transplants survive much longer, some indefinitely. One-third of renal transplants have survived indefinitely, and when rejection did occur, it led to discovery of non-SLA-linked genes that promoted immunemediated rejection. Acceptance of vascular grafts has been shown to induce tolerance to minor histocompatibility loci, and when performed prior to skin grafting, skin graft survival time was extended. Reversing this process (skin graft first) apparently sensitizes the host to reject vascular grafts hyperacutely. The swine-to-baboon xenotransplantation model holds the promise of future technology transfer to enable swine-to-human donation. Lack of long-term success has generated multiple strategies to minimize rejection. Hyperacute rejection of organs occurs within minutes to hours and is mediated by complement when pig organs are perfused with human or nonhuman primate blood (Saadi and Platt, 1999). High titers of natural "xenoantibodies" in the human (or experimentally, in the nonhuman primate) target carbohydrate antigens, such as Gal(c~I-3),Gal(c~I4),GlcNAc-R, expressed on donor tissue endothelium, resulting in complement activation and subsequent vascular injury and in severe cases, disseminated intravascular coagulation (DIC). One strategy has been to eliminate natural xenoantibodies from the circulation of baboons using a-Gal immunoaffinity columns or extracorporeal perfusion through pig liver; depletion is temporary, on the order of days to weeks (Kozlowski et al., 1998). Sachs unsuccessfully attempted to identify an inbred swine genotype of low a-Gal expression with the objective of using selective breeding of pigs to decrease a-Gal expression (Chae

KATHYE. LABERET AL. et al., 1999). Others have suggested screening specific donors

for carbohydrate antigen expression levels as a means to diminish antigenic stimulation of the recipient (Papalois et al., 1999). Brenner et al. (2000) reported that nonspecific depletion of the majority of recipient immunoglobulins of all isotypes by column immunoapheresis significantly improved graft survival of pig hearts in baboons. In addition to the antibody-mediated hyperacute rejection, delayed rejection of xenografts through cell-mediated responses develops over 3 to 4 days, involving activation of endothelial cells of the graft as in the acute rejection response (Brouard et al., 1999). Activation leads to loss of thrombomodulin and adenosine triphosphate diphosphohydrolase (ATPDase), which leads to prothrombosis, proinflammatory gene activation increasing the expression of adhesion molecules, prothrombotic factors, and cytokines. Adoptive cell transfer experiments in immunodeficient rodents have demonstrated that engrafted human CD4+T cells mediate rejection of porcine xenografts (Friedman et al., 1999) as do natural killer cells and monocytes (Sandrin and McKenzie, 1999). Early attempts to promote immunologic tolerance have included bone marrow ablation of the recipient, therapy with complement inhibitors (cobra venom factor or soluble complement receptor I), preadsorption of natural antibodies, immunosuppressive drug regimens, antithymocyte globulin, and splenectomy (Schmoeckel et al., 1999). An attractive approach that has shown early promise is the induction of donor-specific tolerance using bone marrow transplantation to create chimeras. Tolerance to fully MHC-mismatched allografts has been demonstrated in mice and primates after first creating a mixed allogeneic hematopoietic chimerism by engrafting donor bone marrow cells into the recipient. However, this hematopoietic chimerism has been difficult to achieve in the discordant pig-toprimate xenogeneic model (Sablinski et al., 1999), most likely due to species-specific differences in regulatory cytokines and elements of the stromal microenvironment (Emery et al., 1999). Representative of a typical experimental protocol and illustrative of the complexities involved, recipient primates underwent whole-body irradiation on days 6 and 5 prior to infusion of pig bone marrow. Primate antipig xenoantibodies were immunoadsorbed by extracorporeal perfusion of recipient blood through a pig liver immediately before the intravenous infusion of porcine marrow (day 0). In addition to cyclosporine for 4 weeks and 15deoxyspergualin for 2 weeks, recombinant pig stem-cell factor and interleukin 3 were given for 2 weeks. Recipient primates required 4 weeks to recover from pancytopenia from whole body irradiation, and antipig IgM and IgG antibodies were temporarily depleted by the liver perfusion for 12 to 14 days. About 2% of the myeloid progenitors in the bone marrow of the recipient were of pig origin, and the chimeric animal was less responsive by mixed lymphocyte reaction to pig-specific stimulators. This was the first report of long-term survival of discordant xenogeneic bone marrow in a primate recipient. Others have re-

15. BIOLOGYAND DISEASES OF SWINE ported on the poor function of porcine hematopoietic cells in primate marrow microenvironments. Warrens et al. (1998) found differences between swine and human bone marrow cultures in function of two well-characterized ligands known to be important in hematopoiesis, CD44 and very late antigen-4 (VLA-4), but they concluded that the differences were not significant enough to explain lack of effective porcine hematopoiesis in the primate marrow, suggesting that other unknown interactions may be important. Gene therapy using retroviral transfer to minimize rejection has been investigated. In a mouse model, inhibition of xenoantibody production was accomplished by retroviral transfer into mouse bone marrow of a gene encoding the enzyme that synthesizes swine carbohydrate antigens (Bracy et al., 1998). Gene therapy to express swine SLA class II antigens on baboon autologous bone marrow cells has had limited success (Ierino et al., 1999). Transcription of the transgene was transient, and xenografts were rejected after 8 to 22 days. This experiment was important because it demonstrated that transfer and expression of xenogeneic class II transgenes can be achieved in baboons, and this therapy may prevent late T cell-dependent responses to porcine xenografts, which include induced non-a-Gal IgG antibody responses. Porcine thymic grafts in immunodeficient mice have been found to support normal development of polyclonal, functional human T cells, and these T cells were specifically tolerant to SLA antigens of the porcine thymus donor, suggesting thymic transplantation may be an approach to achieve tolerance in pig-to-human xenotransplantation (Nikolic et al., 1999). Transgenic science is the most promising approach to prevent rejection of xenotransplants. Transgenic expression of human a(1,2)-fucosyltransferase in different porcine cells and tissues, including the vascular endothelium, modifies the cell surface carbohydrate phenotype of the xenogeneic donor cell, resulting in the expression of the human universal donor O antigen and a concomitant reduction in the expression of the antigenic Gal(ctl-3)Gal epitope (Costa et al., 1999). Transgenic expression of CD59, a human complement regulatory protein, has promoted survival of swine lungs in a pig-to-primate model (Kulick et al., 2000; Yeatman et al., 1999). Swine endothelium has been genetically engineered to express other human complement regulatory proteins such as human decay-accelerating factor (Waterworth et al., 1998), and membrane cofactor protein (MCP/CD46) (Perez de la Lastra et al., 1999), both shown to be important in a swine model of xenogeneic lung and cardiac injury. A major issue in xenotransplantation research is minimizing the risk for acquired zoonoses, particularly in recipients already immunosuppressed by illness and chemotherapy. Importantly, immunosuppression may take away the expected barrier of "species-specificity" of a potential agent. In addition to the anticipated risks associated with normal flora, environmental contaminants, and true pathogens, there is concern about the unknown risks of viral latency, viral recombination, and en-

629

dogenous retroviruses (Levy et al., 2000), which in the pig are known to infect human cells in vitro (Weiss et al., 1999; Tacke et al., 2000). In order to minimize risks, donor animals must be free of potential zoonoses and other complicating diseases (Ye et al., 1994). The pig as a model has the advantage that it can be produced under gnotobiotic conditions, and management of newborn piglets from hysterectomy to donation has been described (Munoz et al., 1999). Swindle has suggested that the term "xenograft-defined flora" rather than SPF be used to designate the appropriate health status of donor animals in order to avoid confusion with existing standards (Swindle, 1998). There were 237 papers published on the use of swine in xenotransplantation between 1999 and mid-2000, which illustrates the intensity of research on the potential use of pigs in this vital area of human health. This amount of effort also represents the challenges to be overcome before xenotransplantation becomes a practical reality.

III.

A.

DISEASES

Infectious Diseases

If research pigs are typically purchased in relatively small numbers from herds with defined health status, newly introduced animals are adequately quarantined and conditioned, and husbandry conditions are optimum, the incidence of infectious disease in the research laboratory should be minimal. Nonetheless, veterinarians responsible for swine health need to be familiar with both classical swine diseases and the health problems that can emerge from opportunistic agents in animals stressed by experimental manipulation. Many of the diseases discussed below are in fact rare in the majority of modern commercial pigs and will not be found in the commercially supplied miniature swine herds of high health status. Implementing treatment of infectious problems should be considered cautiously and is best reserved for those problems that are expected to resolve quickly with minimal impact on the research use or health status of the research herd as a whole. In the following discussion of infectious diseases, drugs of choice are identified, and the reader is referred to veterinary texts (Friendship, 2000; Hawk and Leary, 1995) for specific doses. Not all drugs mentioned are labeled for use in swine; thus if these drugs are used, the veterinarian must determine a dose from experience with other species and must ensure that treated domestic swine are not used for food. 1. Polysystemic Diseases a.

Salmonellosis

Etiology. There are over 2400 serotypes in the genus Salmonella, and each is conventionally referred to as a separate

630 species. All members of this genus are motile, non-spore-forming, facultative anaerobic gram-negative bacilli possessing peritrichous flagellae. There are 3 serotypes that are typically etiologic agents of clinical disease in swine and numerous others that are occasionally associated with disease. Salmonella choleraesuis var. kunzendorf is the most frequent serotype causing disease in swine, and infection is usually manifested as septicemia and/or pneumonia. Salmonella typhimurium is the second most common isolate and typically causes enterocolitis. Salmonella typhisuis is associated with localized epizootics characterized by chronic wasting, caseous lymphadenitis, diarrhea, and pneumonia. It is very common to isolate more than one serotype from an individual pig; however, it is unusual that primary disease would be caused by a serotype other than S. choleraesuis or S. typhimurium (Schwartz, 1999).

Clinical signs and differential diagnoses. Signs characteristic of Salmonella septicemia include respiratory signs of cough, dyspnea, pneumonia, and cyanosis of the ears and ventral abdomen. Lethargy, anorexia, pyrexia of 105~176 (40.5 ~ 41.6~ and sometimes jaundice followed by watery yellow diarrhea are also evident. This form is usually caused by S. choleraesuis and can affect all ages, with swine aged 3 weeks to 5 months being the most commonly affected group. It can cause abortion in breeding sows. A differential list should include erysipelas and Streptococcus suis as other causes of septicemia. The enteric form is characterized by an acute or chronic enterocolitis that is sporadically hemorrhagic. The initial diarrhea is usually watery and yellow and lasts less than a week but may recur. Anorexia, pyrexia, and dehydration are seen concurrent with diarrhea. Death may be the result in severely affected animals, and a distended abdomen due to rectal strictures can be a sequela to the chronic diarrhea. The majority of affected pigs recover; however, some will be carriers and shed the organism for several months. This form is usually caused by S. typhimurium and most commonly affects swine aged 3 weeks to 4 months. A differential list should include other causes of bacterial, viral, and parasitic gastroenteritis in recently weaned swine. Bacterial agents include colibacillosis, porcine proliferative enteropathy (PPE) (Lawsonia intracellularis), and swine dysentery (Serpulina hyodysenteriae). The viral agents include transmissible gastroenteritis virus (TGE) and rotaviral enteritis. The parasitic agents include Trichuris suis and coccidiosis (lsospora suis). Epizootiology and transmission. The source of Salmonella choleraesuis for swine is essentially other swine and environments contaminated by swine. Transmission is both vertical and horizontal by fecal-oral spread or nasal secretions. The incubation period ranges from 2 days to several weeks, and survivors become carriers that shed the bacteria in feces for at least 3 months. Some form of stress, including shipping, food deprivation, concurrent diseases, research protocols, tempera-

KATHYE. LABERET AL. ture fluctuations, mixing pigs from different sources, and overcrowding, usually precedes clinical disease. The stress increases shedding by inapparent carriers. Salmonella choleraesuis is fairly host-specific for swine, but S. typhimurium is not host-specific (Schwartz, 1999). Therefore, other animal species in addition to swine are likely to be sources for S. typhimurium. Feed and feed ingredients have been shown to be a source of serotypes that can cause disease in swine (Harris et al., 1997).

Necropsy. Gross lesions caused by S. choleraesuis are severe pleuropneumonia; cyanosis of the ears, feet, tail, and abdomen; splenomegaly and hepatomegaly; edematous enlarged mesenteric lymph nodes; erosion of the fundic mucosa in the stomach; and a focal to diffuse necrotic typhlocolitis with or without a necrotic ileitis (Turk et al., 1992). Microscopic lesions include paratyphoid nodules in the liver; necrotic lesions involving the intestinal mucosa, submucosa, and lymphoid follicles; and a bronchopneumonia or hemorrhagic pleuropneumonia (Turk et al., 1992; Wilcox and Schwartz, 1992). Pathogenesis. Salmonella choleraesuis invades the mucosa of the ileum and is taken up by macrophages. It produces both Shiga-like and cholera-like endotoxins that are responsible for the microthrombosis and ischemia of vessels in the lamina propria and resulting necrosis of the enterocytes. Diarrhea is the malabsorption type with extensive fluid loss from the necrotic lesions (Wilcox and Schwartz, 1992). Prevention and control. The most practical approach to preventing clinical salmonellosis is to remove stressors to minimize fecal shedding by carriers and to practice good sanitation to minimize exposure to the bacteria. These stressors include concurrent diseases, mixing of swine from different sources, poor environmental control, and nutritional deficiencies. Sanitary, pest-free facilities should be utilized for preparation, handling, and storage of swine feed. Common surface disinfectants that are efficacious for this bacterium include chlorine, iodine, and phenols. A sensitive and specific multiplex polymerase chain reaction (PCR) assay has been developed that will identify salmonellae in feces and intestinal mucosa scrapings as well as in pure cultures (Elder et al., 1997). This offers an alternative to conventional culture techniques and may be useful for confirming a clinical diagnosis or monitoring herd health. Modified live attenuated vaccines for S. choleraesuis are protective and are thought to be effective because they stimulate cell-mediated immunity. An avirulent live S. choleraesuisculture (SC-54) vaccine has shown a protective effect when applied by spraying the teats and udders of nursing sows. This vaccine reduced fecal shedding and mortality in suckling piglets (Burkhart and Roof, 1999). Killed bacterins for S. typhimurium are available and may provide some protection. Medication of feed or water with appropriate antibiotics

631

15. BIOLOGY AND DISEASES OF SWINE

(e.g., carbadox, neomycin) in conjunction with improvements in husbandry, management, and environment may have a prophylactic benefit.

Treatment. Clinical salmonellosis should not be treated because recovered pigs will likely remain carriers and some isolates may be pathogenic for humans. If absolutely necessary, treatments should be based on susceptibility testing. Recommended antibiotics include ceftiofur (Burton, et al., 1996) trimethoprim-sulfonamide, and gentamicin (Cowart, 1995; Poppe et al., 1998; Seyfarth et al., 1997). Research complications.

Salmonella is present at a low subclinical level in the majority of conventional swine herds. Outbreaks of clinical disease are associated with immunosuppression or stress, which probably includes experimental stress. Clinical disease caused by S. choleraesuis has a reported morbidity ranging to 60% and mortality to 30% (Schwartz, 1991), and this would seriously impact almost any research project.

the upper respiratory tracts of healthy pigs. Experimental evidence suggests that the first site of colonization in piglets is the nasal mucosa (Vahle et al., 1995). It is only known to infect swine, and this suggests that introduction by other carrier species is unlikely. The role of H. parasuis as a respiratory pathogen is not well established. It is usually categorized as a secondary invader or predisposing factor; however, some report that it may be a primary etiologic agent in fibrinosuppurative bronchopneumonia (Rapp-Gabrielson, 1999).

Necropsy.

Gross lesions may include cyanosis of the ears and tail, a polyarthritis with one or more swollen joints, fibrinous pleuritis, pericarditis and peritonitis, and a leptomeningitis (Little and Harding, 1971; Nicolet, 19.92). Histopathologic lesions consist of fibrin thickening of alveolar walls, capillary thrombosis of glomerular tufts, fibrinopurulent arthritis and synovitis, a mononuclear epicarditis (Little and Harding, 1971), and a fibrinopurulent leptomeningitis (Nicolet, 1992).

Pathogenesis. Glasser's Disease (Haemophilus, Porcine Polyserositis, Infectious Polyarthritis) Etiology. Haemophilus parasuis is a small gram-negative pleomorphic coccobacilli, which requires nicotinamide adenine dinucleotide (NAD or factor V) and exhibits satellitism when grown near Staphylococcus epidermidis on a blood agar plate. Serovars 2, 4, 5, 12, 13, and 14 are the most common in North America (Rapp-Gabrielson et al., 1997) of the 15 currently recognized. Both pathogenic and nonpathogenic strains of the organism exist, and prior exposure to the nonpathogenic strain can induce protective immunity. Clinical signs and differential diagnoses. This disease typically affects young pigs from 3 weeks to 4 months of age and varies in severity with the level of herd immunity. In conventional herds where H. parasuis is endemic, the clinical signs will be mild with low morbidity. In susceptible herds the clinical signs occur within a week after exposure and consist of pyrexia 104~176 (40~176 anorexia, depression, lameness, neurological signs, dyspnea, and death. A markedly increased WBC and decreased packed cell volume (PCV) have been reported in experimentally infected SPF piglets (Wiegand et al., 1997). Long-term sequelae include abortion and chronic arthritis. Differentials include Mycoplasma hyorhinis and other bacterial septicemic conditions that affect swine. These should include Erysipelothrix rhusiopathiae, Salmonella choleraesuis, and Streptococcus suis. Haemophilus parasuis is one of the earliest isolates to be cultured from the nasal cavities of swine in conventional herds. In endemic herds it can be cultured when animals are 1 week of age and is commonly cultured from

This organism is part of the normal flora in the nasal passages of swine and is an opportunistic pathogen with swine influenza virus.

Prevention and control.

A prevention and control program should include practices to increase immunity by the use of bacterins and reduction of experimental, environmental, and shipping stress. Herd-specific autogenous vaccines should be considered, as it is unlikely that any one commercial bacterin will induce immunity to all of the pathogenic strains in the population (Rapp-Gabrielson et al., 1997). Elimination by early weaning is often unsuccessful because the piglets can be colonized by the time they are 1 week of age (Maclnnes and Desrosiers, 1999). Medicated early weaning can be successful if high doses of both parenteral and oral antibiotics are utilized (RappGabrielson, 1999). Antimicrobial medication of feed or water of groups of swine at risk may be beneficial. An oligonucleotide-specific capture plate hybridization assay has been developed and is reported to be specific and more sensitive than culturing for H. parasuis from lesions and nasal swabs (Calsamiglia et al., 1999). This may prove useful in a herd-health monitoring program or confirmation of a clinical diagnosis.

Treatment. Parenteral antibiotics should be started as soon as clinical signs become evident. Oral antibiotics are less effective. High doses of penicillin should be given to those with clinical signs and any other pigs in the affected group. Several other antibiotics (cephalosporins, fluoroquinolones, potentiated sulfas, tetracyclines, tylosin) are also effective. Resistance to tetracycline, erythromycin, and penicillin in some strains is increasing.

Epizootiology and transmission.

Research Complications.

This disease will confound cardiovascular studies because the chronic form can produce congestive heart failure and fibrinous pericarditis.

632 C.

KATHYE. LABER ET AL.

Erysipelas (Swine Erysipelas, SE)

Etiology. Erysipelothrix rhusiopathiae (formerly E. insidiosa) is a gram-positive bacillus that has 26 serovars. The majority of isolates from swine are serovars 1 and 2. Clinical signs and differential diagnoses. Animals with acute SE may have no clinical signs or present with a combination of classical rhomboid or diamond-shaped urticarial (pink to purple) skin lesions on the ventral abdomen and back; fever of 104~176 (40~176 anorexia; depression; stiff, stilted gait; sitting posture; abortion; and sudden death. The skin lesions that appear 2 - 3 days postexposure are erythematous, raised, and palpable, and measure 1-8 cm across (Amass and Scholz, 1998); they vary in number from a few to numerous. The fever will usually resolve within 1 week. Differentials for the acute form include any bacterial septicemia. The diamondshaped skin lesions are characteristic. There is also a subacute form, which is a mild version of the acute form and may pass unnoticed. Enlarged, stiff joints resulting in slight to non-weight-bearing lameness characterize the chronic form of SE. The hock and carpal joints are usually the most visibly affected in those with chronic arthritis. In some cases cardiac insufficiency manifested by exercise intolerance and sudden death may result. Chronic SE may follow subclinical, subacute, and acute forms, sometimes within 3 weeks. Differentials for chronic SE include other causes of lameness in swine, including Haemophilus polyserositis, mycoplasmal polyserositis, and trauma and other bacterial septicemias such as Actinobacillus suis. Epizootiology and transmission. The domestic pig is the primary reservoir of E. rhusiopathiae, and probably 30-50% of conventional swine are carriers (Wood, 1999; Cowart, 1995). These pigs harbor the bacteria in lymphoid tissues (tonsils, Peyer's patches) and shed it in nasal secretions, saliva, and feces. Individuals with acute SE will shed large quantities into the environment, and those with the chronic form are a long-term source of contamination. Additionally, contact with infected sheep, turkeys, chickens, ducks, and emus is a potential source of infection for swine. Swine older than 3 months and younger than 3 years of age are most likely to develop clinical disease. Passive antibodies obtained from the sow protect the young, and acquired immunity from subclinical infections protects the mature animals. This bacterium typically gains entry into the body through contaminated food and water (oral route) and skin wounds. Necropsy. Acute phase gross lesions are those of a bacteremia and generalized coagulopathy (Wood, 1984). Characteristic rhomboid or rectangular-shaped, slightly raised, firm skin lesions are most commonly found on the skin of the abdomen but also on the thighs, ears, snout, throat, and jowls. There is con-

gestion of the spleen, lungs, and liver, and there may be petechial hemorrhages on the surface of the kidneys, on the atrial myocardium, and within lymph nodes (Wood, 1984, 1992). Microscopic lesions in the acute phase are the result of damage done to endothelial cells in capillaries and venules. In the dermal papillae these lead to fibrin deposition, microthrombi and lymphocytic and plasmacytic perivascular infiltrates, and focal necrosis. Chronic lesions are manifested as a proliferative nonsuppurative synovitis and arthritis that results in enlarged joints, most commonly involving the stifle, hock, and carpal joints (Wood, 1984, 1992).

Pathogenesis. The organisms gain entry to the body via the palatine tonsils or gut-associated lymphoid tissue, and possibly from wounds in the skin. The pathogenesis of the lesions is not completely understood, but neuraminidase produced in large quantities by E. rhusiopathiae may be responsible for the production and deposition of the fibrin and for the vascular stasis (Wood, 1984). Prevention. Initiation of a vaccination program in unvaccinated animals is very worthwhile although neither attenuated vaccines nor bacterins are successful at preventing chronic SE (Cowart, 1995). Immunization with purified protein antigen P64 has been found to be protective in an experimental challenge (Yamazaki et al., 1999). The surface protein SpaA has shown good potential as an antigen for new vaccines (Makino et al., 1998; Imada et al., 1999; Shimoji et al., 1999). Attenuated vaccines can be injected, given orally in drinking water, or delivered by aerosol with special equipment. Antibiotic treatment should be stopped 10 days prior to giving attenuated live vaccines. Experimental evidence has demonstrated that attenuated vaccines are still effective when porcine reproductive and respiratory syndrome (PRRS) is a concurrent infection (Sakano et al., 1997a). Due to the ubiquitous nature of E. rhusiopathiae the ultimate prevention plan is to obtain SPF animals and maintain them in a barrier facility. Control. Management, husbandry, and the environment in the facility should be improved. Chronically infected animals should be eliminated from the facility. Polymerase chain reaction (PCR) assays for the diagnosis of swine erysipelas can be used to advantage in a control program, since culture can be difficult (Brooke and Riley, 1999). Routine use of common disinfectants, including hypochlorite, quaternary ammonium, phenolic, and alkali, is important as these bacteria can survive in the environment for long periods. Treatment. Penicillin is the treatment of choice for the acute form of SE. Tetracyclines (oxytetracycline and chlortetracycline), tylosin, and lincomycin are also effective. In one reported outbreak a combination of procaine penicillin and dihydrostreptomycin given for 3 days worked well (Wabacha et al.,

15. BIOLOGY AND DISEASES OF SWINE 1998). Hyperimmune serum has been used historically and can be effective if given early in the course of the disease, especially in suckling piglets. This will provide about 2 weeks of passive immunity. Anti-inflammatory drugs can be used to treat the arthritis associated with chronic SE (Wood, 1999; Cowart, 1995).

Research complications. Acute SE can potentially complicate research protocols involving small numbers of swine by causing losses due to sudden death. The chronic form will affect orthopedic and cardiovascular studies since proliferative, nonsuppurative arthritis and vegetative proliferation on the heart valves can result. d.

Streptococcosis (Streptococcal Meningitis)

Etiology. Streptococcus suis (Lancefield's group D) is a grampositive oval cocci found as diplococci or short chains. It is a facultative aerobe that produces a zone of hemolysis when grown on blood agar plates. Capsular types 1-8 are most often associated with clinical disease in swine, with type 2 being the most common (Aarestrup et al., 1998a). Clinical signs and differential diagnoses. Manifestations of meningitis are the most characteristic signs of S. suis type 2 infections in swine, and swine aged 5-16 weeks are most commonly affected. Pyrexia to 42.5~ is usually the initial sign, followed by anorexia, depression, ataxia, paddling, opisthotonus, convulsions, and death. Additional signs of S. suis infection include pneumonia, rhinitis, polyarthritis, stillbirths, abscesses, and vaginitis. Endocarditis and myocarditis may develop in some cases, and dyspnea, cyanosis, weight loss, and sudden death are typical clinical signs (Higgins and Gottschalk, 1999; Staats et al., 1997). Differentials include other streptococcal infections, Haemophilus parasuis, Erysipelothrix rhusiopathiae, Salmonella choleraesuis, and salt poisoning or water deprivation. Epizootiology and transmission. Transmission is by carriers and flies between herds; and sows infect newborns during parturition and suckling by direct contact, aerosols, and fomites. Most piglets of carrier sows are colonized before weaning age (Torremorell et al., 1998). This bacterium has been cultured from a variety of other animals, including humans, birds, and wild Sus scrofa (Seol et al., 1998), and theoretically these are potential vectors. Subclinical carriers harbor the organism in their tonsilar crypts, nasal cavity, and reproductive and gastrointestinal tracts. When a carrier is introduced to a susceptible herd, the signs are usually first evident in recently weaned young between 5 and 12 weeks of age. Simultaneous infection with other pathogens, including PRRS and pseudorabies virus, can increase the severity of clinical signs (Cowart, 1995).

633 Necropsy Necropsy findings include lymphadenopathy, fibrinous pleuritis/pericarditis, polyserositis, and vascular congestion of organs (Erickson, 1987). Histopathologic findings include suppurative meningitis, fibrinous cranioventral bronchopneumonia, and suppurative polyarthritis. Another streptococcus species, S. equisimilis, may be recovered from cases of septicemia associated with subsequent development of swollen joints. At necropsy there is a hyperemic synovium and periarticular edema, and there may be periarticular abscesses and fibrosis as well as metaphyseal osteomyelitis and endocarditis. The organism may persist in the joints for 3 - 6 months (Sanford and Higgins, 1992). Pathogenesis. The pathogenesis of S. suis is believed to begin via colonization of the palatine tonsil. It is then spread either by direct migration through the cribriform palate of the ethmoid bone or via a septicemia. Adult animals may be carriers and spread the organism from sows to neonatal piglets via nasal excretions or from the genital tract during farrowing (CliftonHadley et al., 1984; Erickson, 1987). Prevention. Rederivation by hysterectomy or hysterotomy and maintenance in a barrier facility will eliminate S. suis from an infected herd (Higgins and Gottschalk, 1999). Probably the most satisfactory method is depopulation and repopulation with clean animals since it is feasible to eliminate the modes of transmission in research facilities. Antimicrobial therapy and early weaning did not eliminate the tonsillar carrier state (Amass et al., 1996). A PCR assay developed for the detection of strains of serotypes 1 and 2 in tonsillar specimens (Wisselink et al., 1999) and an ELISA based on a purified polysaccharide antigen (Kataoka et al., 1996) have been found to be specific and sensitive and may be useful for monitoring of herd health. Control. Minimization of environmental and experimental stress, good sanitation, prophylactic antibiotics, and use of bacterins will help control clinical disease. Streptococcus suis is susceptible to common disinfectants. Medicated early weaning and segregated early weaning programs are unlikely to be useful because the neonates are colonized very early after birth (Maclnnes and Desrosiers, 1999). Mixing swine from different sources and of different ages should not occur. Oral medication of feed or water has been shown to be beneficial in controlling streptococcal meningitis. Penicillin, ampicillin, and amoxicillin antimicrobials are recommended (Awad-Masalmeh et al., 1999); however, it must determined that clinically efficacious serum concentrations with the particular dose and route of administration would be achieved. Bacterins of different designs, including autogenous and whole-cell, have had variable success. Live avirulent strains (Busque et al., 1997) and vaccines against cell-wall proteins or extracellular proteins, particularly suilysin (Jacobs et al., 1996), have produced protective immunity in swine (Higgins and Gottschalk, 1999).

KATHYE. LABER ET AL.

634 Treatment. Early treatment with a parenteral antibiotic to which the particular herd strain has been shown to be susceptible by testing or prior experience is the treatment of choice. Drugs to consider include ceftiofur (Burton et al., 1996), enrofloxacin, amoxicillin, ampicillin, and penicillin. Resistance to several antibiotics, including tetracycline, tylosin, and sulfonamides, is a developing concern (Aarestrup et aL, 1998b; Rasmussen et al., 1999). Research complications. Direct losses from fatal meningitis will certainly affect all types of research. Cardiovascular studies will be confounded by the development of endocarditis and myocarditis. Streptococcus suis type 2 is zoonotic to humans (Erickson, 1987). e.

Pseudorabies (PR)

Etiology. Pseudorabies, also known as Aujeszky's disease, was not considered important in the United States prior to 1960. However, since that time it has been significantly elevated in stature due to the emergence of new and more virulent strains and changes in husbandry practices that have potentiated its spread among swine. The virus belongs to the alphavirus subfamily of the Herpesviridae. This classification of viruses is known for the ability to establish latent infections, particularly in the sensory ganglia of the nervous system. The virus can affect a variety of animals, including pigs, cattle, sheep, goats, dogs, cats, rodents, macaques, and marmosets. Reports of human infection have been limited and are poorly documented (Kluge et al., 1992). Infection of all animals other than the pig results in death. Pigs are capable of hosting subclinical and latent infections. Clinical signs and differential diagnoses. The clinical signs associated with this disease are primarily related to the age of the swine affected, although the strain of virus and the infectious dose also play a role. The virus predominantly impacts the respiratory and nervous systems with related clinical signs. Neonatal pigs typically respond to exposure with acute signs related to the central nervous system (CNS). Affected pigs will tremble, hypersalivate, stumble, and exhibit nystagmus and opisthotonus, often with epileptiform-like seizures. Because of posterior paresis, the animals may be observed actually sitting like a dog. Other signs include circling and paddling, vomiting, and diarrhea. Once CNS signs start, death usually follows within 2 4 - 3 6 hr, and mortality approaches 100%. As the pigs age, the clinical signs become less severe, fewer pigs develop CNS involvement, and mortality declines. Respiratory signs characterized by sneezing, nasal discharge, and cough become the hallmark of pigs that are infected at greater than 9 weeks of age. Morbidity rate is high, but mortality is low with uncomplicated CNS signs such as muscle tremors that occur only sporadically. The duration of clinical signs is usually 6 - 1 0 days,

with rapid recovery unless the disease has progressed to pneumonia or a secondary bacterial pneumonia has been initiated. Sows and boars also develop primarily respiratory signs, although pregnant animals in the second to third trimester abort and in the first trimester, resorb the fetus. The main differential diagnosis is swine influenza. Epizootiology and transmission. The virus is spread within a herd in various ways. The single most important mechanism contributing to disease spread is the movement of swine who are shedding the viral particles. Transmission can occur via direct contact, insemination, or transplacental transmission. Indirect transmission can occur by inhalation of aerosolized particles or by contact with contaminated surfaces. Fomite transmission is also possible. Infective levels of virus can persist for up to 7 hr in air with relative humidity of 55% (Schoenbaum et al., 1990). Infective levels of viral particles can also be present in tissues of animals that have died from the disease. Consuming infected carcasses or feed that has been contaminated with the virus is another means of transmission. Evidence indicates that avian species are not a significant contributor to the spread of the virus, and the role of insects in the transmission process has not been adequately evaluated (Zimmerman et al., 1989). Animals other than pigs, which are considered dead-end hosts, typically die within 3 days of being infected. Diagnosis. Pseudorabies in older animals can easily be confused with swine influenza, especially if the only clinical signs manifested are respiratory in nature. When CNS signs are exhibited, the clinical diagnosis of PR becomes much clearer. Serology is not the ideal choice for defining acute infections, as there is a delay in humoral antibody development; and interpretation of serologic results can be difficult, especially in younger animals, as maternal antibodies are present until piglets are up to 4 months of age. If serology is used to diagnose an active infection, it is recommended that paired serum samples be collected at a 2-week interval to demonstrate a rise in antibody titer (Kluge et al., 1992). Virus isolation allows for a definitive diagnosis, with brain, spleen, and lung being the organs.of choice. Necropsy. Gross lesions may be minimal or may include a fibrinonecrotic rhinitis; necrotic foci in the tonsils, liver, spleen, and lungs; and an endometritis and necrotizing placentitis (Thomson, 1988). Microscopic lesions includes a nonsuppurative meningoencephalitis and ganglioneuritis involving both gray and white matter, and intranuclear inclusions may be found in neurons, astrocytes, oligodendroglia, and endothelial cells (Thomson, 1988). There is a necrotizing bronchitis and alveolitis, necrotizing tonsilitis, lymphohistiocytic endometritis, and necrotizing placentitis with inclusion bodies in both necrotic cells and epithelial cells around the foci of necrosis (Kluge et al., 1992).

15. BIOLOGY AND DISEASES OF SWINE

Pathogenesis In natural infections, the virus enters via the mucosal epithelium in the nasopharynx and tonsils and then spreads via the lymphatics to regional lymph nodes where it replicates and results in a viremia (Thomson, 1988). The virus also spreads via the axoplasm of the trigeminal, glossopharyngeal, and olfactory nerves to the medulla and pons where it replicates in neurons and then spreads to other parts of the brain and results in latent infection of the trigeminal ganglia (Kluge et al., 1992). Prevention and treatment. Modified live, killed, and genedeleted vaccines with foreign-gene insertions are available to aid in the control of pseudorabies (Mulder et al., 1997). The vaccines protect pigs against clinical signs and mortality but do nothing toward eradicating the disease; the vaccine does not eliminate the virus in infected animals, nor does it prevent animals from becoming infected with the virus. Animals that are vaccinated, however, do shed lesser amounts of virus and have a limited tissue invasion by the organism. The gene-deleted vaccinations offer the advantage of producing vaccinated animals that lack antibody against the specific protein coded for by the deleted gene. This allows the vaccinated pig to be differentiated serologically from an infected pig. Pseudorabies is a reportable disease, and in January 1989, a national pseudorabies virus-eradication program was implemented in the United States (Ormiston, 1988). This program includes test and removal, offspring segregation, and depopulation and repopulation. The majority of industrialized nations support the concept of and are planning eradication programs. 2.

Respiratory Diseases

a.

Atrophic Rhinitis (AR, PAR, NPAR)

Etiology. Toxigenic strains of Pasteurella multocida, Bordetella bronchiseptica, and Haemophilus parasuis are the bacterial agents of this multifactorial disease. Porcine cytomegalovirus (CMV), which is the cause of inclusion body rhinitis, does not cause nasal turbinate atrophy; however, it may damage the nasal mucosa, predisposing it to colonization with one of these bacterial agents. Environmental air pollutants, namely, high ammonia levels (50-100 ppm) and dust (Hamilton et al., 1999), and genetic factors also play a role. Pasteurella multocida strains A and D produce a thermolabile dermonecrotic toxin (DNT), which severely damages the nasal mucosa, producing a progressive form of nasal turbinate atrophy. Bordetella bronchiseptica also produces a heat-labile DNT and alone will produce a moderate self-limiting form of the disease in which damaged tissues may regenerate in time (Tibor, 1999). Haemophilus parasuis reportedly causes a mild turbinate atrophy (Cowart, 1995). Combined infections of toxigenic P. multocida and B. bronchiseptica produce the most severe form. Typically, two or more infectious organisms are required to produce clin-

635 ical disease with permanent nasal distortion and turbinate atrophy. Recently, the term "nonprogressive atrophic rhinitis" (NPAR) has been applied to the form caused by B. bronchiseptica alone, and the term "progressive atrophic rhinitis" (PAR) to P. multocida alone and combined infections of P. multocida and B. bronchiseptica (De Jong, 1999).

Clinical signs and differential diagnoses. The clinical signs of pure B. bronchiseptica infection (NPAR) generally appear in nursery pigs less than 4 weeks of age and consist of sneezing, snuffling, and a mucopurulent nasal discharge. In older pigs these signs are milder or nonexistent. In very young pigs ( 3 4 days old) a severe bronchopneumonia can result. This form is much more rare than the nasal infections. The signs are a mild fever (103~176 or 39.5~176 marked "whooping" cough, and dyspnea, with high morbidity and mortality possible if untreated. This organism is frequently isolated from pneumonic lesions of older pigs; however, its role as a pathogen in this situation is questionable (De Jong, 1999). Other diagnoses to rule out for B. bronchiseptica include CMV and other causes of sneezing and rhinitis. The clinical signs of Pasteurella multocida (PAR) typically begin at 1 to 3 months of age and consist of sneezing and snuffling, which progresses to more violent sneezing with mucopurulent nasal discharge. In some cases epistaxis is seen. Inflammation of the nasolacrimal duct, which causes occlusion of the duct and subsequent tear staining visible at the medial canthus, frequently occurs. The most characteristic clinical sign is the dorsal and/or lateral deviation of the snout as the pig grows. This is caused by the abnormal bone growth, which occurs as a result of unequal nasal turbinate atrophy. Brachygnathia superior (BS) is the most common form seen and is due to slower bone growth in the upper jaw which gives it an upturned appearance. Significant turbinate atrophy can be present without visible snout abnormalities. Commonly, this atrophy is subjectively measured at necropsy by visual scoring of a section at the level of the second premolar. Techniques for objective quantification of this atrophy by digital image analysis or digitization (Gatlin et al., 1996) and computed tomography (Shryock et al., 1998) have been published. The latter method allows morphometric analysis of live pigs. In the more severe cases, whole-body growth rate will be decreased (De Jong, 1999), and this may be due in part to the possibility that the toxin produced affects the growth of the skeletal system (Ackermann et al., 1996). Differentials for PAR include other causes of facial deformities, including paranasal abscesses and breed variations. Epizootiology and transmission. Bordetella bronchiseptica is spread from pig to pig by aerosol droplets, which probably first occurs with snout-to-snout contact between a sow and a newborn piglet. This is followed by horizontal spread among littermates; however, spread can occur at any age. Piglets infected in the first week of life will generally develop more severe lesions

636 than those infected at 4 weeks or later. Those infected at 9 weeks show almost no lesions (De Jong, 1999). The quantity and quality of passive antibody obtained from the sow also affects the severity of lesions. In SPF herds the mode of transmission is often the introduction of new carrier animals to the herd. Bordetella bronchiseptica can be isolated from many domestic and wild species; however, these strains are usually nonporcine and less pathogenic for swine. This bacterium is commonly cultured from most swine herds and is not always associated with disease. Pasteurella multocida infection in SPF herds typically occurs by the introduction of carrier pigs. Once introduced into a seronegative herd, these bacteria will quickly spread by direct contact and aerosols. The pharynx, especially tonsils, and vagina of sows are sources of infection for piglets. Age of first infection inversely affects the severity of lesions; however, older pigs ( 3 - 4 months) will still develop lesions, which is in contrast to infection with B. bronchiseptica (De Jong, 1999).

Necropsy (Pasteurella multocida). There are varying degrees of deformity of the snout and the nasal septum. Distortion and atrophy of the turbinates are most severe in the ventral scroll of the ventral turbinates but also involve the dorsal scroll of the ventral turbinates, dorsal turbinates, and ethmoid turbinates. Microscopic changes include atrophy of the osseous cores of the turbinates and replacement by fibrous connective tissue, metaplasia of respiratory epithelium to stratified squamous, and inflammatory cell infiltrates in the lamina propria (De Jong, 1992). Pathogenesis (Pasteurella multocida). Production of lesions in this disease is dependent on the presence of toxigenic P. multocida and/or B. bronchiseptica colonization of the nasal epithelium with elaboration of toxins (De Jong, 1992). These toxins incite inflammatory cell infiltrates in the lamina propria and cause atrophy of mucosal glands, osteolysis, and replacement of turbinate bones by fibrous connective tissue. Necropsy (Bordetella bronchiseptica). Lesions in young pigs are catarrhal rhinitis, varying degrees of atrophy of the turbinates (most severe in the ventral scroll of the ventral turbinate), and a bilateral bronchopneumonia involving the apical and cardiac lobes (Giles, 1992; Duncan et al., 1966a). Microscopic lesions in the turbinates are metaplasia of the respiratory epithelium to stratified squamous, and an inflammatory cell infiltrate in the lamina propria with atrophy of the osseous cores and replacement by fibrous connective tissue. There is a severe vasculitis, endothelial cell hyperplasia, hemorrhage, and alveolar and perivascular fibrosis in the lung (Duncan et al., 1966b). Pathogenesis (Bordetella bronchiseptica). Bordetella bronchiseptica colonizes the ciliated epithelial cells in the nasal epithelium, where it results in loss of cilia. It also produces a toxin that is believed to penetrate the lamina propria and initi-

KATHYE. LABER ET AL. ate an inflammatory infiltrate and atrophy of the osseous cores (Giles, 1992).

Prevention. Development and maintenance of an SPF swine facility using cesarian section, medicated early weaning, and segregated early weaning are the most satisfactory methods of prevention. The focus should be on assuring freedom from toxigenic P. multocida since this is the most pathogenic etiologic agent of this multifactorial disease. Polymerase chain reaction (PCR) assays directed at the gene that encodes for the dermonecrotic toxin produced by toxigenic strains of P. multocida are reportedly specific and sensitive when used on nasal and tonsillar swabs (Kamp et al., 1996; Lichtensteiger et al., 1996) and colostrum (Levonen et al., 1996). Assays of this type and enzyme-linked immunosorbent assays (ELISAs) may be used for herd-health monitoring, facility biosecurity, and clinical diagnosis. Control. The majority of conventional swine herds are infected with B. bronchiseptica, and a smaller proportion also have strains A and D of P. multocida, the bacterial etiologic agents of atrophic rhinitis, but it is possible to keep these herds free of significant clinical disease through good sanitation, husbandry, and management. Experimental evidence has shown that continuous exposure of piglets to 20 ppm ammonia for 2 weeks will markedly exacerbate P. multocida colonization in the upper respiratory tract (Hamilton et al., 1998). Practices such as disinfection between groups of animals housed in a facility; adequate air changes to reduce ammonia levels; good temperature control; adequate nutrition and pen space; and control of concurrent diseases, dusty conditions, and experimental stress will help. Pasteurella multocida and B. bronchiseptica are sensitive to most common disinfectants. Vaccines for both P. multocida and B. bronchiseptica are available as bacterins and toxoids and are considered effective against atrophic rhinitis (Sakano et al., 1997b). These are generally given to the sow prefarrowing, as improved colostral immunity is considered more important than piglet vaccination. Treatment. A treatment plan for NPAR and PAR should include a combination of environmental and husbandry improvements followed by a vaccination and antibiotic program tailored to the particular facility. One approach is to medicate the feed of sows during the last month of gestation to reduce the bacterial load and source of initial exposure for suckling piglets. The oral antibiotics of choice include tilmicosin (Olson and Backstrom, 1999), sulfonamides, and tetracyclines. Piglets can be given weekly or biweekly parenteral injections of oxytetracycline, potentiated sulfonamides, ceftiofur, or penicillin/streptomycin, preferably based on culture and susceptibility, for the first month of life. Medication of feed or water in older weaned pigs at risk for PAR for periods of at least 4 - 5 weeks will help control clinical signs (De Jong, 1999).

15. BIOLOGY AND

Research complications. The toxin produced by severe infections of toxigenic strains of P. multocida will induce liver and kidney lesions as well as damage nasal turbinates. Bordetella bronchiseptica can induce pneumonic lesions in very young piglets. Therefore, PAR has the potential to affect most chronic research studies. b.

637

DISEASES OF SWINE

Pasteurellosis

Etiology. Pasteurella multocida is a gram-negative coccobacillus and a facultative anaerobe. Capsular serotypes A, B, and D have been reported in swine, with A being the most common in pneumonic lungs and B causing the most severe disease. Serotype B has not been reported in natural outbreaks in Europe and the United States (Pijoan, 1999). The role of toxin production by P. multocida as a virulence factor in pneumonic pasteurellosis is not clear; however, it has a defined role in causing atrophic rhinitis. Clinical signs and differential diagnoses. The predominant signs of the acute form of the disease are dyspnea ("thumping"), cough, anorexia, and fever to 107~ (41.7~ Sudden death is not typical unless rare serotype B strains are involved. Morbidity and mortality are variable, and typically pigs will lose weight and have a decreased rate of growth. The chronic form of the disease is characterized by intermittent cough, thumping, and low fever of 103~176 (39.5~176 or no pyrexia. The acute form is clinically similar to pleuropneumonia (APP) without the frequency of sudden death; the chronic form is similar to mycoplasmal pneumoniae of swine (MPS). Salmonella choleraesuis should also be considered. Metastrongylus elongatus and Ascaris suum are additional differentials for the chronic form (Cowart, 1995; Pijoan, 1999). Epizootiology and transmission. Pasteurella multocida is a common inhabitant of the upper respiratory tract of swine. It can be cultured from the nose and tonsils of healthy pigs from most herds, including SPF herds (Pijoan, 1999). The transmission is by direct contact and aerosols. Necropsy. Gross findings in the lungs are usually confined to the cranioventral aspects of the lobes and include red to gray areas of consolidation, frothy exudate in the trachea, suppurative pleuritis and pericarditis (Pijoan and Fuentes, 1987), pleural adhesions, and pulmonary abscesses (Pijoan, 1992). The histopathologic lesions in the lungs are a severe suppurative bronchopneumonia, pleuritis, and abscess. Pathogenesis. Pasteurella multocida serotype A adheres to ciliated respiratory epithelial cells, while serotype D adheres to nonciliated cells (Pijoan, 1992). It is usually not a primary agent but results in disease when adherence is facilitated by the presence of other agents. Pasteurella multocida has been shown to

produce a toxin that results in necrosis of osteoblasts and stimulation of osteoclastic bone resorption in the nasal turbinates, leading to turbinate atrophy (Dominick and Rimler, 1988).

Prevention. It is essential to identify and treat or manage any concurrent pathogens since P. multocida is usually the secondary agent. Typically, pasteurellosis is a complication of Mycoplasma hyopneumoniae infection. High quality control of environmental air temperature, humidity, and ammonia levels is critical. Control. Vaccination and medicated feed (tetracyclines, tylosins) and water may be beneficial. Any concurrent respiratory pathogens, especially Mycoplasma hyopneumoniae, PRRS, swine influenza, Actinobacillus pleuropneumoniae, and parasites should be controlled. The role of PRRS in exacerbating pasteurellosis is still uncertain and probably has only a mild effect (Carvalho et al., 1997). Treatment. Animals showing clinical signs should be treated with a parenteral antibiotic based on susceptibility testing. Alternatively, oxytetracycline, ceftiofur (Burton et al., 1996), penicillin, florfenicol, enrofloxacin, or doxycycline dosed in the feed has been shown to be effective at controlling pneumonia caused by P. multocida and M. hyopneumoniae (Bousquet et al., 1998). Development of resistance to antibiotics among Pasteurellae is a concern (Hormansdorfer and Bauer, 1998). Research complications. Bronchopneumonia-associated accumulation of purulent fluid in airways will complicate general anesthesia. Severe infections produce fibrinous pleuritis and pericarditis, which will confound most cardiovascular and respiratory system research studies. c.

Pleuropneumonia (APP)

Etiology. Actinobacillus pleuropneumoniae, previously designated as Haemophilus pleuropneumoniae or H. parahaemolyticus, is the cause of pleuropneumonia of swine. Extracellular hemolytic toxins ApxI, ApxlI, and ApxlII are some of the more important virulence factors of the A. pleuropneumoniae strains that produce them (Reimer et al., 1995; Kamp et al., 1997). All serotypes secrete one or more Apx toxins. There are currently 12 recognized serotypes (1-12) (Burkhardt et al., 1999); however, serovars 1, 5, and 7 are isolated most frequently (Cowart, 1995). This bacterium is a gram-negative encapsulated coccobacillary rod, which requires nicotinamide adenine dinucleotide (NAD or factor V) for growth. Primary isolation may be achieved by cross-streaking on a blood agar plate with a staphylococcus, which produces NAD. Termed "satellitism" or "satellite phenomenon," this will allow growth near the staphylococcus colonies. Chocolate agar media, which is supplemented with NAD, is commonly utilized. This organism also

638

KATHYE. LABERET AL.

shows the Christie-Atkins-Munch-Petersen (CAMP) phenomenon or reaction when colonies are grown near a [3 toxigenic Staphylococcus aureus. This consists of an increased zone of hemolysis greater than the partial lysis created by the S. aureus and is due to cytolysins (Taylor, 1999).

Clinical signs and differential diagnoses. The clinical signs of APP can be categorized into peracute, acute, and chronic forms. The peracute form is characterized by sudden death. In the acute form, pigs have fevers of 105~176 (40.6~176 depression, anorexia, cyanosis, severe dyspnea with a marked abdominal component ("thumping"), and sometimes death within 36 hr. The chronic form is characterized by variable cough, decreased rate of body-weight gain, and other complications (pleuritis, abortion, endocarditis, arthritis, abscesses). Serotype 2 has been connected with lameness due to necrotizing osteomyelitis and fibrinopurulent arthritis in 8- to 12-weekold pigs (Jensen et al., 1999). All three forms may be found in the same group of animals. A list of diagnoses to rule out would include other causes of pneumonia, primarily MPS, pasteurellosis, PRRS, Salmonella choleraesuis, and combinations of these agents. Epizootiology and transmission. Transmission is primarily by snout to snout and by aerosol. Recovered swine become chronic carriers and are a source of transmission within the herd and between herds. Pleuropneumonia is more prevalent in facilities that bring in swine from multiple sources on a regular basis. Typically, in herds where APP is endemic, the piglets are infected in the farrowing pen and a carrier sow is the source. All

age groups are affected, and morbidity and mortality are linked to environmental quality, stress, and concurrent infection with other pathogens. The disease is prevalent worldwide, different countries tend to have a different set of serovars, and multiple serovars can be found in one facility. The spread is likely related to the movement of animals since artificial insemination and embryo transfer are unlikely sources of introduction (Taylor, 1999).

Necropsy. The gross findings in pigs with A. pleuropneumoniae are a fibrinous pleuritis, pulmonary edema, and the presence of bloody froth or clotted fibrin plugs in the trachea and bronchi (Fig. 12). The lungs contain bilateral lesions that are dark red and firm with a predominance of lesions in the dorsal aspects of the caudal lobes, and there may be a bloody nasal discharge (Didier et al., 1984; Bertram, 1985; Nielsen, 1973). Histopathologic lesions are a necrotizing, fibrinous, and hemorrhagic pneumonia that is predominantly lymphocytic and histiocytic, as well as a vasculitis with thrombosis of vessels and lymphatics (Didier et al., 1984; Nielsen, 1973). Pathogenesis. Primary damage to the capillary endothelium in alveoli may be the result of endotoxin produced by A. pleuropneumoniae in acute and peracute infections. This results in severe edema and fibrin deposition as well as in thrombosis of capillaries and ischemic necrosis of pulmonary parenchyma (Bertram, 1985). Prevention. The most satisfactory prevention program is to maintain a closed, APP-free herd through strict isolation. Ar-

Fig. 12. Yucatanmicroswinelung infected withActinobacillus (formerlyHaemophilus)pleuropneumoniae.

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15. BIOLOGY AND DISEASES OF SWINE

tificial insemination and embryo transfer can be utilized when introduction of new genetics is required. Alternatively, only known SPF animals that have been validated by serologic testing (ELISA, CF, or PCR) can be added. An ELISA utilizing the A. pleuropneumoniae ApxlI antigen has been shown to be useful for this purpose (Leiner et al., 1999). In addition, PCR on mixed bacterial cultures from swine tonsils may be more sensitive than culture for detection (Gram et al., 1996). Vaccination of seronegative animals prior to introduction and maintaining optimal ambient temperature, ventilation, and humidity are useful for minimizing clinical disease in infected herds (Taylor, 1999). Segregated early-weaning practices can potentially eliminate APP; however, this is difficult because this bacterium is an early colonizer (Maclnnes and Desrosiers, 1999). Depopulation and restocking with hysterectomy-derived SPF animals is the most satisfactory means of prevention.

Control. Vaccines of the killed whole cell, cell-free antigens (Oishi et al., 1995), or subunit type (Burkhardt et aL, 1999) may reduce morbidity and mortality and extent of treatment. Oral immunization with live or inactivated A. pleuropneumoniae serotype 9 has been shown to provide partial clinical protection from aerosol challenge (Hensel et al., 1995). A vaccine strain of A. pleuropneumoniae produced by insertional inactivation of the ApxlI operon can be delivered live intranasally and provide cross-serovar protection (Prideaux et al., 1999). Additional control measures include good husbandry practices, including use of disinfectants and minimization of stress. Treatment. Parenteral antimicrobials, including ceftiofur (Burton et al., 1996), penicillin, tetracyclines, and enrofloxacin, can reduce mortality in the acute stage of the disease. Marked resistance has been reported for amoxicillin, oxytetracycline, and metronidazole (Gutierrez et al., 1995). Medicating feed and water with an antimicrobial at a low minimum inhibitory concentration (MIC) for members of an affected group that are still eating and drinking may be successful. Oral chlortetracycline (CTC) has been found to offer better protection from clinical disease than oxytetracycline (OTC) in experimental APP serotype 1 clinical challenge. This is probably due to the greater bioavailability of CTC in medicated feed (del Castillo et al., 1999). Tilmicosin phosphate added to feed 5 days prior to clinical disease has been shown to lower APP mortality and clinical impression scores (Moore et al., 1996; Bane et al., 1999). A combination of parenteral and oral medication often yields the best results. Antimicrobial therapy will not eliminate the chronic form or carrier animals from the herd (Taylor, 1999). Research complications. APP will affect any research involving the respiratory or cardiovascular systems since pleurisy, pneumonia, and pericarditis may result. The mortality associated with the acute form may terminate most studies.

d.

Mycoplasmal Polyserositis and Arthritis

Etiology. Mycoplasma hyorhinis is probably the easiest of the porcine mycoplasmas to isolate and is a common contaminant of cell culture lines. Clinical signs and differential diagnoses The age group most commonly affected is 3 to 10 weeks of age. Clinical signs typically begin about 1 week after some form of stress or initial exposure to the etiologic agent. The acute signs are lethargy, anorexia, labored respirations, arched back with tucked-up abdomen, lameness, and fever of 104~176 (40~176 These signs abate in about 2 weeks except that the lameness with swollen joints may persist for several months. Experimental M. hyorhinis intranasal inoculation has been shown to cause eustachitis and occasionally otitis media (Morita et al., 1998, 1999). Haemophilus parasuis should be ruled out for this clinical disease. Epizootiology and transmission. This organism is harbored in the respiratory tract of carrier swine, often without clinical disease. The most likely first exposure for baby pigs is from aerosolization or direct contact with nasal secretions from the sow prior to weaning. The organism will spread rapidly through group-housed pigs and typically will not cause clinical disease unless the animals are stressed. Stress will induce a septicemia and the resulting lesions. Necropsy. Acute lesions include a serofibrinous or fibrinopurulent pleuritis, pericarditis, and peritonitis, as well as a serofibrinous arthritis with increased synovial fluid and swollen reddish yellow synovial membranes (Ross et al., 1971). The joints most frequently involved are the stifle joints, but the tibiotarsal, cubital, coxofemoral, and shoulder joints may also be involved (Ross et al., 1971). This agent has also been reported to cause otitis media in swine (Morita et al., 1995). Pathogenesis. Mycoplasma hyorhinis is commonly found in the nasal passages of young pigs and is believed to be an opportunist following a stressful event, with a septicemia and seeding of the organism in the joints (Ross, 1992). Prevention and control. Eliminating stress of any type can best prevent clinical outbreaks of disease. This includes eliminating other diseases, controlling temperature and humidity fluctuations, and avoiding shipping and invasive research protocols. Concurrent infection with M. hyorhinis and PRRSV has been found to cause severe pulmonary lesions with respiratory distress (Kawashima et al., 1996) and underscores the need to eliminate other pathogens. Treatment. Prophylactic treatment of the entire herd by medicating food or water with lincomycin or tylosin may be

KATHYE. LABERET AL.

640 beneficial. Antimicrobial treatment of clinically affected swine is unrewarding (Ross, 1999a; Cowart, 1995). Research complications. Although mortality is low and morbidity typically less than 25%, clinical disease will confound cardiovascular studies and surgical models because it causes pericarditis, pleuritis, and peritonitis. e.

Mycoplasmal Pneumonia: Enzootic Pneumonia, Virus Pig Pneumonia, Mycoplasmal Pneumoniae of Swine (MPS)

Etiology. Mycoplasma hyopneumoniae is a common pathogen that colonizes the ciliated epithelium of the porcine respiratory tract. Mycoplasmas are small (0.2-0.3 mm), lack a cell wall, and are nonmotile, fastidious, gram-negative facultative anaerobes. They belong to the class Mollicutes and are the smallest free-living cells. Clinical signs and differential diagnoses. Although younger pigs may be affected, generally clinical signs are not obvious until pigs are 3 - 6 months of age. Uncomplicated MPS is generally characterized by a reduced growth rate and a chronic cough precipitated by exercise. In some affected animals the cough may not be readily evident. Morbidity is typically high and mortality low unless complicated by concurrent viral infections, secondary bacterial or other mycoplasmal diseases, or stress of any form. It plays an important role in porcine respiratory disease complex (PRDC) when concurrent infection with porcine reproductive and respiratory syndrome (PRRS) occurs (Thacker et al., 1999); however, additional experimental evidence indicates that this is not true for very young (3-weekold) pigs (Van Alstine et al., 1996). In these complicated infections, malaise, anorexia, fever, labored respirations ("thumping"), and possibly death may result (Ross, 1999b). Bacteria that frequently complicate MPS and are clinical differentials include Pasteurella multocida, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Salmonella choleraesuis, and Streptococcus suis (Bousquet et al., 1998; Cowart, 1995). Pneumonias due to other etiologic agents such as swine influenza, Ascaris suum, and Metastrongylus elongatus are additional differentials and complicating agents. Epizootiology and transmission. The spread of MPS is primarily by direct contact with respiratory secretions and aerosols from carrier swine. Generally it is transmitted from infected sows to suckling piglets prior to weaning; however, pigs of all ages are susceptible. It is probably the most common cause of chronic pneumonia in swine, and most conventional herds are affected. Necropsy. Grossly, the lungs contain pale gray or dark red foci of consolidation that are most commonly found in the apical lobes and the cranioventral aspects of the middle, accessory,

and caudal lobes. Additionally, there may be a purulent exudate in the bronchi. Microscopic lesions consist of perivascular, peribronchial, and peribronchiolar infiltrations of large numbers of lymphoreticular cells, which in chronic lesions may include lymphoid nodules (Piffer and Ross, 1984; Ross, 1992). Additionally, differentiation of cuboidal epithelium to pseudostratifled epithelium in bronchioles occurs (Ackerman et al., 1991). Pathogenesis. Mycoplasma hyopneumoniae adhere to the cilia and apical plasma membrane of the respiratory epithelium in the trachea, bronchi, and bronchioles and result in loss of cilia, ciliostasis, and filling of alveoli with cell debris and exudate (Ackerman et al., 1991). Prevention. The most satisfactory form of prevention is to allow only SPF swine into the facility. Mycoplasma hyopneumoniae-free herds may be derived by hysterotomy or hysterectomy, medicated early weaning, or segregated early weaning (Dritz et al., 1996). The success of these techniques should be monitored by a combination of ELISA testing of serum or milk, PCR assay of bronchoalveolar lavage fluids (Baumeister et al., 1998) or lung tissue (Stemke, 1997), clinical observation, and examination of lungs at necropsy. Culture is not usually feasible since mycoplasmas and M. hyopneumoniae, in particular, are difficult to isolate and grow. Control. Control of clinical disease in infected animals is best accomplished by providing optimal environmental conditions with respect to ammonia levels, humidity, temperature control, air changes, overcrowding, and reduction of stress. Protective immunity will develop in swine recovered from MPS, and vaccines are beneficial in some herds (Ross, 1999b). Maternally derived antibodies have been found to inhibit response to M. hyopneumoniae vaccination, and the timing of the dosing to avoid this interference varies from herd to herd (Daniels et al., 1999). Treatment. Antimicrobials, including lincomycin, tetracyclines, especially doxycycline in feed (Bousquet et al., 1998), tiamulin, and several quinolone antibiotics, have been shown to be efficacious in reducing the severity of pneumonia and weight reduction due to MPS. This beneficial effect is generally attributed to controlling complicating bacterial infections. Experimental evidence has shown that doxycycline has greater in vitro activity than oxytetracycline against M. hyopneumoniae, A. pleuropneumoniae, and P. multocida (Bousquet et al., 1997). Recent in vitro susceptibility studies have shown that valnemulin has exceptional activity against M. hyopneumoniae, and perhaps in vivo trials will validate this as another effective antibiotic treatment for MPS (Hannan et al., 1997). Research complications. Uncomplicated infection with M. hyopneumoniae will directly interfere with research involving the

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15. BIOLOGY AND DISEASES OF SWINE

respiratory system, and complicated infections will also interfere with cardiovascular studies since pericarditis may result.

f.

Inclusion Body Rhinitis (IBR)

Etiology. Inclusion body rhinitis, a disease caused by porcine cytomegalovirus, is found throughout the world. The causative viral agent is a member of the subgroup of slow-growing herpesviruses, beta herpesvirinae, which produce cytomegaly with hallmark intranuclear inclusions. The agent is species-specific and is able to induce latent infection, with shedding of the organism occurring even in the presence of circulating antibodies. Clinical signs and research complications. This disease is usually subclinical in pigs more than 3 weeks of age and may even be totally inapparent in young animals if good management practices are being followed. The clinical sequelae typically associated with this disease include unexpected fetal and piglet death, runting, mild rhinitis, pneumonia, and poor weight gain in young pigs. Some piglets may be born anemic, with edema noted around the jaw and tarsal joints. Adult animals that are exposed to this agent for the first time may develop mild anorexia and lethargy. The virus has been documented to modify the host defensive mechanism through inhibiting T-cell function (Kelsey et al., 1977). A differential diagnosis is parvovirus. Epizootiology and transmission. The virus can be recovered from nasal and ocular secretions, urine, and fluids associated with pregnancy, as well as from male reproductive organs. Dissemination of the agent most commonly occurs via nasal secretions and urine. The majority of virus is excreted from animals at 3 - 8 weeks of age; however, reactivation of excretion can occur when the animals are stressed. Lung macrophages are the reservoir of infection. The virus can be transmitted transplacentally, and congenital infection often manifests as fetal/neonatal death and runted pigs with rhinitis (Edington et al., 1977). Diagnosis. The presence of this disease can be confirmed using a serum ELISA. The virus can be isolated from the nasal mucosa, lung, and kidney. Histologic identification of inclusions and cytomegaly in epithelial tissues is also pathognomonic. Differential diagnoses include those diseases that result in an impact on the reproductive system, such as parvovirus and pseudorabies. Necropsy. Gross lesions in piglets are found in the nasal passages, where there is a serous rhinitis in early stages of the disease and a purulent exudate in older lesions (Thomson, 1988). There may also be a sinusitis, and if the disease becomes systemic, there are petechial hemorrhages and edema in the lungs, lymph nodes, subcutaneous tissues, and pericardial and pleural effusions. The kidneys may contain large numbers of petechia, or they may be dark purple (Edington, 1992). Histologic find-

ings characteristic for this disease are the presence of large basophilic intranuclear inclusions in the epithelial cells in both the mucosa and the mucosal glands (Thomson, 1988; Edington, 1976). If the disease has become systemic, there may be a pneumonia and foci of necrosis in the liver, kidney, CNS, and adrenals, with inclusions in capillary endothelium and sinusoidal cells throughout the body (Thomson, 1988).

Pathogenesis. The virus appears to enter the body through the mucosa, where it replicates inside the epithelial cells of the mucosal, Harderian, and lacrimal glands. The subsequent viremia results in seeding of mucosal glands, renal tubular epithelium, hepatocytes, duodenal epithelium, and in neonates or fetal pigs, the reticuloendothelial cells and capillary endothelium (Edington, 1992, 1976). Prevention and treatment. Supportive therapy to prevent the occurrence of secondary bacterial infections is always helpful in the face of a viral disease outbreak. Caution should always be taken when introducing new animals into an established grouping as new animals may expose susceptible animals or may stress existing groupings to stimulate resurgence of a latent infection. g.

Swine Influenza

Etiology. Swine influenza, first identified in 1918, is caused by a type A influenza virus. The agent is distributed worldwide, and antibodies to the virus are found in about 45% of the sampled pig populations. The influenza A viruses belong to the family of RNA viruses, Orthomyxoviridae. The type A viruses are further classified based on the glycoprotein spikes that extend from the viral particle (hemagglutinin [H] and neuraminidase [N]). The antigenic characteristics of these spikes provided the basis for dividing these viruses into subtypes. Antigenic comparison of the H1N1 swine viruses has shown, in contrast to human strains, that there has been little antigenic variation over the last 50 years (Sherrar et aL, 1989). This could be attributed to the fact that the virus is able to propagate in an ever-present population of nonimmune pigs. Strain H3N2 is also very prevalent in swine. Clinical signs and differential diagnoses. This disease typically spreads rapidly throughout susceptible herds, with morbidity close to 100%. The animals appear very ill with signs including anorexia, labored open-mouthed breathing, and a strong reluctance to move. The animals have fever, conjunctivitis, rhinitis, and nasal discharge, and will exhibit a barking cough. Despite the apparent severe clinical signs, the animals typically recover rapidly after 5 - 7 days of developing clinical signs. Mortality is usually less than 1%. Occasionally, abortions, stillbirths, and infertility have been reported to occur concomitantly with infection; however, studies have shown that the virus was

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not directly responsible (Brown et al., 1982.) The main differentials include bacterial pneumonias.

contaminated sources and preventing contact with birds and infected humans.

Diagnosis.

Treatment. Although not field-tested, amantadine has been shown to reduce the febrile response and the shedding of virus in experimentally infected pigs. This therapeutic drug is used for treatment and prevention of influenza in humans. Solid nursing care, avoidance of stress, and antibiotics to prevent secondary bacterial infections are suggested.

A definitive diagnosis can be made through isolation of the virus by swabbing nasal mucosa, or by demonstration of seroconversion. Serologic diagnosis requires paired serum samples (disease onset, then 4 weeks later) demonstrating an increase in antibody titer. Diagnosing weanling pigs via serology is difficult as maternal antibody persists up to 4 months. Young pigs carrying maternal antibody may still be infected and shed viral particles.

Epizootiology and transmission.

Swine influenza typically appears as a result of population movement, i.e., new animals entering the herd. Outbreaks can be described as explosive, rapidly spreading through all animals within a grouping. Once the virus gains purchase within a population of swine, the disease is likely to recur unless the grouping is totally depopulated. The primary route of transmission is via direct contact with the viral particles that are found in high concentrations in nasal secretions. There is no evidence that supports a carrier state, and the widespread occurrence and persistence of the virus is attributed to its continued passage to young susceptible animals or animals that have lost protective antibody titers obtained from previous infections. The H1N1 viruses have a very wide host range, including humans and birds, and interspecies transmission readily occurs. The H1N1 viruses can cause acute respiratory disease in humans, with evidence indicating that this virus, transmitted from pigs, was responsible for the 1918 influenza epidemic that killed an estimated 20 million people throughout the world. The more recent cases of documented H1N1 transmission have been in younger people, resulting in morbidity but no mortality. These cases also implicate birds, especially turkeys, as important sources of the virus. The strain of influenza virus identified in humans, H3N2, has also been shown to be infective to pigs, resuiting in significant disease.

Necropsy.

There is a fibrinous to mucopurulent exudate in the nasal passages, trachea, bronchi, and bronchioles (Thomson, 1988), and there are dark red to purple firm foci of consolidation in the apical and cardiac lobes of the lung and interlobular edema (Easterday and Hinshaw, 1992). Microscopic lesions are those of a necrotizing bronchitis, bronchiolitis, and bronchointerstitial pneumonia, and the airways are filled with cell debris and neutrophils (Thomson, 1988).

Pathogenesis.

The virus enters via the epithelium throughout the respiratory tract. There may be secondary infection by Haemophilus or Pasteurella.

h.

Verminous Pneumonia (Verminous Bronchitis)

Etiology.

Natural infections of swine with Metastrongylus spp. include one or more of M. salmi, M. pudendotectus, or M. elongates apri, with the latter being the most common. Adults are white, with males averaging 25 mm in length and females, 50 mm. Their eggs are oval, 4 0 - 5 0 mm in diameter, and larvated. Adult Ascaris suum (ascarids) are pinkish yellow nematodes. Males are 15-25 cm in length and females, 2 0 - 4 0 cm. The eggs are oval, 4 0 - 6 0 mm in width and 5 0 - 8 0 mm in length (Corwin and Stewart, 1999).

Clinical signs and differential diagnoses. The clinical signs consist of dyspnea, "thumping," and decreased weight gain. Icterus can be seen if ascarids migrate into the common bile duct. Differentials should include all bacterial, mycoplasmal, and viral causes of pneumonia in swine. Epizootiology and transmission.

Metastrongylus elongatus

(lungworm) has an indirect life cycle and requires an earthworm as an intermediate host. Several species, including Lumbricus terrestris, Helodrilus foetidus (Lapage, 1968b), and Eisenia foetida (Kumar, 1978), can serve this function (Lapage, 1968b). Eggs are coughed up from the lungs, swallowed, and excreted in the feces. Swine eat an earthworm that contains infective larvae, which then migrate to the mesenteric lymph nodes and on to the right heart and lungs. They mature in the bronchi and bronchioles of the diaphragmatic lung lobes. The prepatent period is 28 days. Ascarids have a direct life cycle and therefore can be a problem even in indoor facilities. Ingested larvated eggs hatch in the small intestine and invade the wall of the cecum and colon (Murrell et al., 1997); the larvae then migrate through the liver and lungs. In the lungs the larvae enter the alveoli and migrate up the airways. They are coughed up and swallowed and then return to the small intestine where they molt into adults. The prepatent period ranges from 40 to 53 days. The presence and migration of these two parasites exacerbate the clinical signs and disease of other viral and bacterial pneumonias of swine.

Prevention and control.

Currently, there are several vaccines licensed for use in the United States and Europe. Other means of control include preventing influx of animals from unknown

Necropsy.

Adult M. elongatus can be found in the trachea, bronchi, or bronchioles, and larvae may be found in the lung

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parenchyma at necropsy (Jones and Hunt, 1983). Characteristically, mucoid plugs containing adults and eggs obstruct the bronchioles in the diaphragmatic lobes, producing atelectasis (Corwin and Stewart, 1999). Adult A. suum are found in the small intestine, including the common bile duct, and white focal hepatic lesions (scarring) indicative of ascarid migration and sometimes called "milk spots" are typically found at necropsy (Wagner and Polley, 1997). Larval migration through the lungs produces hemorrhage, inflammation, emphysema, and secondary bacterial pneumonia (Corwin and Stewart, 1999).

Stewart et al., 1996; Saeki et al., 1997), ivermectin, the benzimidazoles (fenbendazole, albendazole, oxfendazole), pyrantel, piperazine, levamisole, dichlorvos, and hygromycin B. Doramectin SQ has been shown to have persistent activity of at least 7 days against a challenge with embryonated A. suum eggs (Lichtensteiger et al., 1999). Metastrongylus elongatus is susceptible to doramectin (Logan et al., 1996; Yazwinski et al., 1997), ivermectin, benzimidazoles, and levamisole (Cowart, 1995). Antibiotic therapy may be indicated to treat secondary or primary bacterial pneumonia in swine showing respiratory signs.

Pathogenesis. Metastrongylus elongatus larvae migrate through the lung parenchyma, causing alveolar hemorrhage followed by inflammation and consolidation of the lungs. This consists predominantly of leukocytes of which the majority are eosinophils. Maturing larvae continue migrating to the bronchioles and bronchi as they mature into adults, where they copulate and lay eggs which produces more irritation and inflammation and further lung consolidation (Jones and Hunt, 1983). Secondary bacterial pneumonia can also result, and Pastuerella and Staphylococcus spp. were common isolates in one report (Copland, J. W., 1976). Migrating A. suum create liver lesions, which are seen grossly as white spots that peak at about 1 week postinfection and heal in 3 - 8 weeks (Roepstorff, A., 1998). The pathogenesis of the lung lesions is similar to that of M. elongates; however, the larvae are coughed up and then swallowed and mature into adults in the small intestine.

Research complications. If untreated, these infections will damage the lungs, liver, and other tissues during migration.

Prevention and control. In indoor research facilities, the life cycles of both these parasites can be broken by frequent and thorough sanitation procedures that minimize contact with feces. The provision of bedding material is associated with a higher incidence of A. suum infection than in bedding-free housing systems (Dangolla et al., 1996). Neopredisan (p-chlorom-cresol) disinfectant has been shown to be a very efficacious ovicide and larvicide for A. suum (Mielke and Hiepe, 1998). This coupled with a strategic or continuous anthelmintic treatment program should eliminate clinical disease. If outdoor pens are utilized, housing on concrete or bringing the animals indoors to prevent access to earthworms and to facilitate sanitation and anthelmintic treatment is worthwhile. Feral Sus scrofa in the United States and Europe have been found to have A. suum and Metastrongylus spp. (Gipson et al., 1999; Henne et al., 1978) and are a potential reservoir in areas where contact is possible. It is recommended that health monitoring for these pathogens be done on any wild populations. An indirect ELISA for anti-A, suum IgG is more sensitive and probably provides a more realistic assessment of the prevalence than fecal examination for oocysts (Roepstorff, 1998). Treatment. Ascaris suum is susceptible to numerous anthelmintics, including avermectins (doramectin (Logan et al., 1996;

3.

Gastrointestinal Diseases

Young swine commonly develop diarrhea associated with shipping stress, changes in diet, primary or mixed infection with a variety of enteric pathogens, or the perioperative use of antibiotics that may upset the balance of normal gut microbiota (Table IV). The morbidity and mortality associated with enteritis make clinically affected pigs unsuitable for experimental use, and residual lesions in recovering animals may interfere with experimental assessment of the gastrointestinal tract. The following is a summary of the infectious diarrheas that may be encountered when young swine are managed within research facilities. The reader is referred to comprehensive texts for detailed information on historical perspective, epidemiology, diagnosis, and treatment of these diseases (Leman, 1992; Hawk and Leary, 1995). a.

Swine Dysentery

Swine dysentery is a severe mucohemorrhagic diarrhea of pigs of postweaning age. Etiology. Brachyspira hyodysenteriae, a gram-negative anaerobic spirochete, is the primary etiologic agent of swine dysentery and is one of five Brachyspira spp. known to infect swine (Boye et al., 1998). Because disease is less severe when gnotobiotic pigs are experimentally infected, other anaerobic microorganisms normally found in the lower bowel are believed to contribute to lesion development. Additionally, nutritional factors may be important; diets rich in rapidly fermentable carbohydrates may exacerbate clinical signs (Pluske et al., 1996). Diagnosis of B. hyodysenteriae infection can be confirmed by culture or PCR (Atyeo, 1998). Clinical signs and differential diagnoses. Rarely, swine dysentery may cause peracute death without premonitory signs. More commonly, severe diarrhea and fever with accompanying dehydration, weight loss, and weakness develop over several days. Diarrhea of acute onset is usually watery with large amounts

Table IV Common Infectious Causes of Enteritis in Newborn to Postweaning Age Swine Disease

Clinical signs

Age

Etiology

Gross lesions Nonspecific dilation and congestion of small intestine, blood-tinged contents Large bowel edema, hyperemia, mucofibrinous exudate on mucosa

Colibacillosis

Acute death, watery to whiteyellow or hemorrhagic diarrhea

Newborn to postweaning

Escherichia coli ETEC, EPEC (AEEC), EHEC

Swine dysentery

Watery, mucoid, and hemorrhagic diarrhea, rarely acute deaths

1 week and older

Serpulina hyodysenteriae

Proliferative enteropathy

Acute death, mucoid to hemorrhagic diarrhea

Postweaning

Lawsonia intracellularis

Clostridial enteritis

Acute death, severe hemorrhagic diarrhea

Newborn to postweaning

Clostridium perfringens

Salmonella enterocolitis

Watery, yellow diarrhea with fever, anorexia, depression

Postweaning

Salmonella typhimurium, S. choleraesuis

Transmissible gastroenteritis

Vomiting, severe diarrhea, high mortality

Any age

TGE virus

Rotavirus

Profuse watery, white/yellow diarrhea

Most severe within days of birth

Porcine rotavirus

Balantidiasis

Asymptomatic to severe ulcerative enterocolitis Asymptomatic to anorexia with diarrhea

Any age

Balantidium coli

Any age

Giardia intestinalis

Giardiasis

Coccidiosis

Asymptomatic to severe diarrhea

1-2 weeks of age

Isospora suis

Whipworms

Asymptomatic to severe mucoid or hemorrhagic diarrhea with nortality

Postweaning

Trichuris suis

Small intestinal threadworm

Asymptomatic to severe diarrhea with mortality

Nursing pigs

Strongyloides ransomi

Gross thickening of distal ileum, cecocolic junction, cecum, edema, exudate Severe hemorrhagic involvement of small intestine, gas, bloody fluid in abdomen Focal or diffuse necrotic typhlocolitis, enlarged mesenteric lymph nodes, other organ involvement Thin-walled small intestine distended with yellow fluid Nonspecific dilation of small and large intestine with yellow to gray watery fluid Variable, secondary to other primary diseases None to nonspecific enteritis None, severe cases may have fibrinonecrotic membrane in jejunum and ileum Edema, nodules containing exudate, fibrinonecrotic membrane, hemorrhage, anemia, adult. worms attached to mucosa Nonspecific, presence of adult worms in small intestine

Histologic lesions Congestion, hemorrhage, acute inflammation, villous atrophy, adherent bacteria Mucosal edema, mucofibrinous enteritis with superficial erosions, hemorrhage Hyperplasia of glands and epithelium, intracellular bacteria on EM Necrotic villi, adherent grampositive bacilli, profuse hemorrhage Necrosis of enterocytes, inflammatory infiltrates, thrombi, lymphoid atrophy or hyperplasia Villous atrophy ulceration of Peyer's patch dome epithelium Villous atrophy

Ciliated trophozoites, flask-shaped ulcers None to nonspecific enteritis, adherent commashaped flagellates Villous atrophy, villous fusion, hyperplasia of crypts, necrosis Migrating larva in submucosa, adult worms attached to mucosa Encysted larvae

Diagnosis Culture, serotyping

Culture, WarthinStarry positive spirochetes in colonic crypts, PCR In situ hybridization, tissue culture isolation, electron microscopy Culture, toxin assays on cecal contents, Gram stain of mucosal smears Culture, clinical signs, necropsy lesions

Rising serum titers, viral isolation, PCR Rising serum titers, viral isolation, PCR, in situ hybridization Histology, fecal direct smears Histology, fecal direct smears Fecal flotation

Fecal flotation, necropsy

Fecal flotation, necropsy

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of mucus accompanied by flecks of blood and white, mucofibrinous exudate. Pigs with chronic diarrhea may pass red to black soft stools that contain mucus. Nursing pigs are typically not affected but may develop catarrhal enteritis without hemorrhage. Hemorrhagic diarrhea in piglets that are newborn to several weeks of age could also be caused by Clostridium perfringens. In older pigs, other causes of hemorrhagic enteritis include Salmonella spp., Lawsonia intracellularis, and Trichuris suis. Mixed infections with Yersinia pseudotuberculosis, Salmonella typhimurium, or B. pilosicoli commonly result in more extensive lesions, affecting the cecum as well as the colon, and may prolong recovery time from swine dysentery (Thomson et al., 1998). Brachyspira pilosicoli is a newly recognized species of pathogenic intestinal spirochete (Duhamel et al., 1998) that causes porcine colonic spirochetosis, a nonfatal diarrheal disease that affects pigs during the growing and finishing stages of production.

Epizootiology and transmission. In natural outbreaks of swine dysentery, B. hyodysenteriae is transmitted by fecal-oral contact, either by direct contact between naive and infected pigs or by use of contaminated housing, equipment, or clothing. The organism will survive up to 60 days in moist ground or feces but is readily eliminated by disinfection in the absence of organic material. Recovered pigs may continue to shed B. hyodysenteriae in their feces. Necropsy. Pigs that have died from swine dysentery are dehydrated and may have rough or fecal-stained coats. The gross lesions vary in distribution but are confined to the large bowel (Hughes et al., 1977). Early lesions include reddening and edema of the gut wall, mucosa, and mesenteric lymph nodes, as well as the presence of a fibrinous, blood-flecked membrane covering the mucosa (Harris et al., 1997). The exudate in the lumen is red-brown and watery and contains exudate (Hughes et al., 1977). Older lesions are less edematous, but there is a thick pseudomembrane composed of fibrin, mucus, and blood covering the mucosa (Harris and Lysons, 1992). Microscopic lesions consist of elongated colonic crypts, hyperplasia of goblet cells, and necrosis of sheets of epithelial cells that results in damage to exposed capillaries and exudation of fluid, fibrin, blood, and inflammatory cells from the lamina propria (Hughes et al., 1977). Large amounts of this exudate accumulate in the mucosal crypts and on the mucosal surface, forming a fibrinous pseudomembrane. Large number of spirochetes can be found in the crypts as well as in the lumen. Pathogenesis. Brachyspira hyodysenteriae is very efficient at penetrating mucus or other material and attaching to the colonic epithelium. These organisms do not invade the gut wall below the lamina propria. The organism has been shown to produce a hemolysin that is cytotoxic and an endotoxin. The diarrhea is the result of colonic malabsorption from failure of colonic ep-

ithelial cells to transport sodium and chloride from the lumen to the blood (Schmall et al., 1983). The mechanism of diarrhea is therefore very different from that of Salmonella, Shigella, and Escherichia coli (Schmall et al., 1983). The dehydration and fluid loss are due to the failure to reabsorb the pig's endogenous secretions (Harris and Lysons, 1992).

Prevention. Swine dysentery is usually introduced to a facility by the purchase of an asymptomatic carrier pig. Wild rodents are also reservoirs. Pigs should be purchased from herds SPF for B. hyodysenteriae or alternatively, from herds in which drugs or vaccines that may only suppress the infection are not used. Control. In the biomedical research setting, pigs affected with swine dysentery should be quarantined and treated or euthanatized. Sanitation of the facility and associated equipment along with review of rodent control and vendor health status should be adequate for avoidance of re-introduction. Valuable pigs can be segregated by health status and treated with antibiotics. Nursing pigs are protected by colostrum from previously infected sows and can be a source of Brachyspira-free pigs if weaned early and housed in a clean facility. Treatment. If indicated, therapy should consist of fluid and electrolyte replacement along with antibiotics. Carbadox, tiamulin, and lincomycin have all been reported to be effective in treatment and/or prevention of swine dysentery. Research complications. The morbidity and mortality associated with swine dysentery make clinically affected pigs unsuitable for experimental use. b.

Proliferative Enteropathy

Based on inflammatory and proliferative lesions found at necropsy in the terminal ileum, proliferative enteropathy (PE) of the pig has been historically referred to as porcine intestinal adenomatosis, terminal or regional ileitis, regional enteritis, intestinal adenoma, porcine proliferative ileitis, and muscular hypertrophy with stenosis of the ileum. Proliferative enteropathy affects multiple species, and the comparative aspects have been reviewed (Cooper and Gebhart, 1998).

Etiology. Proliferative enteropathy had long been associated with the presence of abundant intracellular Campylobacter-like organisms (CLO) in enterocytes that were antigenically distinct from known Campylobacter species (McOrist and Lawson, 1989). Due to its obligate intracellular parasitism of enterocytes, this CLO has been difficult to work with because it can be grown only in tissue culture. Sequence analysis of 16S ribosomal RNA identified the causative bacterium as a close relative of Desulfovibrio desulfuricans, and the bacterium has since

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been definitively identified as the microaerophilic bacterium, Lawsonia intracellularis (McOrist et al., 1995b). Clinical signs and differential diagnoses. Clinical disease attributed to PE is most often observed in the postweaned pig between 6 and 20 weeks of age. Infected animals may show clinical signs ranging from none to severe hemorrhagic diarrhea following a stress such as shipping or weaning. Failure to grow at a normal rate may be the only clinical sign that is detectable antemortem. More severe necrotic enteritis, most commonly found in young adult swine rather than in those just weaned, may present as acute death, anemia secondary to acute hemorrhagic diarrhea, or a more chronic form associated with passage of black tarry feces. Differential diagnoses for hemorrhagic diarrhea in piglets that are newborn to several weeks of age should include Clostridium perfringens and B. hyodysenteriae. Other causes of hemorrhagic enteritis, particularly in older pigs, include Salmonella spp., Lawsonia intracellularis, and Trichuris suis. Fluorescent in situ hybridization targeting 16S ribosomal RNA in formalin-fixed tissue has been used for specific and fast detection of Lawsonia intracellularis in enterocytes from pigs affected by PE (Boye et al., 1998). Epizootiology and transmission. Proliferative enteropathy is worldwide in distribution and affects other species, including the hamster, dog, fox, ferret, horse, rat, and rabbit. Consequently, other animals, such as rodents, could be the sources of new infection. Lawsonia is shed in feces, and transmission is by fecal-oral contact. In endemic areas, 15 to 30% of the herds are estimated to be affected with a 5 to 20% infection rate within a herd. There is risk of environmental contamination, but little is known about how long Lawsonia can remain infectious outside of the animal. Necropsy. The gross lesions of PE are found in the ileum, cecum, and the most proximal one-third of the spiral colon and consist of a markedly thickened gut wall and the mucosa containing multiple transverse or longitudinal folds (Rowland and Lawson, 1992); there may be polyps in the colon. Microscopic lesions consist of markedly elongated branching crypts lined by immature epithelial cells and lack goblet cells. Varying numbers of silver-staining organisms that also exhibit acid-fast staining with a modified Ziehl-Neelsen stain are found free in the apical cytoplasm of the lining cells (McOrist, 1995b). Inflammatory response in the lamina propria may be minimal. The gross lesions of proliferative hemorrhagic enteropathy are confined to the ileum and rarely involve the large bowel. These consist of a thickened, reddened mucosa that does not contain erosions but may be covered by a fibrinous membrane, and the lumen may contain blood clots (Love and Love, 1979). Colon contents may be black and tarry (Rowland and Lawson, 1992). Histologic findings include extensive degeneration and necrosis of the ileal epithelium, crypt abscesses, and extensive accumu-

lation of proteiriaceous fluids in the lamina propria of the villi, resulting in distortion of the villi (Love and Love, 1979). Lesions of regional ileitis consist of a very firm thickened wall of the ileum, which has multiple foci of erosions and ulcerations of the mucosal surface and marked hypertrophy of the outer muscle layers (Rowland and Lawson, 1992). Pathogenesis. Lawsonia intracellularis is an obligate intracellular organism. Animals become infected as a result of consuming fecal-contaminated material (McOrist et al., 1995b). The organisms enter the immature, proliferating crypt epithelial cells and multiply within the apical cytoplasm. The infected crypt cells fail to mature and are not shed, so the crypts become elongated and tortuous (Rowland and Lawson, 1992). Prevention. Swine should be purchased from a vendor with a herd-health history that is free of clinical PE. Newly introduced pigs should be quarantined and housed separately to avoid contact with feces of other swine that may be shedding Lawsonia. Control. Clinically affected pigs should be quarantined and treated or euthanatized, based on severity of disease and the intended use of the animal. Control efforts should include sanitation of equipment and the housing area, review of rodent control, and treatment with antibiotics of pigs at risk of clinical disease (McOrist et al., 1996). Treatment. Proliferative enteropathy can be self-limiting with spontaneous improvement after several weeks. Antibiotics are commonly used to control clinical signs. Treatment of this disease is problematic because of the lack of in vivo or in vitro data on antibiotic sensitivities of Lawsonia. In tissue culture, penicillin, erythromycin, difloxacin, virginiamycin, and chlortetracycline were the most effective antibiotics, followed by tiamulin and tilmicosin (McOrist et al., 1995a). Tylosin phosphate can be effective for prevention and for treatment of PE (McOrist et al., 1997). Research complications. The morbidity and mortality associated with PE make clinically affected pigs unsuitable for experimental use. Lesions typically resolve over time.

c.

Colibacillosis

Enteric colibacillosis is the most important diarrheal disease of newborn to postweaning-age swine (Leman, 1992). Diarrhea attributable to colibacillosis is commonly observed in neonates born to nonimmune sows or in piglets housed in heavily contaminated environments. Susceptible animals include recently weaned piglets that are experiencing the waning of passive maternal immunity, are being stressed by new housing, or are adjusting to dietary changes.

15. BIOLOGYAND DISEASES OF SWINE Etiology. Colibacillosis is caused by pathogenic Escherichia coli, a gram-negative facultative anaerobic rod. The species E. coli includes members that are normal gut flora as well as enteric pathogens that are further classified by antigenic serotype: somatic (O), capsular (K), flagellar (H), and fimbrial adhesins (F). Pathogenic E. coli also possesses one or more virulence factors encoded on either the bacterial genome or plasmids. Various classifications associated with different modes of pathogenesis include enterotoxigenic strains of E. coli (ETEC), which produce heat-stabile (ST) or heat-labile (LT) enterotoxins. Enteropathogenic E. coli (EPEC), also referred to as attaching and effacing strains (AEEC), which attach to the enteric epithelium using fimbrial adhesins, efface the microvilli and invade the epithelial cells. Strains of E. coli that cause hemorrhagic gastroenteritis are referred to as enterohemorrhagic E. coli (EHEC) (Tzipori et al., 1989). As an example, neonatal diarrhea in piglets from 0 to 4 days of age is commonly caused by an enterotoxigenic (ETEC) strain of E. coli that possesses F4-type fimbrial adhesins, produces ST or LT enterotoxin, and belongs to the classical serotypes O149, 08, O147, or O157. Clinical signs and differential diagnoses. Colibacillosis presents as diarrhea that will vary in severity based on the virulence factors present in the E. coli strain involved and the age and immune status of the piglets. Severe dehydration, metabolic acidosis, and weight loss may accompany the diarrhea, or peracute deaths without diarrhea may be seen. Neonatal colibacillosis can develop within hours of birth if piglets are born to gilts in contaminated environments and is characterized by either clear, watery diarrhea or loose stools that vary in color from white to brown. Severe outbreaks are associated with high morbidity and mortality in neonates; older pigs have less severe disease. Hemorrhagic gastroenteritis from colibacillosis can occur peracutely (sudden death) or acutely (rapid decline with severe diarrhea) in previously healthy unweaned or recently weaned pigs. The differential diagnoses for yellow to white, watery diarrhea in piglets that are newborn to several weeks of age should include colibacillosis, salmonellosis, transmissible gastroenteritis virus, rotavirus, nematodiasis, and coccidiosis. Epizootiology and transmission. Clinical disease results from interaction between the causative bacteria, adverse environmental conditions, and select host factors. Newborn pigs encountering large numbers of E. coli carrying the appropriate virulence factors will develop colibacillosis if colostrum is not available because of competition for teats, if the sow develops agalactia, or if the sow is not immune to E. coli. Some pigs are inherently resistant to colibacillosis because they lack receptors on their epithelial cell brush borders to which the fimbriae bind (Baker et al., 1997). Necropsy. Gross lesions of the edema disease syndrome may include marked edema of the mesenteric lymph nodes and

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mesentery, the wall of the stomach, large intestine, subcutaneous lymph nodes, eyelids, subcutaneous tissues, lungs, liver, and gallbladder. The mucosa of the small intestine and stomach may contain extensive hemorrhages (Bertschinger and Pohlenz, 1983). Microscopic lesions in the lamina propria of intestinal villi include vascular congestion with some hemorrhages into the lumen, endothelial swelling, perivascular edema, necrosis of the tunica media, microthrombi, subendothelial deposition of fibrin, an inflammatory infiltrate consisting of neutrophils and macrophages, and villus atrophy (Bertschinger, 1983). Some ETEC isolates can be found adhered to the mucosal epithelium on the villi, while other isolates colonize the crypts of Lieberkuhn (Bertschinger et aL, 1992). Pathogenesis. Enteropathogenic E. coli has numerous virulence factors. The AEEC porcine organisms attach to enterocyte microvilli by means of an attachment factor and then efface the microvilli and invade the epithelial cells (Gyles and Thoen, 1993). The ETEC organisms contain a mucopolysaccharide capsule that is antiphagocytic and not very antigenic. The K1 polysaccharide enhances bacterial resistance to complement mediated killing by inhibiting the alternative pathway to complement activation. The very long O chain polysaccharide chains in the cell wall bind the membrane attack complex resuiting from complement activation distant from the cell membrane so that it cannot lyse the cell (Gyles and Thoen, 1993). Specialized fimbriae, K88(F4), K99(F5), F6, and F41, permit the adherence and colonization of the enterocytes. The ETEC also produce heat-labile toxins, which elevate levels of adenylate cyclase leading to the efflux of Na and C1 ions and water out of the cell. In addition, SLT-IIe, a heat-labile, Shiga-like toxin, binds to and damages vascular endothelium, resulting in edema, hemorrhage, and thrombosis (Gyles and Thoen, 1993). The heat-stable toxins Sta and Stb cause elevated levels of prostaglandins in enterocytes, which may enhance diarrhea (Gyles and Thoen, 1993). Prevention. Farrowing management should be "all in, all out" to provide for adequate sanitation between litters. In problem herds, gilts and sows should be immunized with a commercial vaccine or an autologous bacterin during gestation. Control. To minimize environmental stress, piglets should be housed on warm floors to prevent hypothermia and in clean conditions to avoid ongoing ingestion of bacteria. Housing should be draft-free and maintained at temperatures of 30 ~ to 34~ for very young pigs. Nursing pigs will derive protection from colostrum feeding from immune sows. Vaccination of breeding stock is indicated if problems are recurrent. Treatment. A broad-spectrum antibiotic should be started pending culture and sensitivity results. Most isolates are sensitive to amikacin, gentamicin, spectinomycin, and enrofloxacin.

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Oral fluid therapy consisting of electrolyte replacement solutions containing glucose should be instituted to correct dehydration, energy depletion, and ongoing fluid and electrolyte losses.

The bacillus is transferred from sow to pigs and between pigs by fecal-oral contact. Clostridium perfringens exists in the environment as a vegetative form or as spores that persist for a least a year.

Research complications.

Necropsy.

Morbidity and mortality from colibacillosis in neonatal pigs clearly interfere with their experimental use. Once recovered, animals should be clinically normal.

d.

Clostridial Enteritis

Clostridial infection of the intestinal tract of young swine commonly results in a necrotic enteritis with high mortality.

Etiology.

Clostridium perfringens type A is a normal inhabitant of the swine intestine and causes an enteritis generally associated with low mortality. In contrast, fatal necrotic enteritis is caused by C. perfringens type C. Clostridium perfringens is an encapsulated, gram-positive bacillus that produces a variety of enterotoxins that are responsible for clinical signs and lesions (Buogo et al., 1995). Clinical signs and differential diagnoses. Clinical manifestations of infection with C. perfringens will depend on the immune status of the swine herd and the age of naive exposed piglets. Clinical disease may be peracute with death of piglets aged 12 to 36 hr that may or not have developed hemorrhagic diarrhea. Acute disease is characterized by 2 days of reddish brown diarrhea containing gray, necrotic debris, with ensuing death by 3 days of age. Subacute disease develops as persistent nonhemorrhagic diarrhea that is yellow initially and then changes to clear liquid with flecks of necrotic debris. Piglets with subacute disease remain alert but commonly die from dehydration by 5 to 7 days of age. Chronic enteritis may involve intermittent or persistent diarrhea for several weeks, with feces yellow-gray in color and mucoid in consistency. Pigs may die after several weeks of clinical signs or be culled based on failure to grow. Differential diagnoses for hemorrhagic diarrhea in piglets that are newborn to several weeks of age should include C. perfringens and Brachyspira hyodysenteriae. Other causes of hemorrhagic enteritis, particularly in older pigs, include Salmonella spp., Lawsonia intracellularis, and Trichuris suis. Epizootiology and transmission.

Clostridum perfringens is usually introduced by purchase of a carrier sow or by use of contaminated housing, equipment, or clothing. Although disease is most common in pigs aged 12 hr to 7 days and peaks in incidence at 3 days of age, disease has also been observed in older pigs aged 2 to 4 weeks and in postweaning pigs. Disease is explosive, with 100% mortality in pigs born to nonimmune sows. Subsequent litters are protected by maternal immunity.

Piglets may be found dead, or hemorrhagic diarrhea may be noted in acute and peracute cases. Death is the result of a severe necrotizing enteritis. The affected segments of gut vary from involvement of only several centimeters to extension from a point 14 cm distal to the pylorus to the cecum. The affected gut wall is dark red to black, and there may be gas bubbles. Enteric lymph nodes are red. The mucosa is dark red, and the intestinal contents in affected segments contain hemorrhagic and necrotic debris. There may also be blood-tinged ascitic fluid. Microscopic lesions consist of severe necrosis of villi and crypts, and severe and extensive hemorrhages throughout the lamina propria and mucosa. There may be a necrotic membrane composed of bacteria, sloughed epithelium, fibrin, and inflammatory cells lying over the submucosa (Taylor, 1992).

Pathogenesis.

Disease due to C. perfringens type A occurs when large numbers of organisms build up in the jejunum and ileum and produce an alpha (a) toxin that can cause necrosis of enterocytes, leading to a profuse loss of both electrolytes and fluids. These organisms do not invade the enterocytes. Sporulating forms also produce an enterotoxin that forms a complex with a specific cell-membrane protein and alters the cell-membrane permeability, leading to loss of sodium, chloride, and fluid while inhibiting glucose uptake (Nilo, 1993). Clostridium perfringens type C organisms produce a trypsinsensitive beta (13)toxin that is responsible for much of the necrotizing lesions. These organisms attach to enterocytes and result in initial loss of microvilli on the enterocytes at the tips of the villi and damage to terminal capillaries, with increased capillary permeability. This is followed by a rapid, progressive necrosis of the remaining villus enterocytes, the crypt cells, and mesenchymal structures in the lamina propria and muscularis mucosa (Nilo, 1993). Some organisms may penetrate to the muscle layers and produce emphysema of the gut wall and thrombosis of vessels (Taylor, 1992).

Prevention. Routine vaccination of sows will prevent disease (Kelneric et al., 1996). Sows can be vaccinated with a toxoid at the time of breeding or midgestation and then again 2 weeks prior to farrowing. Piglets from immune sows will be protected by colostrum.

Control. Clinically ill pigs should be isolated and treated and the premises sanitized. Individual piglets and pregnant swine that are at risk from recent exposure should be vaccinated with toxoid. Medicated feed has been shown to control clinical signs (Kyriakis et al., 1996).

15. BIOLOGY AND DISEASES OF SWINE

Treatment. Once clinical signs develop, disease is extensive and often unresponsive to therapy. Oral antimicrobials such as ampicillin given soon after birth and repeated daily for the first 3 days of life may prevent clinical disease. Pigs with severe diarrhea should receive supplemental fluids containing glucose and electrolytes. Research complications. Clostridial enteritis causes acute death and severe morbidity arr/ong survivors. Overgrowth of C. perfringens from perioperative use of antibiotics may cause acute losses and interrupt surgical studies. e.

Salmonella Enterocolitis

Salmonellosis can be subclinical or present with multiorgan involvement, including septicemia, pneumonia, meningitis, lymphadenitis, abortion, and enterocolitis (Leman, 1992). Enterocolitis can be acute or chronic. Salmonellosis is a zoonotic disease.

Etiology. Salmonella enterocolitis is usually attributed to Salmonella typhimurium and less frequently, S. choleraesuis. Clinical signs and differential diagnoses. Signs of Salmonella enterocolitis begin with watery, yellow diarrhea associated with fever, anorexia, and dehydration and will last several days to a week, sometimes with intermittent relapses. Diarrhea containing blood or mucus is not a prominent feature as in diseases such as swine dysentery. The differential for watery, yellow diarrhea in weaned pigs should include salmonellosis, colibacillosis, transmissible gastroenteritis, coccidiosis, and nematodiasis. Prevention. Pigs proven to be clinically ill or shedding Salmonella should not be maintained in a research facility because of chronic fecal shedding and the zoonotic risks associated with husbandry and research use. Control Infected swine may shed Salmonella for 5 months or more. Clinically affected swine should be euthanatized and the facility sanitized. Treatment. Treatment is contraindicated because of the shedding status of clinically recovered animals and the zoonotic risks associated with husbandry and research use. Research complications. Infected stock should be depopulated and the facility sanitized. Ideally, research use would not resume until it can be demonstrated that new stock will remain free of infection. f

Transmissible Gastroenteritis

Transmissible gastroenteritis (TGE) is a viral enteritis associated with vomiting, severe diarrhea, and high mortality in piglets less than 2 weeks old.

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Etiology Transmissible gastroenteritis virus (TGEV) is a pleomorphic enveloped virus containing a positive-sense, singlestranded RNA genome. This virus is one of four members of the family Coronaviridae (Sirinarumitr, 1997) that are known to naturally infect pigs: TGEV, hemagglutinating encephalomyelitis virus, porcine respiratory coronavirus, and porcine epidemic diarrhea virus (Kweon et al., 1997), which has been reported only in Europe and Taiwan. The TGE virus is antigenically related to feline infectious peritonitis virus and canine coronavirus. Swine can be experimentally infected with both cat and dog coronaviruses, and likewise, dogs and cats can be experimentally infected with TGEV, suggesting that cats and dogs may be reservoirs for swine TGEV. Clinical signs and differential diagnoses. Anorexia, vomiting, and/or diarrhea develop within days in susceptible animals of all ages, particularly in the winter. Nursing pigs develop transient vomiting and yellowish diarrhea, with dehydration and rapid loss of weight. Malodorous diarrhea will contain milk curds. Piglets less than 2 weeks old experience high mortality secondary to dehydration from enteritis, which can be compounded by agalactia if the sow is also ill. Differential diagnoses for yellow to white watery diarrhea in piglets that are newborn to several weeks of age should include colibacillosis, transmissible gastroenteritis, rotavirus, and coccidiosis. Epkzootiology and transmission. Epizootic TGE develops within days when the majority of animals are susceptible. A pattern of enzootic TGE will follow if viral challenge exceeds protection afforded by maternal immunity or as passive immunity wanes in the postweaning period. In herds with enzootic TGE, older animals will be asymptomatic, but diarrhea will develop in pigs 1 to 2 weeks old. Usually morbidity and mortality are lower, making diagnosis more difficult and requiring discrimination between other common causes of neonatal diarrhea, such as rotavirus and colibacillosis. Necropsy. Gross lesions are confined to the gastrointestinal tract and consist of a stomach distended with milk; foci of hemorrhages on the diaphragmatic side of the mucosa, varying in size from several millimeters to 2 cm in diameter; and a distended thin-walled small intestine, which is filled with watery material and curds of undigested milk. The piglets are usually severely dehydrated, and there is no chyle in the lymphatic channels in the mesentery (Hooper and Haelterman, 1966, 1969). The most striking microscopic lesion is the severe villus atrophy in the jejunum and ileum, which causes a massive loss of mature absorptive enterocytes. The villus crypt ratio is 1:1, compared to a normal of about 7:1 (Hooper and Haelterman, 1966, 1969). The enterocytes are vacuolated and low-cuboidal or flattened, there is lymphoid depletion of Peyer's patches, and there is a minimal inflammatory response in the lamina propria (Hooper and Haelterman, 1969). Virus particles can be found in

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the cytoplasm of villus enterocytes, M cells, lymphocytes, and macrophages within Peyer's patches (Saif and Wesley, 1992).

Pathogenesis. The virus multiplies in villus enterocytes, which are then sloughed as the villi begin to shrink. Normally, crypt cells divide rapidly to produce a constant supply of enterocytes that develop their absorptive enzyme capacity as they move onto the villus. In TGE this process is impeded due to the massive loss of enterocytes from villus atrophy, and the remaining crypt cells lack these enzyme systems. The secretory flux exceeds the ability to resorb. In addition, the hyperplasia of crypt cells resuits in increased secretory flux, which further adds to the diarrhea (Hooper and Haelterman, 1966; Moon, 1978). Prevention. Naive swine should not be introduced into potentially contaminated environments or into established herds known to harbor enzootic TGEV. Vaccination of boars, gilts, and sows will moderately reduce clinical signs. Control. Entrance of TGEV into production herds in the winter months is difficult to prevent because of probable reservoir transmission by wild birds, which should be excluded in laboratory animal housing. A moratorium on purchase of new animals and vaccination of reproductive stock will eventually contain an outbreak as the herd develops immunity. Treatment. There is no specific treatment for piglets infected with TGEV. Supportive care with fluids containing glucose and electrolytes is indicated. Antibiotics may be protective against concurrent primary or opportunistic bacterial pathogens. Research complications. Clinical signs of TGEV are severe enough to make animals unsuitable for experimental use unless sufficient time is available for clinical recovery. g.

Porcine Rotavirus

Porcine rotavirus is a major cause of morbidity and mortality from acute diarrhea in very young pigs, particularly if piglets are colostrum-deprived or raised under gnotobiotic conditions in which the herd is free of natural infection (Bridger et al., 1998).

Etiology. Four (A, B, C, E) of seven serogroups (A-G) of rotavirus have been described in swine, but group A rotavirus appears to be the most common. Within these serogroups, rotaviruses fall into two major serotypes based on expression of two surface antigens, VP4 and VP7. Rotaviruses are nonenveloped with a double-stranded RNA genome. They are stable in the environment and are relatively resistant to effects of temperature, pH, and disinfectants. Clinical signs and differential diagnoses. Disease is most severe in naive pigs first exposed at 1 to 5 days of age. Typical signs include anorexia, lethargy, some vomiting, and profuse

watery diarrhea that is white to yellow in color and contains flocculent material. In pigs that will recover, consistency of feces slowly returns to normal after 3 to 5 days of diarrhea. Death loss due to dehydration can reach 50-100% of affected piglets. Clinical signs and losses are less severe if exposure occurs after piglets are 7 days of age, and infection is commonly subclinical if it occurs after they are 21 to 28 days of age. Disease is usually mild and self-limiting if other enteric pathogens are absent. If rotaviral infection is detected in clinically ill pigs of postweaning age, mixed infection with other agents such as TGEV should be suspected. Differential diagnoses for yellow to white, watery diarrhea in piglets that are newborn to several weeks of age should include rotavirus, colibacillosis, transmissible gastroenteritis, coccidiosis, and nematodiasis.

Epizootiology and transmission. Rotaviral infection is enzootic in most swine herds, and clinical disease will be apparent only if viral challenge exceeds the capacity of passive maternal immunity. Piglets born to gilts are at greater risk than those farrowed by older sows, who are more likely to have naturally high virus neutralizing titers that protect the nursing piglets. Rotaviruses are resistant to environmental extremes and disinfection. Subclinical infection may persist in adult animals, with periodic shedding. Necropsy. Gross lesions are confined to the small bowel. The wall of the distal half of the small intestine is typically thin and dilated and contains watery material, while the mesenteric lymph nodes are tan and small (Paul and Stevenson, 1992). The cecum and colon are dilated, with watery contents similar to those in the small intestine. Gross lesions in pigs over 21 days of age are variable or absent. Microscopic lesions include degeneration and loss of enterocytes on the tips of the villi, increased thickness of the lamina propria due to large numbers of neutrophils and mononuclear cells, reduction in villus height from the duodenum to the ileocecal juncture, and fusion of villi due to exposed lamina propria in villus cores (Pearson and Me Nulty, 1977). Pathogenesis. Rotaviruses replicate in the cytoplasm of enterocytes and M cells overlying Peyer's patches, as well as in the lining epithelium of the colon and cecum (Butler and Moxley, 1988). The diarrhea is the result of destruction of enterocytes on the tips of the villi and villus atrophy, which reduces the mucosal surface area available for resorption. An osmotic diarrhea ensues due to decreased resorption of sodium, water, and disaccharides in the jejunum and ileum, which causes a hyperosmolarity to the intestinal contents (Graham et al., 1984). Prevention. Because porcine rotavirus is enzootic in most herds, exclusion is difficult. Management should concentrate on minimizing the viral challenge for susceptible pigs through good sanitation and boosting passive immunity by exposing replacement gilts to feces from the herd prior to their first parturition.

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Control Modified live- and inactivated-virus vaccines are commercially available for immunization of sows and nursing pigs. Immunity is serotype-specific, and the duration is unknown. Treatment. No specific treatment is available. To minimize losses, supportive therapy should include replacement fluids containing glucose and electrolytes, antibiotics to treat or prevent secondary bacterial infections, and warm, clean housing. Research complications. Morbidity and mortality of porcine rotaviral infection will impact studies using very young piglets and will probably be subclinical in postweaning animals. h.

Prevention. Herd-health management that minimizes the risk of enteritis from any cause will help prevent clinical balantidiasis. Control Clinically ill pigs should be isolated and treated or necropsied to rule out other predisposing causes of enteritis. Treatment. Balantidiasis can be successfully treated with metronidazole, tetracycline, and diiodohydroxyquin. Research complications. Although B. coli is usually nonpathogenic, severe ulcerative enterocolitis can develop. Because of zoonotic potential, it may be advisable to euthanatize piglets shedding B. coli in high numbers.

Balantidiasis

Because Balantidium coli invades necrotic tissue, clinical balantidiasis may be a sequela to other primary causes of enteritis. Balantidiasis is a zoonotic disease.

Etiology. Balantidiasis is caused by trophozoites of Balantidium coli, a ciliated protozoan that colonizes the cecum and anterior colon of swine, usually as a commensal. Trophozoites are large (25 x 150 ~tm), ciliated ovoid structures containing a macronucleus and micronucleus in addition to contractile and food vacuoles. Trophozoites of B. coli isolated from pigs affected by acute disease and from pigs with subclinical balantidiasis, as well as trophozoites cultured in vitro, have been shown to differ in nucleic acid content, suggesting clinical disease may be associated with different strains of B. coli (Skotarczak and Zielinski, 1997). Clinical signs. Infection with B. coli may present as an acute typhlitis or colitis or more commonly, the host is colonized without apparent effect. Infection can cause severe ulcerative enterocolitis, which can be fatal. Clinical signs include weight loss, anorexia, weakness, lethargy, watery diarrhea, tenesmus, and rectal prolapse. Epizootiology and transmission. Infection with B. coli is contracted by ingestion of trophozoites or cysts that are shed in feces. Most infections are subclinical, so if clinical enteritis can be associated with B. coli, other infectious agents or management problems that may be cofactors in disease development should be investigated.

i.

Coccidiosis

Swine can be naturally infected with three genera of coccidia. While disease is commonly absent or subclinical, significant morbidity and mortality can result from severe diarrhea in neonatal piglets.

Etiology. Eimeria spp., Cryptosporidium parvum, and Isospora suis are three genera of coccidia that infect swine and other mammals. There are eight species of Eimeria that infect up to 95% of the swine housed in the United States on dirt lots. Eimeria spp. are considered to be nonpathogenic in swine. Cryptosporidium parvum typically causes subclinical infection in swine that are 6 to 12 weeks of age. Clinical disease is associated with nonhemorrhagic diarrhea and either no histologic lesions or mild villous atrophy. Clinical neonatal coccidiosis is caused by the intracellular parasite, L suis, and is the most important protozoal disease of nursing piglets that are 1 to 2 weeks of age (Lindsay et aL, 1997). Clinical signs and differential diagnoses. Isospora suis causes clinical disease in nursing piglets that are 1 to 2 weeks old. Yellowish to gray diarrhea that varies in consistency from watery to pasty will develop, although piglets will usually continue to nurse. Weight loss and dehydration secondary to coccidiosis can be exacerbated by concurrent infections with other parasites, bacteria, or viruses. The differential should include colibacillosis, Clostridium perfringens, transmissible gastroenteritis, rotavirus, and Strongyloides ransomi.

Necropsy. Balantidium coli is not considered a primary pathogen in pigs (Manwell, 1968) but may be a secondary invader. Balantidium coli has been shown to invade lesions caused by Oesophagostomum, Trichuris suis (Beer and Lean, 1973), and some types of colitis produced by infectious agents.

Epizootiology and transmission. Isospora suis is transmitted by fecal-oral contact. Warm temperatures and high humidity associated with indoor farrowing favor rapid sporulation of oocysts. Infection rates of sows vary from 1 to 3%, although carrier sows are not the major source of new infections. Contaminated environments pose the greatest risk to naive piglets.

Pathogenesis. Secondary invasion occurs when the integrity of the colonic mucosa is compromised.

Necropsy. The gross lesions are confined to the jejunum and ileum and consist of a necrotic enteritis involving the entire

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thickness of the mucosa. A yellow fibrinonecrotic pseudomembrane may be present over foci of mucosal ulceration. Microscopic lesions consist of moderate to severe segmental villous atrophy and a necrotic enteritis. The variable reduction in villous heights ranges from slight to severe, and the villous enterocytes are flattened and irregularly shaped. There may be crypt epithelial hyperplasia, and the lamina propria is condensed and infiltrated with large numbers of mononuclear cells. The least involved sections of the mucosa contain varying stages of coccidia in vacuoles in the enterocytes on the distal two-thirds of the villi (Eustis and Nelson, 1981).

Pathogenesis. Ingestion of sporulated oocysts by the pig permits development to sporozoites in the intestinal lumen. These invade enterocytes and form trophozoites, which then form merozoites, resulting in rupture of the cell membranes when they are released into the intestinal lumen. Prevention. Piglets should be purchased from vendors with an established herd-health profile that is free of coccidiosis. Newly received piglets should be routinely quarantined and tested for coccidia by fecal flotation. Control. Coccidiosis can be controlled by "all in, all out" husbandry and thorough cleaning of housing areas, which should include removal of organic debris, chemical disinfection, steam cleaning and/or change of flooring from solid concrete to woven wire or Tri-bar. Coccidiostatic drugs can be administered for control of clinical disease. Treatment. Piglets should be individually dosed orally with amprolium or furazolidone. Sulfonamides and trimethoprimsulfa are also effective (Lindsay et al., 1997). Drug therapy may only delay the onset of clinical signs. Electrolyte and waterbalance disturbances should be treated with either oral or parenteral fluids. Research complications. Morbidity is high, but mortality is usually low to moderate in piglets affected by neonatal coccidiosis. Growth may be stunted. j.

Giardiasis

Etiology. Giardia trophozoites are commonly found in domestic swine and belong to the Giardia intestinalis group (Olson et al., 1997). Trophozoites colonize the surface of intestinal crypts of the small intestine from the duodenum to the ileum, with maximum numbers in the cranial part of the upper jejunum (Koudela et al., 1991). Giardiasis is a zoonotic disease. Clinical signs and differential diagnoses. Clinical signs include anorexia, depression, and formless feces. Giardia may be the primary cause of enteritis or may be found coincidental to other causes of enteritis (see Table IV).

Epizootiology and transmission. Giardia is commonly present in domestic swine (Olson et al., 1997). Giardia cysts are intermittently shed in feces and transmitted to other pigs by fecal-oral contact. Necropsy. No pathologic lesions were found in the small intestines of groups of pigs experimentally infected with Giardia intestinalis (Koudela et al., 1991). Detection of these can be done using Giemsa-stained fecal smears or Giemsa-stained histologic sections. Pathogenesis. Transmission is via the fecal-oral route. Most organisms are found in the jejunum, with fewer present in the duodenum and ileum (Koudela et al., 1991). Prevention. Sanitation protocols should include removing feces daily or housing pigs on slatted floors to minimize fecal contact. Control. Giardia exists as a commensal in the vast majority of domestic swine. Clinical enteritis can be controlled by quarantine and treatment. Treatment. Metronidazole is commonly used for 5 days to control giardiasis. Diagnostic steps to rule out other causes of enteritis are indicated. Research complications. Giardiasis can cause debilitation from diarrhea and dehydration but usually responds readily to treatment that is both supportive and specific (metronidazole). Attention to zoonotic aspects of giardiasis is warranted. k.

Nematodiasis

Young swine can be infected with the nematodes Hyostrongylus rubidus, Globocephalus urosubulatus, Macracanthorhynchus hirudinaceus, Oesophagostomum spp., Ascaris suum, Trichuris suis, and Strongyloides ransomi (Leman, 1992). Only Trichuris suis and Strongyloides ransomi will be discussed here because the other parasites are either discussed elsewhere (Ascaris), they require intermediate hosts (Macracanthorhynchus), or infection is associated with pasture maintenance (Hyostrongylus, Globocephalus, Oesophagostomum spp.), which renders them unlikely to be common problems in laboratory animal research facilities.

i. Trichuris suis Etiology. The swine whipworm, Trichuris suis, colonizes the small intestine and cecum. Besides causing morbidity and possibly mortality in young, postweaning swine, this parasite can infect humans and other primates. Clinical signs and differential diagnoses. Trichuris suis may cause anorexia, mucoid to hemorrhagic diarrhea, dehydration, and in severe infections, death. Differential diagnoses for hem-

15. BIOLOGYAND DISEASES OF SWINE orrhagic diarrhea in piglets that are newborn to several weeks of age should include colibacillosis, Clostridium perfringens, and Brachyspira hyodysenteriae. Other causes of hemorrhagic enteritis, particularly in older pigs, include Salmonella spp. and Lawsonia intracellularis.

Epizootiology and transmission. Bipolar, thick-shelled eggs are intermittently shed in feces. After 3 to 4 weeks in the environment, eggs are infective for as long as 6 years. Ingested eggs hatch in the small intestine and cecum, with newly released larvae penetrating cells lining the crypts. Larvae gradually migrate from the lamina propria into the submucosa over several weeks. After a series of molts, adult worms can be found at necropsy with their anterior end buried in the mucosa and the posterior end free in the intestinal lumen. Prepatency is 6 to 7 weeks, and the life span of the adult worm is 4 to 5 months. Necropsy. There is evidence of a profuse bloody diarrhea, dehydration, emaciation, and growth retardation (Batte et al., 1977; Beer and Lean, 1973). Gross lesions are found primarily in the cecum and colon. The wall of the large intestine is thickened, the mesentery may be thickened and appear as bands between coils of gut, and there may be foci of hemorrhages on the serosal surface (Beer and Lean, 1973). The mesenteric lymph nodes are enlarged and congested. The lumen of the gut is filled with bloody fluid, and there is a hemorrhagic catarrhal colitis and typhlitis, with portions of the mucosa being replaced by a yellow crumblike, fibrinonecrotic membrane (Batte et al., 1977; Beer and Lean, 1973). Microscopic examination reveals parasites embedded in the mucosa between villi and in crypts, which may be cystic, or they may have penetrated to the muscularis mucosa (Batte and Moncol, 1972). Enterocytes with degenerative changes are present surrounding the embedded parasites, and the lamina propria is infiltrated by large numbers of mononuclear cells. Foci of hemorrhage may be found in the mucosa, as well as ulcers, which are covered by a thick fibrinonecrotic material (Beer and Lean, 1973). The severe watery and bloody diarrhea results in severe dehydration and death. Scanning electron microscopy (EM) has shown that the penetration of the mucosa by the tunneling parasite results in formation of nodules and disruption of mucosal integrity. Damage caused to the mucosa permits colonization by pathogenic bacteria and Balantidium coli. Prevention. Trichuris suis eggs passed in feces require 3 to 4 additional weeks to develop to an infectious stage; hence, indoor housing with good sanitation that includes regular removal of feces and organic debris should prevent environmental contamination and reinfection. Control. Newly received swine should be tested for Trichuris by fecal flotation and treated with anthelmintics during the quarantine period. Housing areas and equipment should be steam-cleaned to destroy eggs and infective larvae.

653 Treatment. Effective anthelmintics for trichuriasis include fenbendazole, dichlorvos, and levamisole hydrochloride, all of which are formulated for administration in feed. Although ivermectin is considered to be efficacious for elimination of Ascaris, Oesophagostomum, and Metastrongylus, it is less effective for Trichuris. Research complications. Severe infection with Trichuris will cause bloody scours in young pigs, with associated morbidity and some mortality. ii. Strongyloides ransomi Etiology. Strongyloides ransomi is the small intestinal threadworm of swine that is most prevalent in warm climates and causes morbidity in suckling pigs (Leman, 1992). Clinical signs and differential diagnoses. Transcolostral infection of newborn pigs can cause clinical signs of diarrhea, with secondary dehydration and potential death within the first 2 weeks of life. Poor growth commonly results. The differential diagnosis for nonhemorrhagic diarrhea in piglets aged upward of 14 days should include colibacillosis, salmonellosis, rotavirus, transmissible gastroenteritis, giardiasis, coccidiosis, and nematodiasis. Epizootiology and transmission. Larvae of S. ransomi can infect pigs in utero as well as by the oral, percutaneous, and transcolostral routes. Eggs shed in feces hatch within hours to release larvae that are directly infective within 24 hr or develop into males and females that then reproduce, resulting in more larvae within 72 hr. Larvae encysted in the mammary gland of the sow gain access to newborn piglets via colostrum. Necropsy. Pigs may be dehydrated or may be stunted and unthrifty. Pigs less than 10 days old may die. Adult forms of the parasite are found in the small intestine, and ova are present in the feces. There are no pathognomonic findings in parasitized pigs. Pathogenesis. Transmission of ineffective larvae may occur prenatally, with larval migration into the small intestine shortly after birth. Transcolostral, percutaneous, and oral routes of infection also occur. Prevention. Breeding animals should receive anthelmintics to control the shedding of S. ransomi eggs and the transmitting of larvae through colostrum. Removing feces daily or housing on slatted floors should minimize exposure of neonates to infective larvae. Control Treatment with ivermectin several weeks before farrowing will prevent transmission of S. ransomi to piglets. Treatment. Young swine can be treated with paste formulations of thiabendazole. Other effective drugs are ivermectin and levamisole.

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Research complications. Strongyloides ransomi is an important cause of parasitic debilitation in nursing pigs in the southeastern United States. Routine diagnostic screening and timely use of anthelmintics should minimize any impact on research. 4.

Circulatory Disease

Epe rythrozoonosis Etiology. Eperythrozoon suis, a rickettsia of the family Rickettsiaceae, tribe Rickettsieae, is the etiologic agent for this disease in swine and is host-specific. These are obligate, intracellular, coccoid to coccobacillary organisms (0.2-2 ~tm) that are found within or attached to the outer surface of erythrocytes and in the plasma. They change size and shape as they mature, which gives the microscopic appearance of infection by two different rickettsiae. They stain well with Giemsa but not with Gram stain. Clinical signs and differential diagnoses. The acute form is usually seen in suckling or newly weaned piglets or other pigs that have been stressed, and consists of fever of 40~176 anemia, jaundice, pale mucous membranes, cyanosis of the ears, allergic skin reactions, weakness, and poor weight gain. All ages of swine can be clinically affected; however, the very young are most likely to be clinically affected. Acutely affected sows will become anorexic and febrile, and will have decreased milk production and poor maternal behavior. Vulvar and mammary gland edema may also be seen in sows. The chronic form affects older pigs and is usually subclinical, or it may adversely affect reproductive parameters in sows (Solignac et al., 1996). The reproductive problems include anestrus, low conception rates, abortions, weak piglets, and small litters. Mortality due to eperythrozoonosis is extremely low. Differentials include iron deficiency anemia and other causes of anemia in piglets, other infectious diseases, and toxicity producing icterus or anemia.

isms can be found within RBCs in a smear of peripheral blood, where they appear as 0.8-1 ~tm diameter rings with a pale center (Thomson, 1988). Microscopic lesions in other organs include hemosiderosis in hepatocytes and Kupffer's cells, fatty degeneration and centrolobular necrosis of hepatocytes (Splitter, 1950), and a hyperplastic bone marrow (Smith, 1992).

Pathogenesis. Arthropod transmission from pig to pig is believed to be the mode of spread, but this has not been proven. In utero and oral transmission have been reported. Prevention and control. Control measures include eliminating ectoparasites, never reusing needles, and sterilizing surgical instruments thoroughly. Additionally, any concurrent disease should be eliminated, and environmental, nutritional, and experimental stress reduced. The most satisfactory prevention is to allow only known Eperythrozoon suis-free swine into a facility. A PCR assay for E. suis in blood has been developed, which may be useful for herd-health monitoring as well as clinical diagnosis (Messick et al., 1999). In acute cases, a fresh blood smear can be stained with Giemsa to visualize the organisms. Treatment. Oxytetracycline either parenterally or in food or water will control the clinical signs but does not eliminate the rickettsiae. Iron dextran should be given to each clinically affected pig. In severely anemic animals, administration of whole blood may be beneficial. Additionally, any form of environmental, experimental, or physical stress should be eliminated. Research complications. Eperythrozoonosis causes an autoimmune hemolytic anemia, which will be precipitated or exacerbated by the stress of experimental protocols. This also predisposes affected animals to respiratory and gastrointestinal disease, which will further confound research protocols. 5.

Skin Diseases

a.

Exudative Epidermitis (EE): Greasy Pig Disease

Epizootiology and transmission. The reservoir for E. suis is domestic swine, and serologic studies have not detected it in wild swine (Heinritzi, 1999). However, current serologic tests will not detect every latent carrier. The various species of this parasite are considered host-specific. Transmission is mechanical by blood-sucking arthropods, primarily lice, or reuse of blood-contaminated needles and surgical or tattoo instruments. It can be directly transmitted by the oral route when swine lick fresh wounds or any fluids containing blood.

Etiology. Staphylococcus hyicus is a gram-positive cocci that produces an exfoliative toxin (SHET). Three antigenically different exfoliative toxins (ExhA, ExhB, ExhC) have been identified (Andresen, 1998) and are thought to correlate with clinical disease (Tanabe et al., 1996). All three of these toxins are metalloproteins (Andresen, 1999a,b). A fourth toxin has recently been identified, and its proposed designation is SHETB (Sato et al., 1999).

Necropsy. Gross findings include icterus, distended gallbladder filled with gelatinous bile, splenomegaly, pale mucous membranes, watery blood, swollen edematous lymph nodes, ascites, and hydrothorax, and the liver may be swollen and yellowbrown (Smith, 1992; Splitter, 1950). One or more of the organ-

Clinical signs and differential diagnoses. The early clinical signs of EE are lethargy and erythematous skin in a variable number of pigs in a litter. Pigs aged 5 days to 2 months are susceptible, and older pigs are more resistant. The signs progress to an exudative dermatitis characterized by exfoliation and crust-

655

15. BIOLOGY AND DISEASES OF SWINE ing, which begins in the groin, in the axillae, behind the ears, and in areas of damaged skin. Anorexia, dehydration, and poor weight gain are characteristic; however, pyrexia and pruritis are not typical signs. Erosions at the coronary band of hooves and vesicles or ulcers in the mouth and on the tongue and snout are common findings. The dermatitis may progress to cover the majority of the body in 3 - 5 days and becomes exfoliative and crusty. Severely affected members of the litter may die in 24 hr to 10 days, and others may not be affected or be chronically affected with small, localized patches of EE. Adult animals may be mildly affected with small areas of EE on their backs and sides (Wegener and Skov-Jensen, 1999; Cowart, 1995). Staphylococcus hyicus has also been reported to be an etiologic agent for arthritis in piglets less than 12 weeks old (Hill et al., 1996). Differential diagnoses should include swine pox, mange, ringworm, and pityriasis rosea.

Epizootiology and transmission.

Staphylococcus hyicus in carrier swine is harbored in the nasal cavity and conjunctiva, and vagina of sows and prepuce of boars. Outbreaks are seen following introduction of a carrier animal to a nonimmune herd. The newborn piglets are probably infected during parturition, and cross-contamination can occur when weanlings from different litters are group-housed. This bacterium is very persistent in the environment, and aerosol transmission is possible. Damage to the skin by abrasions from pen surfaces, fighting, mange mites, and concurrent vesicular diseases facilitates entry of S. hyicus. Spread by other species is of little concern (Wegener and Skov-Jensen, 1999). The morbidity can reach 20%, and mortality can reach 80% in affected piglets (Cowart, 1995). Necropsy.

The skin in the area of the erosive lesions may be reddened, edematous or thickened, and covered with an exudate composed of sebum, serum, and sweat (Jones, 1956). These lesions are most commonly found on the ears, around the eyes, on the ventral thorax, and on the abdomen. Microscopic findings are the presence of both a superficial and deep pyoderma that may extend to involve the subcutis, with multiple coalescing foci of necrosis of the stratum corneum; and the presence of a brownish exudate, as well as the formation of rete pegs by the hyperplastic stratum germinativum (Jones, 1956; Thomson, 1988; Taylor, 1992).

Pathogenesis.

Staphylococcus hyicus is an opportunist that readily invades traumatic wounds of the skin. Prevention and control.

Autogenous bacterins made from strains cultured from a particular herd and given to nonimmune sows are useful to protect the litters of newly introduced sows. The exfoliative toxin and the bacterial cells should be included as antigens when the vaccine is made. An indirect ELISA or phage typing can be utilized to select a toxigenic strain for vaccine production (Andresen, 1999a). The environment should be

improved, including temperature and humidity control and, especially, sanitation of the farrowing pens. The sows can be washed with appropriate antibacterials (chlorhexidine or povidone-iodine shampoos) prior to parturition, and any sharp or abrasive surfaces removed from the pens. The animals should be checked to be sure that they are free of mange and lice, as these may irritate the skin. Isolation of affected animals and housing animals in socially compatible groups to avoid fighting may help. Reviewing the diet to be sure that it meets NRC minimum requirements is indicated.

Treatment. Treatment with antibiotics will reduce the severity of the dermatitis and aid recovery. This bacteria is potentially susceptible to several antibiotics; however, plasmid-mediated resistance is common. The choice should be based on sensitivity testing whenever possible. The list includes trimethoprimsulfonamides, cephalosporins, lincomycin, amoxicillin, erythromycin, tylosin, and penicillin. Topical treatment of the affected skin with antibiotics and antiseptic shampoos or dips in conjunction with the antibiotics are also beneficial. Treatment is most effective when started early in the course of the disease, and severely affected young piglets may be slow to recover (Wegener and Skov-Jensen, 1999; Cowart, 1995). Research complications.

Exudative epidermitis will complicate most studies involving young piglets due to the potentially significant morbidity and mortality.

b.

Swine Pox

Etiology. Swine pox virus is the only member of the genus Suipoxvirus, family Poxviridae. The virus is found worldwide, primarily in herds with poor sanitation, and chiefly affects pigs less than 4 months of age.

Clinical signs. The lesions associated with this virus mimic other pox diseases. Initially, macules form (reddening); followed by 1-6 mm diameter papules (reddening with edema); then vesicles (fluid within the lesion); then pustules (umbilicated, ischemic); and finally, crusts (brown to black in color). The progression of the lesions occurs over a 3- to 4-week period. Younger animals are affected more severely than adults, and they may have lesions covering the entire body surface. Older animals tend to have lesions in more focal locations. If vector transmission has occurred, the location of the lesions follows the vector preferences, i.e., the pig louse attacks the lower parts of the body, while flies feed predominantly over the top of the body. Adults have lesions primarily on their belly, udder, ears, snout, and vulva. Epizootiology and transmission.

The virus, although worldwide in distribution, exists primarily in herds where poor sanitation is practiced. The reservoir is infected swine, as the virus

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is host-restricted. The virus may persist in an active form in dry skin scabs for up to 1 year. Horizontal transmission may occur via nasal and oral secretions coming in contact with abraded skin. The primary method of transmission, however, is the pig louse. Flies and mosquitoes can also carry the viral particles. Once the virus is established within a herd, it usually persists. Outbreaks can result in high morbidity if young animals are present, although mortality is very low.

Necropsy. Gross lesions are most commonly found on the ventral and lateral abdomen and chest and the medial aspects of the legs, and only in severe cases involve the oral cavity and main airways (Jubb et al., 1985). Early lesions consist of erythematous macules and papules, and later lesions progress to pustules and scabbing. Microscopic findings are related to viral replication in the stratum spinosum, causing hydropic degeneration, intercellular edema, necrosis of epithelial cells, and formation of pustules that involves the full thickness of the epidermis with 1-3 eosinophilic intracytoplasmic poxvirus inclusion bodies in epithelial cells (House and House, 1992). Pathogenesis. The virus gains entry to the body by traumatic injuries and by bites from the pig louse, as well as from flies. It replicates in the cells of the stratum spinosum and is spread from cell to cell in the epidermis (House and House, 1992). A viremia is believed to occur and results in transplacental infection and disease in neonates (Jubb et al., 1985). Diagnosis. The diagnosis is primarily made by identifying the typical lesions in the typical locations. Differential diagnoses include any of the vesicular diseases, pityriasis rosea, allergic skin reactions, sunburn, or staphylococcal or streptococcal epidermitis. The presence of intracytoplasmic inclusion bodies along with central nuclear clearing in affected epithelial cells is a hallmark sign of this disease. Prevention and treatment. Supportive care should be given to prevent secondary bacterial skin infections. Affected animals should be isolated, and sanitation and pest control should be improved. c.

Mange (Scabies)

Etiology. Sarcoptes scabiei var. suis is the cause of sarcoptic mange in swine. This is probably the most significant ectoparasite of swine. This mite is 0.5 mm in length, has 4 pairs of legs, and completes its entire life cycle within the layers of the epidermis. The time necessary for an egg to hatch and develop into a mature egg-laying female is 10-25 days. This is one of the more common swine diseases, but it is frequently overlooked, probably because the clinical signs may be perceived as normal and losses are not readily apparent. Demodectic mange caused by Demodex phylloides can also occur in swine; however, it is a rarity.

Clinical signs and differential diagnoses. There are two clinical forms of sarcoptic mange in swine. The acute pruritic or allergic hypersensitive form affects younger, growing pigs. This is characterized by an intensely pruritic, erythematous papular dermatitis on the ventral abdomen, flank, and rump. Pigs with this form will rub the affected areas, often causing hair loss, abrasions, and thickened, keratinized skin. A reduced growth rate will be seen if the dermatitis is severe (Davies, 1995). It is difficult with this form to find the mites on skin scrapings. The chronic or hyperkeratotic form is typically found in mature sows and boars. Thick, crusty scabs begin on the pinnae and spread to the neck and head, and contain numerous mites that are relatively easy tO find on skin scrapings. Mortality is unlikely unless concurrent disease is severe. Differentials should include causes of dermatitis in swine, such as exudative epidermitis, dermatomycosis, swine pox, parakeratosis, niacin and biotin deficiencies, sunburn, photosensitization, and insect bites (Cargill and Davies, 1999). Epizootiology and transmission. Mange infestations are fairly common in small conventional swine herds in the United States. Nursing piglets first obtain the mites from an infected sow through direct contact. These breeding sows with hyperkeratotic encrustations in their ears are the primary reservoirs of S. scabiei var. suis mites. Group housing of pigs, especially from various sources, will facilitate spread of mites. Spread of mites through environmental contamination is less likely but still possible, as mites can survive off the host for a few days or even longer if ambient temperatures are cool. Herd-to-herd transmission is by introduction of a carrier pig; other species are not known to harbor this mite. Necropsy. Papular dermatitis will be seen in growing swine with or without positive skin scrapings for the sarcoptid mites. The papules, which are manifestations of the hypersensitivity reaction, contain eosinophils, mast cells, and lymphocytes (Cargill and Davies, 1999) and have an associated eosinophilic perivasculitis (Hollanders and Vercruysse, 1990). In mature animals, thick, crusty scabs with positive skin scrapings are typical findings. Histologic sections will reveal offending mites burrowing to the level of the deep stratum corneum and stratum malpighii, producing hyperkeratosis and acanthosis (Jones and Hunt, 1983). Pathogenesis. Young pigs or newly exposed older animals become pruritic due to a hypersensitivity response to the mites burrowing into the dermis and laying eggs (Davis and Moon, 1990). This generally occurs several weeks postinfection. The first 3 weeks postinfection, the females burrow into the skin and a covering of keratinized encrustations develops. These crusts fall off after 7 weeks, and the mites leave the burrows (Morsy et al., 1989). This is seen clinically as rubbing and development of encrustations containing mites, followed by papular dermatitis as hypersensitivity develops. If the rubbing is severe, persist-

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15. BIOLOGYAND DISEASES OF SWINE ent hyperkeratosis will develop. The hyperkeratotic form is seen more often in mature animals (Cargill and Davies, 1999).

Prevention. Allowing only mange-free SPF animals into the facility is the most effective and satisfactory method of prevention. Sarcoptes scabiei can be eliminated by hysterotomy rederivation. Alternatively, mange-free herds can be made by elimination of mites with acaricides such as ivermectin. It is feasible to maintain a herd free of S. scabiei if a good biosecurity and surveillance program is developed (Cargill et al., 1997). Control. Swine with unusually severe chronic hyperkeratosis should be culled from the group if possible. Every pig in the group should be treated twice, with a 1- to 2-week interval. This should be followed by thorough cleaning of the environment, removing any bedding, and spraying the area with an acaricide. The success of the control program can be monitored by using an ELISA for serum antibody levels to S. scabei (Hollanders et al., 1997; Bornstein and Wallgren, 1997; Wallgren and Bornstein, 1997; Jacobson et al., 1999; Zimmermann and Kircher, 1998), using periodic skin scrapings, and monitoring for prevalence of scratching and papular dermatitis lesions (Davies et al., 1996). Treatment. Ivermectin is effective orally or subcutaneously (Hollanders et al., 1995; Cargill and Davies, 1999) and should be repeated in 14 days. Doramectin intramuscularly has also been reported to be very effective (Cargill, 1996; Logan et al., 1996; Saeki et al., 1997; Yazwinski et al., 1997). Experimental evidence has demonstrated that doramectin has a greater persistent efficacy than ivermectin (Arends et al., 1999). There are several other acaricides, including amitraz, phosmet, and diazinon, that are also effective. Two or more treatments at 1- to 2week intervals are usually necessary to eliminate these mites. Research complications. Sarcoptic mange should not result in direct loss of animals in a study since this disease is rarely associated with mortality unless there is concurrent disease. The intense rubbing associated especially with the acute form is a potential threat to surgical incisions and implants in these models. d.

Lice (Pediculosis)

Etiology. Haematopinus suis females are 4 - 6 mm in length and males, 3.5-4.75 mm. These lice belong to the order Phthiraptera and suborder Anoplura, or sucking lice, which possess specialized mouth parts to penetrate swine skin and feed on their blood. It is the only species of louse that affects swine (Lapage, 1968a). Clinical signs and differential diagnoses. Pruritus, poor growth, and anemia in young pigs are the clinical signs. These lice can be found almost anywhere on the body but have a

predilection for the skin on the flank area, neck, axilla, groin, and the inner ears. Their eggs, or nits, are 1-2 mm in length and attach to the hair shafts.

Epizootiology and transmission. Transmission is by direct pig-to-pig contact, as this louse is host-specific and will not survive very long (less than 2 - 3 days) off the host. The life cycle is 23-32 days and is entirely in and on the skin. It is considered a vector for swine pox and Eperythrozoon suis. Necropsy. The entire life cycle of H. suis occurs on the skin surface; therefore, adults can be visualized around the inner ear, face, and neck without special techniques. The nits can be found attached to hair shafts. Allergic dermatitis and mechanically induced skin lesions with hemorrhage may been found on some affected pigs (Nickel and Danner, 1979). Pathogenesis. The three instars of the nymph stage and egglaying females suck blood, causing irritation and pruritus, manifested clinically as rubbing, and possibly as anemia in heavy infestations of young pigs. Prevention, control and treatment. The most reasonable and effective means of lice prevention is to allow only swine known to be lice-free into the research facility. Feral populations of Sus scrofa have been found to be reservoirs for H. suis (Gipson et al., 1999), and certainly contact with domestic populations should be prevented. However, this condition is easier to treat than scabies. The same treatments that are effective for mites also work well for lice. These include several sprays, dips, dusts, and oral and injectable ectoparasiticides. Most are very effective when given as 2 treatments 2 weeks apart. Treatments that are typically administered as sprays include malathion, methoxychlor, permethrin, diazinon, and coumaphos. Phosmet, fenvalerate, and amitraz are typically administered as pour-on solutions for swine treatment. The avermectins (primarily doramectin) (Logan et al., 1996) and ivermectin are available as an oral or injectable treatment and are also effective for ascarids and lungworms. Research complications. Severe infestations may cause anemia in young swine, and the rubbing may damage surgical incisions. Furthermore, the use of potentially toxic treatments to remove the lice may interfere with some research studies. 6.

Reproductive Diseases

a.

Brucellosis

Etiology. Brucella suis, particularly biovars 1, 2, and 3, is the only species of Brucella that causes systemic infection and clinical disease, including infertility, in swine. Biovar 3 is currently the most common cause of this disease in swine. Morphologically, this genus is a nonmotile, non-spore-forming, small gramnegative aerobic bacillus or coccobacillus.

658 Clinical signs and differential diagnoses. The clinical signs of B. suis infection vary with the herd and range from no obvious disease to the classical signs, which include abortion, infertility, metritis, orchitis, lameness, spondylitis, and posterior paralysis. Clinical disease in piglets of weaning age usually consists of spondylitis and posterior paralysis (MacMillan, 1999). Differentials include other causes of infertility and abortion in swine, such as porcine parvovirus and leptospirosis. Epizootiology and transmission. Domestic swine populations are the primary sources for B. suis. The European hare (Lepus capinensis) is a carrier for biovar 2 and has been linked to brucellosis in European swine facilities. Feral pigs are also reservoirs in areas where contact with domestic swine can occur. Brucella suis biovar 2 has been reported in wild boars in Germany (Heinritzi et aL, 1999). Transmission is most frequently through contaminated discharges from infected swine being ingested by a susceptible animal. This takes place from direct contact with aborted fetuses and fetal membranes or with contaminated food or water. Additionally, nursing piglets frequently become infected while suckling infected sows. Brucella suis is present in semen of infected boars and can be spread by natural breeding or artificial insemination. Necropsy. Gross lesions are variable in site and extent but generally consist of one or more abscesses, and there may be erosions of mucous membranes (MacMillan, 1992) and seminal vesiculitis (Deyoe, 1967). Aborted fetuses may appear normal, or there may be edema or evidence of a suppurative placentitis. Microscopic lesions consist of inflammatory cell infiltrates in the endometrium, uterine glands, and placentas (MacMillan, 1992); suppurative seminal vesiculitis (Deyoe, 1967); pyogranulomatous foci in the liver; caseous necrotic foci adjacent to growth plate cartilages in the vertebrae; and abscesses in the kidneys, spleen, ovaries, lungs, brain, and other tissues (MacMillan, 1992). Pathogenesis. The precise mechanisms by which this organism attaches to and penetrates mucosal epithelium is not known. However, once the organisms have penetrated to the submucosa, they travel to the local lymph nodes, gain entrance to macrophages and neutrophils, and multiply, resulting in a bacteremia with seeding of organisms in other lymph nodes, the genital tract, placenta, joint fluids, and bone marrow (MacMillan, 1992). Differential diagnoses should include leptospirosis and any bacterial agent that may cause abortion. Prevention and control. The best prevention is to allow only brucellosis-free swine from validated herds into a facility. A control program in infected herds with valuable genetics might be achieved by test and elimination of all seropositives. This is difficult because of problems with specificity and sensitivity of

KATHYE. LABER ET AL. serologic assays (Ferris et al., 1995). This plan works only if there are very few animals in the herd actually infected. The most satisfactory method is to depopulate infected herds and repopulate with validated animals. If a closed herd is maintained with a good biosecurity program, it is feasible to keep it brucellosis-free. Brucellosis is a zoonotic and reportable disease in the United States.

Treatment. Infected swine should be euthanatized. Antimicrobials are unlikely to eliminate the bacteria from swine, and vaccines that can safely produce a lasting immunity have not been developed. Fortunately, the incidence of brucellosis among domestic swine in the United States has declined to a very low level. Research complications. Research protocols involving any aspect of swine reproduction are at highest risk for brucellosis. Brucella suis is one of the most common species implicated in cases of human brucellosis. Investigators and veterinarians performing necropsies on infected animals are at risk for becoming infected. b.

Leptospirosis

Etiology. The etiologic agent for this disease in swine consists of several serovars of Leptospira interrogans. All are gramnegative, motile aerobic spirochetes. The serovar pomona is the most common cause of clinical leptospirosis in swine, and the serovar bratislava is commonly found in serologic surveys and sometimes correlated with clinical disease. There are several other serovars, which are typically maintained in other mammalian hosts but are occasionally found to infect swine. These include icterohaemorrhagiae from the brown rat (Rattus norvegicus); sejroe from small rodents; hardjo from cattle; canicola from dogs; grippotyphosa from wildlife; and tarassovi from opossums, skunks, and raccoons (Ellis, 1999 and Cowart, 1995). Clinical signs and differential diagnoses. The acute form is characterized by a mild transient anorexia, listlessness, diarrhea, and pyrexia that resolves within a week and usually goes unrecognized. Rarely seen are piglets < 12 weeks of age infected with strains from the serogroup icterohaemorrhagiae, hemoglobinuria, and jaundice. The chronic form is characterized by late-term abortions, stillbirths, and weak newborn piglets. This is particularly true of serovar pomona infection. Infertility of the sow is seen following infections due to serovar bratislava; however, reproductive performance following abortions due to pomona is not affected (Ellis, 1999). Differential diagnoses include parvovirus, brucellosis, and pseudorabies. Epizootiology and transmission. Transmission from animal to animal is by direct or indirect contact with a carrier animal, which harbors the leptospires in the renal tubules or genital

15. BIOLOGYAND DISEASES OF SWINE tract. Leptospires are shed from carrier animals in urine and genital fluids into the environment. Feral swine are potential sources of serovars pomona and bratislava for outdoor facilities where contact can occur (Saliki et al., 1998; Mason et al., 1998). Venereal transmission is thought to be the mode of spread for serovar bratislava because sows and boars harbor it in the reproductive tract and urinary excretion is relatively low. Survival of the bacteria out of the host is favored by warm, moist conditions. Entry into the new host is through mucous membranes of the eye, snout, mouth, and genital tract or through damaged skin. Swine are typically maintenance hosts for serovars of the serogroups pomona, australis (serovars bratislava and muenchen), and tarassovi. Infection with other serovars is considered incidental. Typically, only a limited number of serovars will be endemic in a given area and host species (Ellis, 1999). Necropsy. There may be petechial or echymotic hemorrhages in the lungs and kidneys, which may be swollen, along with small gray lesions on the renal cortex. Microscopic lesions in the kidney include an interstitial nephritis with inflammatory cells, lymphocytes, and plasma cells. Glomeruli may be swollen or atrophic and cellular casts may be found in the lumen of renal tubules lined by atrophic epithelial cells (Ellis, 1992). Pathogenesis. The route of infection is believed to be via the mucous membranes of the mouth, nasal passages, eye, and vagina. A bacteremia develops that results in seeding of Leptospira organisms in most organs, including the liver, the pregnant uterus, and the proximal renal tubules, where they persist, multiply, and are voided for varying periods in the urine (Thomson, 1988). Prevention and control. A biosecurity program that prevents potential vectors, such as rodents and feral swine, from making direct or indirect contact with the swine in the facility is essential to prevent introduction and minimize spread. The microscopic agglutination test (MAT) is commonly utilized for serologic monitoring of herds. Artificial insemination can be used to advantage to prevent spread or introduction of serovar bratislava. Vaccination with bacterins will reduce the incidence of infection but not eliminate the disease from the herd. Immunity is short-lived, which necessitates revaccination at least every 6 months (Ellis, 1999; Cowart, 1995). Treatment. Medicating feed for periods of 4 weeks or more with oxytetracycline or chlortetracycline will help control clinical signs until a vaccination program can be established. Individual dosing of pigs with dihydrostreptomycin-penicillin G, oxytetracycline, erythromycin, or tylosin may help eliminate serovar pomona from the renal tubules (Alt and Bolin, 1996; Ellis, 1999).

659 Research complications. Leptospirosis will interfere with studies involving swine reproduction or fetal surgery, due to the increased rate of late-term abortions associated with the chronic form of the infection. c.

Parvovirus

Etiology Porcine parvovirus (PPV) is a disease of swine characterized by embryonic and fetal infection and death when susceptible sows and gilts are exposed to the virus during the first 70 days of gestation. The infection typically causes no observable clinical signs in the infected female, and its major impact on animal health relates to the agent's ability to interfere with live births. Porcine parvovirus is one of the major infectious causes of embryonic and fetal death (Mengeling et al., 1991). The disease is caused by a single-stranded DNA virus classified in the genus Parvovirus, family Parvoviridae. All isolates of the virus found in swine have been antigenically similar, if not identical, and are also antigenically related to other members of the genus. Although the viral particles have actually been identified in pig feces, there is no evidence implicating that PPV replicates in the intestinal crypt epithelium or causes enteric disease in swine, as the agent does in other species (Brown et al., 1980). Clinical signs. Acute infection of both postnatal and pregnant dams is subclinical; however, the pigs will have a transient, mild leukopenia within 10 days after the initial exposure. The only clinical sequela to exposure is maternal reproductive failure. Dams can cycle back into estrus, farrow fewer pigs per litter, or farrow a large proportion of mummified fetuses. Typically, an epizootic of PPV starts as a subclinical infection and culminates with the delivery of mummified fetuses, usually at or near term. Most of the infected fetuses have a crown-rump length of 17 cm or less because those infected after day 70 are able to respond to the viral assault and survive (Mengeling et al., 1993). Infertility, abortion, stillbirth, neonatal death, prolonged gestations, and reduced neonatal viability have also been attributed to PPV. There is no evidence that PPV impacts on either fertility or libido of boars (Thacker et al., 1987). Epizootiology and transmission. Porcine parvovirus is ubiquitous among swine worldwide. In general, infection is enzootic in most herds, and with rare exception, sows are immune. Also, gilts usually contract PPV before conception and develop an active immunity that persists through life. Disease occurs when there is a large population of gilts that have not developed immunity prior to conceiving. Gilts are most commonly infected via the oronasal route, and prenatal pigs are infected via the transplacental route. Nursing pigs absorb protective PPV antibody from colostrum. These titers diminish to levels that are not protective when the

660 piglets are 3 - 6 months of age. The significance of the passively acquired antibody is that it interferes with the development of active immunity until the 3- to 6-month mark (Paul and Mengeling, 1980). The major reservoir for PPV is environmental. The virus is thermostable and resistant to many disinfectants. It has been shown that pigs transmit PPV for about 2 weeks after exposure, but the pens they were housed in remained infectious for up to 4 months (Mengeling and Paul, 1986). It is also possible that immunotolerant carriers of PPV, resulting from early in utero infection but not death, are carriers (Johnson, 1973). Boars may also play a role in dissemination of the disease. During acute infection with the agent, the virus can be shed in semen. Virus can also be isolated from scrotal lymph nodes up to 35 days postexposure.

Necropsy. Gross lesions are confined to the placenta, which may be edematous and have white, chalklike deposits (Joo et al., 1977) and stunted fetuses with prominent blood vessels on their surfaces, petechial hemorrhages, edema, enlarged dark liver and kidneys, serosanguinous fluid in body cavities, and mummification (Joo et al., 1977; Hogg et al., 1977). Microscopic findings in the fetuses include vasculitis with hypertrophy of endothelial cells; and perivascular accumulations of mononuclear cells around vessels in the gray matter and white matter in the cerebrum, brain stem, and meninges, in the interstitial area around glomeruli, the portal areas of the liver, and the placenta (Joo et al., 1977; Hogg et al., 1977). Pathogenesis. Viral infection of the pregnant sow results in a viremia and allows transplacental passage of the virus to the fetuses (Joo et al., 1976; Mengeling, 1975). Once infected, a fetus can then transmit the virus to other fetuses (Mengeling, 1975). Differential diagnoses. Differential diagnoses should include porcine reproductive and respiratory syndrome (PRRS), brucellosis, and leptospirosis. Diagnosis. Porcine parvovirus is one of the primary diagnostic considerations when swine exhibit embryonic or fetal death. Gilts are the population primarily at risk. The lack of maternal illness, abortions, or fetal developmental anomalies differentiate this disease from other causes of reproductive failure. In addition, identifying mummified fetuses that have a crown-rump length of -< 17 cm is a strong indicator that PPV is the infectious agent at play. The definitive diagnosis can be made by identifying viral antigen by immunofluorescent (IF) microscopy from sections of fetal tissues. Serologic testing for antibodies (i.e., ELISA) are recommend only when tissues from mummified fetuses are not available. Results from serum are of value if antibody is not detected or if samples are collected at intervals that document seroconversion for PPV. Since PPV is ubiquitous, the presence of

KATHYE. LABERET AL. antibody in a single sample is meaningless. Detection of antibody in sera of fetuses/stillborns before they nurse is evidence of in utero infection, as the maternal antibody does not cross the placenta (Chaniago et al., 1978).

Prevention and treatment. There is no treatment for the reproductive failure associated with PPV. Prevention involves either naturally infecting gilts with PPV or vaccinating them prior to pregnancy. Through herd-management practices, natural infections can be promoted. Seronegative gilts can be housed with seropositive sows. Vaccines are used extensively in the United States. They are administered several weeks before conception but after the disappearance of passively acquired colostral antibody. In essence, the window for vaccination is small in herds keyed for production. Vaccination for boars is also recommended. d.

Porcine Reproductive and Respiratory Syndrome

Etiology. A new swine disease was first identified in the United States in the late 1980s. Hallmark signs included reproductive disorders, high piglet mortality, and respiratory disease seen in a wide age range of animals. The disease became known officially as PRRS (porcine reproductive and respiratory syndrome) but is often referred to in the literature as SIRS (swine infertility and respiratory syndrome). The disease has now spread into many countries and has escalated into one of the major causes of reproductive losses and respiratory disease in swine. The causative agent is a single-stranded RNA virus classified in the order Nidovirales, family Arteriviridae, genus Arterivirus. This agent shares structural and functional organization with others in the genus, including lactate dehydrogenaseelevating virus, equine arteritis virus, and simian hemorrhagic fever virus. These viruses in general are known to have high rates of mutation. The isolates found in the United States (VR-2332) and Europe (Lelystad) have genomic and serologic differences, although it is believed they merged from a common ancestor. The U.S. isolates differ genomically but cross-react serologically. Current efforts are being directed toward determining if infection with a single isolate provides immunologic cross protection. Recent infections in vaccinated herds lend suspicion that immunization does not provide protection across all isolates (Collins et al., 1992, 1997). Clinical signs. The clinical presentation of PRRS infection depends on the age of the pig and the gestation status when infected. In addition, the clinical presentation can vary depending on complicating infections with viruses or bacteria. Late gestational abortions typically occur when animals are infected during the third trimester and can occur sporadically or sweep throughout the population of animals. Other reproductive manifestations that have been documented include delayed parturi-

15. BIOLOGY AND DISEASES OF SWINE

tion and premature farrowing resulting in mummified or stillborn fetuses. Clinical signs in infected females vary from none to anorexia, fever, pneumonia, agalactia, red/blue discoloration of ears and vulva, subcutaneous edema, and a delayed return to estrus. Clinical signs in PRRSV-infected newborn pigs also vary in frequency and severity. Dyspnea and tachypnea are the most characteristic clinical signs, with other signs including periocular and eyelid edema, conjunctivitis, blue discoloration of the ears, diarrhea, and CNS signs. Mortality can reach 100%. As the pigs reach postweaning age, the clinical signs shift to include fever, pneumonia, failure to thrive, and significant mortality caused by otherwise non-life-threatening concurrent bacterial infections. The susceptibility and resulting impact of secondary bacterial infections in pigs infected with PRRSV depends on the PRRSV isolate, the swine genetic composition, management practices, and environmental factors. Subclinical infections occur commonly as the pig continues to mature, with the only indication of infection being seroconversion to the virus. Occasionally a transient fever and inappetence or loss of libido can be observed. Hematologic parameters congruent with infection include a decrease in lymphocytes, neutrophils, and monocytes at 4 days postinfection, with a concurrent increase in band neutrophils. Four-week-old pigs had decreased RBC counts, hemoglobin levels, and hematocrits (Rossow et al., 1994). Differential diagnoses include parvovirus, pseudorabies, and leptospirosis. Epizootiology and transmission. This virus is spread predominantly through direct contact between infected and naive pigs, although the route of fetal PRRSV infection has not been identified. The virus is carried in blood, oropharyngeal fluids, semen, feces, and urine. Transmission by aerosolization is possible, though routinely occurs only over short distances. The virus establishes a foothold by infecting macrophages located within mucosal surfaces. The virus infection is limited to domestic swine with the exception of a single report occurring in mallard ducks (Zimmerman et al., 1995). The agent is considered to be a "slow spreader." The virus can be identified in semen prior to seroconversion as well as after cessation of viremia, indicating that virus isolation from serum and serology may not be an adequate indicator of infection status in boars. The disease does persist in infected swine in a transmissible, viable state, often without stimulating antibody production, thereby making serologic screening for the disease inaccurate. Pigs subclinically infected with PRRSV are thought to be the key factor in disease transmission within herds (Rossow, 1998). Diagnosis. The viral infection is most accurately diagnosed through the demonstration of PRRS by virus isolation, fluorescent antibody examination, immunohistochemistry, or PCR in

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concert with clinical signs and characteristic histologic lesions. Exposure to the virus can be documented through the use of serology testing for anti-PRRSV antibodies; however, if pigs are vaccinated with the modified live-PRRSV vaccine, the current serologic tests cannot differentiate between vaccine virus and field PRRSV isolates. It is also important to note that pigs vaccinated with the modified live vaccine can transmit vaccine virus to naive pigs, resulting in infection and seroconversion of the naive animal (Rossow, 1998). The virus can most easily be located in lung tissue, lymphoid tissue, heart, brain, nasal turbinates, and reproductive organs. Again, it is important to note that modified live-PRRSV vaccine virus can also be identified from these tissues, and pathogenic PRRSV isolates must be differentiated from the vaccine virus. Necropsy. Gross lesions in young piglets include mottled lungs with tan foci of consolidation; lymphadenopathy of the mesenteric and middle iliac nodes, which are tan and may contain cysts; moderately enlarged and rounded hearts; and clear fluid in the pericardial space and abdominal cavity. Microscopic lesions consist of a multifocal lymphohistiocytic myocarditis; an interstitial pneumonia with mononuclear cell infiltrates, resulting in septal thickening; peribronchial and peribronchiolar lymphohistiocytic cuffing; hypertrophy and hyperplasia of type II pneumocytes; and filling of alveolar spaces with necrotic and normal macrophages. There is also follicular hypertrophy, hyperplasia, and necrosis in lymphoid tissues and a mild lymphohistiocytic choroiditis with cuffing of vessels in the meninges, choroid plexus, and brain (Halbur et al., 1995). Lesions in fetuses consist of myocarditis with fibrosis, arteritis, and encephalitis (Rossow et al., 1996a). Pathogenesis. The virus has been demonstrated in urine, feces, nasal secretions, semen, saliva, and serum, and all are potential routes of exposure (Rossow, 1998). The virus has been shown to enter via the nasal epithelium, bronchial epithelium, and tonsillar and pulmonary macrophages, followed by replication in macrophages, with a subsequent viremia (Rossow et al., 1996b). Migration of infected macrophages across the placenta may be one of the mechanisms for transplacental infection of fetuses. Prevention and control. Vaccination of pigs with a modified live-PRRSV vaccine has protected pigs from clinical disease when the pigs were challenged with heterologous PRRSV isolates; however, other reports have shown that the vaccine is not universally protective against all isolates of PRRSV. Efforts should be made to obtain pigs from sources that are free of PRRSV. Pigs coming from different sources should be isolated from each other. Treatment. Once pigs show signs of disease, supportive therapy should be implemented. This can include antibiotics to

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control concurrent bacterial infections and vitamin and food supplements until animals regain their appetite.

B.

a.

Metabolic/Nutritional Diseases

Porcine Stress Syndrome

Etiology. Porcine stress syndrome (PSS) refers to a cascade of physiologic events and clinical signs that occur in pigs that have a mutation in the calcium-release channel protein (ryanodine receptor [RYR]). This mutation results in a hypersensitive triggering mechanism of the calcium-release channel in skeletal muscle sarcoplasmic reticulum in response to various stressors, such as gas anesthetics or stressful environmental conditions. The lack of proper calcium control within the membranous portions of the sarcoplasmic reticulum and mitochondria is thought to initiate the cascade of events that results in the syndrome (O'Brien et. al., 1991; Fujii et al., 1991). Stress-susceptible pigs are also known to overrespond to stressful stimuli, with excessive [3-adrenergic receptor stimulation, lower rates of lactate, alanine, and aspartate conversion to carbon dioxide by the liver, abnormal phosphorus metabolism, and a much higher cortisol and thyroxine turnover rate. Animals carrying the genetic defect are found throughout the world with a frequency varying between 0 to 89% among herds (Webb et al., 1992). Genotypic analyses have indicated that the mutation arose from a single founder animal. The mutation is found in five major breeds of swine: Landrace, Yorkshire, Duroc, Pietrain, and Poland China. However, there are reports of this disease occurring in other breeds, including miniature potbellied pigs (Claxton-Gill et al., 1993). The mode of inheritance is autosomal recessive with variable penetrance. This syndrome has also been reported in humans, dogs, cats, and horses. Clinical signs. In a laboratory setting, development of PSS has most commonly been associated with exposure to halothane and succinylcholine; however, methoxyflurane, enflurane, and isoflurane have all been shown to be capable of eliciting a reaction in susceptible swine. The course of the disease is variable, ranging from abatement of clinical signs when anesthesia is stopped to fatality. Initial signs include tachycardia, tachypnea, muscle rigidity, and hyperthermia. Clinicopathologic changes include metabolic acidosis, myoglobinemia, hyperkalemia, and hyperglycemia. These metabolic derangements frequently lead to cardiovascular collapse and death. In addition to the typical manifestation, nonrigid and normothermic forms have been described. Signs of this disease are less pronounced in young pigs and those that are heterozygous for the trait. In nonanesthetized pigs, stressful situations will lead to the early signs of the disease, which include muscle and tail tremors. Progression of the syndrome leads to dyspnea, blanched and reddened areas on the

skin, increased body temperature, and cyanosis. Muscle rigidity and cardiovascular collapse follow. Necropsy. Pigs exhibiting this syndrome present with a very rapid development of rigor mortis. In addition, many of the animals will have muscles that appear very pale and are very soft, almost watery in texture, due to the high lactic acid content in muscles that occurs postmortem. Antemortem histologic changes have not been identified in these animals. Prevention and control. The disease is best controlled by identifying those animals who carry the genetic mutation and eliminating them from the breeding stock. A readily available, inexpensive DNA-based test can be used to screen for the mutation (O'Brien et al., 1993). Treatment. Early recognition of the disease is the key to treatment. Anesthetic delivery should be discontinued immediately and 100% oxygen delivered. Additional treatment includes sodium bicarbonate to combat the metabolic acidosis and hyperkalemia. Active cooling of the animal may be done by ice packing and IV administration of cooled fluids, or by gastric and/or rectal lavage with iced saline. Dantrolene, an agent that prevents PSS by decreasing release of calcium from the sarcoplasmic reticulum while allowing calcium uptake to continue, is highly effective in stopping the progression of the syndrome when administered at the onset of signs. After the crisis is alleviated, the animal must be monitored closely for 48 hr; redevelopment of the syndrome in response to minor stressors can occur. Dantrolene can also be given as preventive therapy in animals known to be susceptible. b.

Salt Poisoning

Etiology. Salt poisoning, also known as sodium ion toxicosis, is a condition that can easily occur in swine. It can be caused directly by the animal consuming excessive amounts of sodium. This happens infrequently, as animals are rarely presented with feed that has excessively high sodium content. However, feeding milk by-products such as whey, which has a high sodium content, has been shown to cause the disease. By far, the most common initiator for the condition is water deprivation. Usually, signs are initiated after a minimum of 24 hr of deprivation, but the condition can also occur after just a few hours of deprivation. Clinical signs. Initially, the animal presents as being very thirsty and constipated. Central nervous system (CNS) involvement, which may be delayed for several days after the insult, follows. The pigs will appear tense and apprehensive, with ears pricked and staring ahead with the head slightly elevated. The nose will then twitch, the eyes will close, and a rhythmic chomping of the jaws follows. Animals may also appear blind

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15. BIOLOGYAND DISEASES OF SWINE and deaf. Pigs near death may paddle continuously. If the condition occurs because of excessive salt consumption rather than water deprivation, vomiting and diarrhea may be part of the presentation.

Pathogenesis.

Salt poisoning is caused by hyperosmolarity of the CNS. When the animal rehydrates, the osmotic pressure causes water to be drawn into the CNS, resulting in swelling and edema.

Diagnosis and pathology.

The diagnosis can easily be made if the clinical signs are matched with known water deprivation. Histologic evaluation reveals eosinophilic cuffing of the meningeal and cerebral vessels. Supporting findings include gastritis, constipation, or enteritis. A laminar subcortical polioencephalomalacia may occur if pigs are subacutely affected. The animal may present with hypernatremia; however, if the animal has had a chance to rehydrate, this finding will not be present. Differential diagnoses include pseudorabies, hog cholera, and edema disease. Other causes of toxicoses, such as food poisoning, should also be considered.

Treatment.

Unfortunately, treatment is generally ineffective, and in fact, the condition is likely to be exacerbated by rehydration.

c.

Gastric Ulcers

Epizootiology.

Although this condition has been identified for decades, the definitive pathogenesis is unknown. Nutritional factors, such as increased concentrations of copper and unsaturated fatty acids, and decreased concentrations of tocopherols and selenium, have been shown to induce the condition. Fasted pigs exposed to stressful environmental conditions had a higher incidence of ulcers compared to controls. An increased incidence of ulceration was produced when pigs were fed finely ground diets. Many species of bacteria and fungi have been isolated from ulcer lesions, but none have been shown to be causative. One study investigated the prevalence of Gastrospirillum suis in pigs with gastric ulcer but found no correlation between its presence and the occurrence of ulceration (Barbosa et al., 1995). Further investigations are needed to better define the etiology of this condition.

Pathology: Gross.

The pars oesophagea contains no glands and is covered by stratified squamous epithelium continuous with the esophagus. In a healthy animal, this surface appears white and smooth. Lesions can first be detected as a roughened, irregular surface. Ulceration follows, with a disruption in the epithelium that may be small, discrete, and single to multiple, large, and irregular. Blood or blood clots can be seen at the ulceration site, as well as in the stomach or in the gastrointestinal tract. If the subacute/chronic form of the condition is present, chronic ulceration usually ensues. This is characterized by the presence of fibrous tissue and the contraction of the area of ulceration.

Etiology.

Gastric ulceration in pigs refers to a condition in which ulceration of a specific region of the pig's stomach, the pars oesophagea, occurs. This condition has been diagnosed with increasing frequency since the 1950s, with the distribution being worldwide and varied in occurrence. To date, the pathogenesis of the disease remains speculative.

Clinical signs and differential diagnoses. The clinical signs vary depending on duration of the ulceration. In the peracute form, apparently healthy animals are simply found dead. In the acute form, pigs will become pale and weak, with an increased respiratory rate. Vomiting of blood and passage of bloody, tarry feces are seen. In the subacute or chronic form, the animal will be anemic and anorexic, with passage of dark feces that may be intermittent or persistent. Occasionally, the only sign observed may be the passage of dark, hard feces. Pigs of either sex and any breed may be affected. Usually, single pigs are affected, and body temperature is normal or slightly subnormal. Anemia can be detected hematologically if the chronic/subacute form is present. Differential diagnoses include swine dysentery, Salmonella choleraesuis, transmissible gastroenteritis, and intestinal hemorrhagic syndrome. These diseases can be differentiated relatively easily, as they impact on groupings of animals and result in high body temperatures, except in the hemorrhagic syndrome.

Pathology: Microscopic.

The pars oesophagea in the pig is covered by stratified squamous epithelium. In the early stages of the ulcer formation, parakeratosis of the epithelium occurs. Occasionally, infiltration of some polymorphonuclear cells occurs, but usually inflammatory cells are absent. The epithelium is weakened, and erosion of the tissue eventually occurs as a result of the parakeratosis. Once the underlying tissues are exposed to the gastric juices, diffuse necrosis and bleeding characteristic of any ulcer occurs. Chronic ulcer.s develop as fibrous connective tissue forms in the underlying lamina propria. The muscularis mucosae may hypertrophy or may degenerate and be replaced by collagenized fibrous tissue. Occasionally, the ulcer may penetrate the serosa.

Prevention.

Providing pigs with appropriate feed is a prudent measure to take toward disease prevention. The diet should be more coarsely ground (not less than 700 ~tm in size), not contain excessive unsaturated fatty acids, and have the right balance of vitamin E and selenium. Stressful conditions such as overcrowding, fasting, and unstable social groupings should be avoided.

Treatment. Early stages of ulceration are not typically identified, so treatment is often not initiated until the condition has

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progressed to a point where treatment is ineffective. Options include administering nonabsorbable antacids, and vitamin E and selenium, as well as H-2 blockers (cimetidine, Zantac, etc.).

sites. Thrombi dislodged from catheters during flushing can result in infarcts in multiple tissues, including the kidney.

Differential diagnosis. The differential diagnosis should include foreign body reactions to the biomaterials.

C.

Iatrogenic Diseases

Catheter Infections Etiology. A wide variety of either venous or arterial vascularaccess lines are commonly used in swine and maintained for variable periods. Bacteria can be easily introduced into these lines if strict adherence to sterile technique is not observed during flushing. Improper maintenance of the catheter can also result in seeding of thrombi. Clinical signs. Swine with a catheter infection will be febrile, have decreased appetite, and have a discharge from around the vascular access port. Necropsy. A suppurative exudate may be present around the external access port or around subcutaneous implants. The entire catheter tract should be dissected to observe for any gross evidence of infection. Cultures should be taken of any suspicious sites. There may be a suppurative pneumonia with consolidation, suppurative emboli in multiple organs, renal infarcts (Fig. 13), or infarcts in other organs. Microscopic lesions may include a cellulitis, myositis, suppurative pneumonia, suppurative emboli in one or more organs, or infarcts in the kidneys or other organs. Pathogenesis. Bacteremia with seeding of multiple organs can result in septic emboli in the lungs, kidney, spleen, and other

Fig. 13. Renalinfarcts in a pig kidney.

Prevention and control. Prevention and control consist of strict adherence to sterile technique in flushing and adequate flushing of lines that have high enough concentrations of anticoagulants to prevent thrombus formation. Treatment. Blood cultures or cultures taken from around the implant may identify the infectious agent responsible, and a sensitivity test should provide information needed to select appropriate antibiotics. Research complications. Catheter infections are themselves research complications that may result in the animal being terminated from a study or euthanatized due to persistent febrile state or compromised function of one or more organs.

D.

Neoplastic Diseases

It has been touted that neoplasms occur with less frequency in pigs than in other domestic animals; however, this commonly held belief may be influenced by the fact that the majority of the pig population is slaughtered before reaching an age when cancer would normally appear with any significant incidence. The tumors that are reported are those seen in young pigs, with the most common tumors being lymphosarcoma, embyronal nephroma, and melanoma. Lymphosarcomas affect primarily younger animals but can affect mature animals of either sex. Most cases are classified as multicentric; thymic is the next most frequent classification. Infiltration of the liver, spleen, and kidney predominates. Histologically, pigs typically exhibit lymphocytic lymphosarcomas; however, lymphoblastic, histiocytic, and mixed types do occur. Embryonal nephromas affect pigs under 1 year of age, with a predominance in females. The tumor arises in the kidney parenchyma, is typically unilateral, and may spread to the lungs and liver. Histologically, the classifications that occur most commonly are nephroblastic and epithelial. Melanomas occur as congenital lesions with exceptionally high frequency in Sinclair miniature swine (85% incidence at 1 year of age) and in Duroc and Hormel breeds. The disease is occasionally seen in other breeds as well. The tumors can be single or multiple and may affect the skin only or may involve metastasis to multiple internal organs. Initially, the skin tumor appears as a flat black spot that becomes a raised nodule. The tumor initiates as a focus of melanocytic hyperplasia within the basal layer. Spontaneous regression, thought to be caused by the cytotoxic effects of infiltrated tumor-specific T lymphocytes, occurs in the vast majority of cases.

15. BIOLOGY AND DISEASES OF SWINE

E. 1.

Miscellaneous

Umbilical/Inguinal Hernia

Inguinal and umbilical hernias are two of the most common developmental defects found in swine, with the occurrence of inguinal hernias exceeding that of umbilical. Inguinal hernias occur mainly in male pigs, with increasing presence by 5 weeks of age. It may occur singularly or bilaterally. Incidence is controlled by culling affected animals, as the condition is believed to be inherited polygenically; however, a recent study concluded that umbilical lesions such as omphalitis or umbilical abscesses are associated with herniation (Searcy-Bernal et al., 1994). The occurrence of umbilical hernias varies with breed and sex but can reach 1.2%.

2.

Starvation

Starvation is one of the main causes of noninfectious neonatal mortality. In one study, it accounted for 43% of postnatal deaths. Neonatal pigs are deficient in hepatic gluconeogenesis; consequently, they are very susceptible to hypoglycemia with short periods of milk deprivation (36 hr). Factors contributing to this condition include congenital abnormalities, trauma caused by the female, chilling, and female hypogalactia. Infectious hypogalactia (also referred to as M M A for mastitis, metritis, and agalactia) is a condition in which one or more of the mammary glands are inflamed and infected with gram-negative bacteria. Endotoxin release interferes with the release of prolactin and subsequently prevents milk letdown. Oxytocin, antibiotics, and anti-inflammatory drugs are helpful. Tranquilizers such as chlorpromazine have also been shown to have lactogenic effects.

3.

Iron Deficiency Anemia

Iron deficiency anemia of the microcytic hypochromic type occurs commonly in suckling pigs that are not supplemented after birth. The pig fetus has minimal capacity for iron storage, and sow's milk contains low concentrations of iron. Unsupplemented piglets will exhibit rough hair coat and elevated respirations. Necropsy findings include a dilated heart and pulmonary edema. It is recommended that piglets receive 1 0 0 - 3 0 0 mg of supplemental iron between the time of birth and the time they begin to eat solid food. Injections are preferred over oral supplements, as oral administration of iron may upset the balance of gastrointestinal flora.

4.

Behavioral Problems/Fight or Traumatic Injuries (Tail, Ear, and Flank Biting)

Tail, vulva, ear, and flank biting is an inconsistent but widespread problem of group-housed swine. The clinical out-

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comes are damaged areas of skin that can become infected, leading to abscesses (Huey, 1996), poor performance (Wallgren and Lindahl, 1996), suppurative arthritis (Cowart, 1995), and possible mortality. The basis for this behavior probably stems from the natural tendency of pigs to root and chew to obtain food in their natural environment. In confined group housing, pen mates, for lack of anything better, take the place of natural rooting structures. Iron deficiency anemia has been considered a possible cause for this behavior in piglets. Inadequate ambient temperature control, ventilation, pen and feeder space, and access to water (Rizvi et al., 1998) are also considered contributing factors. However, the most plausible explanation is the lack of sufficient environmental enrichment. Corrective measures should include access to rooting materials, including wood-chip bedding, straw, and "indestructible" polyethylene balls. When groups are formed, the animals should be of similar age and size and of numbers appropriate for the space. Tail docking is also considered a preventive measure since this reduces the incidence of tail biting (Hemsworth, 1999).

REFERENCES

Aarestrup, E M., Jorsal, S. E., and Jensen, N. E. (1998a). Serologicalcharacterization and antimicrobial susceptibility of Streptococcus suis isolates from diagnostic samples in Denmark during 1995 and 1996. Vet. Microbiol. 60(1), 59-66. Aarestrup, E M., Rasmussen, S. R., Artursson, K., and Jensen, N. E. (1998b). Trends in the resistance to antimicrobial agents of Streptococcus suis isolates from Denmark and Sweden. Vet. Microbiol. 63(1), 71-80. Ackerman, M. R., Debey, M. C., and Debey, B. M. (1991). Bronchiolar metaplasia and Ulex europaeus agglutinin 1 (UEA-1) affinity in Mycoplasma hyopneumoniae infected lungs of 6 pigs. Vet. Pathol. 28, 533-535. Ackermann, M. R., Register, K. B., Stabel, J. R., Gwaltney, S. M., Howe, T. S., and Rimler, R. B. (1996). Effect of Pasteurella multocida toxin on physeal growth in young pigs. Am. J. Vet. Res. 57(6), 848-852. Alt, D. P., and Bolin, C. A. (1996). Preliminary evaluation of antimicrobial agents for treatmentof Leptospira interrogans serovarpomona infection in hamsters and swine. Am. J. Vet. Res. 57(1), 59-62. Amass, S. E, and Scholz, D. A. (1998). Acute nonfatal erysipelas in sows in a commercial farrow-to-finish operation. J. Am. Vet. Med. Assoc. 212(5), 708-709. Amass, S. E, Wu, C. C., Clark, L. K. (1996). Evaluation of antibiotics for the elimination of the tonsillarcarrier state of Streptococcus suis in pigs. J. Vet. Diagn. Invest. 8(1), 64-67. Andresen, L. O. (1998). Differentiation and distribution of 3 types of exfoliative toxin produced by Staphylococcus hyicus from pigs with exudativeepidermitis. FEMS lmmunol. Med. Microbiol. 20(4), 301-310. Andresen, L. O. (1999a). Developmentand evaluation of an indirect ELISA for detection of exfoliativetoxin ExhA, ExhB, or ExhC produced by Staphylococcus hyicus. Vet. Microbiol. 68(3-4), 285-292. Andresen, L. O., (1999b). Studies on the effect of divalent metal ions on exfoliative toxins from Staphylococcus hyicus: Indications of ExhA and ExhB being metalloproteins. FEMS Immunol. Med. Microbiol. 23(4), 295-301. Arends, J. J., Skogerboe, T. L., and Ritzhaupt, L. K. (1999). Persistentefficacy of doramectin and ivermectin against experimental infestations of Sarcoptes scabiei var. suis in swine. Vet. Parasitol. 82(1), 71-79.

666 Atyeo, R. F., Oxberry, S. L., Combs, B. G., and Hampson, D. J. (1998). Development and evaluation of polymerase chain reaction tests as an aid to diagnosis of swine dysentery and intestinal spirochaetosis. Lett. Appl. Microbiol. 26, 126-130. Awad-Masalmeh, M., Kofer, J., Schuh, M., and Hinterdorfer, E (1999). Serotypes, virulence factors, and sensitivity against antibiotics of S. suis strains isolated from clinically healthy as well as diseased pigs of Austrian swine herds. Wien. Tierarztl. Monatsschr. 86(8), 262-269. Baker, D. R., Billey, L. O., and Francis, D. H. (1997). Distribution of K88 Escherichia coli-adhesive and nonadhesive phenotypes among pigs of 4 breeds. Vet. MicrobioL 54, 123-132. Bane, D. P., Clark, L. K., Knox, K. E., Wu, C. C., and Watkins, L. E. (1999). Evaluation of the recrudescence of Actinobacillus pleuropneumoniae following Pulmotil treatments in nursery and grower pigs. In "Allen D. Leman Swine Conference" (W. C. Scruton and S. Class, eds.), Vol. 26, Suppl. p. 17. Vet Outreach Programs, Univ. of Minnesota, Minneapolis. Barbosa, A. J. A., Silva, J. C. P., Nogueira, A. M. M. E, Paulino, E., Jr., and Miranda, C. R. (1995). Higher incidence of gastrospirillum in swine with gastric ulcer of the pars oesophagea. Vet. Pathol. 32, 134-139. Barman, N. N., Bianchi, A. T., Zwart, R. J., Pabst, R., and Rothkotter, H. J. (1997). Jejunal and ileal Peyer's patches in pigs differ in their postnatal development. Anat. Embryol. (Berl.) 195, 41-50. Batte, E. G., and Moncol, D. J. (1972). Whipworms and dysentery in feeder pigs. J. Am. Vet. Med. Assoc. 161, 1226-1228. Batte, E. G., McLamb, R. D., Muse, K. E., Tally, S. D., and Vestal, T. J. (1977). Pathophysiology of swine trichuriasis. Am. J. Vet. Res. 38, 1075-1079. Baumeister, A. K., Runge, M., Ganter, M., Feenstra, A. A., Delbeck, E, and Kirchhoff, H. (1998). Detection of Mycoplasma hyopneumoniae in bronchoalveolar lavage fluids of pigs by PCR. J. Clin. Microbiol. 36(7), 19841988. Beer, R. J. S., and Lean, I. J. (1973). Clinical trichuriasis produced experimentally in growing pigs: Part 1: Pathology of infection. Vet. Rec. 93, 189-195. Bertram, T. A. (1985). Quantitative morphology of peracute pulmonary lesions in swine induced by Haemophilus pleuropneumoniae. Vet. PathoL 22, 598-609. Bertschinger, H. U., and Pohlenz, J. (1983). Bacterial colonization and morphology of the intestine in porcine Escherichia coli enterotoxemia (edema disease). Vet. Pathol. 20, 99-110. Bertschinger, H. U., Fairbrother, J. M., Nielson, N. O., and Pohlenz, J. E (1992). "External Parasites, Diseases of Swine," 7th ed., pp. 487-497. Iowa State Univ. Press, Ames. Bobbie, D. L., and Swindle, M. M. (1986). Pulse monitoring, intravascular and intramuscular injection sites in pigs. In "Swine in Biomedical Research" (M. E. Tumbleson,) pp. 273-277. Plenum Press, New York. Bollen, P. J. A., Hansen, A. K., and Rasmussen, H. J. (2000). "The Laboratory Swine." CRC Press, Boca Raton, Florida. Bornstein, S., and Wallgren, P. (1997). Serodiagnosis of sarcoptic mange in pigs. Vet. Rec. 141(1), 8-12. Bousquet, E., Morvan, H., Aitken, I., and Morgan, J. H. (1997). Comparative in vitro activity of doxycycline and oxytetracycline against porcine respiratory pathogens. Vet. Rec. 141(2), 37-40. Bousquet, E., Pommier, P., Wessel-Robert, S., Morvan, H., Benoit-Valiergue, H., and Laval, A. (1998). Efficacy of doxycycline in feed for the control of pneumonia caused by Pasteurella multocida and Mycoplasma hyopneumoniae in fattening pigs. Vet. Rec. 143(10), 269-272. Boye, M., Jensen, T. K., Moiler, K., Leser, T. D., and Jorsal, S. E. (1998). Specific detection of Lawsonia intracellularis in porcine proliferative enteropathy inferred from fluorescent rRNA in situ hybridization. Vet. Pathol. 35, 153-156. Bracy, J. L., Sachs, D. H., and Iacomini, J. (1998). Inhibition of xenoreactive natural antibody production by retroviral gene therapy. Science 281, 1845-1847. Brady, P. S., Ku, P. K., Ullrey, D. E., and Miller, E. R. (1978). Evaluation of an amino acid-iron chelate hematinic for the baby pig. J. Anim. Sci. 47(5), 1135-1140.

KATHY E. LABER ET AL.

Braun, W. (1993). Reproduction in miniature pet pigs. In "Care and Management of Miniature Pet Pigs" (D. E. Reeves, ed.), pp. 27-39. Vet Practice Publ., Santa Barbara, California. Brenner, P., Reichenspurner, H., Schmoeckel, M., Wimmer, C., Rucker, A., Eder, V., Meiser, B., Hinz, M., Felbinger, T., Muller-Hocker, J., Hammer, C., and Reichart, B. (2000). Ig-therasorb immunoapheresis in orthotopic xenotransplantation of baboons with Landrace pig hearts. Transplantation 69, 208-214. Bridger, J. C., Tauscher, G. I., and Desselberger, U. (1998). Viral determinants of rotavirus pathogenicity in pigs: Evidence that the fourth gene of a porcine rotavirus confers diarrhea in the homologous host. J. Virol. 72, 6929-6931. Brooke, C. J., and Riley, T. V. (1999). Erysipelothrix rhusiopathiae: Bacteriology, epidemiology, and clinical manifestations of an occupational pathogen. J. Med. MicrobioL 48(9), 789-799. Brouard, S., Gagne, K., Blancho, G., and Soulillou, J. P. (1999). T cell response in xenorecognition and xenografts: A review. Hum. Immunol. 60, 455468. Brown, T. T., Jr., Paul, P. S., and Mengeling, W. L. (1980). Response of conventionally raised weanling pigs to experimental infection with a virulent strain of porcine parvovirus. Am. J. Vet. Res. 41, 1221-1224. Brown, T. T., Jr., Mengeling, W. L., and Pirtle, E. C. (1982). Failure of swine influenza virus to cause transplacental infection of porcine fetuses. Am. J. Vet. Res. 43, 817- 819. Buller, C. R., and Moxley, R. A. (1988). Natural infection of porcine ileal dome M cells with rotavirus and enteric adenovirus. Vet. Pathol. 250, 516-517. Buogo, C., Capaul, S., Hani, H., Frey, J., and Nicolet, J. (1995). Diagnosis of Clostridium perfringens type C enteritis in pigs using a DNA amplification technique (PCR). Zentralbl. Veterinarmed. B. 42, 51-58. Burkhardt, D., Barker, M., Laugesen, T., Lenz, K., and van den Bosch, H. (1999) Actinobacillus pteuropneumoniaes (APP) subunit vaccine provides cross protection against virulent APP challenge. In "Allen D. Leman Swine Conference" (W. C. Scruton and S. Claas, eds.), Vol. 26, Suppl., pp. 45-46. Vet Outreach Programs, Univ. of Minnesota, Minneapolis. Burkhart, K., and Roof, M. (1999). Efficacy of Salmonella choleraesuis vaccine, avirulent live culture, SC-54, using spraying as an alternative method of vaccine delivery. In "Allen D. Leman Swine Conference" (W. C. Scruton and S. Claas, eds.), Vol. 26, Suppl., p. 47. Vet Outreach Programs, Univ. of Minnesota, Minneapolis. Burton, P. J., Thornsberry, C., Yee, Y. C., Watts, J. L., and Yancy, R. J. (1996). Interpretive criteria for antimicrobial susceptibility testing of ceftiofur against bacteria associated with swine respiratory disease. J. Vet. Diagn. Invest. 8(4), 464-468. Busque, P., Higgins, R., Caya, E, and Quessy, S. (1997). Immunization of pigs against Streptococcus suis serotype 2 infection using a live avirulent strain. Can. J. Vet. Res. 61(4), 275-279. Butler, C. R. and Moxley, R. A. (1988). Natural infection of porcine ileal dome M cell with rotavirus and enteric adenovirus. Vet. Pathol. 25, 516517. Calsamiglia, M., Pijoan, C., Solano, G., and Rapp-Gabrielson, V. (1999). Development of an oligonucleotide-specific capture plate hybridization assay for detection of Haemophilus parasuis. J. Vet. Diagn. Invest. 11(2), 140145. Cargill, C. F., Pointon, A. M., Davies, P. R., and Garcia, R. (1997). Using slaughter inspections to evaluate sarcoptic mange infestation of finishing swine. Vet. Parasitol. 70(1-3), 191-200. Cargill, C., Davies, P., Carmichael, I., Hooke, E, and Moore, M. (1996). Treatment of sarcoptic mite infestation and mite hypersensitivity in pigs with injectable doramectin. Vet. Rec. 138(19), 468-471. Cargill, C., and Davies, P. R. (1999). In "External Parasites, Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 669-683. Iowa State Univ. Press, Ames. Carvalho, L. F., Segales, J., and Pijoan, C. (1997). Effect of porcine reproductive and respiratory syndrome virus on subsequent Pasteurella multocida challenge in pigs. Vet. Microbiol. 55(1-4), 241-246.

15. BIOLOGY AND DISEASES OF SWINE Chae, S. J., Kramer, A. D., Zhao, Y., Am, S., Cooper, D. K., and Sachs, D. H. 1999. Lack of variation in alphaGal expression on lymphocytes in miniature swine of different genotypes. Xenotransplantation 6, 43-51. Chaniago, T. D., Watson, D. L., Owen, R. A., and Johnson, R. H. (1978). Immunoglobulins in blood serum of fetal pigs. Aust. Vet. J. 54, 30-33. Claxton-Gill, M. A., Cornick-Seahorn, J. L., Gamboa, J. C., and Boatright, B. A. (1993). Suspected malignant hyperthermia syndrome in a miniature pot-bellied pig anesthetized with isoflurane. J. Am. Vet. Med. Assoc. 2tl3, 1434-1436. Clifton-Hadley, F. A., Alexander, T. J. L., Upton, I., and Duffus, W. P. H. (1984). Further studies on the subclinical carrier state of Streptococcus suis type 2 in pigs. Vet. Rec. 114, 513-518. Collins, J. E., Benfield, D. A., Christianson, W. T., Harris, L., ChristopherHennings, J., Shaw, D. P., Goyal, S. M., McCullough, S., Morrison, R. B., Joo, H. S., Gorcyca, D., and Chladek, D. (1992). Isolation of swine infertility and respiratory syndrome virus in North America and experimental reproduction of the disease in gnotobiotic pigs. J. Vet. Diagn. Invest. 4, 117-126. Collins, J., Dee, S., Halbur, P., Keffaber, K., Lautner, B., McCaw, M., Rodibaugh, M., Sanford, E., and Yeske, P. (1997). Recent PRRS outbreaks. Am. Assoc. Swine Pract. News. Jan., 1. Cooper, D. M., and Gebhart, C. J. (1998). Comparative aspects of proliferative enteritis. J. Am. Vet. Med. Assoc. 212, 1446-1451. Copland, J. W. (1976). A study of acute "short-wind" (pneumonia) in village pigs of Papua, New Guinea. Trop. Anim. Health Prod. 8(4), 187-194. Corwin, R. M., and Stewart, T. B. (1999). Internal parasites. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 717-724. Iowa State Univ. Press, Ames. Costa, C., Zhao, L., Burton, W. V., Bondioli, K. R., Williams, B. L., Hoagland, T. A., Ditullio, P. A., Ebert, K. M., and Fodor, W. L. (1999). Expression of the human alphal,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serummediated cytolysis. FASEB J. 13, 1762-1773. Cowart, R. P. (1995). "Respiratory Diseases/Pleuropneumonia. An Outline of Swine Diseases: A Handbook." Iowa State Univ. Press, Ames. Dangolla, A., Willeberg, P., Bjorn, H., and Roepstorff, A. (1996). Associations of Ascaris suum and Oesophagostomum spp. infections of sows with management factors in 83 Danish sow herds. Prev. Vet. Med. 27(3-4), 197209. Daniels, C. S., Thacker, B., Sornsen, S., and Thacker, E. (1999). Survey of Mycoplasma hyopneumoniae maternally derived antibodies in suckling pigs. In "Allen D. Leman Swine Conference" (W. C. Scruton and S. Claas, eds.), Vol. 26, Suppl., p. 41. Vet Outreach Programs, Univ. of Minnesota, Minneapolis. Davies, H. S., Taylor, J. E., White, D. J., and Binns, R. M. (1978). Major transplantation antigens of pig kidney and liver. Comparisons between the whole organs and their parenchymal constituents. Transplantation 25, 290-295. Davies, K. A., Chapman, P. T., Norsworthy, P. J., Jamar, E, Athanassiou, P., Keelan, E. T., Harrison, A. A., Binns, R. M., Haskard, D. O., and Walport, M. J. (1995). Clearance pathways of soluble immune complexes in the pig. Insights into the adaptive nature of antigen clearance in humans. J. Immunol. 155, 5760-5768. Davies, P. R. (1995). Sarcoptic mange and production performance of swinem review of the literature and studies of associations between mite infestation, growth rate, and measures of mange severity in growing pigs. Vet. Parasitol. 60(3-4), 249-264. Davies, P. R., Bahnson, P. B., Grass, J. J., Marsh, W. E., Garcia, R., Melancon, J., and Dial, G. D. (1996). Evaluation of the monitoring of papular dermatitis lesions in slaughtered swine to assess sarcoptic mite infestation. Vet. Parasitol. 62(1-2), 143-153. Davis, D. P., and Moon, R.. D. (1990). Density of itch mite, Sarcoptes scabiei (Acari: Sarcoptidae), and temporal development of cutaneous hypersensitivity in swine mange. Vet. Parasitol. 36(3-4), 285-293. Davis, W. C., Zuckermann, E A., Hamilton, M. J., Barbosa, J. I., Saalmuller, A., Binns, R. M., and Licence, S. T. (1998). Analysis of monoclonal antibod-

667 ies that recognize gamma delta T/null cells. Vet. Immunol. Immunopathol. 60, 305-316. Day, B. N. (1980). Parturition. In "Current Therapy in Theriogenology" (D. A. Morrow, ed.), pp. 1064-1067. Saunders, Philadelphia. De Jong, M. E (1992). Progressive atrophic rhinitis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 414-433. Iowa State Univ. Press, Ames. De Jong, M. E (1999). Progressive and nonprogressive atrophic rhinitis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'A1laire, and D. J. Taylor, eds.), 8th ed., pp. 355-384. del Castillo, J. R. E., Hannon, M. R., Martineau, G. P., and Besner, J. G. (1999). Comparative efficacy of chlortetracycline and oxytetracycline administered in feed against experimental pleuropneumonia in pigs. In "Allen D. Leman Swine Conference" (W. C. Scruton and S. Claas, eds.), Vol. 26, Suppl., p. 13. Vet Outreach Programs, Univ. of Minnesota, Minneapolis. Deyoe, B. L. (1967). Pathogenesis of 3 strains of Brucella suis in swine. Am. J. Vet. Res. 28, 951-957. Dial, G. D., Marsh, W. E., Poison, D. D., and Vaillancourt, J.-P. (1992). Reproductive failure. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 88-137 Iowa State Univ. Press, Ames. Didier, P. J., Perino, B. S., and Urbance, J. (1984). Porcine Haemophilus pleuropneumonia: Microbiologic and pathologic findings. J. Am. Vet. Med. 184, 716-719. Dominick, M. A., and Rimler, R. B. (1988). Turbinate osteoporosis in pigs following intranasal inoculation of purified Pasteurella toxin: Histomorphometric and ultrastructural studies. Vet. Pathol. 25, 17-27. Dritz, S. S., Chengappa, M. M., Nelssen, J. L., Tokach, M. D., Goodband, R. D., Nietfeld, J. C., and Staats, J. J. (1996). Growth and microbial flora of nonmedicated, segregated, early weaned pigs from a commercial swine operation. J. Am. Vet. Med. Assoc. 208(5), 711-715. Duhamel, G. E., Kinyon, J. M., Mathiesen, M. R., Murphy, D. P., and Walter, D. (1998). In vitro activity of 4 antimicrobial agents against North American isolates of porcine Serpulina pilosicoli. J. Vet. Diagn. Invest. 10(4), 350356. Duncan, J. R., Ross, R. E, Switzer, W. P., and Ramsey, E K. (1966a). Pathology of experimental Bordetella bronchiseptica infection in swine. Atrophic rhinitis. Am. J. Vet. Res. 27, 457-466. Duncan, J. R., Ramsey, E K., and Switzer, W. P. (1966b). Pathology of experimental Bordetella bronchiseptica infection in swine. Pneumonia. Am. J. Vet. Res. 27, 467-472. Easterday, B. C., and Hinshaw, V. S. (1992). Swine influenza. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 349-357. Iowa State Univ. Press, Ames. Edington, N. (1976). Relationship of porcine cytomegalovirus and B. bronchiseptica to atrophic rhinitis in gnotobiotic piglets. Vet. Rec. 98, 42-45. Edington, N. (1992). Cytomegalovirus. In "Disease of Swine" (A. D., Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 250-256. Iowa State Univ. Press, Ames. Edington, N., Watt, R. G., Plowright, W., Wrathall, A. E., and Done, J. T. (1977). Experimental transmission of porcine cytomegalovirus. J. Hyg. 78, 243251. Einarsson, S. (1980). Artificial insemination. In "Current Therapy in Theriogenology" (D. A. Morrow, ed.), pp. 1021-1024. Saunders, Philadelphia. Elder, R. O., Duhamel, G. E., Mathiesen, M. R., Erickson, E. D., Gebhart, C. J., and Oberst, R. D., (1997). Multiplex polymerase chain reaction for simultaneous detection of Lawsonia intracellularis, Serpulina hyodysenteriae, and Salmonellae in porcine intestinal specimens. J. Vet. Diagn. Invest. 9(3), 281-286. Ellis, W. A. (1992). Leptospirosis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 529-536. Iowa State Univ. Press, Ames. Ellis, W. A. (1999). Leptospirosis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 483-493. Iowa State Univ. Press, Ames.

668 Emery, D. W., Holley, K., and Sachs, D. H. (1999). Enhancement of swine progenitor chimerism in mixed swine/human bone marrow cultures with swine cytokines. Exp. Hematol. 27, 1330-1337. Erickson, E. D. (1987). Streptococcosis. J. Am. Vet. Med. Assoc. 191, 13911393. Eustis, S. L., and Nelson, D. T. (1981). Lesions associated with coccidiosis in nursing piglets. Vet. Pathol. 18, 21-28. Ferris, R. A., Schoenbaum, M. A., and Crawford, R. P. (1995). Comparison of serologic tests and bacteriologic culture for detection of brucellosis in swine from naturally infected herds. J. Am. Vet. Med. Assoc. 207(10), 1332. Fisher, T. F. (1993). Miniature swine in biomedical research: Applications and husbandry considerations. Lab. Anim. 22, 47-50. Friedman, T., Shimizu, A., Smith, R. N., Colvin, R. B., Seebach, J. D., Sachs, D. H., and Iacomini, J. (1999). Human CD4§ cells mediate rejection of porcine xenografts. J. Immunol. 162, 5256-5262. Friendship, R. M. (2000). Antimicrobial drug use in swine. In "Antimicrobial Therapy in Vet Medicine" (J. F. Prescott, J. D. Baggot, and R. D. Walker, eds.), 3rd ed., pp. 602-616. Iowa State Univ. Press, Ames. Fujii, J., Otsu, K., Zorzato, F., deLeon, S., Khanna, V. K., Weiher, J. E., O'Brien, P. J., and MacLennan, D. H. (1991). Identification of a mutation in the porcine ryanodine receptor associated with malignant hyperthermia. Science 253, 448-451. Gatlin, C. L., Jordan, W. H., Shryock, T. R., and Smith, W. C. (1996). The quantitation of turbinate atrophy in pigs to measure the severity of induced atrophic rhinitis. Can. J. Vet. Res. 60(2), 121-126. Gianello, P., Soares, M., Latinne, D., and Bazin, H. (1998). Xenotransplantation. In "Handbook of Vertebrate Immunology" (P. P. Pastoret, P. Griebel, H. Bazin, and A. Govaerts, eds.), pp. 619-629. Academic Press, New York. Giles, C. J. (1992). Bordetellosis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 436-445. Iowa State Univ. Press, Ames. Gipson, P. S., Veatch, J. K., Matlack, R. S., and Jones, D. P. (1999). Health status of a recently discovered population of feral swine in Kansas. J. Wildl. Dis. 35(3), 624-627. Graham, D. Y., Sackman, J. W., Estes, M. K. (1984). Pathogenesis of rotavirus induced diarrhea. Digest Dis. Sci. 29, 1028-1035. Gram, T., Ahrens, P., and Nielsen, J. P. (1996). Evaluation of a PCR for detection of Actinobacillus pleuropneumoniae in mixed bacterial cultures from tonsils. Vet. Microbiol. 51(1-2), 95-104. Griniston, B. (1988). Pseudorabie committee: USDA report. In "Proceedings," 92nd Annual Meeting, U.S. Animal Health Association, pp. 294-295. Gutierrez, C. B., Barbosa, J. I. R., Tascon, R. I., Costa, L., Riera, P., and Ferri, E. F. R. (1995). Serologic characterization and antimicrobial susceptibility of Actinobacillus pleuropneumoniae strains isolated from pigs in Spain. Vet. Rec. 137(3), 62-64. Gyles, C. L., and Thoen, C. O. (1993). Pathogenesis of bacterial infections in animals. 2nd ed. Iowa State Univ. Press, Ames. Halbur, P. G., Paul, P. S., Frey, M. L., Landgraf, J., Eernisse, K., Meng, X. J., Lum, M. A., Andrews, J. J., and Rathje, J. A. (1995). Comparison of the pathogenicity of two porcine reproductive and respiratory virus isolates with that of the Leystad virus. Vet. Pathol. 32, 648-660. Hamilton, T. D., Roe, J. M., Hayes, C. M., and Webster, A. J. (1998). Effects of ammonia inhalation and acetic acid pretreatment on colonization kinetics of toxigenic Pasteurella multocida within upper respiratory tracts of swine. J. Clin. Microbiol. 36(5), 1260-1265. Hamilton, T. D., Roe, J. M., Hayes, C. M., Jones, P., Pearson, G. R., and Webster, A. J. (1999). Contributory and exacerbating roles of gaseous ammonia and organic dust in the etiology of atrophic rhinitis. Clin. Diagn. Lab. Immunol. 62(2), 199-203. Hannan, P. C., Windsor, H. M., and Ripley, P. H. (1997). In vitro susceptibilities of recent field isolates of Mycoplasma hyopneumoniae and Mycoplasma hyosynoviae to valnemulin (econor), tiamulin, and enrofloxacin and the in vitro development of resistance to certain antimicrobial agents to Mycoplasma hyopneumoniae. Res. Vet. Sci. 63(2), 157-160.

KATHY E. LABER ET AL.

Harris, D. L., and Lysons, R. L. (1992). Swine dysentery. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 699-616. Iowa State Univ. Press, Ames. Harris, I. T., Fedorkacray, P. J., Gray, J. T., Thomas, L. A., and Ferris, K. (1997). Prevalence of Salmonella organisms in swine feed. J. Am. Vet. Med. Assoc. 210(3), 382. Hawk, C. T., and Leary, S. L. (1995). "Formulary for laboratory animals." Iowa State Univ. Press, Ames. Heinritzi, K. (1999). Eperythrozoonosis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 413-418. Iowa State Univ. Press, Ames. Heinritzi, K., Aigner, K., Erber, M., Kersjes, C., and von Wangenheim, B. (1999). Brucellosis and Aujeszky's disease in a wild boar. Case report. Tierarztl. Prax. Ausg. G. Grosstiere Nutztiere 27(1), 41-46. Hemsworth, P. H. (1999). Behavioral problems. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 648-649. Iowa State Univ. Press, Ames. Henne, E., Nickel, S., and Hiepe, T. (1978). Parasites in the GDR. 1. Studies on helminth occurrence in European wild pigs (Sus scrofa). Angewandte Parasitol. 19(1), 52-57. Hensel, A., Stockhofezurwieden, N., Petzoldt, K., and Lubitz, W. (1995). Oral immunization of pigs with viable or inactivated Actinobacillus pleuropneumoniae serotype 9 induces pulmonary and systemic antibodies and protects against homologous aerosol challenge. Infect. Immun. 63(8), 3048-3053. Higgins, R., and Gottschalk, M. (1999). Streptococcal diseases. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 563-578. Iowa State Univ. Press, Ames. Hill, B. D., Corney, B. G., and Wagner, T. M. (1996). Importance of Staphylococcus hyicus ssp. hyicus as a cause of arthritis in pigs up to 12 weeks of age. Aust. Vet. J. 73(5), 179-181. Hogg, G. G., Lenghaus, C., and Forman, A. J. (1977). Experimental porcine parvovirus infection of foetal pigs resulting in abortion, histologic lesions, and antibody formation. J. Comp. Pathol. 87, 539-549. Hollanders, W., and Vercruysse, J. (1990). Sarcoptic mite hypersensitivity: A cause of dermatitis in fattening pigs at slaughter. Vet. Rec. 126(13), 308310. Hollanders, W., Harbers, A. H. M., Huige, J. C. M., Monster, P., Rambags, P. G. M., and Hendrikx, W. M. L. (1995). Control of Sarcoptes scabiei var. suis with ivermectin - - influence on scratching behavior of fattening pigs and occurrence of dermatitis at slaughter. Vet. Parasitol. 58(1-2), 117127. Hollanders, W., Vercruysse, J., Raes, S., and Bornstein, S. (1997). Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of sarcoptic mange in swine. Vet. Parasitol. 69(1-2), 117-123. Hooper, B. E., and Haelterman, E. O. (1966). Concepts of pathogenesis and passive immunity in transmissible gastroenteritis of swine. J. Am. Vet. Med. Assoc. 149, 1580-1586. Hooper, B. E., and Haelterman, E. O. (1969). Lesions of the gastrointestinal tract of pigs infected with transmissible gastroenteritis. Can. J. Comp. Med. 33, 29-36. Hormansdorfer, S., and Bauer, J. (1998). Resistance of bovine and porcine Pasteurella to florfenicol and other antibiotics. Berl. Munch. Tierarztl. Wochenschr. 111, (11-12), 422-426. House, J. A., and House, C. A. (1992). Swine pox. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 358-361. Iowa State Univ. Press, Ames. Huang, C. A., Loft, T., Arn, J. S., Koo, G. C., Blake, T., and Sachs, D. H. (1999). Characterization of a monoclonal anti-porcine CD3 antibody. Xenotransplantation 6, 201-212. Huey, R. J. (1996). Incidence, location, and interrelationships between the sites of abscesses recorded in pigs at a bacon factory in Northern Ireland. Vet. Rec. 138(21), 511-514. Hughes, R., Olander, H. J., Kanitz, D. L., and Qureshi, S. (1977). A study of

15. BIOLOGY AND DISEASES OF SWINE

swine dysentery by immunofluorescence and histology. Vet. Path. 14, 490507. Ierino, E L., Gojo, S., Banerjee, P. T., Giovino, M., Xu, Y., Gere, J., Kaynor, C., Awwad, M., Monroy, R., Rembert, J., Hatch, T., Foley, A., Kozlowski, T., Yamada, K., Neethling, F. A., Fishman, J., Bailin, M., Spitzer, T. R., Cooper, D. K., Cosimi, A. B., LeGuern, C., and Sachs, D. H. (1999). Transfer of swine major histocompatibility complex class II genes into autologous bone marrow cells of baboons for the induction of tolerance across xenogeneic barriers. Transplantation 67, 1119-1128. Imada, Y., Goji, N., Ishikawa, H., Kishima, M., Sekizaki, T. (1999). Truncated surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae serotype la elicits protection against challenge with serotypes 1a and 2b in pigs. Infect. Immun. 67(9), 4376-4382. Jacobs, A. A., van den Berg, A. J., and Loeffen, P. L. (1996). Protection of experimentally infected pigs by suilysin, the thiol-activated haemolysin of Streptococcus suis. Vet. Rec. 139(10), 225-228. Jacobson, M., Bornstein, S., and Wallgren, P. (1999). The efficacy of simplified eradication strategies against sarcoptic mange mite infections in swine herds monitored by an ELISA. Vet. Parasitol. 81(3), 249-258. Jensen, T. K., Boye, M., Hagedorn-Olsen, T., Riising, H. J., and Angen, O. (1999). Actinobacillus pleuropneumoniae osteomyelitis in pigs demonstrated by fluorescent in situ hybridization. Vet. Pathol. 36(3), 258-261. Johnson, R. H. (1973). Isolation of swine parvovirus in Queensland. Aust. Vet. J. 49, 257-259. Jones, L. D. (1956). Exudative epidermitis of pigs. Am. J. Vet. Res. 17, 179193. Jones, T. C., and Hunt, R. D. (1983). Diseases caused by parasitic helminths and arthropods. In "Veterinary Pathology," pp. 778-879. Lea and Febiger, Philadelphia. Joo, H. S., Donaldson-Wood, C. R., and Johnson, R. H. (1976). Observations on the pathogenesis of porcine parvovirus infection. Arch. Virol. 51, 123-129. Joo, H. S., Donaldson-Wood, C. R., Johnson, R. H., and Campbell, R. S. E (1977). Pathogenesis of porcine parvovirus infection: Pathology and immunofluorescence in the foetus. J. Comp. Pathol. 87, 383-391. Jubb, K. V. E, Kennedy, P. C., and Palmer, N. (1985). "Pathology of Domestic Animals," 3rd ed., p. 470. Academic Press, Orlando, Florida. Kamp, E. M., Bokken, G. C., Vermeulen, T. M., De Jong, M. E, Buys, H. E., Reek, E H., and Smits, M. A. (1996). A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs. J. Vet. Diagn. Invest. 8(3), 304-309. Kamp, E. M., Stockhofezurwieden, N., Vanleengoed, L. A. M. G., and Smits, M. A. (1997). Endobronchial inoculation with Apx toxins of Actinobacillus pleuropneumoniae leads to pleuropneumonia. Infect. Immun. 65(10), 4350-4354. Kataoka, Y., Yamashita, T., Sunaga, S., Imada, Y., Ishikawa, H., Kishima, M., and Nakazawa, M. (1996). An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody against Streptococcus suis type 2 in infected pigs. J. Vet. Med. Sci. 58(4), 369-372. Kawashima, K., Yamada, S., Kobayashi, H., and Narita, M. (1996). Detection of porcine reproductive and respiratory syndrome virus and Mycoplasma hyorhinis antigens in pulmonary lesions of pigs suffering from respiratory distress. J. Comp. Pathol. 114(3), 315-323. Kelneric, Z., Naglic, T., and Udovicic, I. (1996). Prevention of necrotic enteritis in piglets by vaccination of pregnant gilts with a Clostridium perfringens type C and D bacterin-toxoid. Vet. Med. (Praha) 41, 335-338. Kelsey, D. K., Olsen, G. A., Overall, J. C., and Glasgow, L. A. (1977). Alteration of host defense mechanisms by murine cytomegalovirus. Infect. Immun. 18, 754-760. Kluge, J. P., Beran, G. W., Hill, H. T., and Platt, K. B. (1992). Pseudorabies (Aujeszky's disease). In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 312-323. Iowa State Univ. Press, Ames. Koudela, B., Nohynkova, E., Vitovec, J., Pakandl, M., and Kulda, J. (1991).

669 Giardia infection in pigs: Detection and in vitro isolation of trophozoites of the Giardia intestinalis group. Parasitology 102(Part 2), 163-166. Kozlowski, T., Ierino, F. L., Lambrigts, D., Foley, A., Andrews, D., Awwad, M., Monroy, R., Cosimi, A. B., Cooper, D. K., and Sachs, D. H. (1998). Depletion of anti-Gal(alpha)l-3Gal antibody in baboons by specific alpha-Gal immunoaffinity columns. Xenotransplantation 5, 122-131. Kulick, D. M., Salerno, C. T., Dalmasso, A. P., Park, S. J., Paz, M. G., Fodor, W. L., and Bolman, R. M. (2000). Transgenic swine lungs expressing human CD59 are protected from injury in a pig-to-human model of xenotransplantation. J. Thorac. Cardiovasc. Surg. 119(4), 690-699. Kumar, V., Geerts, S., and Mortelmans, J. (1978). Localization of larvae of Metastrongylus apri (Gmelin, 1790) Vostokov, 1905, the lungworm of pigs, in the annelid Eisenia foetida (Savigny, 1826). Acta Zool. PathoL Antverp. 70, 201-210. Kweon, C. H., Lee, J. G., Han, M. G., and Kang, Y. B. (1997). Rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction. J. Vet. Med. Sci. 59, 231-232. Kyriakis, S. C., Sarris, K., Kritas, S. K., Tsinas, A. C., and Giannakopoulos, C. (1996). Effect of salinomycin in the control of Clostridium perfringens type C infections in sucklings pigs. Vet. Rec. 138, 281-283. Lapage, G. (1968b). Order Strongyloidea. In "Veterinary Parasitology," 2nd ed., p. 227. Thomas, Springfield, Illinois. Lapage, G. (1968a). Order Phthiraptera. In "Veterinary Parasitology," 2nd ed., pp. 634-663. Thomas, Springfield, Illinois. Leiner, G., Franz, B., Strutzberg, K., and Gedach, G. F. (1999). A novel enzyme-linked immunosorbent assay using the recombinant Actinobacillus pleuropneumoniae ApxlI antigen for diagnosis of pleuropneumonia in pig herds. Clin. Diagn. Lab. Immunol. 6(4), 630-632. Leman, A. D., Straw, B. E., Mengeling, W. L., D'Allaire, S., and Taylor, D. J., eds. (1992). Diseases of Swine. Iowa State Univ. Press, Ames. Levonen, K., Frandsen, P. L., Seppanen, J., and Veijalainen, P. (1996). Detection of toxigenic Pasteurella multocida infections in swine herds by assaying antibodies in sow colostrum. J. Vet. Diagn. Invest. 8(4), 455-459. Levy, M. E, Crippin, J., Sutton, S., Netto, G., McCormack, J., Curiel, T., Goldstein, R. M., Newman, J. T., Gonwa, T. A., Banchereau, J., Diamond, L. E., Byrne, G., Logan, J., and Klintmalm, G. B. (2000). Liver allotransplantation after extracorporeal hepatic support with transgenic (hCD55/hCD59) porcine livers: Clinical results and lack of pig-to-human transmission of the porcine endogenous retrovirus. Transplantation 69, 272-280. Lewis, A. J., and Southern, L. L. (2000). "Swine Nutrition," 2nd ed. CRC Press, Boca Raton, Florida. Lichtensteiger, C. A., Steenbergen, S. M., Lee, R. M., Poison, D. D., and Vimer, E. R. (1996). Direct PCR analysis for toxigenic Pasteurella multocida. J. Clin. Microbiol. 34(12), 3035-3039. Lichtensteiger, C. A., DiPietro, J. A., Paul, A. J., Neumann, E. J., and Thompson, L. (1999). Persistent activity of doramectin and ivermectin against Ascaris suum in experimentally infected pigs. Vet. Parasitol. 82(3), 235-241. Lindsay, D. S., Dubey, J. P., and Blagburn, B. L. (1997). Biology of Isospora spp. from humans, nonhuman primates, and domestic animals. Clin. Microbiol. Rev. 10, 19-34. Little, T. W. A., and Harding, J. D. J. (1971). The comparative pathogenicity of two porcine Haemophilus species. Vet. Rec. 88, 540-545. Loeb, W., and Quimby, E, eds. (1999). "The Clinical Chemistry of Laboratory Animals." 2nd ed. Taylor and Francis, Philadelphia. Logan, N. B., Weatherley, A. J., and Jones, R. M. (1996). Activity of doramectin against nematode and arthropod parasites of swine. Vet. Parasitol. 66(1-2), 87-94. Love, D. N., and Love, R. J. (1979). Pathology of proliferative hemorrhagic enteropathy in pigs. Vet. Pathol. 16, 41-48. Lunney, J. K., Pescovitz, M. D., and Sachs, D. H. (1986). The swine major histocompatibility complex: Its structure and function. Swine Biomed. Res. 3, 1821-1835. Maclnnes, J. I., and Desrosiers, R. (1999). Agents of the "suis-ide diseases" of

670 swine: Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis. Can. J. Vet. Res. 63(2), 83-89. MacMillan, A. P. (1992). Brucellosis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 446-453. Iowa State Univ. Press, Ames. MacMillan, A. P. (1999). Brucellosis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, D. J. Taylor, eds.), 8th ed., pp. 385-393. Iowa State Univ. Press, Ames. Makino, S., Yamamoto, K., Murakami, S., Shirahata, T., Uemura, K., Sawada, T., Wakamoto, H., and Morita, Y. (1998). Properties of repeat domain found in a novel protective antigen, SpaA, of Erysipelothrix rhusiopathiae. Microb. Pathog. 25(2), 101-109. Manwell, R. D. (1968). "Introduction to Protozoology," pp. 434-438. Dover, New York. Mason, R. J., Fleming, P. J., Smythe, L. D., Dohnt, M. E, Norris, M. A., and Symonds, M. L. (1998). Leptospira interrogans antibodies in feral pigs from New South Wales. J. Wildl. Dis. 34(4), 738-743. McOrist, S., and Lawson, G. H. (1989). Reproduction of proliferative enteritis in gnotobiotic pigs. Res. Vet. Sci. 46, 27-33. McOrist, S., Mackie, R. A., and Lawson, G. H. (1995a). Antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy. J. Clin. Microbiol. 33, 1314-1317. McOrist, S., Gebhart, C. J., Boid, R., et al. (1995b). Characterization of Lawsonia intracellularis, the obligate intracellular bacterium of porcine proliferative enteropathy. Int. J. Syst. BacterioL 45, 820-825. McOrist, S., Smith, S. H., Shearn, M. E, et al. (1996). Treatment and prevention of porcine proliferative enteropathy with oral tiamulin. Vet. Rec. 139, 615-618. McOrist, S., Morgan, J., Veenhuizen, M. E, et al. (1997). Oral administration of tylosin phosphate for treatment and prevention of proliferative enteropathy in pigs. Am. J. Vet. Res. 58, 136-139. Mengeling, W. L. (1975). Porcine parvovirus: Frequency of naturally occurring transplacental infection and viral contamination of fetal porcine kidney cultures. J. Am. Vet. Med. Assoc. 36, 41-44. Mengeling, W. L., and Paul, P. S. (1986). The relative importance of swine and contaminate premises as reservoirs of porcine parvovirus. J. Am. Vet. Med. Assoc. 188, 1293-1295. Mengeling, W. L., Lager, K. M., Immerman, J. K., Samarikermani, N., and Beran, G. W. (1991). A current assessment of the relative role of porcine parvovirus as a cause of fetal porcine death. J. Vet. Diagn. Invest. 3, 33-35. Mengeling, W. L., Paul, P. D., and Lager, K. M. (1993). Virus-induced maternal reproductive failure of swine. J. Am. Vet. Med. Assoc. 203, 1268-1272. Messick, J. B., Cooper, S. K., and Huntley, M. (1999). Development and evaluation of a polymerase chain reaction assay using the 16S rRNA gene for detection of Eperythrozoon suis infection. J. Vet. Diagn. Invest. 11(3), 229236. Mielke, D., and Hiepe, T. (1998). The effectiveness of different disinfectants based on p-chloro-m-cresol against Ascaris suum eggs under laboratory conditions. Berl. Munch. Tierarztl. Wochenschr. 111(7-8), 291-294. Moiler, K., Jensen, T. K., Jorsal, S. E., et al. (1998). Detection of Lawsonia intracellularis, Serpulina hyodysenteriae, weakly beta-haemolytic intestinal spirochaetes, Salmonella enterica, and haemolytic Escherichia coli from swine herds with and without diarrhoea among growing pigs. Vet. Microbiol. 62, 59-72. Moon, H. W. (1978). Mechanisms in the pathogenesis of diarrhea: A review. J. Am. Vet. Med. Assoc. 172, 443-448. Moore, G. M., Basson, R. P., and Tonkinson, L. V. (1996). Clinical field trials with tilmicosin phosphate in feed for the control of naturally aquired pneumonia caused by Actinobacillus pleuropneumoniae and Pasteurella multocida in swine. Am. J. Vet. Res. 57(2), 224-228. Morita, T., Fukuda, H., Awakura, T., Shimada, A., Umemura, T., Kazama, S., and Yagihashi, T. (1995). Demonstration of Mycoplasma hyorhinis as a possible primary pathogen for porcine otitis media. Vet. Pathol. 32, 107-111.

KATHY E. LABER E T AL. Morita, T., Sasaki, A., Kaji, N., Shimada, A., Kazama, S., Yagihashi, T., and Umemura, T. (1998). Induction of temporary otitis media in specificpathogen-free pigs by intratympanic inoculation of Mycoplasma hyorhinis. Am. J. Vet. Res. 59(7), 869-873. Morita, T., Ohiwa, S., Shimada, A., Kazama, S., Yagihashi, T., and Umemura, T. (1999). Intranasally inoculated Mycoplasma hyorhinis causes eustachitis in pigs. Vet. Pathol. 36 (2), 174-178. Morsy, G. H., Turek, J. J., and Gaafar, S. M. (1989). Scanning electron microscopy of sarcoptic mange lesions in swine. Vet. Parasitol. 31 (3-4), 281-288. Mueller, Y. M., Davenport, C., and Ildstad, S. T. (1999). Xenotransplantation: Application of disease resistance. Clin. Exp. Pharmacol. Physiol. 26, 1009-1012. Mulder, W. A. M., Pol, J. M. A., Gruys, E., Jacobs, L., and DeLong, M. C. M. (1997). Pseudorabies virus infections in pigs. Role of viral proteins in virulence, pathogenesis and transmission. Vet. Res. 28, 1-17. Munoz, A., Ramis, G., Pallares, E J., Sanchez, A., Chavez, R., Munitiz, V., Yelamos, J., Ramirez, P., and Parrilla, P. (1999). Management and nutrition of newborn piglets from hysterectomy to donation. Transplant. Proc. 31, 2823-2825. Murrell, K. D., Eriksen, L., Nansen, P., Slotved, H. C., and Rasmussen, T. (1997). Ascaris suum--a revision of its early migratory path and implications for human ascariasis. J. Parasitol. 83(2), 255-260. Murtaugh, M. P. (1994). Porcine cytokines. Vet. Immunol. Immunopathol. 43, 37-44. National Research Council (NRC), Subcommittee on Swine Nutrition (1988). "Nutrient Requirements of Swine," 9th rev. ed., pp. 9, 33. National Academy Press, Washington, D.C. National Research Council (NRC), Subcommittee on Swine Nutrition (1998). "Nutrient Requirements of Swine," 10th rev. ed., pp. 51, 54. National Academy Press, Washington, D.C. Nickel, E. A., and Danner, G. (1979). Experimental studies on the course and the effects of pediculosis in domestic swine. Arch. Exp. Veterinarmed. 333(5), 645-649. Nicolet, J. (1992). Haemophilus parasuis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 526-527. Iowa State Univ. Press, Ames. Nielsen, R. (1973). An outbreak of pleuropneumonia among a group of baconers. Nord. Vet. Med. 25, 492-496. Nikolic, B., Gardner, J. P., Scadden, D. T., Arn, J. S., Sachs, D. H., and Sykes, M. (1999). Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC. J. Immunol. 162, 34023407. Nilo, L. (1993). Enterotoxemic Clostridium perfringens. In "Pathogenesis of Bacterial Infections in Animals" (C. L. Gyles and C. O. Thoen, eds.), pp. 114-123. Iowa State Univ. Press, Ames. Ober, B. T., Summerfield, A., Mattlinger, C., Wiesmuller, K. H., Jung, G., Pfaff, E., Saalmuller, A., and Rziha, H. J. (1998). Vaccine-induced, pseudorabies virus-specific, extrathymic CD4(+)CD8(+) memory T-helper cells in swine. J. Virol. 72, 4866-4873. O'Brien, P. J., Shen, H., Weiler, J,. Ianuzzo, C. D., Wittnick, C., Moe, G. W., and Armstrong, P. W. (1991). Cardiac and muscle fatigue due to relative functional overload induced by excessive stimulation, hypersensitive excitation-contraction coupling, or diminished performance capacity correlates with sarcoplasmic reticulum failure. Can. J. Physiol. Pharmacol. 69, 262-268. O'Brien, P. J., Shen, H., Cory, C. R., and Zang, X. (1993). Use of a DNA-based test for the mutation associated with porcine stress syndrome in 10,000 breeding swine. J. Am. Vet. Med. Assoc. 203, 842-851. Oishi, E., Kitajima, T., Koyama Y., Ohgitani, T., Katayama, S., and Okabe, T. (1995). Protective effect of the combined vaccine prepared from cell-free antigen of Actinobacillus pleuropneumoniae serotypes 1, 2, and 5 in pigs. J. Vet. Med. Sci. 57(6), 1125-1128.

15. BIOLOGY AND DISEASES OF SWINE

Olson, L., and Backstrom, L. (1999). Efficacy of tilmicosin (Pulmotil) for control of swine progressive atrophic rhinitis (AR) involving infection with Pasteurella multocida. In "Allen D. Leman Swine Conference" (W. C. Scruton and S. Claas, eds.), Vol. 26, Suppl., p. 15. Vet Outreach Programs, University of Minnesota, Minneapolis. Olson, M. E., Thodakson, C. L., Deselliers, L., Morck, D. W., and McAllister, T. A. (1997). Giardia and cryptosporidium in Canadian farm animals. Vet. Parasitol. 68, 375-381. Panepinto, L. M. (1986). Character and management of miniature swine. In "Swine in Cardiovascular Research" (H. C. Stanton and J. H. Mersmann, eds.), pp. 11-24. CRC Press, Boca Raton, Florida. Panepinto, L. M., Phillips, R. W., Norden, S. W., Pryor, P. C., and Cox, R. (1983). A comfortable minimum stress method of restraint for Yucatan miniature swine. Lab. Anim. Sci. 33, 95-97. Papalois, V. E., Goldberg, L. C., Lee, J., Preston, R. C., Hakim, N. S., Cairns, T., and Taube, D. H. (1999). Donor selection for xenotransplantation: Detection of Gal-alphal-3Gal on different porcine organs. Int. Surg. 84, 258261. Paul, P. S., and Mengeling, W. L. (1980). Effect of vaccinal and passive immunity on experimental infection of pigs with porcine parvovirus. Am. J. Vet. Res. 41, 1376-1379. Paul, P. S., and Stevenson, G. W. (1992). Rotavirus and reovirus. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 331-347. Iowa State Univ. Press, Ames. Pearson, G. R., and McNulty, M. S. (1977). Pathological changes in the small intestine of neonatal pigs infected with a pig reovirus-like agent. J. Comp. Pathol. 87, 363-375. Perez de la Lastra, J. M., Hanna, S. M., and Morgan, B. P. (1999). Distribution of membrane cofactor protein (MCP/CD46) on pig tissues. Relevance to xenotransplantation. Immunology 98, 144-151. Pescovitz, M. D. (1998). Immunology of the pig. In "Handbook of Vertebrate Immunology" (P. P. Pastoret, P. J. Griebel, H. Bazin, and A. Govaerts, eds.), pp. 373-419. Academic Press, New York. Piffer, I. A., and Ross, R. F. (1984). Effect of age on susceptibility of pigs to Mycoplasma hyopneumoniae pneumonia. Am. J. Vet. Res. 45, 478-481. Pijoan, C. (1992). Pneumonic pasteurollosis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 552-559. Iowa State Univ. Press, Ames. Pijoan, C. (1999). Pneumonic pasteurellosis. In "Diseases of Swine" (B. E. Straw, A. D. Leman, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 511-520. Iowa State Univ. Press, Ames. Pijoan, C., and Fuentes, M. (1987). Severe pleuritis associated with certain strains of Pasteurella multocida in swine. J. Am. Vet. Med. Assoc. 91, 823826. Pluske, J. R., Siba, P. M., Pethick, D. W., Durmic, Z., Mullan, B. P., and Hampson, D. J. (1996). The incidence of swine dysentery in pigs can be reduced by feeding diets that limit the amount of fermentable substrate entering the large intestine. J. Nutr. 126, 2920-2933. Poppe, C., Smart, N., Khakhria, R., Johnson, W., Spika, J., and Prescott, J. (1998). Salmonella typhimurium DT104: A virulent and drug-resistant pathogen. Can. Vet. J. 39(9), 559-565. Prideaux, C. T., Lenghaus, C., Krywult, J., and Hodgson, A. L. (1999). Vaccination and protection of pigs against pleuropneumonia with a vaccine strain ofActinobacillus pleuropneumoniae produced by site-specific mutagenesis of the ApxlI operon. Infect. Immun. 67(4), 1962-1966. Rapp-Gabrielson, V. J. (1999). Haemophilus parasuis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 475-481. Iowa State Univ. Press, Ames. Rapp-Gabrielson, V. J., Kocur, G. J., Clark, J. T., and Muir, S. K. (1997). Haemophilus parasuis: Immunity in swine after vaccination. Vet. Med. 92(1), 83-90. Rasmussen, S. R., Aarestrup, E M., Jensen, N. E., and Jorsal, S. E. (1999). Associations of Streptococcus suis serotype 2 ribotype profiles with clini-

671 cal disease and antimicrobial resistance. J. Clin. Microbiol. 37(2), 404408. Reeves, D. E. (1993). Neonatal care of miniature pet pigs. In "Care and Management of Miniature Pet Pigs" (D. E. Reeves, ed.), pp. 41-45. Vet Practice Publ., Santa Barbara, California. Reimer, D., Frey, J., Jansen, R., Veit, H. P., and Inzana, T. J. (1995). Molecular investigation of the role of ApxI and ApxlI in the virulence of Actinobacillus pleuropneumoniae serotype 5. Microb. Pathog. 18(3), 97-209. Rizvi, S., Nicol, C. J., and Green, L. E. (1998). Risk factors for vulva biting in breeding sows in southwest England. Vet. Rec. 143(24), 654-658. Roepstorff, A. (1998). Natural Ascaris suum infection in swine diagnosed by coprological and serological (ELISA) methods. Parasitol. Res. 84(7), 537543. Ross, R. F. (1992). Mycoplasmal diseases. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, D. J. Taylor, eds.), 7th ed., pp. 537-550. Iowa State Univ. Press, Ames. Ross, R. E (1999a). Mycoplasmal diseases. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 501-503. Iowa State Univ. Press, Ames. Ross, R. E (1999b). Mycoplasmal diseases/mycoplasmal pneumonia of swine. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 495-501. Iowa State Univ. Press, Ames. Ross, R. E, Switzer, W. P., and Duncan, J. R. (1971). Experimental production of Mycoplasma hyosynoviae arthritis in swine. J. Am. Vet. Med. Assoc. 32, 1743-1749. Rossow, K. D. (1998). Review article. Porcine reproductive and respiratory syndrome. Vet. Pathol. 35, 1-20. Rossow, K. D., Bautista, E. M., Goyal, S. M., Molitor, T. W., Murtaugh, M. P., Morrison, R. B., Benfield, D. A., and Collins, J. E. (1994). Experimental porcine reproductive and respiratory syndrome virus infection in one, 4, and 10-week old pigs. J. Vet. Diagn. Invest. 6, 3-12. Rossow, K. D., Laube, K. L., Goyal, S. M., and Collins, J. E. (1996a). Fetal microscopic lesions in porcine reproductive and respiratory syndrome virus induced abortion. Vet. PathoL 33, 95-99. Rossow, K. D., Benfield, D. A., Goyal, S. M., Nelson, E. A., Christopher-Hennings, J., and Collins, J. E. (1996b). Chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs. Vet. Pathol. 33, 551-556. Rowland, A. C., and Lawson, G. H. K. (1992). Porcine proliferative enteropathies. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, D. J. Taylor, eds.), pp. 560-569. Iowa State Univ. Press, Ames. Saadi, S., and Platt, J. L. (1999). Role of complement in xenotransplantation. Clin. Exp. Pharmacol. PhysioL 26, 1016-1019. Saalmuller, A., Pauly, T., Lunney, J. K., Boyd, P., Aasted, B., Sachs, D. H., Arn, S., Bianchi, A., Binns, R. M., Licence, S., Whyte, A., Blecha, E, Chen, Z., Chu, R. M., Davis, W. C., Denham, S., Yang, H., Whittall, T., Parkhouse, R. M., Dominguez, J., Ezquerra, A., Alonso, E, Horstick, G., Howard, C., Sopp, P., Kim, Y. B., Lipp, J., MacKay, C., Magyar, A., McCullough, K., Arriens, A., Summerfield, A., Murtaugh, M., Nielsen, J., Novikov, B., Pescovitz, M. D., Schuberth, H. J., Leibold, W., Schutt, C., Shimizu, M., Stokes, C., Haverson, K., Bailey, M., Tlaskalova, H., Trebichavsky, I., Valpotic, I., Walker, J., Lee, R., and Zuckermann, F. (1998). Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens. Vet. Immunol. Immunopathol. 60, 207-228. Sablinski, T., Emery, D. W., Monroy, R., Hawley, R. J., Xu, Y., Gianello, P., Lorf, T., Kozlowski, T., Bailin, M., Cooper, D. K., Cosimi, A. B., and Sachs, D. H. (1999). Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors. Transplantation 67, 972-977. Saeki, H., Fujii, T., Fukumoto, S., Kagota, K., Taneichi, A., Takeda, S., and Tsukaguchi, M. (1997). Efficacy of doramectin against intestinal nematodes

6

7

2

K

A

T

and sarcoptic mange mites in naturally infected swine. J. Vet. Med. Sci. 59(2), 129-132. Saffron, J., and Gonder, J. C. (1997). The SPF pig in research. ILAR J. 38, 2831. Saif, L. J., and Wesley, R. D. (1992). Transmissible gastroenteritis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, D. J. Taylor, eds.), 7th ed., pp. 362-385. Iowa State Univ. Press, Ames. Sakano, T., Shibata, I., Namimatsu, T., Mori, M., Ono, M., Uruno, K., and Osumi, T. (1997a). Effect of attenuated Erysipelothrix rhusiopathiae vaccine in pigs infected with porcine reproductive respiratory syndrome virus. J. Vet. Med. Sci. 59(11), 977-981. Sakano, T., Okada, M., Taneda, A., Mukai, T., and Sato, S. (1997b). Effect of Bordetella bronchiseptica and serotype D Pasteurella multocida bacterintoxoid on the occurrence of atrophic rhinitis after experimental infection with B. bronchiseptica and toxigenic type A P. multocida. J. Vet. Med. Sci. 59(1), 55-57. Saliki, J. T., Rodgers, S. J., and Eskew, G. (1998). Serosurvey of selected viral and bacterial diseases in wild swine from Oklahoma. J. Wildl. Dis. 34(4), 834-838. Sandrin, M. S., and McKenzie, I. E (1999). Recent advances in xenotransplantation. Curr. Opin. Immunol. 11, 527-531. Sanford, S. E., and Higgins, R. (1992). Streptococcal diseases. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 588-598. Iowa State Univ. Press, Ames. Sato, H., Watanabe, T., Murata, Y., Ohtake, A., Nakamura, M., Aizawa, C., Saito, H., and Maehara, N. (1999). New exfoliative toxin produced by a plasmid-carrying strain of Staphylococcus hyicus. Infect. Immun. 67(8), 4014-4018. Sawatsky, J. (1993). Behavior of miniature pet pigs. In "Care and Management of Miniature Pet Pigs" (D. E. Reeves, ed.), pp. 7-12. Vet Practice Publ., Santa Barbara, California. Schmall, L. M., Argenzio, R. A., and Whipp, S. C. (1983). Pathophysiologic features of swine dysentery: Cyclic nucleotide-independent production of diarrhea. Am. J. Vet. Res. 44, 1309-1315. Schmoeckel, M., Bhatti, F. N., Zaidi, A., Cozzi, E., Chavez, G., Wallwork, J., White, D. J., and Friend, E J. (1999). Splenectomy improves survival of HDAF transgenic pig kidneys in primates. Transplant. Proc. 31, 961. Schoenbaum, M. A., Zimmerman, J. J., Beran, G. W., and Murphy, D. E (1990). Survival of pseudorabies virus in aerosol. Am. J. Vet. Res. 51, 331-333. Schwartz, K. J. (1991). Diagnosing and controlling Salmonella choleraesuis in swine. Vet. Med. 86, 1041-1048. Schwartz, K. J. (1999). Salmonellosis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 535-551. Iowa State Univ. Press, Ames. Searcy-Bernal, R., Gardner, I. A., and Hird, D. W. (1994). Effects of and factors associated with umbilical hernias in a swine herd. J. Am. Vet. Med. Assoc. 204, 1660-1664. Seol, B., Naglic, T., and Vrbanac, I. (1998). Isolation of Streptococcus suis capsular type 3 from a young wild boar (Sus scrofa). Vet. Rec. 143(24), 664. Seyfarth, A. M., Wegener, H. C., and Frimodt-Moller, N. (1997). Antimicrobial resistance in Salmonella enterica subsp, enterica serovar typhimurium from humans and production animals. J. Antimicrob. Chemother. 40(1), 67-75. Sherrar, M. G., Easterday, B. C., and Hinshaw, V. S. (1989). Antigenic conservation of HIN2 swine influenza viruses. J. Gen. Virol. 70, 3297-3303. Shimoji, Y., Mori, Y., and Fischetii, V. A. (1999). Immunological characterization of a protective antigen of Erysipelothrix rhusiopathiae: Identification of the region responsible for protective immunity. Infect. Immun. 67(4), 1646-1651. Shryock, T. R., Losonsky, J. M., Smith, W. C., Gatlin, C. L., Francisco, C. J., Kuriashkin, I. V., Clarkson, R. B., and Jordan, W. H. (1998). Computed axial tomography of the porcine nasal cavity and a morphometric comparison of the nasal turbinates with other visualization techniques. Can. J. Vet. Res. 62(4), 287-292.

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E. LABER ET AL.

Sirinarumitr, T., Paul, E S., Halbur, E G., et al. (1997). An overview of immunological and genetic methods for detecting swine coronaviruses, transmissible gastroenteritis virus, and porcine respiratory coronavirus in tissues. Adv. Exp. Med. Biol. 412, 37-46. Skotarczak, B., and Zielinski, R. (1997). A comparison of nucleic acid content in Balantidium coli trophozoites from different isolates. Folia Biol. (Krakow) 45, 121-124. Smith, A. R. (1992). Eperythrozoonosis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 470-474. Iowa State Univ. Press, Ames. Solignac, T., Nicolas, Y., and Fourchon, E (1996). Eperythrozoonosis in swineidentification in French swine herds. Rev. Med. Vet. 147(2), 131. Splitter, E. J. (1950). Eperythrozoon suis: The etiologic agent of icteroanemia or an anaplasmosis-like disease in swine. Am. J. Vet. Res. 11, 324-330. Staats, J. J., Feder, I., Okwumabua, O., and Chengappa, M. M. (1997). Streptococcus suis--past and present. Vet. Res. Commun. 21(6), 381-407. Stanton, H. C., and Mersmann, H. J. (1986). "Swine in Cardiovascular Research." CRC Press, Boca Raton, Florida. Stemke, G. W. (1997). Gene amplification (PCR) to detect and differentiate mycoplasmas in porcine mycoplasmal pneumonia. Lett. Appl. Microbiol. 25(5), 327-330. Stewart, T. B., Fox, M. C., and Wiles, S. E. (1996). Doramectin efficacy against gastrointestinal nematodes in pigs. Vet. Parasitol. 66(1-2), 101-108. Swindle, M. M. (1983). "Basic Surgical Exercises Using Swine." Praeger Press, Philadelphia. Swindle, M. M. (1986). Surgery and anesthesia. In "Swine in Biomedical Research" (M. E. Tumbleson, ed.), pp. 233-433. Plenum Press, New York. Swindle, M. M. (1992). "Swine as Models in Biomedical Research." Iowa State Univ. Press, Ames. Swindle, M. M. (1998a). Defining appropriate health status and management programs for specific-pathogen-free swine for xenotransplantation. Ann. N.Y. Acad. Sci. 862, 111-120. Swindle, M. M. (1998b). "Surgery, Anesthesia, and Experimental Techniques in Swine." Iowa State Univ. Press, Ames. Swindle, M. M., Smith, A. C., Laber-Laird, K., and Dungan, L. (1994). Swine in biomedical research: Management and models. ILAR News 36, 1-5. Swindle, M. M., Thompson, R. E, Smith, A. C., Keech, G. B., Carabello, B. A., Radtke, W., Fyfe, D., and Gillette, E C. (1996). The Yucatan miniature pig model of ventricular septal defect. In "Advances in Swine in Biomedical Research" (M. Tumbleson and L. Schook, eds.), Vol. 2, pp. 613620. Plenum Press, New York. Tacke, S. J., Kurth, R., and Denner, J., (2000). Porcine endogenous retroviruses inhibit human immune cell function: Risk for xenotransplantation? Virology 268, 87-93. Tanabe, T., Sato, H., Sato, H., Watanabe, K., Hirano, M., Hirose, K., Kurokawa, S., Nakano, K., Saito, H., and Maehara, N. (1996). Correlation between occurrence of exudative epidermitis and exfoliative toxin-producing ability of Staphylococcus hyicus. Vet. Microbiol. 48(1-2), 9-17. Taylor, D. J. (1992). Exudative epidermitis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 522-525. Iowa State Univ. Press, Ames. Taylor, D. J. (1999). Actinobacillus pleuropneumoniae. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 343-354. Iowa State Univ. Press, Ames. Taylor, D. J., and Bergeland, M. E. (1992). Clostridial infections. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 454-464. Iowa State Univ. Press, Ames. Thacker, B. J., Joo, H. S., Winkelman, N. L., Leman, A. D., and Barnes, D. M. (1987). Clinical, virologic, and histopathologic observation of induced porcine parvovirus infection in boars. Am. J. Vet. Res. 48, 763-767. Thacker, E. L., Halbur, E G., Ross, R. E, Thanawongnuwech, R., and Thacker, B. J. (1999). Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia. J. Clin. Microbiol. 37(3), 620-627.

15. BIOLOGY AND DISEASES OF SWINE Thome, M., Hirt, W., Pfaff, E., Reddehase, M. J., and Saalmuller, A. (1994). Porcine T-cell receptors: Molecular and biochemical characterization. Vet. Immunol. Immunopathol. 43, 13-18. Thomson, R. G. (1988). "Special Vet Pathology," p. 544. B. C. Decker, Toronto, Canada. Thomson, J. R., Smith, W. J., and Murray, B. P. (1998). Investigations into field cases of porcine colitis with particular reference to infection with Serpulina pilosicoli. Vet. Rec. 142, 235-239. Tibor, M. (1999). Biological characteristics of BordeteUa bronchiseptica. Magy. Allatorvosok Lapja 121(5), 267-274. Torremorell, M., Calsamiglia, M., and Pijoan, C. (1998). Colonization of suckling pigs by Streptococcus suis with particular reference to pathogenic serotype 2 strains. Can. J. Vet. Res. 62(1), 21-26. Tumbleson, M. E., ed. (1986). Swine in Biomedical Research. Vols. 1-3. Plenum Press. New York. Tumbleson, M. E., and Schook, L. B., eds. (1996). Advances in Swine in Biomedical Research, Vols. 1-2. Plenum Press, New York. Turk, J. R., Fales, W. H., Maddox, C., Miller, M., Pace, L., Fischer, J., Kreeger, J., Johnson, G., Turnquist, S., Ramos, J. A., and Gosser, H. S. (1992). Pneumonia associated with Salmonella choleraesuis infection in swine: 99 cases (1987-1990). J. Am. Vet. Med. Assoc. 201, 1615-1616. Tzipori, S., Gibson, R., and Montanaro, J. (1989). Nature and distribution of mucosal lesions associated with enteropathogenic and enterohemorrhagic Escherichia coli in piglets and the role of plasmid-mediated factors. Infect. Immun. 57, 1142-1150. Vahle, J. L., Haynes, J. S., and Andrews, J. J. (1995). Experimental reproduction of Haemophilus parasuis infection in swinemclinical, bacteriologic, and morphologic findings. J. Vet. Diagn. Invest. 7(4), 476-480. Van Alstine, W. G., Stevenson, G. W., and Kanitz, C. L. (1996). Porcine reproductive and respiratory syndrome virus does not exacerbate Mycoplasma hyopneumoniae infection in young pigs. Vet. Microbiol. 49(3-4), 297303. Wabacha, J. K., Gitau, G. K., Nduhiu, J. M., Thaiya, A. G., Mbithi, P. M. F., and Munyua, S. J. M. (1998). An outbreak of urticarial form of swine erysipelas in a medium-scale piggery in Kiambu district, Kenya. J. S. Afr. Vet. Med. Assoc. 69(2), 61-63. Wagner, B., and Polley, L. (1997). Ascaris suum prevalence and intensityman abattoir survey of market hogs in Saskatchewan. Vet. Parasitol. 73(3-4), 309-313. Wallgren, P., and Bornstein, S. (1997). The spread of porcine sarcoptic mange during the fattening period revealed by development of antibodies to Sarcoptes scabiei. Vet. Parasitol. 73(3-4), 315-324. Wallgren, P., and Lindahl, E. (1996). The influence of tail biting on performance of fattening pigs. Acta Vet. Scand. 37(4), 453-460. Warrens, A. N., Simon, A. R., Theodore, P. R., Sachs, D. H., and Sykes, M. (1998). Function of porcine adhesion molecules in a human marrow microenvironment. Transplantation 66, 252-259. Waterworth, P. D., Dunning, J., Tolan, M., Cozzi, E., Langford, G., Chavez, G., White, D., and Wallwork, J. (1998). Life-supporting pig-to-baboon heart xenotransplantation. J. Heart Lung Transplant. 17, 1201-1207. Webb, A. J., Carden, A. E., Smith, C., and Imlah, P. (1992). Porcine stress syndrome in pig breeding. In "Proceedings of 2nd World Congress Genetically Applied Livestock Production," Vol 5., pp. 588-604. Madrid.

673 Wegener, H. C., and Skov-Jensen, E. W. (1999). Exudative epidermitis. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 469-474. Weiss, R. A., Griffiths, D., Takeuchi, Y., Patience, C., and Venables, P. J. (1999). Retroviruses: Ancient and modern. Arch. Virol. Suppl. 15, 171-177. Whary, M. T., Zarkower, A., Confer, F. L., and Ferguson, E G. (1995). Agerelated differences in subset composition and activation responses of intestinal intraepithelial and mesenteric lymph node lymphocytes from neonatal swine. Cell Immunol. 163, 215-221. Wiegand, M., Kielstein, P., Pohle, D., and Rassbach, A. (1997). Examination of primary SPF swine after experimental infection with Haemophilus parasuis. Clinical signs, changes in hematological parameters and in the parameters of the cerebrospinal fluid. Tierarztl. Prax. 25(3), 226-232. Wilcox, B. P., and Schwartz, K. J. (1992). Salmonellosis. In "Disease of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 570-582. Iowa State Univ. Press, Ames. Wisselink, H. J., Reek, E H., Vecht, U., Stockhofe-Zurwieden, N., Smits, M. A., and Smith, H. E. (1999). Detection of virulent strains of Streptococcus suis type 2 and highly virulent strains of Streptococcus suis type 1 in tonsillar specimens of pigs by PCR. Vet. Microbiol. 67(2), 143-157. Wood, R. L. (1984). Swine erysipelasma review of prevalence and research. J. Am. Vet. Med. Assoc. 184, 944-949. Wood, R. L. (1992). Erysipelas. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 7th ed., pp. 475-486. Iowa State Univ. Press, Ames. Wood, R. L. (1999). Erysipelas. In "Diseases of Swine" (A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor, eds.), 8th ed., pp. 419-430. Iowa State Univ. Press, Ames. Yamazaki, Y., Sato, H., Sakakura, H., Shigeto, K., Nakano, K., Saito, H., and Maehara, N. (1999). Protective activity of the purified protein antigen of Erysipelothrix rhusiopathiae in pigs. Zentralbl. Veterinarmed., Reihe 46(1), 47-55. Yazwinski, T. A., Tucker, C., Featherston, H., Johnson, Z., and Woodhuels, N. (1997). Endectocidal efficacies of doramectin in naturally parasitized pigs. Vet. Parasitol. 70(1-3), 123-128. Ye, Y., Niekrasz, M., Kosanke, S., Welsh, R., Jordan, H. E., Fox, J. C., Edwards, W. C., Maxwell, C., and Cooper, D. K. (1994). The pig as a potential organ donor for man. A study of potentially transferable disease from donor pig to recipient man. Transplantation 57, 694-703. Yeatman, M., Daggett, C. W., Lau, C. L., Byrne, G. W., Logan, J. S., Platt, J. L., and Davis, R. D. (1999). Human complement regulatory proteins protect swine lungs from xenogeneic injury. Ann. Thorac. Surg. 67, 769-775. Zimmerman, J. J., Berry, W. J., Beran, G. W., and Murphy, D. P. (1989). Influence of temperatures and age on the recovery of pseudorabies virus from house flies. Am. J. Vet. Res. 50, 1471-1474. Zimmerman, J., Yoon, K. J., Wills, R., McGinley, M., and Sanderson, T. (1995). PRRS virus in avian species. Int. Symp. PRRS. 2, 29. Zimmermann, W., and Kircher, P. (1998). Continuous serologic study and sanitation inspection of Sarcoptes scabiei var. suis infection: Preliminary resuits. Schweiz. Arch. Tierheilkd. 140(12), 513-517.

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Chapter 16 Nonhuman Primates Bruce J. Bernacky, Susan V. Gibson, Michale E. Keeling, and Christian R. Abee

I. II.

III.

Introduction .................................................

676

Taxonomy

677

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A.

C h a r a c t e r i s t i c s C o m m o n to All P r i m a t e s

B.

Geographic Distribution

C.

Nomenclature ............................................

678

D.

T e r m s U s e d in Identification o f P r i m a t e s

678

E.

D i s t i n g u i s h i n g Features o f P r i m a t e Infraorders

Biology A.

......................

................................... ...................... .................

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Callitrichidae: M a r m o s e t s and T a m a r i n s . . . . . . . . . . . . . . . . . . . . . . .

B. Aotus spp.: O w l M o n k e y s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Saimiri spp.: Squirrel M o n k e y s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Macaca mulatta: R h e s u s M o n k e y s . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Macacafascicularis: C y n o m o l g u s M o n k e y s . . . . . . . . . . . . . . . . . . . E Papio spp.: B a b o o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Pan spp.: C h i m p a n z e e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV.

V.

VI.

Principles of Colony Man ag emen t

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679 680 680 686 692 698 703 706 711 715

A.

Housing

B.

Enrichment Programs

C.

Restraint Techniques

D.

Biosafety

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720

E.

Sanitation . . . . . . . . .......................................

721

F.

Environmental Controls ....................................

723

G.

Records

723

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717 718

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Medical Management

715

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724

A,

Preventive Medicine

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724

B.

Clinical T e c h n i q u e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

727

Diseases ....................................................

730

A.

Bacterial D i s e a s e s

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730

B.

Mycotic Diseases .........................................

744

C.

Viral D i s e a s e s

746

D.

Parasitic D i s e a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

757

E.

Nutritional Diseases .......................................

757

F.

Miscellaneous Disorders

775

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...................................

References ..................................................

LABORATORY ANIMAL MEDICINE, 2nd edition

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777

Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

676

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

I.

INTRODUCTION

Nonhuman primates have a unique role in biomedical research because of their close phylogenetic relationship to human beings. Their susceptibility to human infectious agents, similarities in physiological responses, developmental biology, and response to experimentally induced diseases are critically important to the advancement of biomedicine. Nonhuman primates are also among the most scarce, costly, and sentient of animal models used in research. During the past 30 years there has been a significant increase in our knowledge of the biology and care of these valuable animals. This chapter is intended to provide veterinarians, colony managers, and research scientists with an overview of the natural history, biology, clinical management, husbandry, and diseases of the seven most commonly used primate genera. There are a number of nonhuman primate medical management, biology, and husbandry publications that should be examined when specific needs exceed the limits of this chapter (Keeling, 1975; Richter et al., 1984; Bennett et al., 1998). The chapter on primates in "Laboratory Animal Medicine" (1984) remains a valuable general reference to be consulted in conjunction with this chapter. More current American College of Laboratory Animal Medicine (ACLAM) series publications include the volumes titled "Nonhuman Primates in Biomedical Research: Biology and Management" (1995)and "Nonhuman Primates in Biomedical Research: Diseases" (1998). These publications together provide an excellent information resource on the veterinary care, husbandry, and management of nonhuman primates used in research. Although there are approximately 200 species in the order Primates, this chapter emphasizes species within the seven genera most commonly used in research. Among the Old World primates, rhesus monkeys (Macaca mulatta), cynomolgus monkeys (M. fascicularis) and baboons (Papio spp.); and among the New World primates, squirrel monkeys (Saimiri spp.), owl monkeys (Aotus spp.), marmosets (Callithrix spp.), and tamarins (Saguinus spp.) are discussed in detail. One great ape, the common chimpanzee (Pan troglodytes), is also covered. Although direct correlation cannot always be made, much of the information presented for these nonhuman primates may be applicable to other species that are closely related taxonomically. During the last quarter century, the number of nonhuman primates imported into the United States has declined significantly from 126,857 in 1968 (Johnsen, 1995) to a domestic supply and imports total of 22,591 in 1984 (Wolfle, 1983). From June 1990 to September 1991 only 12,245 Old World monkeys, including cynomolgus, rhesus, and African green monkeys, were imported into the United States (Centers for Disease Control and Prevention [CDCP], 1991). Numbers of New World monkey species imported during this period were not provided. Factors contributing to this decline include bans on importation of non-

human primates as pets in the United States, placement of many nonhuman primates on the Endangered Species list, bans on exportation from countries of origin and the accompanying conservation movement, and the efforts of animal activist groups to sway public opinion against the use of nonhuman primates in research. More contemporary factors impacting nonhuman primate production and use include efforts by the research community to more efficiently use nonhuman primate resources. There has been a heightened sensitivity and more intense review of nonhuman primate studies by Institutional Animal Care and Use Committees. The Washington Regional Primate Research Center publication "Primate Supply Clearinghouse" has made significant contributions to conservation by recycling animals to studies that would not be confounded by their prior use. Reliable statistics are difficult to obtain, but most data indicate a current average use of 50,000-60,000 nonhuman primates for the past several years (Hackerman, 1988; U.S. Department of Agriculture [USDA], 1998). This is not a reflection of numbers imported that can be compared with figures from prior years. Usage numbers today are based primarily upon importation from well-managed, limited-production colonies within the countries of origin and domestic production colonies in the United States. The domestic breeding programs initiated in the 1970s have become the major supply source of rhesus monkeys, baboons, chimpanzees, and squirrel monkeys. The United States has been dependent on domestic production of chimpanzees since it became signatory to the Convention on International Trade In Endangered Species of Wild Fauna and Flora (CITES) Treaty in 1975. Chimpanzee production peaked in the late 1980s and early 1990s. The National Center for Research Resources chimpanzee breeding program recorded 381 births between 1986 and 1994 (National Institutes of Health [NIH], 1994). Currently there is a 5-year moratorium on chimpanzee production following a National Research Council (NRC) recommendation (Institute for Laboratory Animal Research [ILAR], 1997). Specific pathogen-free (SPF) production colonies are rapidly being developed (Buchl et al., 1997) and, based on current demands, will become the standard resource for rhesus monkeys. Despite significantly increasing the cost of research using nonhuman primates, the engines driving these new trends in domestic production of more well defined nonhuman primates are personnel safety and the need for better-defined animals in sophisticated, molecular-based biomedical research. Such research demands high-quality nonhuman primate models. In spite of efforts by groups opposed to the use of nonhuman primates in research, the contributions to date and future potential for improving human health and welfare provide compelling justification for continued use of biologically defined and genetically characterized nonhuman primate models. Their close phylogenetic relationship to human beings, their similarity in susceptibility to diseases, and their subsequent similarity in immune responses place nonhuman primates in a unique po-

16. NONHUMANPRIMATES

677

sition among currently available animal models. Public concern over the use of nonhuman primates in biomedical research, combined with their high maintenance costs and relative scarcity, mandates high standards of care and judicious use of these species.

II.

TAXONOMY

The taxonomic classification of nonhuman primates continues to change as new species are discovered. Twenty-two new species of New World monkeys alone have been identified since 1990 (Rylands et al., 2000). As new species are identified and genetic and evolutionary relationships are redefined, new taxonomic classification schemes are proposed and debated. Tarsiers, a species that shares char~icteristics of both prosimians and anthropoid primates, is a specific example of change and discussion in taxonomic classification. As described by Napier and Napier (1967), tarsiers belonged to the suborder Prosimii, infraorder Tarsiiformes, and family Tarsiidae. In the first edition of "Laboratory Animal Medicine" (Richter et al., 1984), tarsiers were classified as a separate superfamily Tarsioidea, family Tarsiidae within the suborder Prosimii. In 1985, Napier and Napier removed tarsiers from the suborder Prosimii and created a separate suborder Tarsioidea, which contained only tarsiers (Napier and Napier, 1985). This classification put tarsiers on equal footing with prosimians and anthropoids and set them apart as an intermediate group between the two. This classification scheme was used by Whitney (1995) in "Nonhuman Primates in Biomedical Research: Biology and Management." To further complicate matters, there are taxonomists that divide the order Primates into two distinct classes: Strepsirhini, which includes only prosimians; and Haplorhini, which includes tarsiers and anthropoid primates. Strepsirhini and Haplorhini are synonymous with the traditional suborders Prosimii and Anthropoidea, with the exception that tarsiers are classified as haplorhines (Fleagle, 1999). Whether tarsiers should be classified as a separate suborder from other prosimians continues to be debated. For the purposes of this chapter, the authors have elected to use the classification of Fleagle (1999), which includes tarsiers within the suborder Prosimii but separates them from other prosimians into the infraorder Tarsiiformes, a return to the classification scheme of Napier and Napier (1967). As Fleagle (1999) eloquently points out, "In the science of classifying organisms, systematics, we attempt to apply the tidy Linnean system to the untidy, unlabeled world of animals..." The classification used in this book is the result of one such attempt. Precise identification of primates used in research is an essential part of the care and management of nonhuman primates.

Because some laboratory species were obtained from the wild or are the offspring of animals obtained from importers many years ago, precise identification may not be documented or may not be correct. Thus, laboratory personnel should have a basic working knowledge of primate taxonomy. Identification is important for experimental reasons since some species and subspecies within the same genus respond differently to experimental use. Mixing species or subspecies within experimental groups could generate inaccurate data because of confounding genetic differences. Environmental and nutritional requirements also vary among primate species; therefore, identification may be important for optimizing the husbandry, management, and veterinary care programs. Finally, some species are listed as endangered by the CITES treaty. Because the United States and most other countries are CITES signatories, all nonhuman primates must be precisely identified. There are strict regulations on export, import, movement, and use of some species as designated in the CITES appendices. The taxonomic classification of Wilson and Reeder (1993), as used by CITES and international regulatory authorities and in concordance with Fleagle (1999), is used for classification from family to genus and species level in the tables in this chapter.

A.

Characteristics Common to All Primates

The defining characteristics of taxa within the order Primates were first published by St. George Mivart in 1873. Mivart (1873) described a primate as "an unguiculate, claviculate, placental mammal with orbits encircled by bone; three kinds of teeth at least at one time of life; brain always with a posterior lobe and a calcarine fissure; the innermost digits of at least one pair of extremities opposable; hallux with a fiat nail or none; a well-marked caecum; penis pendulous; testes scrotal; always two pectoral mammae." Nonhuman primates constitute the most diverse taxonomic group of those presented within the chapters of this text. They range in size from pygmy marmosets weighing less than 200 gm to gorillas weighing more than 160 kg, and they are indigenous to all continents except Australia and Antarctica.

B.

Geographic Distribution

With few exceptions, nonhuman primate species are found in the tropics, ranging from 25 ~ north latitude to 30 ~ south latitude. Habitats range from tropical rain forests to semiarid savannas to desert steppes. Primates are indigenous to regions within Africa, Asia, South America, Central America, and extreme southern Europe. Although most species are found primarily in tropical rain forest and savanna habitats, two species, Macaca mulatta and M. fuscata, range as far north as Beijing, China, and the island of Honshu in Japan (approximately 41 ~

678

BRUCE J. BERNACKY, SUSANV. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

north latitude) (Napier and Napier, 1967). A number of species and subspecies are now classified by CITES as either threatened or endangered, primarily due to habitat encroachment from destructive agricultural practices, real estate development, and poaching. The most highly specialized species are most at risk because they have the most strict habitat requirements.

C.

Nomenclature

The classification of extant taxa of nonhuman primates continues to change as more information is accumulated, making classification increasingly complex as new species and subspecies are described. Members of some genera continue to be separated into new species or genera based on information obtained from tools such as those used by molecular geneticists. The classification of nonhuman primates from order to superfamilies is summarized in Table I. Prosimian, platyrrhine, and catarrhine primates are classified to the genus level in Table II. The classification of. genera most commonly used in biomedical research is found in Section III, Biology.

D.

Terms Used in Identification of Primates

The following terms are frequently used by primatologists to describe primate groups. Old World monkey and New World monkey are used to identify genera found in Africa and Asia or South and Central America, respectively. The term neotropical primate is considered to be interchangeable with New World monkey to describe primates indigenous to the Americas. Prosimian is used to describe all taxa within the suborder Prosimii, such as lemurs and tarsiers. The word prosimian, from the Latin root word meaning "before monkeys," refers to their relatively more primitive phylogenetic position within the order. Prosimian primates are not considered to be "monkeys." Simian is used as an adjective or noun to describe monkeys and apes. Tarsier is used to describe primates of the genus Tarsius that share characteristics of prosimians and simians. Like prosimi-

ans, tarsiers are nocturnal, have large eyes and mobile ears, have "toilet claws" on the foot, and have a two-part mandible. Unlike prosimians, tarsiers lack a naked rhinarium and dental comb. Like anthropoids (simians), tarsiers have upright lower incisors and a dry, furry nose (Napier and Napier, 1985). Monkey is the common name that describes all species of nonhuman primates except prosimians and apes. Monkeys are distinguished from apes by the presence of an external tail. Macaque is the common name for primates belonging to the genus Macaca. This genus includes rhesus monkeys (M. mulatta) and cynomolgus monkeys (M. fascicularis), two of the most commonly used species in biomedical research. Baboon refers to primates belonging to the genus Papio. Great ape is a term used to identify the apes within the family Hominidae. The great apes include chimpanzees, bonobos, gorillas, and orangutans. Great apes are distinguished from monkeys by a number of anatomic features, including lack of tail. Great apes are capable of bipedalism, although quadrupedal locomotion is common. Lesser ape is the term used to identify members of the family Hylobatidae. Lesser apes include those species referred to as gibbons and siamangs. Lesser apes are smaller than great apes and are almost entirely arboreal, whereas great apes such as chimpanzees and gorillas spend a large part of their time on the ground. Lesser apes are true brachiators, using their arms to swing from branch to branch as their primary means of locomotion. Lesser apes also lack an external tail. Callitrichid is used as an adjective or noun to describe species in the family Callitrichidae, which includes marmosets and tamarins. Marmoset is the common name used to identify New World primates belonging to the genus Callithrix within the family Callitrichidae. Tamarin is the common name used to identify New World primates belonging to the genera Saguinus and Leontopithecus within the family Callitrichidae. A prehensile tail is found in some genera of neotropical primates. The prehensile tail has a tactile pad similar to that found on the tactile surface of fingers and palms of hands; it is used as an additional appendage for clinging and hanging from tree limbs. The primate can wrap and constrict its tail in a manner

Table I ClassificatiOn of NonhumanPrimates

a

Order

Primates

Suborder

Prosimii

Infraorder Superfamily

I

Lemuriformes

I

Lorisoidea

Anthropoidea Tarsiiformes

I

Lemuroidea

I

Tarsioidea

I

I

Platyrrhini

I

Ceboidea

Catarrhini

I

,

aFrom Fleagle (1999).

I

Cercopithecoidea

Hominoidea i

679

16. NONHUMAN PRIMATES

Table II Classification of Prosimian, Platyrrhine, and Catarrhine Primates a Prosimian Primates

Superfamily

Lorisoidea

I

Lemuroidea

I

Family

Loridae

Genera

Arctocebus Loris Nycticebus Perodicticus

I

Galagonidae

I

Cheirogaleidae

I

I

Euoticus Galago Galagoides Otolemur

I

Megaladapidae

Lemuridae

I

Allocebus Cheirogaleus Microcebus Phaner

Tarsioidea

I

I

Lepilemur

Eulemur Hapalemur Lemur Varecia

I

I

Indriidae

Daubentoniidae

I

I

Avahi Daubentonia Indri Propithecus

I

Tarsiidae

I

Tarsius

Platyrrhine Primates

Superfamily Family

Ceboidea

I

I

Callitrichidae

I Subfamily

Callitrichinae

Genera

Callimico Callithrix Leontopithecus Saguinus

I

Cebidae I

I

Aotinae

I

I

Callicebinae

Cebinae

I

Aotus

I

Callicebus

I

Pitheciinae

I Cacajao

Cebus Saimiri

I

I

Alouattinae

Atelinae

I Alouatta

Chiropotes Pithecia

I Ateles Brachyteles Lagothrix

Catarrhine Primates

Superfamily

Cercopithecoidea

Hominoidea

I

Family

I

Cercopithecidae

I

Subfamily

Cercopithecinae

Genera

Allenopithecus Cercocebus Cercopithecus Chlorocebus Erythrocebus Lophocebus Macaca Mandrillus Miopithecus Papio Theropithecus

I

I

Hylobatidae

Hominidae

Hylobates

Gorilla Pan Pongo

I

Colobinae

I Colobus Nasalis Presbytis Procolobus Pygathrix Semnopithecus Trachypithecus

aFrom Fleagle (1999).

resembling that of an elephant's trunk. Prehensile tails are not found in any Old World monkey taxa. A pseudoprehensile tail is found in some genera of neotropical primates. The term pseudoprehensile tail refers to the ability of the animal to grasp and cling with the tail; however, the tail does not possess a tactile pad. Cheek pouches are specialized pouches found in genera within the family Cercopithecidae. These specialized structures are extensions of the cheeks that extend below each ramus of the mandible. Cheek pouches allow the animal to quickly store food for eating at a later time. Ischial callosities are specialized pads that cover the surface of the ischium and facilitate sitting. Ischial callosities are found

in Old World monkeys and lesser apes. These structures are not found in New World monkeys.

E.

Distinguishing

Features of Primate Infraorders

Nonhuman primates have been classified in part by phenotypic features such as pelage color, pattern and distribution, anatomic characteristics, and geographic distribution. The principal descriptor used to differentiate the infraorders Platyrrhini and Catarrhini is the spacing and orientation of the nares. The term catarrhine, meaning "narrow, turned-down nose," has been used extensively in the literature to describe Old World

680

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

m o n k e y s a n d apes. T h e t e r m platyrrhine, m e a n i n g "flat n o s e , " refers to the flattened m u z z l e w i t h b r o a d l y spaced, laterally

9. Dental formulas: Cebidae: 4 incisors + 2 canines + 6 premolars + 6 molars x 2 = 36 Callitrichidae: 4 incisors + 2 canines + 6 premolars + 4 molars X 2 = 32 (except genus Callicebus with 36 as in Cebidae). 10. All have hemochorial placentas.

flared nares f o u n d in N e w W o r l d m o n k e y s . A l t h o u g h these t e r m s t r a n s l a t e d f r o m the L a t i n r o o t w o r d s literally r e f e r to the nose, they actually d e s c r i b e the p o s i t i o n i n g o f the nares or o p e n ings o f the nostrils. N o n h u m a n p r i m a t e s do not p o s s e s s a p r o m i n e n t bony, c a r t i l a g i n o u s n o s e as do h u m a n beings. T h e f o l l o w ing c h a r a c t e r i s t i c s m a y be u s e d to h e l p differentiate p r o s i m i a n , p l a t y r r h i n e , and c a t a r r h i n e p r i m a t e s .

1. Distinguishing Features of Prosimians 1. Indigenous to Africa, India, and Southeast Asia. 2. Possess a naked, moist snout called a rhinarium with a fissured, fixed upper lip resembling the rhinarium of dogs and cats (Fig. 1). 3. All are nocturnal except the genera Lemur, Varecia, Hapalemur, Indri, and Propithecus. 4. Possess a "toilet claw" on the second digit of the foot for grooming. 5. Possess a toothcomb for grooming, which is formed from the lower incisors. 6. Possess a sublingual structure for cleaning the toothcomb. 7. The mandible is in two parts joined at the midline by cartilage. 8. Dental formula: 4 incisors + 2 canines + 6 premolars + 6 molars x 2 = 36 (except Indridae with 30). 9. All have epitheliochorial placentas except Tarsiidae, which has hemochorial.

2.

Distinguishing Features of Platyrrhine Primates 1. 2. 3. 4. 5. 6. 7. 8.

Indigenous to tropical South and Central America. Muzzle is flattened with broadly spaced, laterally flared nares (Fig. 1). Some species possess prehensile or pseudoprehensile tails. Do not possess cheek pouches or ischial callosities. Require vitamin D 3 in their diet; ingested vitamin D2 is not bioavailable. All have estrous cycles, except Cebus spp., which menstruate. All are arboreal. All are diurnal except Aotus spp. (the only nocturnal simian primate).

3.

Distinguishing Features of Catarrhine Primates 1. Indigenous to Africa, Asia, and extreme southern Europe (introduced to Gibraltar). 2. Muzzle is elongate (varying in degree among genera) with narrowly spaced, turned-down nares (Fig. 1). 3. Some species possess ischial callosities for sitting. 4. Cheek pouches for storing food are found in some genera of Cercopithecidae. 5. All have menstrual cycles. 6. Some species are adapted to terrestrial living while others are primarily arboreal. 7. All can utilize vitamin D2 in their diet. 8. Dental formula: 4 incisors + 2 canines + 4 premolars + 6 molars X 2 = 32 9. All are diurnal. 10. All have hemochorial placentas.

III.

A.

BIOLOGY

Callitrichidae: Marmosets and Tamarins

1. Introduction The neotropical primates, marmosets and tamarins, constitute the m o s t p r i m i t i v e s i m i a n p r i m a t e species. T h e y are disting u i s h e d f r o m other n o n h u m a n p r i m a t e s b y their s m a l l size, w h i c h r a n g e s f r o m 150 to 6 0 0 g m ; their dental f o r m u l a ; c l a w s

Fig. 1. Distinguishing features of prosimians, New World monkeys, and Old World monkeys. Pictured from left to right are examples of a prosimian (Lemur catta), a New World monkey (Saimiri spp.), and an Old World monkey (Macaca mulatta). Note the rhinarium with a fissured, fixed upper lip in the lemur. The squirrel monkey has broadly spaced, laterally flared nares. The rhesus monkey has an elongate muzzle with narrowly spaced, turned-down nares.

681

16. NONHUMAN PRIMATES

or falcula instead of nails on the digits; and a high frequency of twinning, approximately 80%. They have a specialized nail on the first digit of each foot, the hallux, which is opposable, while the thumb is not. Callitrichids are scent markers and have two distinct marking glands. Circumgenital glands are welldeveloped sebaceous glands overlying enlarged apocrine glands that cover the labia majora and pudendum in the female and the scrotum in the male. Sternal glands located on the anterior chest are less distinct and may be focal or diffuse in structure. Callitrichids are unique in that usually only one adult female in an extended breeding group reproduces; subordinate females, usually offspring of the breeding female, may or may not have an estrous cycle. All callitrichid twins and many singletons appear to be permanent chimeras. In normal twinning, there are placental vascular anastomoses and continuous placental hematopoiesis (Jollie et al., 1975), resulting in stem cell crossover between developing fetuses. 2.

Taxonomy

The Callitrichidae contain one genus of marmosets, Callithrix (Fig. 2), and two genera of tamarins, Saguinus and Leontopithecus (Fig. 3). A list of the species is found in Tables III and IV. Leontopithecus spp. are not listed as they are not used in biomedical research. In general, marmosets are smaller than tamarins. Marmosets have procumbent incisor teeth that are the same length as the canine teeth. This dental arrangement enables them to gnaw holes in trees and eat gums and exudates, a staple of their diet. Only marmosets and tamarins in the genera

Fig. 3. Saguinusoedipus, cotton-toptamarin adult with infant. (Photograph from Suzette Tardiff. Courtesy of the Marmoset Research Center, Oak Ridge Associated Universities.)

Callithrix and Saguinus will be discussed further, as these species are most commonly used in research. 3.

Fig. 2. Callithrix jacchus, the common marmoset. (Photograph from Donna Layne. Courtesy of the Department of Biological Sciences, Kent State University.)

Natural History

Marmosets and tamarins prefer different habitats although their ranges may overlap. Marmosets are found throughout most of Brazil, primarily in savanna/forest habitats. The common marmoset (Callithrix jacchus) prefers secondary or disturbed forests or edge habitats. Tamarins of the genus Saguinus are found throughout much of the lowland neotropical rain forest from Panama to Bolivia to northeastern Brazil and prefer primary or secondary forest. Size of territory varies considerably with species and within species: from 1 hectare for common marmosets (Stevenson, 1977) to 3 0 - 5 0 hectares for Saguinus nigricollis (Izawa, 1978). Daily travel for a marmoset group is about 500-1000 meters. Availability of food supply is

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

682

Table III

C. flaviceps

Buffy-headed marmoset

I

Brazil

C. geoffroyi

Geoffroy's tufted-ear marmoset White-fronted marmoset

II

Brazil

lated and related adults and offspring (Garber, 1993). All-male groups and solitary males have been observed. The mating system is usually polyandry; multiple males copulate with the reproductive female. Polygyny and m o n o g a m y also occur (Garber, 1993; Kinzey, 1997). Group members sleep in huddles together in one of a n u m b e r of familiar nesting trees within their territory. Callitrichids are diurnal; group activity usually begins 1-1.5 hr after sunrise. Approximately one-fifth of the day will be spent traveling and up to one-third of the day foraging. Marmosets and tamarins are omnivorous, feeding on insects, fruits, nectars, tree exudates (gum and sap), and whatever small animals they can capture. Marmosets spend considerable time consuming tree exudates and gums either by gnawing the bark

C. humeralifer

Tassel-eared marmoset

II

Brazil

Tamarin Taxonomy, CITES Status, and Distribution

C. jacchus

Common marmoset

II

C. kuhlii

Syn. C. jacchus kuhlii Manicor6marmoset

II ?

Brazil Brazil Brazil

C. penicillata

Black-pencilled marmoset

II

Brazil

S. bicolor

Pied tamarin Bare-faced tamarin

I

Brazil, Peru (?)

C. pygmaea

Pygmy marmoset

Bolivia, Brazil, Colombia, Ecuador, Peru

S. fuscicollis

Rio Napo tamarin Brown-headed tamarin Saddle-back tamarin

II

Bolivia,Brazil, Colombia, Ecuador, Peru

S. geoffroyi

Rufous-naped tamarin Cotton-top marmoset Pinche marmoset Liszt monkey

I

Colombia, Costa Rica, Panama

S. imperator

Emperor tamarin

II

S. inustus

Mottle-faced tamarin Dusky tamarin

II

Bolivia,Brazil, Peru Brazil, Colombia

S. labiatus

Red-chested tamarin Red-bellied tamarin White-lipped tamarin White-footed tamarin Negro tamarin Red-handed tamarin

II

Marmoset Taxonomy, CITES Status, and Distribution Genus Callithrix

Common name(s)

CITES status

a

Distribution

C. acariensis b

Rio Acari marmoset

?

Brazil

C. argentata

Silvery marmoset Black-tailed marmoset

II

Bolivia, Brazil, Paraguay

C. aurita

Buffy-tufted-ear marmoset White-eared marmoset

I

Brazil

C. manicorensis b

Table IV

I, II

a I, Species listed in CITES Appendix I are threatened with extinction (endangered); II, Species listed in CITES Appendix II are not currently threatened with extinction but may become so unless trade is strictly regulated. From CITES (2000) and Wilson and Reeder (1993). bFrom Van Roosmalen et al. (2000).

probably the single most important determinant for group territory size. Callitrichids are territorial and may patrol and defend their territory vigorously from other groups (Dawson, 1977). Group size ranges from 2 to 15, with the mean group size for most species falling in the range of 4 to 7 individuals. Larger groups of 4 0 + are rarely seen and are assumed to be temporary associations of several family groups (Izawa, 1978). Variation in group size is also a reflection of emigration and immigration of transients, usually subadults (Dawson, 1977). Group composition is dependent on the species, and several compositions are possible. Initially, m a r m o s e t and tamarin groups were thought to have a m o n o g a m o u s breeding pair and offspring of different ages. It is now known that social structure and organization are more complex in these species. In C. j a c c h u s , group composition may be m u l t i m a l e - m u l t i f e m a l e , one m a l e - m u l t i f e m a l e , or one f e m a l e - m u l t i m a l e . Usually, c o m m o n m a r m o s e t groups contain only one breeding pair, which are the highest-ranking animals within the group. The group may contain one to two immigrants and several sets of offspring. Tamarins have a multim a l e - m u l t i f e m a l e social structure, and groups consist of unre-

Genus Saguinus

S. leucopus S. midas

CITES status a

Common name(s)

I Ii

Distribution

Bolivia, Brazil, Colombia, Peru Colombia Brazil, French Guiana, Guyana, Suriname Brazil, Peru Brazil, Colombia, Ecuador, Peru

S. mystax

Mustached tamarin

II

S. nigricollis

Black-and-redtamarin

II

S. oedipus

Cotton-headed tamarin Geoffrey's tamarin Cotton-top tamarin Rufous-naped tamarin

I

Colombia, Panama

S. tripartitus

Golden-mantled saddleback tamarin

II

Ecuador, Peru

a I, Species listed in CITES Appendix I are threatened with extinction (endangered); II, Species listed in CITES Appendix II are not currently threatened with extinction but may become so unless trade is strictly regulated. From CITES (2000) and Wilson and Reeder (1993).

16. NONHUMANPRIMATES or by consuming exudates released by other trauma to the tree (Coimbra-Filho and Mittermeier, 1977). Tamarins eat more fruits but also consume tree exudates released by trauma to the bark (Garber, 1980); they lack the dentition to gnaw on trees to release sap. In S. fuscicollis, the dominant female may control access to an exudate source on a damaged tree (Garber, 1993). Exudates are rich in sugars and minerals, especially calcium. Consumption of plant exudates may vary with seasonal availability of other foodstuffs. 4.

Reproduction

Callitrichids are unusual in that the onset of puberty in females can be influenced by the type of social setting. Puberty or sexual maturation begins in female tamarins living in their family group at 15-17 months of age (Ziegler et al., 1987). However, females removed from the family group at 9 months of age and paired with an unrelated male begin cycling at 10-11 months of age. Young adult female tamarins, 2 0 - 2 8 months of age, have suppressed fertility while living in their family groups, as indicated by low acyclic levels of luteinizing hormone and estradiol. Cyclicity occurs rapidly when females are removed from the family and paired with an unrelated male, with ovulation occurring as early as 8 days after pairing (Ziegler et al., 1987). Hearn (1977) found that subordinate female C. j a c c h u s in peer groups will not cycle until removed from the group. Suppression of cyclicity is caused by scent marking by the dominant adult female. Usually only the dominant female in peer groups will reproduce (Epple, 1970b). In some instances, normal ovarian cycling has been detected in daughters in wild cotton-top tamarin (S. oedipus) family groups, resulting in pregnancy (Savage et al., 1997). Male C. j a c c h u s do not reach adult levels of plasma testosterone until approximately 18 months of age (Hearn and Lunn, 1975), even though they are probably capable of impregnating females prior to this age (Epple and Katz, 1980). The ovarian cycle of the common marmoset has been reported to average 28.6 ___ 1.0 days (Hearn, 1983; Kholkute, 1984) to 30.1 __+ 3.8 days (Harding et al., 1982). The follicular phase of the cycle lasts about 8.5 days and the luteal phase, 1 9 21 days. There is no detectable change in vaginal cytology. The estrous cycle can be followed by changes in peripheral plasma hormone levels or hormone levels in urine or feces (Hodgen et al., 1976; Hodges et al., 1979). A marked rise in plasma progesterone within 1 day postovulation is a useful indicator of ovulation. In the common marmoset, there is no lactational anestrus, and Hearn and Lunn (1975) have reported that estrus occurs as early as 3 days postpartum. Ovulation occurs 10-18 days postpartum, and sexual encounters increase (Dixson and Lunn, 1987). The estrous cycle is shorter in tamarins. In S. fuscicollis, the ovarian cycle lasts 17.3 days and in S. oedipus, 15.2 days. There is no postpartum estrus in tamarins. Gestation ranges from 140 to 150 days for the species used in

683

research. Pregnancy can be diagnosed as early as 2 weeks by measurement of plasma or urine placental chorionic gonadotropin (Hodges et al., 1979). Diagnostic ultrasound has been used to follow pregnancy, and multiple examinations were well tolerated with no increase in abortions (Jaquish et al., 1996). Female callitrichids are polyovulatory and dizygotic twinning is the rule, but singleton births, triplets, and even quadruplets occur. Reproductive information on selected species is presented in Table V. In most captive colonies of marmosets and tamarins, there is no seasonality to births, although these animals are seasonal breeders in the wild. Most deliveries occur at night. Neonates receive little help from their dam and must climb to her back and nipple. Infants are usually carried on the dorsum. Survivability of triplets and quadruplets is poor. Hand rearing of infants (Wolfe et al., 1972), twice-a-day supplemental feeding of infants left with the parents (Ziegler et al., 1981), and rotation of infants off the parents for 24 hr periods of diet supplementation (Hearn and Burden, 1979) have all been used to increase survivability of triplets or quadruplets. Blood chimerism occurs in callitrichids due to placental vascular anastomoses. Freemartinism does not occur because the callitrichid placenta possesses an effective aromatizing enzyme Table V

Reproductive Parameters for Marmosets and Tamarins

Species Callithrix jacchus

Gestation period (days) 148 _+4.3a

Saguinus 145-152d fuscicollis

S. mystax

140-150e 140-150e

S. oedipus

140

S. labiatus

Birth weights mean (g)

Age at sexual maturity (months)

Interbirth interval (days)

F: 31.1 _ 3.8 b M: 31.7 +_ 1.9

F: 20-24 a

39.9 (34-52.7)d

23e

40j

F: 15-17e 330-600 f M: 17-18 187-274 F: 15-17 in 207-576, natal groupg m e a n 3 3 3 h F: 10-11 208.5 ___16.3i new pair captive

M: 15

153-337, mean 214c 155-160 d ca. 365 ferale 146-383 d captive

From Hearn (1977). bFrom Peters and Guerra (1998). cFrom Jaquish et al. (1996). dInterbirth intervals are decreased when infants are removed from pairs and raised by hand. From Wolfe et al. (1975). eFrom Goldizen (1987). fFrom Soini (1993). gSexual maturity of young females occurs more rapidly when removed from natal group and paired with an unrelated male. From Ziegler et al. (1987). h F e r a l . From Savage et al. (1997). iFrom French (1983). JFrom Richter et al. (1984). a

684

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

system, which can convert androgens to estrone (Ryan et al., 1981). Since many singletons are the result of a twin being resorbed, they are commonly chimeric; however, nonchimeric singletons are found (Gengozian and Batson, 1975). Reproductive capacity of callitrichids exceeds that of any other simian primate because of postpartum estrus and twinning. Records of over 20 infants per reproductive female in several species have been reported (Richter et al., 1984). Sex ratios of offspring are 1:1, and twins normally show the predicted 1:2:1 ratio (Gengozian et al., 1977). Pregnancies occur throughout the year. Weaning may begin as early as 45 days and is usually complete by 60 days. In addition to supplementation and methods for feeding infants from multiple births, hand rearing of callitrichid infants may be attempted because of parental neglect (Pook, 1976) or for research purposes (Wolfe et al., 1972). Hand rearing can be difficult, with a high failure rate, behavioral aberrations in surviving individuals, and low costeffectiveness (Richter et al., 1984). First pregnancies in callitrichids may occur as early as 1 year of age; however, in view of normal growth and behavior requirements, planned mating at 2 years of age is preferable. There is a high rate of infant-rearing failure among primiparous captive-born females in some species. Most infant loss appears to be the result of failure of young females to accept and nurse their young. Young females are more likely to raise offspring successfully if paired with experienced males. Many research colonies retain young animals with their families through at least one subsequent cycle of infant rearing so that they might learn parenting skills. Virtually all family members, including juveniles, participate in infant carrying. Other important aspects of infant rearing include active and passive food sharing with infants by adults and juveniles. Young callitrichids are ambulatory as early as 2 weeks, but they do not venture far from adults much before 4 weeks of age. 5.

Laboratory Management

Marmosets and tamarins are usually housed as pairs or in small family groups. Although multiple male and multiple female breeding groups occur in the wild, most captive species are raised as extended families or monogamous pairs with a single breeding female. Females are removed from the natal group and paired after they have participated in rearing younger siblings. Environmental temperatures for callitrichids in the laboratory may need to be relatively warm. Studies on thermoregulation in the cotton-top tamarin indicate that tamarins do not acclimate to a temperate environment and are metabolically stressed at an ambient temperature less than 32~ (89~ (Stonerook et al., 1994). Cold stress associated with low ambient temperatures in captive environments has been proposed as a possible stressor leading to the development of chronic colitis in this species (Stonerook et al., 1994).

Large cages with branches, ropes, or other substrates for climbing are preferable and have been used successfully in breeding colonies (ILAR, 1998). Callitrichids spend little time on the floor of the cage. Single, narrow high cages are preferable to wide, low cages placed over each other in a two-tiered system. Callitrichids housed in the lower cage of a two-tiered system are reported to have decreased activity levels and poorer reproductive performance than those housed in the upper tier (ILAR, 1998). Feeding areas or stations should be above the floor and multiple stations provided if more than one animal is in the cage. Lactating females may dominate feeding stations and consume most of the preferred food if additional feeding sites are not made available to accommodate other group members (Tardif and Richter, 1981). One factor contributing to "wasting marmoset syndrome," a chronic wasting disease of multiple etiologies, has been limited access to food due to guarding by the dominant animal in the cage. Social, especially sexual, and territorial messages are conveyed by scent marking (Epple, 1973). Both sexes mark by rubbing the genitalia, pubis, or chest on the object being marked. Both sexes also mark with urine. Therefore, it is important to have materials within the cage, preferably wood or fiber, for scent marking. Wood or fiber structures, such as rope, can be replaced as they wear out in 2 - 3 months, or as necessary to maintain sanitation (ILAR, 1998). If the item for scent marking is to be sanitized, it should be on an alternate schedule from regular cage sanitation to allow some marked surfaces to remain within the living area at all times. Callitrichids require a flat surface for sleeping, preferably in a nest box where a curled posture can be assumed to conserve body heat (Richter et al., 1984). The nest box should have a viewing area at one end for observation and a closable opening into the cage. Animals can be herded into the nest box, the opening closed, and the nest box removed and used as a transfer cage. Breeding families may need a larger nest box (25 X 25 X 50 cm), and the opening to the interior of the cage from the box should permit easy access by adults carrying young (Richter, 1984). The nest box should be located as high in the cage as possible. Nest boxes should be sanitized every other week, preferably on an alternate schedule from sanitization of the cages to allow for retention of social odor or scent marks (ILAR, 1998). Allogrooming, play, and aggression are part of normal callitrichid behavior. Allogrooming is observed more among sexually mature individuals, while play is seen among infants and subadults. Aggression can be seen among any family members, but is usually nondestructive (Wolters, 1977) and brief. Destructive agonistic behavior can result in injury and may require permanent separation of involved individuals. The introduction of mature adults into breeding groups in captivity is destabilizing and may result in fighting (Epple, 1970a).

16. NONHUMAN PRIMATES 6.

685 7.

Nutrition

Marmosets and tamarins have a dietary requirement for vitamin D 3 and ascorbic acid. Animals housed indoors without any exposure to natural sunlight may require a supplemental source of ultraviolet B radiation to prevent the development of rickets (Gacad et aL, 1992). Dietary iron levels found in standard New World primate diets have been associated with the development of hepatic hemosiderosis in marmosets (Miller et al., 1997). Although some laboratories have maintained colonies on unsupplemented single-item diets (Wirthand Buselmaier, 1982), most callitrichid husbandry programs feed varied diets in an effort to improve general health status. Examples of varied diets can be found in Hampton (1964), Brand (1981), G. King (1975), and Levy and Artecona (1964); these are similar in content. Varied diets must be fed in a disciplined manner to ensure a nutritionally balanced diet. Individual monkeys need to be observed closely to determine that they eat a balanced diet and not only a preferred item, such as fruit. Some investigators supplement with high-protein foods such as cottage cheese, quail or chicken eggs, minced meat, mealworms, and/or neonatal mice. The health status of the colony producing neonatal mice must be known, as lymphocytic choriomeningitis virus has been transmitted to marmosets and tamarins fed infected mice (Montali et al., 1993).

Normal Values

Normal daytime rectal temperature for callitrichids varies between 38.5 ~ and 40.0~ (Richter et al., 1984). The wide range may reflect the excitability of the species, rigors involved in catching, and nocturnal torpor. Callitrichids develop a distinct torpor with hypothermia (34.0~ during sleep (Hetherington, 1978). Normal hemograms for three species of callitrichids are given in Table VI. Detailed hematologic reference values are available for C. j a c c h u s (Hawkey et al., 1982) and for S. labiatus (Wadsworth et al., 1982; Hawkey et al., 1982). Mean plasma volume in S. oedipus and S. labiatus is 43.77 m l / k g body weight, and the mean blood volume is 82.1 m l / k g as determined by Anderson et aL (1967). Small numbers of nucleated red blood cells are commonly seen in callitrichid blood smears. Blood chemistry values for several species of callitrichids are presented in Table VII. 8.

Research Uses

Marmosets and tamarins are used in a variety of biomedical research areas, including pharmacology, neurophysiology, and viral oncology, and as models of infectious and noninfectious disease. Callithrix j a c c h u s is the species most widely used in

Table VI

Marmoset and Tamarin Hemogramsa Hemogramsb (mean values) Value

Unit

A

Red blood count (RBC) Hemoglobin Hematocrit MCV MCH MCHC Reticulocyte Leukocytes Bands Segmented neutrophils Lymphocytes Monocytes Eosinophil Basophil Platelets Fibrinogen Prothrombin time Partial PT with kaolin

106/1111113 gm/dl % Ixm 3

pg % % 103/rnln % % % % % % 103/1111113 mg/dl sec sec 3

B

6.59 15.5 45 69 23 34 3.5 12.6 0.4 43 49 5.0 1.2 0.1

6.86 15.1 45 67 22 34 4.3 12.8 1.7 28 67 2.1 0.6 0.3

- -

~

--m

~ m __

C 6.90 15.5 48 69 22 32 3.5 7.3 ~ 55 43 0.4 0.5 1.3 490 300 -__

D

Conversion factor

SI Units

6.95 17.0 52 74 25 33 2.6 11.9 ~ 42 54 2.8 0.9 0.7 -~ 7.1 32

10 0.155 m 1.0 0.0155 0.01 106 106 106 106 106 106 106 106 106 0.0293 ~ m

106-1012/liter mM/liter

6

fl fmole m 109/liter 109/liter 106/liter 106/liter 106/liter 106/liter 106/liter 106/liter 106/liter ~14/liter

aFrom Richter et al. (1984). bA, Sixteen hr fasted adult Saguinus oedipus; n, 10 (C.B. Richter); B, 16 hr fasted adult Callithrix jacchus; n, 10 (C.B. Richter); C, Clinically normal C. jacchus; n, 27-30. Adapted from Hawkey et al. (1982); D, Young adult S. labiatus; n, 33-39. Adapted from Wadsworth et al. (1982). Note. MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PT, prothrombin time.

686

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Table VII Blood Chemistry Values for Marmosets and Tamarins a Value b Compound

Units

A

B

Glucose Glycohemoglobin Uric acid Blood urea nitrogen Urea Creatinine Total bilirubin Direct bilirubin Indirect bilirubin Total protein Albumin Globulin Alkaline phosphatase SGOT SGPT Lactate dehydrogenase (LDC) ~/-Glutamyl transpeptidase (GGT) Choline esterase Calcium Inorganic phosphate Sodium Potassium Chloride Magnesium CO2 Serum iron

mg/dl % mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl gm/dl gm/dl gm/dl IU/liter IU/liter IU/liter IU/liter

134 2.4 3.0 31 n ~ 0.4 ~ m 6.6 3.9 2.7 5.4 177 109 883

126 1.6 4.1 27 ~ ~ 0.5 0.2 0.3 7.0 3.8 3.2 2.4 160 60 799

IU/liter

11

pHU mg/dl mg/dl mEq/liter mEq/liter mEq/liter mEq/liter mEq/liter ~g/dl

~ 9.4 6.0 ~ ~ ~ ~ ~ 118

. m 9.5 6.9 ~ m n ~ ~ 143

C

D

150 . n m 51.8 0.9 0.6 n ~ 7.1 3.9 3.2 606 182 32 . .

228 . 1.8 9.7 m 1.2 ~ 0.1 0.1 ~ ~ 3.6 29.8 99 .

. 2.7 9.0 ~ ~ ~ 0.1 0.1 ~ ~ 3.9 11.9 113 .

.

SI Units

0.0555

mM/liter

0.0595 0.714 0.167 76.3 17.1 17.1 17.1 10 10 10 ~ ~ . .

mM/liter mM/liter mM/liter ~M/liter ~M/liter la3//liter p~M/liter gm/liter gm/liter gm/liter

.

.

.

.

0.6 10.4 5.5 169 5.7 114 2.4 13.6 ~

Conversion factor

157

.

. ~ 10.2 ~ 158 4.2 ~ ~ m ~

E

. . 0.3 10.0 4.7 161 6.0 109 2.1 18.7 ~

. ~ 0.50 0.323 1.0 1.0 1.0 1.0 1.0 0.179

mM/liter mM/liter mM/liter mM/liter mM/liter mM/liter mM/liter p~M/liter

aFrom Richter et al. (1984). bA, >--10 fasted Saguinus oedipus. From Richter et al. (1984); B, ->10 fasted Callithrix jacchus. Selected healthy adults. From Richter et al. (1984); C, S. labiatus; mean age, 21 months. Adapted from Wadsworth et al. (1982); D, White-lip tamarins, adult. Adapted from Holmes et al. (1967); E, S. oedipus, adults. Adapted from Holmes et al. (1967). Note. SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase.

b i o m e d i c a l research. C o m m o n m a r m o s e t s have b e e n used as m o d e l s o f Parkinson's disease ( G i b b e t al., 1987) and as m o d e l s o f allergic e n c e p h a l o m y e l i t i s , w h i c h m i m i c s several facets o f

genus living as pairs or in small f a m i l y groups (Fig. 4). T h e y are not s e x u a l l y dimorphic. T h e A o t u s genus appears to have

m u l t i p l e sclerosis in h u m a n s ( G e n a i n and Hauser, 1996, 1997).

e v o l v e d f r o m a diurnal a n c e s t o r b e c a u s e the eyes retain vestigial features characteristic o f diurnal vision, such as a retinal fovea.

T h e y are also m o d e l s for i d i o p a t h i c h e m o c h r o m a t o s i s (Bulte

T h e eyes are large and g l o b e l i k e with a m o r e spherical lens than

et al., 1997). T h e c o t t o n - t o p tamarin, S. oedipus, w h i c h is an en-

f o u n d in diurnal m o n k e y s . T h e altered lens shape e n a b l e s re-

d a n g e r e d species, is used in r e s e a r c h as a m o d e l of c h r o n i c co-

fraction and focus of an i m a g e on the retina at low light levels.

litis and c o l o n cancer. S a g u i n a s labiatus, the w h i t e - l i p p e d or

U n l i k e other n o c t u r n a l m a m m a l s , A o t u s spp. lack a t a p e t u m lu-

r e d - b e l l i e d tamarin, and S. mystax, the m u s t a c h e d tamarin, are

cidum. T h e retina contains both rods and cones, with a m a r k -

used in viral hepatitis studies.

edly d e c r e a s e d n u m b e r o f cones c o m p a r e d to other primates.

A o t u s spp. are superior to diurnal N e w W o r l d m o n k e y s in seeing and f o l l o w i n g m o v i n g objects, and spatial r e s o l u t i o n at low

B. 1.

Aotus spp.: Owl Monkeys

Introduction

light levels (Wright, 1989, 1994). O w l m o n k e y s have a low basal m e t a b o l i c rate, 1 8 - 2 4 % below the p r e d i c t e d value for a 1 k g m a m m a l ( L e M a h o et al., 1981). T h e low basal m e t a b o l i c rate is b e l i e v e d to be an adapta-

Aotus, or owl m o n k e y s , are small ( a p p r o x i m a t e l y 1 kg), noc-

tion o f n o c t u r n a l m a m m a l s that allows survival with less e n e r g y

turnal simian primates. T h e y are an arboreal, m o n o g a m o u s

c o n s u m p t i o n ( C r o m p t o n et al., 1978). In contrast, the squirrel

16. NONHUMAN PRIMATES

687

Fig. 4. Aotus lemurinus griseimembra, gray-necked owl monkey family group with 2 adults and a juvenile. Note the lack of sexual dimorphism in the adults. (Photograph from Media Productions. Courtesy of the Department of Comparative Medicine, University of South Alabama.)

monkey, a diurnal New World monkey of similar body size, has a metabolic rate 10% above the predicted value. Owl monkeys belong to a single genus, yet they have a wide variation in diploid chromosome number, from 46 to 56 (Table VIII). Karyotypic variation may also occur within the species, as in A. l e m u r i n u s and A. v o c i f e r a n s . Apparently in the wild, some Aotus populations can experience a degree of chromosomal/karyotypic variation without speciation or crossinfertility. In particular, A. l e m u r i n u s and A. v o c i f e r a n s populations in Colombia and Panama, respectively, have chromosomal polymorphisms that do not pose a barrier to fertility or fitness (Ma, 1981; Ma et al., 1985; Ma and Harris, 1989). 2.

Table VIII

Karyotypes of Species of Aotus a Karyotype Gray-necked group m V

II III IV VIII

IX Red-necked group I

Taxonomy

As with several species discussed in this chapter, there is debate about taxonomic designations for members of the genus Aotus. Hershkovitz (1983) describes two phenotypic groups of Aotus composed of nine allopatric species distinguished by karyotype, pelage patterns, and neck color (Table VIII). The gray-necked species group of owl monkeys contains A. b r u m b a c k i and A. l e m u r i n u s l e m u r i n u s (karyotypes VIII and IX); A. l e m u r i n u s g r i s e i m e m b r a (karyotypes II, III, and IV); and A. t r i v i r g a t u s and A. v o c i f e r a n s (karyotype V) (Ma et al., 1976,

VII VII VI VI a

Species (subspecies) A. A. A. A. A. A. A.

brumbacki vociferans griseimembra b griseimembra b griseimembra b lemurinus c lemurinus C

A. A. A. A. A.

nancymaae nigriceps nigriceps boliviensis d boliviensis d

Diploid chromosome number 50 46 54 53 52 55 56 54 51

52 49 50

Modified from Hershkovitz (1983).

bAotus lemurinus griseimembra. CAotus lemurinus lemurinus. aAotus azarae boliviensis; according to Dr. Orestes J. Colillas, the karyotype of three female A. azarae azarae from Formosa and Chaco, Argentina, as de-

termined by Dr. Marta Mudry de Pargament, of the National Research Council of Argentina, is the same as that of A. boliviensis.

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

688

1978; Hershkovitz, 1983). The red-necked group, which is derived from the gray-necked group, includes A. nancymaae (karyotype I); A. nigriceps (karyotype VII); A. boliviensis (karyotype VI identical to A. azarae); A. miconax; and A. infulatus(Ma et aL, 1976, 1980; Hershkovitz 1983). The taxonomic designations of Hershkovitz are those most commonly used in biomedical research, specifically in describing animals used in malaria research. Taxonomic designations proposed by Wilson and Reeder (1993) and used by CITES appear in Table IX. Ford (1994) has proposed that owl monkeys be classified into seven species: A. trivirgatus and A. vociferans (gray-necked); and A. micronax, A. nancymaae, A. nigriceps, A. azarae azarae, and A. infulatus (red-necked). Owl monkeys are distributed widely from western Panama to northern Argentina. In general, the gray-necked group occurs north of the Amazon River and the red-necked group is found south. Phenotypically, gray-necked owl monkeys have an agouti or gray neck identical in color to the dorsum and torso of the monkey, an intrascapular crest of hair, and elongate pectoral glands with feathering of the periglandular hair. Red-necked

Table IX

Owl MonkeyTaxonomy,CITES Status, and Distribution Genus Aotus

Common name(s)

CITES a status

Distribution

A. azarai

Syn. A. trivirgatus azarai

II

Argentina, Bolivia, Paraguay

A. brumbacki

Brumback's night monkey

II

Colombia

A. hershkovitzi

Syn. A. trivirgatus hershkovitzi

II

Colombia

A. infulatus

Syn. A. trivirgatus infulatus

II

Brazil

A. lemurinus

Syn. A. trivirgatus lemurinus

II

Colombia, Costa Rica, Ecuador, Panama

A. miconax

Andean night monkey

II

Peru

A. nancymaae

Syn. A. trivirgatus nancymaae

II

Brazil, Peru

A. nigriceps

Syn. A. trivirgatus nigriceps

II

Brazil, Peru

A. trivirgatus

Night monkey Douroucouli Owl monkey

II

Brazil, Colombia, Ecuador, Peru, Venezuela

A. vociferans

Syn. A. trivirgatus vociferans

II

Brazil, Colombia

owl monkeys have monochromatic phelomelanin hairs (red neck) that are the same color as the ventrum of the body, an intrascapular whorl, and a rounded pectoral gland with surrounding hairs whorled (Hershkovitz, 1983). Differences in degree of susceptibility or immunity to experimental malarial infection and differences in serum proteins correspond to the red- or gray-necked group type. Aotus lemurinus griseimembra is highly susceptible to Plasmodium falciparum; A. nancymaae is less susceptible to some strains and resistant to others (Schmidt, 1978). Both of these Aotus spp. are susceptible to P. vivax (Schmidt, 1978). Differences in response to P. falciparum infection have been reported for owl monkeys collected in Colombia and Panama (Rossan et al., 1985). The Panamanian monkeys had lower mortality rates for the first 30 days of infection, higher maximum parasitemias, and earlier attainment of peak parasitemia after patency than the owl monkeys of Colombian origin. Duration of primary attacks and recrudescence were shorter in Panamanian monkeys (Rossan et al., 1985). These differences in response to malarial infection vary according to karyotype; the Panamanian monkeys are A. lemurinus lemurinus, 2N = 55/56, and the Colombian monkeys are A. lemurinus griseimembra, 2N = 52/53/54 (Ma et al., 1978; Hershkovitz, 1983). In addition, lymphocytes from different species of owl monkeys have differing responses to mitogen stimulation with phytohemagglutinin, concanavalin A, and pokeweed mitogen (Taylor and Siddiqui, 1978). Karyotyperelated differences in albumin and globulin fractions have been reported (Reardon et al., 1979). These results highlight the necessity of having owl monkeys of known karyotype for research. 3.

a I, Species listed in CITES Appendix I are threatened with extinction (endangered); II, Species listed'in CITES Appendix II are not currently threatened with extinction but may become so unless trade is strictly regulated. From CITES (2000) and Wilson and Reeder (1993).

Natural History

In the wild, Aotus spp. live as monogamous pairs within an extended family that may include 2 - 3 offspring (Aquino and Encarnacfon, 1986; Aquino et al., 1990), usually an infant, juvenile, and subadult (Wright, 1994). At approximately 3 years of age, the subadult leaves the family group to find a mate and establish its own territory. Aggression between the parents and subadult is not a primary cause of dispersement (Wright, 1994). In A. nancymaae, noncycling or breeding offspring of both sexes are tolerated within the family group, whereas in A. vociferans, only male offspring remain with the group (Aquino et al., 1990). The adult male and offspring actively participate in infant care. Owl monkeys are found in a wide variety of forest habitats from sea level up to 3000 meters. Average territory size ranges from 5 to 9 hectares depending on whether the monkeys live in a tropical dry forest (5 hectares) or tropical rain forest (Wright, 1994). Owl monkeys travel at night. Available light affects night ranging patterns with monkeys traveling about twice as far on moonlit nights as on moonless nights. Owl monkeys are also more socially active on moonlit nights. The group exits the sleeping site at dusk and returns at dawn. They sleep as family

16. NONHUMAN PRIMATES

groups within tree holes or vine tangles, and frequently use the same tree (Wright, 1994). A o t u s spp. defend their territory by vocalization, posturing, chases, and fights. Intergroup aggression occurs at bordering fruit trees and is preceded by loud whooping vocalization by both sexes (Moynihan, 1964). Whoops are accompanied by piloerection and stiff-legged jumping. Chases, wrestling, and fighting accompanied by whoops, followed with fights lasting no longer than 10 min, are the norm (Wright, 1978, 1994). Groups then retreat within their respective territories. Owl monkeys utilize both urine and glandular secretions for scent marking. Scent marking plays an important role in sexual recognition and intermale aggression, but it has little effect on male sexual behavior (Dixson, 1983). Scent marking by either sex does not increase with female estrus. Scent marking does increase during intergroup encounters (Wright, 1994). Owl monkeys are principally frugivorous but consume a varied diet of fruits, young leaves, flowers, and insects, and lesser amounts of bird eggs, small birds, and mammals. Composition of the diet can vary with the season and forest type. Increased amounts of flowers and insects are eaten in the spring, and increased amounts of leaves are eaten in the winter in the dry forest. Little seasonal difference in eating patterns is found in the rain forest (Wright, 1994). 4. Reproduction

The owl monkey exhibits no change in external genitalia or predictable changes in vaginal cornification during the 15- to 16-day ovarian or estrous cycle (Cicmanec and Campbell, 1977; Bonney et al., 1980; Dixson, 1983). Menstruation does not occur. Estrone concentrations rise sharply from ovulation, peak 5 days later, and then decline to basal levels by day 13. Increases in plasma progesterone were observed 24 hr after the estrone rise, peaking at day 8 and declining to basal levels by day 11. Urinary excretion of estrone and pregnanediol3a-glucuronide, a major progesterone metabolite in the owl monkey, followed changes in the plasma levels of these hormones (Bonney et al., 1979). Peak plasma steroid levels during the cycle are extremely high (3.59 _ 0.066 ng/ml estrone; 250.48 ___ 11.37 ng/ml progesterone), as is seen in other New World monkeys (Dixson, 1983). Unlike the squirrel monkey in which copulations are limited to the day of ovulation when females are maximally receptive to males (Williams et al., 1988), female owl monkeys remain sexually attractive and receptive to males throughout the ovarian cycle (Dixson, 1983). When owl monkey pairs were caged singly and test-mated for 30 min a day throughout two or more cycles, no changes in copulatory behavior, scent marking, or grooming, based on the stage of the ovarian cycle, were observed. During test pairing, copulatory behavior and scent marking occur more frequently than when the monkeys are housed continuously as pairs. In addition, agonistic behaviors such as "arching," a hostile display, and "tail rubbing," a form

689

of scent marking, occur more frequently in test matings than in family groups. Levels of plasma testosterone in adult male owl monkeys have a circadian rhythm with peak levels occurring in the light period of the light cycle (resting period), and lowest levels during the dark (active period) (Dixson and Gardner, 1981). These daily changes in testosterone are the opposite of those of macaques and lemurs in terms of light cycle, but the same in terms of activity cycles (Goodman et al., 1974; Dixson and Gardner, 1981). Partially arrested spermatogenesis has been described in captive adult male owl monkeys (Hunt et al., 1975; N. King, 1975). The condition may occur at any time of the year. Histologic examination of testicular biopsies reveal few spermatozoa, degenerate-appearing spermatocytes, and orange-brown pigment deposits between the tubules. Vitamin E deficiency has been proposed as the cause of this defective spermatogenesis (Hunt et al., 1975; N. King, 1975). However, many of these animals are fertile and may be successful breeders (Dixson, 1983). Based on one report of a timed pregnancy, the gestation period for owl monkeys is 133 days (Hunter et al., 1979). Births have occurred as early as 138 days following initial pairing (Dixson, 1983). Estimates of the gestation length have been as high as 148-159 days (Elliot et al., 1976; Meritt, 1976). Urinary chorionic gonadotropin is detectable from 5 to 6 weeks following conception until 1 week before parturition (Hall and Hodgen, 1979). Interbirth intervals reported by Dixson (1983) average 253 days and range from 166 to 419 days. Malaga et al. (1997) reported a diminishing interbirth interval in multiparous pairs with 11 months between the first and second births, 9.9 months until the third birth, and 8.8 months between subsequent parturitions. The mean time from pairing to first parturition was 14.6 _ 7.7 months (Malaga et al., 1997). There is no postpartum estrus. Unlike squirrel monkeys, captive owl monkeys have no seasonal breeding or birth seasons. Infants weigh 90-105 gm at birth and have a well-developed pelage except for the abdomen and inner surface of the limbs. Most births are singletons; twinning occurs rarely. Most of the time, newborns cling to the parent in a ventrolateral position, climbing up to the nipple to suckle (Meritt, 1976; Cicmanec and Campbell, 1977; Dixson, 1983). Ventrolateral clinging is the major resting position of the infant until it reaches 3 - 4 weeks of age, at which time dorsal clinging becomes the preferred position (Dixson, 1983). Dorsal clinging and transfer from female to male parents may be observed as soon as the first day. Infants begin to get off the parents from 3 to 6 weeks of age, and start to eat solid food from 35 to 60 days (Dixson, 1983). Both parents play a role in carrying and caring for their offspring, a similar involvement of the male as has been reported for the Callitrichidae (Epple, 1975; Ingram, 1977). For the first week, the female carries the infant most of the time (Cicmanec and Campbell, 1977; Dixson and Fleming, 1981). Thereafter, the male carries the infant, and the infant transfers to its mother to nurse. Unlike macaques, owl monkeys rarely remain on the nipple except to nurse. As the infant matures, the female may

690

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

begin to reject the infant following nursing by biting at its extremities. The infant promptly moves to the male or to a sibling. After the infant reaches 8 weeks of age, the male may also begin to reject the infant. Similar patterns of carrying and rejection behavior are observed in marmosets (Ingram, 1977). From weeks 4 to 5, owl monkey infants spend increasing time off either parent, and by 18 weeks the infant moves independently, returning to a parent, usually the male, only if stressed (Dixson and Fleming, 1981). Puberty begins between 300 and 400 days of age in both males and females (Dixson et al., 1980; Dixson, 1983). A useful indicator of pubertal development is the growth of the subcaudal scent-marking gland at the base of the tail. Growth of this gland corresponds to increasing testosterone levels in the male. Owl monkeys are probably sexually mature at 18 to 24 months of age. In family groups there is no increase in aggression as offspring progress through puberty. Male parents interact more frequently than female parents with older offspring, particularly in grooming behavior (Dixson, 1983). Reproductive success in the owl monkey is poor compared to that of Old World primate species. In a large breeding colony of owl monkeys, 168 pairs produced 368 conceptions in 4 years, an average of 0.7 conceptions per pair per year or 1 birth every 17 months per pair (Malaga et al., 1997). Fewer than 50% of pairs produced offspring during a given year. Seventy-eight percent of conceptions resulted in live births. Neonatal mortality was 6.2% during the first week of life, and survivability to 3 months of age was 85.9% (Malaga et al., 1997). 5.

fers. Handling and restraint are potentially upsetting not only to the animal being handled but also to others within the room. If necessary, owl monkeys may be captured within the nest box and the box and animal removed from the colony room. The animal can then be removed and handled in a separate room as warranted. A nest box provides owl monkeys with a structure that simulates a tree-hole nest and can be placed in or attached to each cage. Small animal airline transport cages in which the floor has been replaced with hardware cloth or other wire mesh, the door removed, and a perch provided are effective thermoneutral nest boxes that can be attached to the outside of the cage. Feces and urine fall through the mesh to the floor. Eighteen-inch sections of 8-inch diameter polyvinyl chloride (PVC) pipe with access holes and an interior perch, or PVC T-sections, are inexpensive nest boxes that can be placed either on the floor or hung within the cage. Nest-box and cage sanitation should occur on alternate weeks. This provides stabilization of olfactory cues within the monkey's environment. In addition, because owl monkeys are scent markers, suitable pieces of hardwood may be used as perches within the cage. It is desirable that these perches also be sanitized on a different schedule than cages. Owl monkeys may be housed singly, as pairs, or as family groups. Careful attention must be paid to juvenile animals to ensure that they are removed from family groups prior to any aggression. Usually when pairs start to reject a juvenile animal, they will prevent it from entering the nest box. Attempts have been made with some success to house same-sex pairs together when breeding is undesirable (Weed and Watson, 1998).

Laboratory Management

Housing and maintenance of the owl monkey should be designed to meet its unique needs as a nocturnal neotropical primate. Room temperature should range from 75 ~ to 80~ with the higher temperature preferable. Provision of a tranquil setting with a minimum of extraneous noise appears to improve adaptation to captivity and survival. Use of disposable, absorptive fiberboard sheets beneath the cages in the refuse pans is an alternative to daily hosing of cages (C. Abee, personal communication, 2000). Fiberboard sheets are changed every other day. Red filters or gray filters are placed over light fixtures in many laboratories to provide diminished lighting. Owl monkeys are provided a 12:12 light-dark cycle that is offset from the normal day so that monkeys can be observed during the active "night" cycle. Diminished illumination is better than absolute dark, as normal feeding, locomotion, and social behaviors occur more frequently when some illumination is present (Erkert, 1976; Wright, 1994). Provision of an automatic night-light is an alternative to red-light illumination during the dark cycle. Owl monkeys are sensitive to changes in routine and personnel. Visitors should be excluded or minimized. Unnecessary handling should be avoided by the use of transfer boxes or tunnels to facilitate cage changes. Owl monkeys are easily trained to jump into a transfer box or into a clean cage for cage trans-

6.

Nutrition

Little is known of the dietary requirements ofAotus spp. They are assumed to be similar to other New World primates. As such, they require dietary supplementation with vitamin D 3 and vitamin C. In most owl monkey colonies, New World monkey formulation is fed. Owl monkeys have a decreased incidence of diarrhea when fed a high-fiber diet formulation manufactured for Old World monkeys (S. Gibson, unpublished data, 2000). This diet preparation is usually formulated with vitamin D 3. No deficiencies occurred, and reproductive indices remained the same during a 2.5-year experience of feeding a high-fiber diet to Aotus spp. The natural high-fiber diet of fruit, leaves, and insects may explain why a high-fiber commercial diet is effective in this species. In some colonies, the commercial diet is moistened with fruit juice prior to feeding to soften the diet and increase its acceptance. Owl monkeys readily accept a variety of fruit and vegetable foodstuffs, including bananas, oranges, grapes, celery, squash, yams, carrots, green tomatoes, and green beans. These items should be fed to supplement, but not replace, the commercial monkey diet. The gray-necked owl monkey species (karyotypes II, III, IV, V, and VII) are susceptible to vitamin E-responsive hemolytic anemia. In some colonies, monthly injections of supplemental

691

16. NONHUMAN PRIMATES

Table X Hematological Data for Owl Monkeys a K-I b (N=254) Analyte RBCs (10 6/1TI1) Hgb (g/dl) VPRC (%) Platelets (103/ml) MCV (gm3) MCH (pg) MCHC (%) WBC (103/ml) Segs (103/ml) Lymphs (103/ml) Monos (103/ml) Eosin (103/ml) Basos (103/ml)

X

K-II (N=62) SD

6.2 16.3 49.7 431 79.8 26.2 32.8 10.1 3.0 6.1 0.4 0.4 0.2

0.6 1.6 4.4 119 3.5 1.6 1.1 3.2 2.0 2.6 0.3 0.5 0.2

K-III (N=55)

X

SD

X

5.3 14.3 43.6 342 82.6 27.0 32.8 12.2 3.4 6.9 0.3 1.4 0.2

0.5 1.5 4.2 m 3.0 1.4 0.9 4.4 1.8 2.2 0.3 1.1 0.2

5.3 14.0 43.6 333 82.3 26.5 32.2 14.2 4.3 8.1 0.3 1.4 0.1

K-V (N=35) SD

0.9 2.3 7.0 117 4.0 1.6 0.8 11.2 2.2 2.5 0.2 1.5 0.2

X

SD

6.4 17.1 52.2 295 81.3 26.6 32.8 8.8 2.6 5.5 0.3 1.6 0.1

0.5 1.3 m 90 4.1 1.6 0.6 3.8 1.2 1.5 0.3 1.1 0.1

aFrom Baer (1994). bKaryotype designation.

Note. RBCs, red blood cells; Hgb, hemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood count; Segs, neutrophils; Lymphs, lymphocytes; Monos, monocytes; Eosin, eosinophils; Basos, basophils.

v i t a m i n E have b e e n u s e d to p r e v e n t this p r o b l e m ( S e h g a l et al., 1980). Oral v i t a m i n E a d m i n i s t r a t i o n alone is ineffective.

S e h g a l et al., 1980; M r e m a et al., 1987; M a l a g a et al., 1990). R e p o r t e d differences a m o n g l a b o r a t o r i e s are p r o b a b l y due to o w l m o n k e y k a r y o t y p e , differences in h u s b a n d r y and diet, and

7.

assay techniques. Tables X and X I p r o v i d e h e m a t o l o g i c and

Normal Values

clinical c h e m i s t r y data on four k a r y o t y p e s o f owl m o n k e y s N o r m a t i v e h e m a t o l o g i c and s e r u m c h e m i s t r y data for o w l

m a i n t a i n e d at the s a m e facility u n d e r similar c o n d i t i o n s (Baer,

m o n k e y s have b e e n p u b l i s h e d b y several authors (Porter, 1969;

1994). All m o n k e y s tested w e r e clinically h e a l t h y adults. Tables

Table Xl Serum Chemistry Data for Owl Monkeys a K-I b (N =254) Analyte Total bilirubin (mg/dl) Cholesterol (mg/dl) Creatinine (mg/dl) Glucose (mg/dl) Calcium (mg/dl) Alkaline phosphatase (IU/liter) Phosphorus (mg/dl) ALT (IU/liter) SUN (mg/dl) Total protein (g/dl) Albumin (g/dl) GGT (IU/liter) Sodium (mEq/liter) Potassium (mEq/liter)

K-II (N =57)

K-III (N=53)

K-V (N =35)

X

SD

X

SD

X

SD

X

SD

0.8 150 1.0 139 10.4

0.4 46 0.4 35 1.0

0.5 91 1.0 153 9.3

0.2 34 0.2 39 0.9

0.5 111 1.1 150 9.2

0.2 44 0.4 47 0.9

0.7 99 1.0 172 9.6

0.2 34 0.3 40 0.7

494 4.0 47 15 8.3 4.4 17 152 3.8

469 1.5 37 5.4 0.9 0.5 14 5 0.7

183 4.4 44 15 8.0 3.8 20 156 4.6

151 1.5 34 6.7 0.7 0.5 14 9 1.6

143 4.6 49 17 8.1 3.7 23 154 4.8

87 1.5 35 11 1.1 0.5 18 6 2.0

364 4.8 59 15 8.2 4.6 26 148 3.3

381 1.5 34 8.9 0.5 0.4 21 3 0.7

aFrom Baer (1994). bKaryotype designation.

Note. ALT, alanine aminotransferase; SUN, serum urea nitrogen; GGT, y-glutamyltransferase.

692

BRUCE J. BERNACKY, SUSAN V. GIBSON, M I C H A L E E. KEELING, AND CHRISTIAN R. ABEE

Table XII Normal Urine Parameters in Owl Monkeys a Urine

Mean __+S.E.M.

Range

Specific gravity Volume (ml/kg/day)

1.010 ___0.001 81.9 ___7.5

1.002-1.023 9-303

Urinalysis Semiquantitative values Protein multistix Glucose Ketones Bilirubin Urobilinogen Occult blood pH Appearance

0 -3+ 0-1+ 0 -2+ 0 0 0-4+ 6-8.5 Light-dark yellow; clear- cloudy

Microscopic urinalysis

View

Red blood cells White blood cells Epithelial cells Casts Crystals

Rare, few ( 3 - 5 / h p f ) Rare, occasional Rare, occasional squamous Occasional granular Few, moderate triple phosphate; rare calcium oxalate Occasional renal tubular epithelial cells

Miscellaneous aFrom Baer (1994).

Note. S.E.M., standard error of the mean.

of normal urine parameters (Table XII) and mean adult body weights (Table XIII) of owl monkeys from the same facility are also provided. 8.

R e s e a r c h Uses

Owl monkeys have been used extensively in the study of malaria, including maintenance of various malarial strains, antigen production, studies of host-vector relationships, and

Table XIII Mean Adult Owl Monkey Body Weight a Karyotype I II III V

Male 957 880 898 838

___ 124 ___98 ___ 109 ___ 118

N 130 55 52 18

Female 885 864 907 751

__+ 120 +__ 111 + 99 + 91

N

Range

124 48 37 17

567-1232 531-1281 529-1237 572-1077

aData given in gm (mean ___SD) listed by karyotype and sex. Data collected at Battelle Pacific Northwest Laboratories. From Baer (1994).

parasite life cycles (Aikawa et al., 1988). Owl monkeys are susceptible to human and nonhuman primate malarias and can transmit these infections to mosquitoes. Thus, the owl monkey is an excellent animal model for human malaria and its treatment. The owl monkey has also been identified as a model for cutaneous and visceral leishmaniasis (Chapman et al., 1981; Broderson et al., 1986), for studies of oncogenic viruses, particularly herpesviruses (Barhona et al., 1976), and nononcogenic viruses such as hepatitis A (LeDuc et al., 1983). The unique characteristics of the owl monkey eye have made it a valuable animal in vision research (Jacobs et al., 1979).

C.

Saimiri spp." Squirrel Monkeys

1. Introduction

Squirrel monkeys (Saimiri spp.) are the most commonly used neotropical primates in biomedical research in the United States. Physical characteristics such as small size and ease of handling contribute to their desirability as research subjects (Fig. 5). The mean body weight of adult squirrel monkeys is less than 1 kg. Some sexual dimorphism is observed in squirrel monkeys, although sex differences are less distinct than in many Old World primates. Male squirrel monkeys are 25 to 30% heavier than females, and canine teeth are larger and longer in males. Both males and females undergo marked seasonal reproductive changes, moving from periods of infertility or anestrus with low levels of circulating steroid hormones to fertility with high levels of hormones. Females have a very short estrous cycle, averaging 9-10 days. Males experience seasonal enlargement of testes concomitant with spermatogenesis and undergo "fatting" prior to breeding season. Squirrel monkeys have circulating levels of free or unbound cortisol approximately 100 times greater than that found in humans or Old World primates (Klosterman et al., 1986), and have been valuable models in molecular studies of the cortisol receptor and the role of chaperone proteins in this phenomenon (Scammell, 2000). Naturally occurring atherosclerosis, which can be potentiated by feeding dietary cholesterol, has made the squirrel monkey a valuable model of this disease. Large numbers of squirrel monkeys were imported to the United States in the 1960s (Cooper, 1968). However, the governments of South America began banning the export of primates indigenous to their countries in the 1970s. The exportation of the Bolivian squirrel monkey (S. boliviensis boliviensis), a species considered especially desirable for malaria vaccine studies, was banned by the Bolivian government in the 1980s. In the mid-1980s, only Peruvian squirrel monkeys (S. boliviensis peruviensis) were available from the wild. Currently, limited numbers of Guyanese squirrel monkeys (S. sciureus sciureus) are also available for importation.

16. NONHUMANPRIMATES

Fig. 5. Saimiri boliviensis boliviensis, youngadultfemaleBoliviansquirrel monkey.(PhotographfromMediaProductions.Courtesyof the Departmentof ComparativeMedicine,Universityof SouthAlabama.) 2.

Taxonomy

Squirrel monkeys were once considered to be a single species (S. sciureus) with several geographically separated subspecies.

However, karyotypic and phenotypic information gathered in the early 1980s led to the conclusion by Hershkovitz (1984) that squirrel monkeys should be classified as a single genus with four species and nine subspecies. Studies done by Assis and Barros (1987), Da Silva et al. (1987), and VandeBerg et al. (1990) support the taxonomic classification of Hershkovitz (Table XIV). The most commonly used phenotypic characteristic for species identification is the shape of the patch of nonpigmented hair above the eyes; squirrel monkeys are divided into two groups based on this characteristic. Those belonging to the S. sciureus, S. oerstedii, and S. ustus groups are classified as "gothic arch" squirrel monkeys as they possess a pointed arch of whitish hair above each eye (Fig. 6). Those belonging to the

693 S. boliviensis group are referred to as "roman arch" squirrel monkeys, characterized by more shallow, semicircular patterns above the eyes. Hershkovitz (1984) confirmed the value of this phenotypic characteristic. Additional phenotypic characteristics include differences in coloration of the hair on the head and body. These differences in coloration can range from subtle to obvious. Squirrel monkeys of the "roman arch" variety usually have black hair crowning their heads, though exceptions exist (Hershkovitz, 1984), while "gothic arch" squirrel monkeys usually have a gray-green, agouti coloration. Saimiri sciureus sciureus, the Guyanese squirrel monkey, also possesses a pattern of pigmented hairs within the patch of whitish hair above each eye that resembles an eyebrow (Ariga et al., 1978). Precise identification of squirrel monkeys requires both phenotypic and karyotypic examination. All squirrel monkey species and subspecies have 44 (diploid) chromosomes; however, they vary in their number of acrocentric autosomes from 5 to 7. By counting the number of acrocentric autosomes and observing the periocular patches, more certain identification can be made (Ariga et al., 1978). Such specific identification of the type of squirrel monkey to be used in a particular experiment is critical in that species and subspecies vary in their susceptibility to both naturally occurring and experimentally induced diseases (Portman et al., 1980; Martin and McNease, 1982; Ausman et al., 1985; Coe et al., 1985). Furthermore, failure to identify and separate Peruvian, Bolivian, and Guyanese squirrel monkeys in breeding colonies may result in interbreeding. The karyotypic variations observed in squirrel monkeys are thought to be due to pericentric inversions in the ancestral karyotype (Jones et al., 1973). Therefore, the progeny of squirrel monkeys that interbreed will be heterozygous for the inversion. This inversion heterozygosity can lead to the production of nonviable gametes due to crossovers at the inversion loop during meiosis. Theoretically, 50% of conceptions in hybrid squirrel monkeys could be nonviable, thus potentially reducing reproductive efficiency in breeding colonies. Also, mixing species and subspecies within experimental groups may create confounding variables caused by differences in responses to experimental manipulation, which could lead to difficulties in interpreting experimental results.

3.

Natural History

Squirrel monkeys are found in the Amazon basin of South America and as isolated populations in Panama and Costa Rica. The Bolivian squirrel monkey (S. boliviensis boliviensis) ranges from extreme western Brazil between the Rio Jurac and Purus, to the upper Rio Madre basin of the upper two-thirds of Bolivia, and the lower third of Peru in the Rio Ucalyli basin. Peruvian squirrel monkeys (S. boliviensis peruviensis) are found in northwestern Peru east of the Andes Mountains. Saimiri sciureus sciureus, or the Guyanese squirrel monkey, is found in

694

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Table XIV

Squirrel MonkeyTaxonomy,CITESStatus,and Distribution Genus Saimiri S. sciureus S. s. sciureus S. s. macrodon S. s. cassiguiarensis S. s. albigena S. boliviensis S. b. boliviensis S. b. peruvensis S. ustus S. oerstedii S. o. oerstedii S. o. citrinellis S. vanzolinii

Common name(s)

CITES a status

Distribution

Common squirrel monkey Guyanese squirrel monkey

II

Brazil, Colombia, Ecuador, French Guiana, Guyana, Peru, Suriname, Venezuela

Syn. S. sciureus boliviensis Bolivian squirrel monkey Peruvian squirrel monkey Syn. S. saimiri ustus Central American squirrel Red-backed squirrel monkey

II

Bolivia, Brazil, Peru

II I

Brazil Costa Rica, Panama

Blackish squirrel monkey

II

Brazil

a I, Species listed in CITES Appendix I are threatened with extinction (endangered); II, Species listed in CITES Appendix II are not currently threatened with extinction but may become so unless trade is strictly regulated. From CITES (2000) and Wilson and Reeder (1993); modified from Hershkovitz (1984).

Guyana, Suriname, French Guiana, and extreme northeastern Brazil (Hershkovitz, 1984). Squirrel monkeys are diurnal and arboreal. They inhabit most types of tropical forest, including wet and dry forest, continuous and secondary forest, mangrove swamps, riparian habitat, and forest fragments (Hermindez-Camacho and Cooper, 1976; Terborgh, 1983; Baldwin, 1985; Boinski, 1987). Their range includes altitudes from sea level to 2000 meters. Squirrel monkeys are highly flexible in their adaptation to different environments; in some areas they have flourished in disturbed habitats (Konstant and Mittermeier, 1982; Boinski, 1987). Squirrel monkeys form large multimale-multifemale troops in the wild, numbering from 50 animals up to 300 (HermindezCamacho and Cooper, 1976; Baldwin, 1985; Boinski, 1987; Mendoza et al., 1991). The sex ratio within wild groups tends

Fig. 6. Phenotypic facial characteristics of Saimiri sp. A squirrel monkey with a "gothic arch" phenotype typical of S. sciureus sp. is pictured on the left. A "roman arch" phenotype characteristic of S. boliviensis sp. is featured on the right.

to be 50: 50. Groups consist of adult females and offspring, subadult females, subadult males, and adult males. Female squirrel monkeys reach sexual maturity at 2.5-3 years of age. Males become subadults at 2.5-3 years of age and generally transfer out of their natal group. In Bolivian, Peruvian, and Guyanese squirrel monkeys, there is a low rate of female transfer and a high rate of male transfer from natal groups. Subadult males may join all-male groups until they become full adults at 5 years of age and can work their way into the male dominance hierarchy of an established mixed-sex group. The adult females generally unify squirrel monkey society. Sexual segregation occurs on a seasonal basis with males staying on the group periphery during nonbreeding season. The majority of social interactions between sexes occurs during the breeding season. Bolivian squirrel monkeys exhibit strong sexual segregation, while Guyanese squirrel monkeys are more sexually integrated (Mendoza et al., 1978a; Boinski, 1987). Sexual segregation may be initiated by active female exclusion of males (Baldwin, 1968; Dumond, 1968; Baldwin and Baldwin, 1981; Coe and Rosenblum, 1974; Mendoza et al., 1978b) or may be the result of intermale social dynamics (Strayer and Harris, 1979; Lyons et al., 1992). Male squirrel monkeys do not participate in infant care. Allomaternal care, or care provided to the infant by an individual other than its dam, is provided by juvenile and adult females. Allomothering begins within the first 2 weeks of infant life. A1lomothering usually involves carrying the infant. Some females that have been unsuccessful during that reproductive year may also nurse the infant they allomother (Williams et al., 1994). Squirrel monkey groups tend to forage as a cohesive unit. They are omnivorous; the diet varies with subspecies and habi-

16. NONHUMAN PRIMATES

695

tat. Bolivian monkeys eat primarily insects (82% of diet) with lesser amounts of fruit, flowers, and seeds. In the dry season, figs are an important diet component. Guyanese squirrel monkeys eat animal prey, including insects, frogs, crabs, and snails (Rowe, 1996). 4.

Reproduction

Squirrel monkeys are seasonal breeders. Both male and female squirrel monkeys undergo hormonally induced physiological changes during the breeding season. The breeding season is approximately 3 months in duration (December-March in the Northern Hemisphere) and consists of a cluster of ovulatory cycles varying between 6 and 12 days in length, with a mean cycle length of 9.5 days (Diamond et al., 1984). Gestation is approximately 150 days, and the interbirth interval in captivity is about 465 days. The breeding season of female squirrel monkeys is characterized by elevations in circulating levels of estradiol and progesterone. Serum concentrations of estradiol in S. boliviensis increase dramatically in cycling females, from peak prebreeding season levels averaging less than 95 pg/ml to levels greater than 1000 pg/ml during the breeding season. Although ovulatory cycles all occur at the same time of year, females do not cycle in synchrony with others in the same social group (Williams et al., 1986). This makes the prediction of cyclic events within a breeding group difficult. Some adult females within breeding groups fail to cycle during portions of the breeding season, which further complicates management of a breeding colony. Ovulation can be verified by laparoscopy or serial ovarian hormone determinations (Aksel et al., 1985; Alexander et al., 1991). Prior to the onset of the breeding season of the female, breeding age males undergo a rapid weight gain, which is distributed primarily over the upper torso (Fig. 7). Peak body weights occur immediately prior to the onset of the breeding season (Williams et al., 1986; Wiebe et al., 1988). This phenomenon is called "fatting" (DuMond and Hutchinson, 1967; DuMond, 1968). Behavior of male squirrel monkeys during the breeding season is characterized by a reduction in responses associated with aggression and an increase in sexually related responses such as genital displays, anogenital inspection, and copulation. These responses correlate positively with elevations in circulating levels of androstenedione (Williams et al., 1986). Anderson and Mason (1977) showed that male squirrel monkeys exposed to females stimulated with exogenous estradiol had increased sexual behavior. Copulations during the breeding season are associated specifically with ovulation; all copulations occur within 24 hr of predicted ovulation, based on daily serum luteinizing hormone determinations (Williams et al., 1986). Serum concentrations of estradiol (E2), progesterone (P), and squirrel monkey chorionic gonadotropin (SMG) can be used to

Fig. 7. Fattedadult male Boliviansquirrel monkey,Saimiri boliviensis boliviensis. Note broad head and heavy shoulders. (Photographfrom Media Pro-

ductions. Courtesyof the Departmentof ComparativeMedicine, Universityof South Alabama.) diagnose early pregnancy in squirrel monkeys. In animals pregnant for less than 25 days, SMG levels are less than 300 ~tg/ml. After 30 days of pregnancy, concentrations increase and fluctuate between 700 and 2000 ~tg/ml. Pregnant squirrel monkeys have fluctuating concentrations of E2 above 300 pg/ml and P above 150 ng/ml (Diamond et al., 1987). Detection of maintained, elevated serum levels of these hormones can be used to ascertain early pregnancies. Early abortions can be a serious problem in squirrel monkey breeding colonies because all females must conceive during the relatively narrow time span of a strict seasonal breeding pattern. Those that abort may not have another opportunity to conceive before the breeding season ends. Early, frequently occult abortions were documented in 25% of pregnancies in which pregnancy was diagnosed and followed by changes in circulating hormone levels (Diamond et al., 1985). Another contributing factor to reduced reproductive efficiency in squirrel monkeys is

696

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

the large fetal mass in comparison to maternal size. A term infant squirrel monkey weighs approximately 18% of the nonpregnant weight of the dam. Large fetal-to-dam size contributes to a high incidence of dystocia and resultant stillbirths in this species. Squirrel monkey infants are usually born in the summer months in the northern hemisphere. Normal squirrel monkey deliveries occur at night with labor lasting 1-2 hr. The infant actively participates in delivery. Once the shoulders are free, the infant reaches up and grabs the ventrum of the dam and assists in pulling itself out. The infant climbs and clings to the dorsal surface of the dam, riding just behind the dam's head. Newborn infants weigh about 100 gm (Table XV), and infants weighing less than 90 gm rarely survive. During the first 2 weeks of life the infant spends most of its time sleeping while clinging to the dorsum of the dam. At 1 month of age, the infant begins to move off the dam. Weaning begins at 3 - 4 months of age, although in captivity some dams nurse their infants until 1 year of age.

5. Laboratory Management As squirrel monkeys are neotropical primates with little body fat, a high metabolic rate, and a relatively large surface area, they need to be maintained in a warm laboratory environment, preferably at 80~ Squirrel monkeys are prone to hypothermia and are stressed if placed in standard stainless steel cages at a room temperature of 72~ Thermoneutral perching, such as PVC pipe, is preferable to metal perches. It is important to provide perching that keeps the animals off the floor. Perches < 3/4 inch in diameter can cause pressure sores and eventually ulcers on the dorsum of the base of the tail. Squirrel monkeys routinely perch and sleep balancing on the base of the tail, with the tail wrapped around over the front and ventrum of the animal. They lack ischial callosities, so sitting puts considerable pressure on the base of the tail. Large-diameter perching will prevent the formation of tail-base ulcers. In addition, squirrel

monkeys scent-mark by urine washing, and cages should contain a substrate suitable for this behavior. Squirrel monkeys are social animals and when feasible should be housed with compatible cohorts. It is possible to house squirrel monkeys as same-sex pairs; however, pairing of adult males must be carefully monitored. Squirrel monkeys do well in large pens or indoor-outdoor enclosures. It is important to provide multiple-level perches and multiple feeding areas in group cages. As arboreal animals, squirrel monkeys will use all levels of perching within the cage. Hide boxes can be used to provide escape areas for animals that are being chased or harassed. Small to large, mixed-sex groups work well in breeding situations. Usually there are 1-2 males for 12-16 females. Singlemale groups can also be effective in breeding when housed with no more than 12 females. Once established, mixed-sex groups are stable with minimal aggression or fighting, except at the beginning of the breeding season. New animals should not be added individually to established groups as aggression and serious injury will result. Squirrel monkey infants may be reared successfully in a nursery. Supplemental heat should be provided by a neonatal isolator or an enclosure placed over a water-recirculating heating pad. Ambient temperature should be maintained at 85 ~F for the first 2 - 3 weeks of life. Surrogates that allow for the normal dorsal clinging posture of the infant on the back of the dam are desirable. Human infant formulas have been used successfully, but the formulation is not the same as that of squirrel monkey milk. Infant squirrel monkeys are inefficient in protein utilization and require approximately 13% of their calories as dietary protein (Ausman et al., 1979). Use of a dry formulation (Zoologic Milk Matrix, Pet-Ag., Inc., Hampshire, Illinois) that is mixed with powdered milk and water more closely approximates squirrel monkey milk and is readily accepted by infants (S. Gibson, personal communication, 2000). Infants should be fed every hour initially for at least 14 hr a day. The feeding interval can be extended as the infant gains weight, if the blood glucose remains

Table XV Squirrel Monkey Birth Weights (Saimiri spp.) Weight + SD (N)

a

Sex Species

Female

Male

Average

106.5 ___21.0 (143)

112.0 ___22.5 (140)

109.2 _+ 21.9 (283)

114.9 +_ 14.5 (24)

118.8 ___8.9 (27)

117.0 ___ 11.9 (51)

129.0 ___ 13.8 (31)

136.5 __+17.1 (25)

132.4 ___ 15.7 (56)

Bolivian

(S. b. boliviensis) Peruvian

(S. b. peruviensis) Guyanese

(S. sciureus)

aMean body weight in grams ___standard deviation (number of infants weighed). Data from live births that survived to at least 3 days of age, 1996-2000. Unpublished data from Squirrel Monkey Breeding and Research Resource, University of South Alabama, 2000.

16. NONHUMANPRIMATES

697

normal ( > 40 mg/dl). By 3 - 4 weeks of age the infant should be able to self-feed from a bottle, and at 1 month of age it can be started on moistened monkey chow. Infants without health problems are best reared by the dam or fostered to another monkey. If infants cannot be fostered, they should be socialized as soon as possible. Play times with other nursery infants or older, tolerant, nonreproductive females can be used to socialize the infant. Nursery infants can be introduced to a multiage social group by 6 months of age. Return of the infant to a social group can prevent or minimize the development of behavioral abnormalities.

7.

Normal Values

Normal values for hematology and serum chemistry for Bolivian squirrel monkeys are presented in Tables XVI and XVII. Samples were collected from healthy individuals that had been in captivity for 5 years. Squirrel monkeys have a relatively small mean corpuscular volume, 60 Bm3, which may require adjustments to hematology units to obtain correct results. Normal adult body weights for Bolivian squirrel monkeys are presented in Table XVIII. 8.

6.

Nutrition

Squirrel monkeys adapt well to commercial monkey diets. Because of a high basal metabolic rate and a gastrointestinal tract that is short compared to that of other monkeys, squirrel monkeys require a diet of high caloric density. Feed should always be present in the cage as they are susceptible to hypoglycemia. Similar to other New World monkeys, squirrel monkeys have a dietary requirement for vitamin D 3 and vitamin C. As little as 1 IU/gm diet of vitamin D 3 is sufficient to prevent the development of rickets (Lehner et al., 1967). Ten milligrams per kilogram body weight per day is sufficient ascorbic acid to correct signs of scurvy (Lehner et al., 1968). Squirrel monkeys have a high dietary requirement for folic acid, approximately 200 ~tg per day. Megaloblastic anemia, low-birth weight infants, and stillbirths have been associated with folic acid deficiency in pregnant squirrel monkeys (Rasmussen et al., 1980). Supplemental foods for dietary diversity and environmental enrichment include carrots, squash, green beans, celery, yams, grapes, roasted peanuts, hard-boiled eggs, mealworms, and crickets.

Research Uses

Although research methods and approaches have changed radically over the past decade with advances in molecular and cellular biology, the use of squirrel monkeys has remained relatively constant, based on the number of literature citations each year. From 1995 through 1998, neuroscience research, including studies of the central nervous system, behavior/learning, and perception have yielded the most publications in the scientific literature (Abee, 2000). Squirrel monkeys are also frequently used in infectious disease, genetics, pharmacology, and toxicology research. The squirrel monkey is an important animal model for malaria vaccine development studies. Because Plasmodium spp., which cause malaria, are host-specific, animals used for studies of human malaria must be susceptible to the same strains of Plasmodium that cause disease in humans. The Bolivian squirrel monkey has been shown to be a superior model for studies of the pathogenesis of P. falciparum Indochina I (Whiteley et al., 1987), developing lesions and signs similar to those reported in the human disease. Bolivian, Peruvian, and Guyanese squirrel monkeys are susceptible to infection with different strains of P. vivax, but they respond differently

Table XVI

Hematologic Data for Bolivian Squirrel Monkeysa Parameter

Sample

Mean

SD

WBC (x 1000/mm 3) RBC (X 106/mm3) Hgb (gm/dl) Hct (%) MCV (p.m3) MCH (Ixg) MCHC (gm/dl)

104 104 103 104 104 103 103

7.9 6.6 12.8 40.9 60.3 19.4 31.6

2.8 0.8 1.2 4.8 6.4 1.4 2.1

Minimum 3.7 3.5 7.9 23 46.7 14.1 22.7

Maximum 25thPercentile 24.4 8.5 16 51.5 70 23.9 35.7

6.0 6.3 12.2 38.6 58.5 18.6 30.5

Median

75th Percentile

7.4 6.6 12.9 41 60.3 19.4 31.7

9.1 7.1 13.6 43.6 62.9 20.3 33.1

aUnpublished data from Squirrel MonkeyBreeding and Research Resource, Universityof South Alabama, 2000. Samples from clinicallynormal squirrel monkeys that had been in the colonyfor 5 years. Data collected in 1985. Note. WBC, white blood cells; RBC, red blood cells; Hgb, hemoglobin; Hct, hematocrit; MCV, mean corpuscularvolume; MCH, mean corpuscularhemoglobin; MCHC, mean corpuscularhemoglobinconcentration.

698

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Table X V I I Serum Chemistry Values for Bolivian Squirrel Monkeys a Analyte

N

Mean

SD

Minimum

Maximum

25th Percentile

Median

75th Percentile

Alkaline phosphatase (PU/liter) GGT (U/liter) Blood urea nitrogen (mg/dl) Lactate dehydrogenase (U/liter) AST (SGOT) (U/liter) ALT (GPT) c (U/liter) Creatine phosphokinase (U/liter) Amylase (U/liter) Cholesterol (mg/dl) Triglycerides (mg/dl) Glucose (mg/dl) Inorganic phosphorus (mg/dl) Albumin (gm/dl) Total bilirubin (mg/dl) Total protein (gm/dl) Calcium (mg/dl) Creatinine (mg/dl) Direct bilirubin (mg/dl) Sodium Potassium CO2

99 99 99 99 97 93 98 94 99 86 100 99 99 87 87 96 97 84 99 99 96

357.9 56.4 38.7 123.2 185.1 184.2 562 515.5 151.4 74.9 102.6 6.9 4.2 0.8 6.9 9.6 0.9 0.2 150.1 5.7 11.1

175.3 150.4 10 111.9 95.3 110.3 1379.8 188.6 64.7 32.7 30.3 7.8 0.6 0.6 1.0 0.9 0.2 0.2 7.2 1.0 3.9

123 5 16.7 9 74 47 54 67 53 28 43 2.2 2.4 0.4 0.6 6.4 0.2 0 131.1 3.9 1.2

939 1329 69.8 721 527 548 9306 1746 542 208 209 18 7.3 4.3 8.7 12 1.6 1.1 169.1 12.0 20

236 21 31.5 60 124 112 85 435 123 51 84 4.3 3.9 0.5 6.6 9.2 0.8 0.06 145.3 5.1 8.8

326 29 37.9 91 159 154 127.5 533 143 66 95 5.9 4.2 0.6 7.1 9.5 0.9 0.1 150.8 5.6 11.3

413 42 45.6 149 190 228 350 570 161 95 120.5 7.1 4.4 0.8 7.5 10.1 1 0.2 154.4 6.2 14.5

aUnpublished data from Squirrel Monkey Breeding and Research Resource, University of South Alabama, 2000. Squirrel monkeys had been in the colony for 5 years and were clinically normal. Data collected in 1985.

Note. GGT, y-glutamyl transferase; AST, aspartate amino transferase; ALT, alanine aminotransferase.

depending on the strain of the parasite used (Galland, 2000). These differences in susceptibility to experimental malaria infections underscore the importance of species identification when using squirrel monkeys. For many years, the squirrel monkey has been recognized as one of the most susceptible nonhuman primate species to experimental infection with Creutzfeldt-Jakob disease (CJD) and other transmissible spongiform encephalopathies (Zlotnik et al., 1974; Brown et al., 1994; Sch~itzl et al., 1997). The susceptibility of squirrel monkeys to experimental CJD infection is be-

Table X V I I I Adult Body Weights for Bolivian Squirrel Monkeys a Weight (gm) Adult Males (Nov-Feb) Males (Mar-Oct) Females

Time Breeding season Nonbreeding season Nonpregnant

10th Percentile

Median

90th Percentile

870

1094

1362

682

892

1062

600

700

798

a Unpublished data from Squirrel Monkey Breeding and Research Resource, University of South Alabama, 2000. Weights obtained from Bolivian squirrel monkeys (Saimiri boliviensis boliviensis) that had been in captivity for at least 5 years. Values will probably vary with housing conditions and diet.

lieved to be genetic; the squirrel monkey P r P gene sequence in squirrel monkeys is 93.8% homologous to the human P r P sequence, which is associated with increased susceptibility to infection in human beings (Sch~itzl et al., 1997). The squirrel monkey has been evaluated as a model for human labor and delivery and for pelvic organ prolapse (POP). In women, the flexed position of the fetus and its rotating course through the birth canal are well documented. Fetal rotation resembling labor and delivery in women has been documented in the squirrel monkey (Stoller, 1995). The similarity of infant delivery in women and squirrel monkeys, and reports of lesions resembling POP in women and in squirrel monkeys (Coates et al., 1995a), suggest that POP in the squirrel monkey may be of a similar etiology. POP afflicts older women, and it is associated with increased parity, with large infants, and with hormonal changes. Similarly, the incidence of POP in squirrel monkeys increases with age and parity, and the severity of the lesions is influenced by seasonal hormonal changes (Coates et al., 1995b). D. 1.

Macaca mulatta: Rhesus Monkeys

Introduction

Rhesus monkeys (Macaca mulatta) are among the least threatened and most biologically diverse monkeys in the world (Southwick and Lindburg, 1986). They are a medium-sized, di-

16. NONHUMANPRIMATES urnal, mostly terrestrial Old World monkey, characterized as having brown to gray fur, lighter undersides, and a mediumlength nonprehensile tail (Fig. 8). Adult females range in weight from 4.4 to 10.9 kg and adult males from 5.5 to 12.0 kg. Their life span is approximately 29 years (Rowe, 1996). Rhesus monkeys have a total of 32 teeth with a dental formula of 2-1-2-3. They have cheek pouches that allow storage of food for mastication at a later time. These pouches originate in the midbuccal area and extend toward the neck. Genetic homology to humans, based on nucleotide base sequences, is 92.7% (VandeBerg, 1995). This genetic similarity, coupled with a susceptibility to the simian immunodeficiency virus (SIV) and the resulting AIDS-like syndrome, makes the rhesus monkey an excellent model for HIV vaccine research and development.

699

forests, in areas extending from Afghanistan to China, Hong Kong, and India, the rhesus monkey has survived in part due to its commensal relationship with humans (Wang and Quan, 1986). It is not uncommon to find large groups of rhesus monkeys inhabiting towns, cities, and temples. In the wild, rhesus monkeys exist primarily in male-dominated, multimale-multifemale groups ranging in size from 10 to 50 members. Females adhere to a strict hierarchical class system with the dominant male changing groups every few years (Rowe, 1996). They are known as a belligerent species. Their most common threat behavior is a wide-open mouth with staring eyes. The submissive animal will scream and bare teeth, or will perform a silent bare-tooth display (Rowe, 1996). 4.

2.

Taxonomy

Rhesus monkeys are a species composed of three subspecies: M. m. mulatta, M. m. vestita and M. m. brachyurus, all of which have a genetic diploid number of 42. Taxonomy, CITES designation, and geographic distribution are presented in Table XIX. 3.

Natural History

Rhesus monkeys inhabit the widest geographical and environmental habitat of any species of nonhuman primate. From elevations surpassing 12,000 feet to lowland tropical rain

Reproduction

Rhesus monkeys are seasonal breeders. The breeding season in captivity is approximately 5 months (mid-September to midFebruary in the Northern Hemisphere), and their gestation period is approximately 164 days (Rowe, 1996). Their menstrual cycle is 28 days with a mean estrous period of 9.2 days (Napier and Napier, 1967). The interbirth interval averages 360 days, and sexual maturity is achieved in females at 3 4 - 4 3 months of age and in males at approximately 38 months of age (JohnsonDelaney, 1994). With vaginal delivery, infant birth weights average 410 gm for females and 450 gm for males (B. Bernacky, unpublished data, 2000).

Fig. 8. Macaca mulatta, adult female rhesus monkeyswith infants. (Photographfrom Christian Abee. Courtesyof the Departmentof ComparativeMedicine, University of South Alabama.)

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

700

Table XIX Macaque Taxonomy, CITES Status, and Distribution a Genus Macaca M. arctoides

Common name(s) Stump-tailed macaque

CITES status

Distribution

II

Bangladesh, Cambodia, China, India, Laos, Malaysia, Myanmar, Thailand, Vietnam

II

Bangladesh, Bhutan, China, India, Laos, Myanmar, Nepal, Thailand, Vietnam

Stumptail macaque Bear macaque M. assamensis

Assamese macaque

M. cyclopis

Taiwan macaque Formosan rock macaque

II

Japan [int], Taiwan

M. fascicularis

Crab-eating macaque Cynomolgus monkey Long-tailed macaque

II

Bangladesh, Brunei, Cambodia, Hong Kong [int], India, Indonesia, Laos (?), Malaysia, Mauritius [int], Myanmar, Palau, Philippines, Singapore, Thailand, USA (?) [int], Vietnam

M. fuscata M. maura

Japanese macaque Moor macaque Celebes macaque Rhesus macaque Rhesus monkey

II II

Hong Kong [int], Japan Indonesia

II

Afghanistan, Bangladesh, Bhutan, China, Hong Kong, India, Laos, Myanmar, Nepal, Pakistan, Thailand, USA [int], Vietnam

M. nemestrina

Pig-tailed macaque Pigtail macaque

II

M. nigra

Sulawesi macaque Celebes black macaque Gorontalo macaque Celebes crested macaque Booted macaque Bonnet macaque Lion-tailed macaque Wanderoo Toque macaque Barbary ape Barbary macaque Short-tailed Tibetan macaque Tibetan macaque Pere David's macaque Tokean macaque Tokean black macaque

II

Bangladesh, Brunei, Cambodia (?), China, India, Indonesia, Laos, Malaysia, Myanmar, Singapore (ex) [int], Thailand, Vietnam Indonesia

II II I

Indonesia India India

II II

Sri Lanka Algeria, Gibraltar [int], Morocco

II

China

II

Indonesia

Assam macaque

M. mulatta

M. ochreata M. radiata M. silenus M. sinica M. sylvanus M. thibetana

M. tonkeana

aI, Species listed in CITES Appendix I are threatened with extinction (endangered); II, Species listed in CITES Appendix II are not currently threatened with extinction but may become so unless trade is strictly regulated. From CITES (2000) and Wilson and Reeder (1993). Note. [int], introduced; (ex), extinct.

5. Laboratory Management

Rhesus monkeys are very adaptable and do well in a laboratory setting. Environmental lighting can be satisfied by providing a 12 hr light-12 hr dark cycle. Their temperature requirements are broad, with an ambient range of 60-85~ Housing methods include in groups, as pairs, or individually. It has been noted that within the laboratory setting the well-being of troopliving primates like the rhesus monkey is enhanced if they are housed in compatible pairs or social groups (Segal, 1989). When forming pairs or groups, consideration must be given to the behavioral influences of age, sex, and social history. A1-

though minor trauma and wounding can occur, a social environment is the preferred setting, especially for younger rhesus monkeys. Adult rhesus monkeys should also be housed socially to encourage species-typical behavior (grooming interaction, play, exercise, manipulation of objects) and decrease the risk of their developing abnormal habits and behaviors (Segal, 1989; Schapiro et al., 1996). Nevertheless, research protocols or other situations may necessitate individual housing. These monkeys should be provided opportunity for olfactory, auditory, and visual stimulation by being housed in rooms with other monkeys (Code of Federal Regulations [CFR], 1999). Strategic rearrangement of caging

701

16. NONHUMAN PRIMATES

within a room can enhance opportunities for stimulation. Inanimate enhancements (toys, structures, feeding devices), although not a replacement for social housing, can relieve boredom and encourage limited activity and exercise. Further benefit can be derived through daily positive interaction with a human caregiver. Many times this type of stimuli can temporarily compensate for short periods of individual housing (CFR, 1999). Extended isolated housing can result in detrimental repetitive behaviors (rocking, self-inflicted trauma, and selfclutching) and should be avoided (Segal, 1989). Orphaned infants can be fostered on replacement mothers, but the success rate is only about 50%. Hand rearing infants can be done utilizing commercially prepared infant formulas but is very time-intensive. The infant is started on a 5% dextrose formula every 2 hr for the first 12 hr. If there is no vomiting, one of the commercial formulas (Primilac, Prosobee) can be started at 50% concentration and be fed every 2 hr for the next 24 hr period. If there is no diarrhea during that 24 hr, the formula concentration can be increased to 75% or full strength. This feeding regime is maintained for 3 weeks at 2 hr intervals, then another 2 weeks at 4 hr intervals. Rice cereal can then be added to the formula for another 2 weeks. By the eighth week most infants will accept a moistened commercial biscuit. The infant should be weaned from the bottle at 6 to 8 weeks of age. The average daily rate of gain for infants of both genders is approximately 6 gm per day (B. Bernacky, unpublished data, 2000). Behavioral differences between the Indian-origin and the Chinese-origin rhesus monkeys have been documented. Starting as neonates, Chinese rhesus monkeys were found to be more temperamental and had a more irritable disposition. This behavior extended into adulthood with animals showing aggression toward humans and conspecifics (Champoux et al., 1994). During development, Chinese male and female juvenile rhesus monkeys were found to be heavier and longer than their Indian counterparts (Champoux et aL, 1997). This pattern reversed itself in adulthood. Also, Chinese rhesus monkeys have been shown to exhibit more adult sexual dimorphism than the Indian rhesus (Clarke and O'Neil, 1999). 6.

Nutrition

In the wild, rhesus monkeys are mainly frugivorous. Their diet has been observed to be 65-70% fruit supplemented with leaves, shoots, roots, bark, fungi, and small invertebrates. The ingestion of soil has been noted to be both a source of nutrients and an aid in digestion (Lindburg, 1980). In captivity they readily adapt to a commercial biscuit diet. These diets are prepared from a variety of ingredients with a guaranteed analysis for crude protein, fat, and fiber. Maintenance rations of 15% protein are adequate for both young growing rhesus monkeys and adults. A 20% protein ration is thought to be of more benefit to a breeding colony (B. Bernacky, personal communication, 2000). The crude fiber of standard diets is typically 6.0 to 8.5%,

but a number of primate facilities are requesting crude fiber measurements of 15% and have reported a decrease in gastrointestinal-related morbidity (R. Carter, personal communication, 2000). The stability of vitamin C in commercial diets has historically been a concern. It is still recommended that the commercial monkey diet be used within 90 days of being manufactured. If biscuits need to be moistened, they should be soaked in fruit juice rather than water because of the propensity of water to deteriorate the vitamin C (LabDiet Product Reference Manual, 1998). The typical daily ration of commercial biscuits for both male and female rhesus monkeys is approximately 2 - 4 % of their body weight. They are usually fed twice daily to help prevent food waste. Providing a variety of novel, supplemental food items is an important form of enrichment that promotes the well-being of rhesus monkeys (ILAR, 1998). Fruits, vegetables, legumes, and seeds are provided to mimic natural foraging substrates and promote species-typical behaviors. Seeds, grains, and legumes can be scattered among bedding substrates (hay, straw, grass) or used in food puzzles to encourage foraging behavior and possibly to reduce aggressive and stereotypic behaviors (Segal, 1989). 7.

Normal Values

Normative hematologic and serum chemistry data for the rhesus monkey have been published by Buchl and Howard (1997). Tables XX-XXIII provide data derived from 527 healthy, domestically bred and reared rhesus monkeys. 8.

Research Uses

Rhesus monkeys are the most common nonhuman primates used in biomedical research. In 1937, they contributed to the identification of the red blood cell Rh factor (Lee, 1993). During the 1950s, they were the laboratory animal models used to investigate, develop, and produce the polio vaccine (Johnsen, 1995). During the 1970s and 1980s, they became the primate models of choice in drug safety and efficacy research. Presently, rhesus monkeys are the preferred models for studying the mechanisms of immunodeficiency diseases. Their susceptibility to SIV and their homology to the human major histocompatibility complex (MHC) class I, II, and TCR genes (Knapp et al., 1997) make them valuable in HIV research. Rhesus monkeys are currently the models of choice for HIV/AIDS vaccine development and study. Rhesus monkeys are also being used extensively in research using a recombinant virus known as simian-human immunodeficiency virus (SHIV). These studies will necessitate improved MHC typing techniques and promote breeding genetically defined rhesus monkeys for use in immunological studies of AIDS vaccine candidates. Other areas of research have included aging, atherosclerosis, alcoholism, diabetes, cancer, and myocarditis (Lee, 1993).

702

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE Table XX Normal Blood Values of Rhesus by Sex, Age, and Gravidity a

Age (years)

Sex

0.05-1 0.05-1 1-2 1-2 2-3 2-3 3-4 3-4 3-4 4-5 4-5 4-5 5-10 5-10 5-10 10+ 10+

F M F M F M F F M F F M F F M F F

Gravidity

Y

Y

Y

Y

N

HCT (%)

HGB (gm/dl)

WBC (>( 103/• 1)

27 27 77 30 50 27 25 11 30 13 20 44 30 44 21 29 22

40.4 __+2.5 41.4 • 3.2 40.1 • 2.4 40.0 • 2.2 39.0 • 2.5 39.7 • 2.0 38.6 _+ 2.3 40.5 • 2.3 40.2 • 2.8 39.5 • 2.5 41.3 • 3.2 41.1 • 2.8 40.3 __+2.6 40.3 • 2.2 42.4 • 2.5 42.1 • 2.6 40.7 • 3.1

12.8 ___0.8 13.1 • 0.8 12.9 • 0.7 12.8 • 0.7 12.6 • 0.8 13.0 • 0.6 12.5 • 0.7 13.0 ___0.8 12.9 • 0.7 12.8 _ 0.7 13.3 ___ 1.0 13.2 • 0.8 12.9 • 0.8 12.9 • 0.7 13.6 • 0.7 13.6 • 0.9 13.5 • 1.1

11.2 • 4.3 9.5 • 3.0 9.8 • 3.4 9.8 • 3.5 9.7 • 3.3 8.9 • 2.1 10.6 • 3.3 8.6 ___ 1.8 10.5 • 2.8 11.6 • 3.1 9.9 • 3.0 10.4 • 2.6 10.3 • 3.3 10.0 • 2.8 11.8 • 2.9 9.6 • 3.1 9.6 • 2.2

RBC ( x 106/-+-1) 6.04 6.18 5.75 5.73 5.49 5.71 5.56 5.73 5.79 5.85 5.91 5.89 5.75 5.79 6.90 6.01 6.81

• • • • • • • • • • • • • • • • •

0.46 0.56 0.36 0.39 0.41 0.31 0.46 0.30 0.42 0.42 0.58 0.35 0.41 0.44 0.34 0.72 0.55

MCV (fl) 67.2 67.1 69.4 69.8 71.1 68.4 69.6 70.8 69.5 67.6 70.2 69.7 70.4 69.8 70.7 70.0 70.3

• 3.9 ___3.0 • 4.2 • 2.6 • 2.6 ___6.1 • 2.9 ___3.1 • 2.4 • 2.5 • 3.1 • 2.8 • 3.6 • 3.5 • 2.2 • 2.6 • 2.5

MCH (pg) 21.3 21.3 22.4 22.4 23.0 22.7 22.6 22.8 22.4 22.0 22.6 22.4 22.4 22.4 22.8 23.7 23.3

• 1.4 ___ 1.1 • 0.9 • 1.0 • 1.1 • 0.9 • 1.0 • 1.2 • 1.1 • 0.8 ___ 1.3 • 0.9 • 1.3 • 1.3 • 0.9 • 1.1 • 1.0

MCHC (gm/dl) 31.6 31.7 32.0 32.0 32.3 32.7 32.4 32.2 31.9 32.6 32.2 32.2 31.9 32.2 32.2 32.4 33.2

• • • • • • • • • • • + • • • • •

0.7 0.8 1.2 0.6 0.6 0.7 0.7 0.5 1.8 0.5 0.6 0.6 0.6 1.0 0.8 1.0 0.8

a From M. D. Anderson Cancer Center, Department of Veterinary Sciences. Samples collected from clinically normal, colony-origin, SPF rhesus monkeys. SPF (specific-pathogen-free), no viral antibody titers to CHV-1, SIV, SRV, or STLV.

Note. CHV-1, Cercopithecine herpesvirus 1; SIV, Simian Immunodeficiency Virus; SRV, Simian Retrovirus; STLV, Systemic-T-Lymphotrophic Virus.

Table XXI Normal White Blood Cell Counts and Corresponding Absolute Values for Rhesus Monkeys a Age (years)

Sex

0.05-1 0.05-1 1-2 1-2 2-3 2-3 3-4 3-4 3-4 4-5 4-5 4-5 5-10 5-10 5-10 10+ 10+

F M F M F M F F M F F M F F M F F

Gravidity

Y

Y

Y

Y

N

WBC (• 103/1xl)

27 27 77 30 50 27 25 11 30 13 20 44 30 44 21 29 22

11.2 • 4.3 9.5 • 3.0 9.8 • 3.4 9.8 • 3.5 9.7 • 3.5 8.9 • 2.1 10.6 • 3.3 8.6 • 1.8 10.5 ___2.8 11.6 __+3.1 9.9 • 3.0 10.4 • 2.6 10.3 • 3.3 10.0 • 2.8 11.8 • 2.9 9.6 ___3.1 9.6 __+2.2

SEG (Ixl) 5999 4794 5959 6369 5639 5118 6601 5646 6034 7678 7637 6374 6922 7301 7911 6536 6706

___3171 • 2490 • 3028 • 2861 • 2890 • 1705 • 2954 • 1728 ___2316 • 2960 • 2778 _+ 2322 __+3160 • 2823 ___3580 • 3262 • 2510

Lymphocytes (l~l)

Monocytes (txl)

Eosinophils (Ixl)

4749 4368 3439 2845 3400 3528 3630 2491 3909 3588 1832 3587 3650 2088 3103 2618 2210

261 208 277 251 324 162 185 343 371 191 272 288 359 324 440 236 326

66 29 41 29 79 29 89 81 119 77 43 128 117 69 144 162 250

• 2665 • 1914 • 1729 • 1609 • 1711 • 1692 • 1540 • 1226 • 1238 • 1102 • 801 • 1589 ___4663 • 712 ___ 1840 • 1072 ___ 1201

___235 • 165 • 269 • 249 __+260 • 151 • 185 ___387 • 391 • 206 • 159 • 247 • 258 • 243 ___296 • 237 + 300

• 101 • 52 • 81 • 49 • 121 • 52 • 150 • 109 ___220 • 105 • 61 • 203 • 198 • 84 ___202 • 242 • 225

Band (1~1) 109 50 82 276 163 30 35 37 94 97 123 55 92 137 216 58 30

• 205 • 92 • 122 • 476 • 335 • 43 • 77 • 62 • 132 • 154 • 191 • 110 ___ 172 • 177 • 333 ___96 • 59

Basophils (1~1) 35 19 4 5 17 6 12 0 5 0 2 7 14 12 3 16 38

• 76 • 42 • 17 • 19 • 38 • 22 • 58 • 0 • 20 ___0 • 11 • 28 _+ 59 • 34 • 15 ___47 • 70

a From M. D. Anderson Cancer Center, Department of Veterinary Sciences. Samples collected from clinically normal, colony-origin, SPF rhesus monkeys. SPF (specific-pathogen-free), no viral antibody titers to CHV-1, SIV, SRV, or STLV.

Note. CHV-1, Cercopithecine herpesvirus 1; SIV, Simian Immunodeficiency Virus; SRV, Simian Retrovirus; STLV, Systemic-T-Lymphotrophic Virus.

703

16. N O N H U M A N P R I M A T E S Table XXH Normal Serum Biochemical Values for Rhesus Monkeys a

Age (years)

Sex

0.05-1 0.05-1 1-2 1-2 2-3 2-3 3-4 3-4 3-4 4-5 4-5 4-5 5-10 5-10 5-10 10+ 10+

F M F M F M F F M F F M F F M F F

Age (years)

Sex

Gravidity N

0.05-1 0.05-1 1-2 1-2 2-3 2-3 3-4 3-4 3-4 4-5 4-5 4-5 5-10 5-10 5-10 10+ 10+

F M F M F M F F M F F M F F M F F

27 27 77 30 50 27 25 11 30 13 20 44 30 44 21 29 22

Gravidity N

Y

Y

Y

Y

Y

Y

Y

Y

27 27 77 30 50 27 25 11 30 13 20 44 30 44 21 29 22

Bilirubin (mg/dl)

ALT (U/liter)

AST (U/liter)

0.2 0.2 0.1 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.1 0.2

39 42 37 38 37 35 36 42 37 31 29 41 35 28 33 40 27

48 47 49 47 44 51 43 32 40 38 31 39 32 29 38 41 42

___0.2 __. 0.2 __. 0.1 _+ 0.1 ___0.1 4- 0.1 ___0.1 ___0.1 ___0.1 4- 0.1 4- 0.1 ___0.1 4- 0.1 4- 0.1 4- 0.2 4- 0.1 4- 0.2

Globulin (gm/dl) 2.8 2.8 2.8 2.6 2.6 2.2 2.8 3,2 2.7 2.9 3.4 2.8 3.3 3.8 3.3 3.4 4.1

_ 0.3 ___0.5 4- 0.7 ___0.5 ___0.3 ___0.5 ___0.4 _+ 0.3 ___0.4 ___0.6 ___0.4 __+0.4 ___0.4 ___0.4 ___0.5 ___0.6 4- 0.4

___9 __. 9 __. 10 + 9 -+- 10 ___ 11 ___ 14 ___42 ___9 ___ 8 4- 8 4- 12 ___7 4- 13 4- 8 4- 12 4- 10

Cholesterol (mg/dl) 165 179 165 161 146 151 141 73 138 152 75 143 150 81 155 180 81

_ 30 ___29 4- 30 4- 30 4- 27 ___22 4- 20 ___35 4- 20 ___30 4- 18 ___23 ___34 ___33 ___22 _ 42 ___24

-+- 11 ___ 10 __. 13 4- 13 ___ 15 ___ 13 4- 12 __. 10 _ 13 ___ 10 4- 7 4- 13 4- 8 4- 10 4- 10 4- 11 4- 12

GGT (U/liter) 75 77 71 67 59 72 47 46 57 49 42 63 77 45 55 41 41

Alkaline phosphatase (U/liter)

4- 18 4- 14 ___ 16 ___ 19 ___ 13 ___ 13 ___7 ___ 12 ___20 ___9 ___ 12 ___ 17 __. 14 4- 14 ___ 16 4- 6 ___8

646 727 578 527 479 529 384 197 541 288 134 456 180 110 203 118 85

___ 136 +_ 148 __. 149 __. 146 ___ 136 ___ 177 + 121 4- 58 ___ 144 ___71 4- 56 _+ 115 4- 63 4- 64 4- 84 4- 40 4- 58

Triglycerides (mg/dl) 44 52 43 41 44 46 45 48 50 55 53 45 45 53 43 58 54

CR (mg/dl)

_ 14 4- 35 4- 11 4- 14 ___ 16 4- 11 ___ 16 ___ 16 ___ 16 4- 23 ___21 ___ 14 ___ 19 ___22 ___ 18 _ 34 ___21

0.6 0.7 0.7 0.7 0.7 0.7 0.8 0.7 0.9 0.9 0.9 1.0 0.9 0.8 1.1 0.9 0.8

4- 0.2 4- 0.2 ___0.1 ___0.1 ___0.1 ___0.1 4- 0.2 _ 0.1 4- 0.1 4- 0.1 4- 0.3 4- 0.2 4- 0.1 4- 0.1 4- 0.1 4- 0.2 4- 0.1

Calcium (mg/dl) 10.5 10.6 10.5 10.3 10.1 9.7 10.4 9.8 10.7 10.4 9.8 10.8 10.7 9.2 10.4 10.2 8.8

Glucose (mg/dl)

BUN (mg/dl)

82 88 74 74 64 60 67 68 73 76 58 77 66 57 67 74 55

18 20 21 21 21 24 19 12 18 21 15 19 19 12 20 21 15

4- 16 __. 20 4- 15 4- 12 4- 14 4- 10 ___ 14 4- 7 ___ 16 4- 18 4- 11 ___ 13 4- 13 4- 12 4- 16 4- 15 ___ 18

PHOS (mg/dl)

_ 1.0 4- 0.8 _ 1.0 4- 0.5 4- 0.7 ___0.7 4- 1.6 ___0.7 ___0.6 ___0.5 ___0.6 ___0.8 ___0.9 ___0.9 ___0.9 _ 1.1 ___0.8

5.5 6.4 5.3 5.1 5.6 6.2 5.1 4.1 5.2 4.4 3.6 4.7 3.9 3.2 3.6 3.5 3.0

-+- 1.2 ___ 1.1 ___ 1.0 4- 0.7 _+ 1.0 ___0.7 4- 0.9 ___0.7 ___0.6 4- 1.1 ___0.9 + 0.9 ___0.9 4- 0.6 ___0.9 __+ 1.1 ___ 1.4

4- 5 4- 5 ___6 __. 5 __. 4 4- 5 ___3 4- 2 ___3 ___3 4- 5 4- 5 ___3 4- 4 4- 3 4- 4 __+3

CK (U/liter) 386 366 436 507 446 423 544 305 491 447 444 425 446 420 413 442 286

4- 220 ___ 147 ___227 ___297 _ 289 4- 276 ___289 4- 93 ___250 4- 163 ___232 4- 184 ___ 187 4- 246 _+ 163 4-4-231 4- 225

Total protein (gm/dl) 7.1 7.3 7.2 7.0 7.1 7.0 7.3 6.9 7.3 7.6 7.0 7.5 7.7 7.2 7.8 7.8 7.4

___0.3 __. 0.5 ___0.8 __. 0.6 4- 0.3 ___0.4 ___0.4 ___0.4 ___0.4 4- 0.4 4- 0.5 4- 0.4 4- 0.5 4- 0.5 ___0.5 4- 0.4 ___0.5

Albumin (gm/dl) 4.3 7.5 4.5 4.5 4.5 4.7 4.5 3.8 4.6 4.6 3.6 4.6 4.5 3.3 4.5 4.4 3.3

LD (U/liter) 467 4- 186 427 ___ 166 443 ___ 180 422 4- 198 434 ___211 542 ___ 173 372 4- 129 433 _+ 197 427 ___222 404 4- 139 305 4- 61 381 + 265 297 4- 83 370 __+ 143 363 ___ 173 393 _ 171 577 ___229

___0.3 ___ 18 _+ 0.3 4- 0.3 ___0.4 4- 0.4 ___ 1.4 ___0.5 ___0.3 4- 0.3 4- 0.5 4- 0.4 4- 0.5 4- 0.4 4- 0.4 4- 0.6 4- 0.3

UA (mg/dl) 0.2 0.3 0.2 0.2 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.2 0.1 0.1

4- 0.1 4- 0.3 ___0.2 4- 0.1 ___0.1 ___0.1 4- 0.1 ___0.1 ___0.1 4- 0.1 ___0.1 4- 0.1 4- 0.1 4- 0.1 ___0.2 ___0.1 + 0.1

aFrom M. D. Anderson Cancer Center, Department of Veterinary Sciences. Samples collected from clinically normal, colony-origin, SPF rhesus monkeys. SPF (specific-pathogen-free), no viral antibody titers to CHV-1, SIV, SRV, or STLV.

Note. CHV-1, Cercopithecine herpesvirus 1; SIV, Simian Immunodeficiency Virus; SRV, Simian Retrovirus; STLV, Systemic-T-Lymphotrophic Virus.

E. Macaca fascicularis: Cynomolgus Monkeys

the nonprehensile

t a i l is e q u a l to t h e h e a d a n d b o d y l e n g t h o r

l o n g e r ( N a p i e r a n d N a p i e r , 1 9 6 7 ) . T h e s p e c i e s is s m a l l e r t h a n

1.

the rhesus monkey

Introduction

w i t h a d u l t f e m a l e s r a n g i n g in w e i g h t f r o m

2 . 5 t o 5 . 7 k g a n d m a l e s f r o m 4 . 7 to 8.3 k g . T h e l o n g e s t l i f e s p a n Cynomolgus the long-tailed

monkeys

(Macaca fascicularis) are k n o w n as

or crab-eating

macaques.

s c r i b e d as h a v i n g g r a y t o r e d - b r o w n

The

s p e c i e s is d e -

fur, l i g h t e r u n d e r p a r t s ,

a

recorded

is 3 7 . 1 y e a r s ( R o w e ,

1996). Cynomolgus

monkeys

have 32 teeth, and cheek pouches. The thumb of the cynomolgus

monkey

pointed crest to the crown of the head, females with a beard, and

grooming,

m a l e s w i t h c h e e k w h i s k e r s ( R o w e , 1 9 9 6 ) ( F i g . 9). T h e l e n g t h o f

Hong, 1995).

has

sexual

true

opposability,

behavior,

and

which

locomotion

aids

in f e e d i n g ,

(Turnquist

and

704

BRUCE J. BERNACKY, SUSAN V. GIBSON, M I C H A L E E. K E E L I N G , AND C H R I S T I A N R. A B E E

Table XXIII Normal Serum Electrolyte Values for Rhesus Monkeys a Age (years)

Sex

0.05-1 0.05-1 1-2 1-2 2-3 2-3 3-4 3-4 3-4 4-5 4-5 4-5 5-10 5-10 5-10 10+ 10+

F M F M F M F F M F F M F F M F F

Gravidity

Y

Y

Y

Y

Chloride (C1-) (mEq/liter)

N 27 27 77 30 50 27 25 11 30 13 20 44 30 44 21 29 22

112 113 113 114 113 112 114 113 113 116 112 113 115 114 113 116 115

Sodium (Na § (mEq/liter)

___3 ___2 _ 4 ___2 • 2 +_ 2 ___2 • 2 ___2 ___3 __+3 ___3 ___3 ___3 ___2 • 3 • 2

148 148 148 148 148 148 147 144 148 147 142 148 148 143 148 150 145

_ 3 ___3 • 3 _ 2 _ 2 ___2 ___2 ___ 3 • 3 ___3 • 3 ___ 3 ___ 3 • 3 ___3 _ 2 _ 3

Potassium (K § (mEq/liter) 4.3 4.3 3.8 4.0 3.8 3.7 4.0 4.0 4.0 3.9 4.2 4.0 4.0 4.1 3.9 4.0 3.9

+__0.6 ___0.6 ___0.6 ___0.5 • 0.4 ___0.3 • 0.3 ___0.4 ___0.3 • 0.2 ___0.6 ___0.4 ___0.5 • 0.4 ___0.3 • 0.4 • 0.3

aFrom M. D. Anderson Cancer Center, Department of Veterinary Sciences. Samples collected from clinically normal, colony-origin, SPF rhesus monkeys. SPF (specific-pathogen-free), no viral antibody titers to CHV-1, SIV, SRV, or STLV. Note. CHV-1, Cercopithecine herpesvirus 1; SIV, Simian Immunodeficiency Virus; SRV, Simian Retrovirus; STLV, Systemic-T-Lymphotrophic Virus.

2.

Taxonomy

Cynomolgus monkeys are a species composed of 10 subspecies. Former names have been M. cynomolgus and M. irus (Rowe, 1996). Like other Old World monkeys, their diploid number is 42.

adults following an act of aggression typically is manifested by an increase in self-grooming, shaking, and scratching. Reconciliation between these adults is shown by the subservient member staring into the eyes and touching the genitals of the dominant member (Rowe, 1996). 4.

3.

Natural History

Cynomolgus monkeys have been found to inhabit coastal, mangrove, swamp, and riverine forests at elevations greater than 6000 feet (Rowe, 1996). Their geographical range is primarily Southeast Asia and includes Indochina, Myanmar, Indonesia, and the Philippines (Rowe, 1996). They have been introduced to the island of Mauritius. Cynomolgus monkeys are found to be less cold tolerant than M. mulatta or M. fuscata (Lindburg, 1980). These diurnal and arboreal primates are also known to be excellent swimmers (Rowe, 1996). Their diet is considered omnivorous due to the consumption of insects, frogs, and crabs, but they can be highly frugivorous. In some instances, fruit has been noted to comprise 90% of their diet (Lindburg, 1980). Their social structure consists of multimale-multifemale groups with a less stringent hierarchical dominance ranking involving males when compared to other macaque species (Rowe, 1996). Groups are often led by a number of high-ranking males. Within the groups, infants of the same age tend to play together, as do infants born to higher-ranking adults. Tension among

Reproduction

Cynomolgus monkeys are not seasonal breeders and are capable of mating and birthing throughout the year. Their gestation period is approximately 160 to 170 days (Rowe, 1996). They have a 28-day menstrual cycle with a mean estrus length of 11 days (Napier and Napier, 1967). The interbirth interval is approximately 390 days. Sexual maturity occurs at approximately 46 months of age for females and 4 2 - 6 0 months of age for males (Delaney, 1994). Infants are born black in color and transition to gray after the first few months (Rowe, 1996). Infant birth weights range from 260 to 310 gm for females and 340 to 400 gm for males (Frost, personal communication, 2000). 5.

Laboratory Management

Laboratory management is similar to that for the rhesus monkey; therefore, the reader is referred to Section III,D,5. 6.

Nutrition

Nutrition is similar to that for the rhesus monkey. The reader is referred to Section III,D,6.

16. NONHUMAN PRIMATES

705

Table XXIV Normal Hematologic and Chemistry Values for Macaca fascicularis a

Test name

Fig. 9. Macaca fascicularis, adult cynomolgus monkey. (Photograph from Pat Frost. Courtesy of the Southwest Foundation of Biomedical Research.)

7.

Normal Values

N o r m a t i v e h e m a t o l o g i c and s e r u m c h e m i s t r y data for the c y n o m o l g u s m o n k e y are p r e s e n t e d in Table XXIV.

8.

Research Uses

C y n o m o l g u s m o n k e y s are the s e c o n d m o s t c o m m o n l y u s e d n o n h u m a n p r i m a t e in b i o m e d i c a l research. M o r e c y n o m o l g u s m o n k e y s are being p u r p o s e - b r e d in the U n i t e d States than ever before (Erwin et al., 1995). C y n o m o l g u s m o n k e y s are the prim a r y m o d e l s used in reproductive b i o l o g y r e s e a r c h ( H e n d r i c k x and D u k e l o w , 1995). T h e y are u s e d in other r e s e a r c h fields as well, i n c l u d i n g cancer, drug, atherosclerosis, c y t o m e g a l o v i r u s ( C M V ) , plague, tuberculosis, and retroviral studies (Lee, 1993).

Glucose Sodium Potassium Chloride BUN Creatinine BUN/Creatinine Calcium Phosphate Total Protein Albumin Globulin Alb/Glob ratio Total bilirubin Alkaline phosphatase LDH Cholesterol SGOT SGPT Triglycerides Uric acid WBC Polysomes Band Lymphocytes Monocytes Eosinophils Basophils Platelet count RBC Male Female HGB Male Female HCT Male Female MCV Male Female MCH Male Female MCHC Male Female

Observed range

Mean

Units

42-111 135-154 3.4-6.3 97-113 5-25 0.5-1.3 5 - 44 8.3-10.9 1.4-6.7 6.5-8.7 2.6-3.4 2.1 - 5.9 0.4-2.4 0.1-0.5 62-680 174-975 42-210 16 - 64 11-88 6-94 0.1-0.3 4.5-18.3 1568-13,986 0-470 999-10,551 0-1175 0 - 999 0-0 195-357

63 147 4.3 107 13 0.8 24 9.7 4.5 7.6 3.9 3.6 1.2 0.2 207 562 106 35 38 30 0.15 10.7 6499 56 3593 309 239 0 269

mg/dl mmol/liter mmol/liter mmol / liter mg/dl mg/dl ratio mg/dl mg/dl gm/dl gm/dl gm/dl ratio mg/dl IU/liter IU/liter mg/dl IU/liter IU/liter mg/dl mg/dl x 103121 Absolute Absolute Absolute Absolute Absolute Absolute x 103/121

80 80 80 80 100 80 80 80 80 80 80 80 80 80 80 80 80 100 100 60 60 80

80

3.5-6.9 4.1-6.7

5.9 5.4

X 106/121

80

9.6-13.3 8.2-12.5

11.7 10.2

gm/dl

80

24.0-41.0 25.0-40.0

35.9 32.0

gm/dl

80

54.0 -75.0 46.0-66.0

61.0 60.0

femtoliter

80

15.7-21.7 14.9-21.6

20.4 19.0

picogram

80

29.1-36.4 29.8-33.8

32.4 31.6

gm/dl

80

a From Charles River Laboratories. Note. BUN, blood urea nitrogen; Alb/Glob, Albumin/Globulin; LDH, lactate dehydrogenase; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase; WBC, white blood cells; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration.

706

BRUCE J. BERNACKY,SUSANV. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

F. Papio spp.: Baboons 1.

Introduction

Baboons are the largest Old World nonhuman primates (Fig. 10). The baboon's large size allows biomedical devices made for humans, such as angioplasty catheters and vascular shunts, to be tested without modification of size. Baboons have marked sexual dimorphism; the male can be up to twice as large as the female. Baboons are diurnal and primarily terrestrial with some species also having an arboreal component. They have an immune system similar to that of humans in that they have the same immunoglobulin G (IgG) subclasses: 1, 2, 3, and 4. This similarity makes them an excellent model for vaccine development.

2.

Three different methods have been used to classify species within the genus Papio. The method used in Table XXV, and in this chapter, shows the genus divided into five species. Four of these species are considered savanna baboons: P. anubis, P. cynocephalus, P. papio, and P. ursinus. The remaining species, P. hamadryas, is sometimes referred to as a desert baboon. In a classification scheme that is currently increasing in usage in the scientific community, there is only one species, P. hamadryas, within the genus Papio, with five subspecies. In this system, the nomenclature would be P. hamadryas anubis, P. hamadryas cynocephalus, and so on. An older form of nomenclature splits the genus into two species: P. cynocephalus and P. hamadryas. In this latter system, the savanna baboons are all subspecies of P. cynocephalus, e.g., P. cynocephalus anubis. 3.

Fig. 10. Papioanubis, adult olive baboon. (Photograph from Maria Lang. Courtesy of the Biologic Resources Laboratory, University of Illinois at Chicago.)

Taxonomy

Natural History

Baboons are widely distributed throughout sub-Saharan Africa from Senegal and Sudan to the Cape of Good Hope, except for the main forested areas. Savanna baboons occupy a wide range of habitats, including semidesert, savanna, scrubland, woodlands, highland grass, and gallery forest. Hamadryas baboons inhabit wooded or subdesert steppe. Availability of food and water may define the size of the home range or territory. For example, baboons in forest habitats where fruit is the major portion of the diet have smaller home ranges and higher population densities than baboons that feed on grass and roots in arid savanna habitats (Melnick and Pearl, 1987). Baboons have strict social hierarchies. The savanna species have a multimale-multifemale social structure. Group sizes usually range from 20 to 40 animals and contain many more females than males. Females remain with their natal group and form the stable core of the social group with a linear type of dominance hierarchy (Altmann et al., 1977). These groups contain more than one male, and there is competition for access to estrous females within the group. Males disperse from natal groups around puberty. Male-male interactions are usually more aggressive than affiliative. Mating access to estrous females is determined as much by the male's length of stay in the social group, alliances with females, and female choice as by male-male competition and dominance (Williams and Bernstein, 1995). In contrast, the hamadryas baboon forms a one-male social unit consisting of one male, several females, and their offspring. The male is the focus of attention in the one-male unit and receives most of the grooming. Males actively herd female members and prevent them from straying too far from the group. Several one-male units, usually two to three, may associate with each other to form a clan (Abegglen, 1984). In clans, the adult males usually are related. Clans frequently travel and forage together. Social interactions occur more frequently within a clan

707

16. NONHUMAN PRIMATES

Table XXV

Baboon Taxonomy,CITES Status, and Distributiona Genus Papio

Common name(s)

CITES status

Distribution

P. anubis

Olive baboon

II

Benin, Brukina Faso, Burundi, Cameroon, Central African Republic, Chad, Congo, Cote d'Ivoire, Eritrea, Ethiopia, Ghana, Guinea (?), Kenya, Libya, Mali, Mauritania (?), Niger, Nigeria, Rwanda, Sierra Leone (?), Somalia (?), Sudan, Tanzania, Togo, Uganda, Zaire

P. cynocephalus

Yellow baboon

II

Angola, Ethiopia, Kenya, Malawi, Mozambique, Somalia, Tanzania, Zaire, Zambia

P. hamadryas

Olive baboon Yellow baboon Guinea baboon Chacma baboon Hamadryas baboon

II

Djibouti, Egypt (ex), Eritrea, Ethiopia, Saudi Arabia, Somalia, Sudan, Yemen

P. papio

Guinea baboon Western baboon

II

Gambia, Guinea, Guinea-Bissau (?), Liberia (?), Mali, Mauritania, Senegal, Sierra Leone

P ursinus

Chacma baboon

II

Angola, Botswana, Lesotho, Malawi, Mozambique, Namibia, South Africa, Swaziland, Zambia, Zimbabwe

aI, Species listed in CITES Appendix I are threatened with extinction (endangered); II, Species listed in CITES Appendix II are not currently threatened with extinction but may become so unless trade is strictly regulated. From CITES (2000) and Wilson and Reeder (1993). Note. (ex), extinct.

than between clans. Several clans, one-male units, and single males can come together to form a stable social unit called the band (Kummer et al., 1981; Sigg et al., 1982; Abegglen, 1984). Social interactions are usually restricted to band members, and bands forage as an autonomous unit. Female bonds are strongest within the band. Troops consist of several bands that come together at sleeping sites or spots of limited resources such as water holes. Baboons lack birth seasonality in the wild and in captivity. Sexual maturity generally occurs at 4 - 5 years of age for females and 4 - 7 years of age for males (Table XXVI). Gestation averages 6 months and interbirth intervals range from 1 to 3 years. Females that experience abortions, stillbirths, or neonatal deaths have shorter interbirth intervals than those that have a surviving live birth. There is lactational amenorrhea. Savanna and hamadryas baboons are general feeders with diets consisting primarily of different parts and types of vegetation and fruit. They consume a variety of plant material, including roots, bulbs, tubers, corms, rhizomes, flowers, leaves and grasses, twigs, bark, seeds, and tree gum. They also eat insects and small vertebrates such as hares, vervets, infant gazelles, and dik-diks (Stolz and Saayman, 1970; Strum, 1975). 4.

Reproduction

Female baboons are capable of breeding continuously throughout the year. The females have prominent perineal sex

skins, which allows assessment of both their ovarian function and their pregnancy status with relative ease. There is a menstrual cycle associated with swelling and color change of the sex skin. The initial turgescence or swelling takes an average of 4 days and is followed by increasing edematous distension until the sexual skin has no wrinkles and develops an intense red color (Hendrickx, 1971). Turgescence lasts 13-21 days depending on the species of baboon (Gauthier, 1999). Maximum turgescence is associated with the hormonal changes that occur with ovulation (Bercovitch, 1985). Initial deturgescence is characterized by loss of color, decreased swelling, and an increase in wrinkles in the perineum. A quiescent stage of about 12 days follows in which the sexual skin has many wrinkles, a dull surface, and little color (Hendrickx, 1971). The length of the menstrual cycle or intermenstrual interval varies among species, between individuals of each species, and with the age of the individual (Birrell et al., 1996; Gauthier, 1999). Gauthier (1999) summarized several reports describing intermenstrual cycle length of wild and captive baboon species. Captive P. papio had the shortest intermenstrual interval, 29.8 _+ 4.1 days; and feral P. anubisthe longest, 40.1 ___6.9 days. Birrell et al. (1996) reported that P. hamadryas females less than 5 years of age and females over 10 years of age had an increased average intermenstrual cycle length of 39 days, whereas animals of prime breeding age, from 5 to 10 years old, had an average cycle length of 34 days. Ovarian cyclicity can be determined by monitoring menses,

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

708

Table XXVI Sexual Maturity, Gestation, and Interbirth Intervals of Baboons in the Wild and in Captivity Sexual maturity (years of age) Species

Male

Female

Gestation (days)

interval (months)

Interbirth

12-34

Papio anubis a

5-7

4.5

180

P. cynocephalus b

4-7

5-6

21 (mean) 11 (unsuccessful birth) 22 (successful birth) 182.2_ 3.3 11 (unsuccessful (SD) birth) 13 (successful birth) 187 18-24 172 12-36, 24 (mean)

P. papio c

3.8 +_ 0.8

P. ursinus d

3.2

P. hamadryas e P. hamadryas f

4.7- 6.8

4 -5, 4.3 (mean) 3.4

175

182.3 _ 4.8

13

(SD) aWild population (Nicholson, 1982; Scott, 1984). bWild population (Altmann et al., 1977). CCaptive population (Gauthier, 1999). aWild population (Devore and Hall, 1965; Harvey et al., 1987). eWild population (Sigg et al., 1982; Harvey et al., 1987). fCaptive population (Birrell et al., 1996).

cyclic changes in sex skin, or hormone determinations of blood, urine, or feces. In singly caged animals, menses may be followed by visual examination of the external genitalia for fresh blood or by the use of vaginal swabs to obtain smears to examine for blood. Visual examination for blood is the least accurate method. Baboons can be trained to present for daily vaginal swabs, which will detect menstruation in approximately 95% of cycles (Hendrickx, 1971). Recording daily changes in the sex skin is a very accurate method of following the ovarian cycle and can be used in baboons that are singly or group-housed. Ovulation usually occurs on day 1 ' 2 before deturgescence (Wildt et al., 1977; Shaikh et al., 1982); the third day prior to deturgescence is the optimal day for mating. Daily blood or urine collection for hormone determinations by enzyme immunoassay (EIA) or radio immunoassay (RIA) can provide a precise date for ovulation but are more time-consuming and expensive methods of determining cyclicity. For investigators monitoring baboon troops in the wild, a combination of daily observation of sex skin, collection of individual fecal samples for future hormone evaluation, and behavioral observation of mounting and copulation can provide an accurate retrospective evaluation of cyclicity and even conception date (Wasser, 1996; Wasser et al., 1998).

Pregnancy can be determined as early as gestation day (G.D.) 15-18 by detection of chorionic gonadotropin in plasma or urine (Hodgen and Neimann, 1975; Shaikh et al., 1976; Fortman et al., 1993). Detection of pregnancy is possible by ultrasound examination by G.D. 18-21 (Herring et al., 1991) and by bimanual palpation by G.D. 20-21 (Hendrickx and Dukelow, 1995). During bimanual palpation, a gloved finger of one hand is inserted in the rectum while the other hand is placed on the abdomen, grasping the uterus. As with ultrasound, this procedure requires that the animal be anesthetized. Artificial insemination (Gould and Martin, 1986), in vitro fertilization (Clayton and Kuehl, 1984), and transfer of cryopreserved embryos (Pope et al., 1986) have all resulted in normal pregnancy and live births in baboons. Gestation in the baboon ranges from 164 to 186 days (Henrickson, 1985). Births are usually of singletons. The single discoid placenta is similar to that of humans. Reported birth weights range from 854 gm (P. cynocephalus) to 1068 gm (P. anubis) (Harvey et al., 1987). McMahan et al. (1976) reported that male P. cynocephalus infants were heavier, mean weight 920 gm, than females, mean weight 830 gm, although the weight ranges of both sexes had considerable overlap. Infants are carried in a ventral-ventral position by the dam when moving. There is no consistent alloparental behavior; on occasion, adult male hamadryas baboons have fostered infants if the dam has died. Infants are usually weaned at 6 months of age. Interbirth interval is decreased in individuals that have unsuccessful pregnancies. 5.

Laboratory Management

Baboons adapt readily to a variety of housing situations, whether standard indoor laboratory cages, indoor/outdoor small-group housing, or large outdoor corrals. Caging should be sufficient to allow for normal posture and movement. Adaptation of single cages with "grooming panels" allows normal grooming behavior between individually housed baboons in adjacent but separate cages (Crockett and Hefferman, 1998). In these modified cages, the possibility of trauma between animals is reduced while contact is still permitted between animals with tethers, indwelling catheters, or other research requirements that preclude social housing. Timed matings are used to produce fetuses of known age and stage of development. Reported conception rates (number of conceptions/number of breedings) vary from 35% (Moore, 1975) to 48% when matings were performed on the third day following deturgescence (Hendrickx and Kraemer, 1969). The annual conception rate using timed matings usually exceeds 70%. Harem breeding has been used successfully in groups of baboons consisting of 1-2 adult males with 20-25 adult females in outdoor cages (Moore, 1975). Breeding groups required several days to establish a social structure. Although fighting occurred, wounds were minor. Pregnant animals were removed

709

16. NONHUMAN PRIMATES Table XXVII Hematologic Reference Ranges in Baboons a Individual caged baboons Value

Gang-caged baboons

Corralled baboons

N

Mean

2 S.D.

2 S.D. range

N

Mean

2 S.D.

2 S.D. range

Mean

2 S.D.

2 S.D. range

White blood cell count ( x 103)

c 89 M 44 F 45

9.6 9.2 10.0

5.8 6.1 5.5

3.8-15.4 3.1-15.3 4.5-15.5

c 62 M 24 F 38

9.3 8.1 10.1

6.5 4.8 6.9

2.8-15.8 3.3-12.9 3.2-17.0

c 30 M15 F15

15.3 14.6 16.0

11.4 12.8 10.2

3.9-26.7 1.8-27.4 5.8-26.2

Red blood cell count (Xl06)

c 90 M 45 F 45

4.95 5.05 4.86

0.64 0.64 0.59

4.31-5.59 4.41-5.69 4.27-5.45

c 62 M 24 F 38

4.88 4.99 4.82

0.78 0.80 0.75

4.10-5.66 4.19-5.79 4.07-5.57

c 30 M15 F15

4.65 4.79 4.51

0.82 0.82 0.75

3.83 -5.47 3.97-5.61 3.76-5.26

Hemoglobin (gm/dl)

c 89 M 44 F 45

12.6 12.9 12.3

1.7 1.5 1.7

10.9-14.3 11.4 - 14.4 10.6-14.0

c 62 M 24 F 38

12.5 12.9 12.2

2.0 2.0 1.9

10.5-14.5 10.9-14.9 10.3-14.1

c 30 M15 F15

11.9 12.4 11.3

2.2 2.1 1.8

9.7-14.1 10.3-14.5 9.5-13.1

Hematocrit (%)

c 89 M 44 F 45

38.2 39.0 37.4

5.0 4.5 5.0

33.2-43.2 34.5-43.5 32.4-42.4

c 62 M 24 F 38

38.3 39.3 37.7

5.5 5.6 5.2

32.8-43.8 33.7-44.9 32.5-42.9

c 30 M15 F15

36.5 38.0 34.9

7.2 7.2 5.9

29.3-43.7 30.8-J15.2 29.0-40.8

Mean cell volume (fl)

c 90 M 45 F 45

77.0 76.9 77.1

5.8 5.2 6.3

71.2-82.8 71.7-82.1 70.8-83.4

c 62 M 24 F 38

78.6 78.9 78.4

5.6 6.6 4.8

73.0-84.2 72.3-85.5 73.6-83.2

c 30 M14 F15

78.4 80.0 77.5

6.7 3.3 6.9

71.7-85.1 76.7-83.3 70.6-84.4

Mean cell hemoglobin (pg)

c 90 M 45 F 45

25.3 25.4 25.3

1.8 1.7 1.9

23.5-27.1 23.7-27.1 23.4-27.2

c 62 M 24 F 38

25.5 25.9 25.3

2.1 2.4 1.8

23.4 -27.6 23.5-28.3 23.5-27.1

c 30 M15 F15

25.6 26.0 25.2

2.5 2.3 2.6

23.1-28.1 23.7-28.3 22.6-27.8

Mean cell hemoglobin concentration (gm/dl)

c 90 M 45 F45

32.9 33.1 32.8

1.3 1.5 1.1

31.6-34.2 31.6-34.6 31.7-33.9

c 62 M 24 F 38

32.5 32.8 32.3

1.4 1.1 1.4

31.1-.33.9 31.7-33.9 30.9-33.7

c 30 M15 F15

32.6 32.7 32.5

1.7 1.7 1.7

30.9-34.3 31.0-34.4 30.8-34.2

Red blood cell distribution width (%)

c 90 M 45 F 45

12.8 12.7 13.0

1.6 1.5 1.7

11.2-14.4 11.2-14.2 11.3-14.7

c 61 M 23 F 37

12.6 12.4 12.6

1.6 1.7 1.3

11.0-14.2 10.7-14.1 11.3-13.9

c 30 M14 F15

13.4 12.7 13.8

3.0 1.7 2.7

10.4-16.4 11.0-14.4 11.1-16.5

Platelet count ( x 106)

c 89 M 44 F 44

316 279 348

165 115 173

151- 481 164-394 175-521

c 60 M 23 F 37

363 344 375

183 194 175

180-546 150-538 200-550

c 30 M15 F15

320 274 367

205 170 199

115-525 104-444 168-566

Mean platelet volume (fl)

c 89 M 45 F 44

8.3 8.2 8.4

1.9 1.9 1.9

6.4-10.2 6.3-10.1 6.5-10.3

c 59 M 23 F 37

9.3 9.1 9.5

2.1 2.5 2.0

7.2-11.4 6.6-11.6 7.5-11.5

c 30 M15 F15

10.2 10.0 10.5

2.2 2.0 2.4

8.0-12.4 8.0-12.0 8.1-12.9

Neutrophils (%)

c 90 M 45 F 45

62 62 61

27 24 29

35-89 38-86 32-90

c 62 M 24 F 38

49 49 48

37 35 39

12-86 14-84 9-87

c 30 M15 F15

77 79 76

29 29 29

48 - 100 50 - 100 47-100

Bands (%)

c 90 M 45 F45

0 0 0

0 0 0

0 0 0

c 62 M 62 F62

0 0 0

0 0 0

0 0 0

c 30 M15 F15

0 0 0

1 1 2

0-1 0-1 0-2

Lymphocytes (%)

c 90 M 45 F 45

36 35 36

27 24 30

9-63 11-59 6-66

c 62 M 24 F 38

49 48 49

37 34 39

12-86 14-82 10-88

c 30 M15 F15

22 22 22

25 24 27

0-47 0-46 0-49

Monocytes (%)

c 90 M 45 F 45

2 2 2

3 3 3

0-5 0-5 0-5

c 62 M 24 F 38

2 2 2

3 3 3

0-5 0-5 0-5

c 30 M15 F15

2 3 2

3 4 3

0-5 0-7 0-5

Eosinophils (%)

c 89 M 44 F 45

1 1 1

2 2 2

0-3 0-3 0-3

c 62 M24 F 38

1 1 1

3 2 3

0-4 0-3 0-4

c 29 M15 F15

0 0 0

1 1 2

0-1 0-1 0-2

Basophils (%)

c 90 M 45 F45

0 0 0

0 0 0

0 0 0

c 62 M 62 F62

0 0 0

1 1 1

0-1 0-1 0-1

c 30 M 30 F 30

0 0 0

1 1 1

0-1 0-1 0-1

aFrom Hainsey et al., 1993; clinically normal, sedated adult female baboons, Papio spp.

710

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE Table XXVIII Clinical Chemical Reference Ranges in Baboons a Value

A/G ratio

Albumin (gm/dl)

Alkaline phosphatase (U/liter) ALT (SGPT) (U/liter)

Amyalse (U/liter)

Anion gap (mmol/liter)

AST (SGOT) (U/liter)

Bilirubin, direct (mg/dl) Bilirubin, total (mg/dl)

Blood urea, nitrogen (BUN) (mg/dl) Calcium (mg/dl)

Carbon dioxide

(CO2) (mmol/liter) Chloride (mmol/liter)

Cholesterol (mg/dl)

N

Mean

2 S.D.

2 S.D. range

c 25 M 15 F 10 c 25 M 15 F 10 c 25 M 15 F 10 c 24 M 15 F 10 c 25 M 15 F 10 c 25 M 15 F 10 c 25 M 15 F 10 c 25 M 15 F 10 c 25 M 15 F 10 c 25 M 15 F 10 c 25 M 15 F 10 c 25 M 15 F 10 c 24 M 15 F 10 c 25 M 15 F 10

1.0 1.1 0.9 3.5 3.7 3.2 248 243 254 45 49 44 243 253 228 29 29 30 39 42 34 0.1 0.1 0.1 0.2 0.2 0.2 14 14 14 9.0 9.1 8.9 24 25 22 99 101 99 99 101 97

0.7 0.7 0.5 1.4 1.4 1.4 303 312 306 22 20 40 155 136 183 11 11 12 20 20 16 0.1 0.1 0.1 0.2 0.2 0.1 5 5 6 1.2 0.9 1.5 5 5 5 8 11 10 57 49 70

0.3-1.7 0.4-1.8 0.4-1.4 2.1-4.9 2.3-5.1 1.8-4.6 0-551 0-555 0-560 23-67 29-69 4-84 88-398 117-389 45-411 18-40 18-40 18-42 19-59 22-62 18-50 0.0-0.2 0.0-0.2 0.0-0.2 0.0-0.4 0.0-0.4 0.1-0.3 9-19 9-19 7-20 7.8-10.2 8.2-10.0 7.4-10.4 19-29 20-30 17-27 91-107 90-112 89-109 42-156 52-150 27-167

Value Creatine kinase (U/liter) Creatinine (mg/dl)

Gamma GT (U/liter)

Globulin (gm/dl)

Glucose (mg/dl)

HDL cholesterol (mg/dl) Iron (lxg/dl)

Lactate dehydrogenase (U/liter) Lipase (U/liter)

Phosphorus (mg/dl)

Potassium (mmol/liter)

Sodium (mmol/liter)

Total protein (mg/dl)

Triglyceride (mg/dl)

N

Mean

2 S.D.

2 S.D. range

c 25 M 15 F10 c 25 M15 F10 c 25 M15 F10 c 25 M15 F10 c 24 M15 F10 c 25 M 15 F10 c 24 M15 F10 c 25 M 15 F10 c 22 M14 F9 c 25 M15 F10 c 25 M15 F10 c 25 M15 F10 c 25 M15 F10 c 25 M15 F10

391 400 379 1.0 1.1 0.8 39 43 32 3.6 3.5 3.7 83 83 88 56 60 50 68 64 84 276 271 282 5 7 4 2.9 3.1 2.6 3.9 4.0 3.9 149 151 147 7.1 7.1 7.0 66 65 68

349 330 401 0.5 0.4 0.3 22 20 21 1.1 1.2 0.9 25 18 48 45 42 48 53 60 79 147 167 119 7 12 8 1.7 1.5 1.8 1.1 1.1 1.2 5 3 6 0.9 0.8 1.1 31 29 36

42-740 70-730 0-780 0.5-1.5 0.7-1.5 0.5-1.1 17-61 23-63 11-53 2.5-4.7 2.3-4.7 2.8-4.6 58-108 65-101 40-136 11-101 18-102 2-98 15-121 4-124 5-163 129-423 104-438 163-401 0-12 0-19 0-12 1.2-4.6 1.6-4.6 0.8-4.4 2.8-5.0 2.9-5.1 2.7-5.1 144-154 148-154 141-153 6.2-8.0 6.3-'7.9 5.9-8.1 35-97 36-94 32-104

aFrom Hainsey et al., 1993" clinically normal, sedated Papio spp.

3 weeks prior to delivery and placed in individual cages for delivery. The live birth rate exceeded 70%. Hamadryas baboons are best housed as single-male-multifemale groups that mimic the one-male units of their social structure in the wild (Else et al., 1986; Birrell et al., 1996). No maternal mortality occurred in these one-male units, unlike in hamadryas groups that had more than one male (Else et al., 1986). In this housing situation, the pregnancy rate was 90%. Abortions occurred in 20.5% of deliveries and stillbirths in 8%; the live birth rate was 71.5% (Birrell et al., 1996).

Large-scale breeding of savanna baboons in a 6-acre corral resulted in approximately 200 infants/year (Goodwin and Coelho, 1982) with an 81% live birth rate. Births occurred in all months of the year, with peak numbers occurring between June and December. 6.

Nutrition

In the wild, seeds, roots, tubers, leaves, bulbs, flowers, and fruit predominate in the diet of baboons. Also, most of the spe-

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Table XXIX

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Pan spp.: C h i m p a n z e e s

Adult Weights for Papio spp.

1.

Introduction

Weight (kg) Species

Adult male

Adult female

P. anubis a P. cynocephalus a P. papio a P. ursinus a P. hamadryas a P. hamadryas b

21

12

20 26 20.4 21.5 16.4 - 19.5

15 13 16.8 9.4 10.5-11.5

aFrom Harvey et al., 1987; feral animals. bFrom Mahaney et al., 1993; captive animals i> 5 years of age; necropsy weights.

cies will consume insects, reptiles, birds, and small mammals (Stolz and Saayman, 1970; Strum, 1975). Papio ursinus living near the sea have been known to consume crabs, mussels, and limpets (Rowe, 1996). In captivity, baboons readily adapt to commercialized diets consisting of a minimum of 15% protein, 4% fat, and 5% fiber (1999 Report of Progress, Southwest Foundation for Biomedical Research). As in other nonhuman primates, they require an exogenous source of vitamins C and D. Fresh fruits and vegetables can supplement the vitamin and mineral content of the ration and serve as enrichment. 7.

Normal Values

The normal hematologic and clinical chemistry values for baboons can be found in Tables XXVII and XXVIII. Adult body weights are listed in Table XXIX. 8.

The chimpanzee is one of three members of the great apes, family Pongidae. Chimpanzees are large, vegetarian nonhuman primates from the African continent. They are tailless, their arms are longer than their legs, they have protrusive lips and prominent ears, and there is a short opposable thumb (Fig. 11). The foot is short compared to trunk length, and the big toe is long and strong. Face and body skin coloration and pelage are unique for each subspecies (Napier and Napier, 1967); in captivity the facial coloration can vary between white, mottled, freckled, and black. The pelage is usually coarse, sparse, and predominantly black. Variation in age and sex will result in white hair tufts in the anal region and chin. The dental formula is 2-1-2-3 for 32 total teeth, with canines that are well developed. The stomach is simple, and the cecum has an appendix, similar to that of humans. Chimpanzees have extensive laryngeal sacs in the neck and axillary spaces. A quadrupedal walk is used, interspersed with short distances of brachiating and standing upright, primarily to increase visual range. Chimpanzees choose to be arboreal 50-75% of the time; they sleep primarily in tree nests newly built each night and seldom less than 15 feet above the ground (Napier and Napier, 1967). The chimpanzee is the largest nonhuman primate currently used in biomedical research, and the only great ape that continues to make significant contributions to improving human health and welfare. In captivity, birth weight will average 1.52.0 kg, and by adulthood, chimpanzees will weigh 40-70 kg.

Research Uses

With a genetic similarity to humans of approximately 89% and a relatively large size capable of providing ample fluid and tissue samples, the baboon has served as an ideal experimental model for over 3 decades (Moore, 1975; Rogers and Hixson, 1997). Known as the laboratory model for coronary heart and chronic lung diseases, the baboon has also been used extensively in research involving atherosclerosis, hypertension, and osteoporosis (Rogers and Hixson, 1997; 1999 Report of Progress, Southwest Foundation for Biomedical Research). Studies involving human spinal disorders have relied heavily on P. anubis as a model. This research has been successful largely because this animal spends a significant amount of time in an erect position (Lauerman, 1992). Currently, research utilizing baboons centers on reproductive physiology; vaccine development for HIV, hepatitis C, and respiratory syncytial virus; and neonatal research, particularly in the field of premature infant development postdelivery and hyaline membrane disease (1999 Report of Progress, Southwest Foundation for Biomedical Research).

Fig. 11. Pan troglodytes, adult chimpanzees. (Courtesy of the Department of Veterinary Sciences, M. D. Anderson Cancer Center.)

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The generation time of chimpanzees approaches that of the human. It is not uncommon for chimpanzees to live 4 0 - 5 0 years in captivity (Bloomsmith et al., 1991). This longevity, and the fact that during their lifetime they pass through five distinct life phases (infant, juvenile, adolescent, sexually mature adult, and aged adult), makes their management and care in captivity very difficult. 2.

Taxonomy and Sources

There are two species (Pan troglodytes and P. p a n i s c u s ) and three subspecies (P. t. troglodytes, P. t. verus, a n d P. t. schweinfurthii) of chimpanzees (Table XXX). Pan troglodytes has traditionally been known as the "common chimpanzee" and has contributed the most to comparative biomedical research. The bonobo (P. p a n i s c u s ) has had more limited research use, primarily in the fields of behavior and cognition. The normal diploid number for the chimpanzee is 48 compared to 46 for the human. Hematology and serum chemistries are similar to those of the human, and there are numerous normal value studies available. Blood types are similar to the ABO typing system of the human; chimpanzees must be crossmatched before transfusions. Although there are still wild populations of chimpanzees in the forested areas from Sierra Leone and Guinea eastward to the River Niger, the numbers continue to decline. The International Species Information System (ISIS) indicates there are about 2500 known captive chimpanzees (Seal, 1986). Approximately 1500 chimpanzees are housed in biomedical research institutions in the United States. The research and breeding pool of

chimpanzees currently owned or supported by the National Institutes of Health (NIH) is about 900. This national research resource is managed by a single multiagency organizational unit, the Chimpanzee Management Program (ChiMP) within the National Center for Research Resources (NCRR), which is assisted by an advisory council. Chimpanzees are classified as an endangered species in the wild and have not been legally imported into the United States since 1975 when the United States became signatory to the CITES treaty. Although their survival in the wild is questionable due to habitat encroachment, poaching for bush meat, and human warring factions in West Africa, domestic breeding programs in the United States have been so successful that chimpanzees in captive breeding colonies have a threatened classification and are no longer considered endangered. All chimpanzees used in research are produced in domestic-production colonies within the United States. In fact, the highly successful breeding program in the United States created a surplus of chimpanzees in 1995. This led the Institute of Laboratory Animal Resources, National Research Council, to recommend a 5-year moratorium on breeding chimpanzees (ILAR, 1997). The biomedical research community has a healthy, well-defined population of genetically heterogeneous, demographically balanced, behaviorally normal, breeding chimpanzees available to meet future research needs. Captive breeding programs have not selectively bred within a subspecies; therefore, the captive population contains very few pure subspecies. The numbers of wild-born chimpanzees of pure subspecies stock in the current captive population are steadily declining.

Table XXX

Chimpanzee Taxonomy,CITES Status, and Distributiona Genus Pan

Distribution

Commonname(s)

CITES status II, I

Congo (?), Zaire

P. troglodytes

Bonobo, dwarfchimpanzee, pygmy chimpanzee Chimpanzee

II, I

P. t. schweinfurthi

Eastern chimpanzee

II, I

P. t. troglodytes

Central chimpanzee

II, I

P. t. verus

Western chimpanzee

II, I

Angola, Benin (ex), Burkina Faso (ex), Burundi, Cameroon, Central African Republic, Congo, Cote d'Ivoire, Equatorial Guinea, Gabon, Gambia [int], Ghana, Guinea, Guinea-Bissau (ex?), Liberia, Mali, Nigeria, Rwanda (ex?), Senegal, Sierra Leone, Sudan, Tanzania, Togo (ex), Uganda, Zaire Burundi, DemocraticRepublic of the Congo, Rwanda, Sudan, Tanzania, United Republic of Uganda Angola, Cameroon,Central African Republic, Congo, Equatorial Guinea, Gabon, Nigeria Cote d'Ivoire, Ghana, Guinea, Guinea-Bissau, Liberia, Mali, Nigeria, Senegal, Sierra Leone

P. paniscus

I, Species listed in CITES Appendix I are threatened with extinction (endangered); II, Species listed in CITES Appendix II are not currently threatened with extinction but may become so unless trade is strictlyregulated; Captivepopulationsin the United States are consideredAppendix II. FromCITES (2000) and Wilson and Reeder (1993). Note. lint], introduced; (ex), extinct. a

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Natural History

No individual has brought more knowledge and understanding of the chimpanzee to the public and the research community than Jane Goodall with her 26-year study of the chimpanzees of Gombe (Goodall, 1986). Her legendary commitment, one of the longest continuous field studies of any living creature, has produced numerous behavioral papers, timeless National Geographic television footage, and several best-selling books (Goodall, 1986). Her work has provided insight into the nonhuman primate that is considered to be the closest phylogenetic relative of human beings. These writings take a reader from the first documented arrival of a chimpanzee to a European shore in 1640 to some of Dr. Goodall's current efforts to get congressional support for chimpanzee sanctuaries in the United States (H.R. 3514 and S.R. 2725). Dr. Goodall is opposed to the use of chimpanzees in biomedical research but her many publications are recommended as essential reading for developing an appreciation of the natural history of the chimpanzee. 4.

Reproduction

Puberty in the female chimpanzee is detected with the onset of cyclic swelling of the anogenital tissue. This swelling may occur 1-1.5 years before menarche and is usually seen at about 8 years of age. Sexual maturity in captivity can be much earlier. The chimpanzee menstrual cycle is approximately 37 days, slightly longer than that of the human. Estrus or sexual receptivity coincides with a 5- to 6-day period of maximum swelling of the anogenital tissue. Ovulation occurs 1 to 6 days before detumescence of the anogenital tissue (Keeling and Roberts, 1972). The chimpanzee normally has a 7.5-8 month gestation, approximately 227-235 days. In captivity, births occur throughout the year. Human pregnancy test kits are dependable in the chimpanzee, and as in women, amenorrhea is indicative of pregnancy. Another indication of pregnancy is a change in the sexcycle swelling. Births are usually single; the twinning rate is comparable to that in humans. The onset of labor is rapid, and parturition usually takes only a few hours. Dystocia is rare because relative to the weight and pelvic dimensions of the female, the average infant birth weight is low. The placenta should be produced within an hour of delivery, and placentophagia is common. Menopause, as defined by human standards, has not been well documented in the literature; however, most captive female breeders are retired after 40 years of age. Puberty in the male chimpanzee is usually seen about the seventh year of life. In captivity, viable sperm production and impregnation can occur much earlier based on the male's exposure to and experience with sexually mature females. These same variables can place a male into the eleventh or twelfth year before he becomes a successful breeder. When socially housed, most sexually mature males will develop successful breeding

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skills, but their individual personalities, social histories, and experiences will heavily influence their long-term breeding proficiency and genetic-pool contribution. Great care must be taken to assure that initial exposures are good experiences for the male and female. A very shy and timid female placed with an aggressive, domineering male can result in serious injury, no breeding, and the development of abnormal behavior. The biomedical research community has established a very successful captive breeding program (NIH, 1994). 5.

Laboratory Management

Even though the chimpanzee has been successfully managed in the biomedical research laboratory for over half a century, the last two decades have brought significant performance-based improvements in their maintenance, care, and welfare. Chimpanzee behavioral, reproductive, and neonatal research has evolved into animal welfare regulatory and accreditation standards that can provide the chimpanzee a good-quality life and assure future propagation of the species, even in a research setting. The captive chimpanzee population is managed as a national resource with an adequate number of experimentally naive breeders being set aside to assure their future propagation. The preferred housing management is compatible, multimale social groups maintained in outdoor enclosures that have accessible shelters for protection from temperature extremes (Riddle et al., 1982). The formation of socially compatible groups of chimpanzees must be approached carefully and with knowledge of specific techniques of successful group formation (Fritz and Fritz, 1979). Group formation can take months, and in some instances, years, to be completely successful. Females should always be allowed to rear their young for a minimum of 2 years, unless the infant's survival is at risk. Institutional Animal Care and Use Committees are highly sensitive to the public's expectations when chimpanzees are used in biomedical research. Many experiments can be designed to allow chimpanzees to be socially housed during studies. Breeding programs are most successful when animals are housed in multimale groups. When this is impossible, modern housing and equipment allow individually housed animals to always see, smell, and hear other chimpanzees. Individual caging is large enough to provide a reasonable activity level and brachiation for short-term studies. A vast array of environmental enrichment techniques have been developed for chimpanzees that must be individually housed. These include physical, food, and cognitive enrichment strategies (Bloomsmith et al., 1990, 1991). Positive-reinforcement training is an effective captive management strategy gradually finding its way into the chimpanzee biomedical research discipline. Training strategies can bring a significant reduction in the daily stress that chimpanzees and husbandry personnel experience when performing routine procedures associated with animal movement, restraint, feeding,

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sanitation, and care. The training also serves as an enrichment activity for the chimpanzee and can bring an investigator new and exciting options in experimental design. Once established, positive-reinforcement training can significantly strengthen the efficiency and cost-effectiveness of routine chimpanzee husbandry and care. 6.

Nutrition

In the wild, chimpanzees are primarily vegetarians, spending a significant part of their time foraging. Commercial diets in captivity are of high enough quality to provide all of their nutritional needs in a primate biscuit; however, all facilities supplement the staple biscuit diet with a variety of fresh fruits, produce, and novel items, including items that are commonly consumed and enjoyed by humans. These practices attempt to fill the psychological rewards of food gathering in the wild. Chimpanzees have no need for meat protein, so feeding meat is unnecessary and rarely practiced. The popularity of foodenrichment strategies (types, delivery, puzzles) introduced in the 1980s continues today and is a well-recognized form of psychological enrichment, but recent observations indicate that moderation and good nutritional policies must be practiced in the efforts that provide food enrichment. There are some indications of more obesity, hypertension, and cardiovascular disease than was previously observed in captive populations (D. Lee, personal communication, 1999). To continue the psychological enrichment benefits that a varied and novel diet offers, the type of fat and levels of calories, sodium, and fiber present in an enriched diet may need to be examined.

7.

Normal Values

There is a significant body of literature available for chimpanzee normal values. The most extensive volume of such information on the chimpanzee is the 1969 six-volume publication "The Chimpanzee," edited by G. H. Bourne. These volumes discuss anatomy, behavior, diseases, pathology, histology, physiology, hematology, serology, growth, reproduction, and captive maintenance. Normal hematology and serum chemistry values are provided in Tables XXXI and XXXII. 8.

Research Uses

Chimpanzees are expensive and difficult to manage in a biomedical research setting. As adults they can be very aggressive. Expenses for maintaining an adult chimpanzee in an enriched environment can range between 15 and 20 dollars (US) a day in a conventional setting and 100 to 200 dollars (US) per day in a biohazard containment setting. However, because of their phylogenetic similarities to the human, chimpanzees have been attractive animal models for limited, sophisticated, nonconsuming types of research. Their use in biomedical research has always evoked public concern and continues to challenge our evolving ethical, legal, and social perspectives. Their significant contributions to biomedical research are undeniable, as is our obligation to maintain these research veterans in a high-quality life setting for their lifetime of 40 to 50 years. The National Research Council has recently recommended that euthanasia not be endorsed as a general means of population control (ILAR, 1997). While most of the research contributions made by chim-

Table X X X I

Normal Chimpanzee Values: Hematologya Value

N

Infant

N

Juvenile

N

WBC (• 103/I.1.1) RBC (• 106/ixl) Hgb (gm/dl) Hct (%) MCV (fl) MCH (pg) MCHC (gm/dl) Platelet count (• 103/1~1) Monocytes (• 103/p,1) Lymphocytes (• 103/1~1) Seg (X 103/txl) Bands (• 103/p,1) Eosinophil (• 103/txl) Basophil (• 103/~1) Retic count (%) Sed rate (mm/hr)

74 57 74 74 58 58 58 57 74 74 74 74 74 74 74 45

10.9 (6.64-16.0) 5.1 (4.7-5.8) 12.8 (11.5-14.7) 39 (35-44) 75 (68-81) 25 (22-27) 33 (32-34) 319 (192-530) 0.2 (0-.6) 5.6 (3.6-10.0) 4.0 (1.9-9.7) 0 (0-.18) 0.1 (0.4-.7) 0 (0-.25) 0.7 (0.1-2.4) 14 (2-36)

67 56 67 67 57 57 57 56 67 67 67 67 67 67 67 49

10.4 (6.93-16.3) 5.0 (4.6-5.4) 13.2 (11.9-15.2) 40 (36-44) 79 (75-84) 26 (25-28) 33.2 (32-34.3) 320 (236-429) 0.3 (49-539) 4.0 (2179-6687) 5.2 (2750-11101) 0 (0-230) 0.1 (0-454) 0 (0-5) 0.1 (.1-.2) 17 (5-36)

60 43 60 60 44 44 44 43 60 60 60 60 60 60 60 37

Adolescent 11.0 (6.4-16.2) 5.1 (4.6-5.8) 13.9 (12.8-16.4) 42 (37-48) 81 (76-85) 27 (25-29) 33.6 (32.5-34.6) 279 (148-363) 0.2 (0-443) 3.9 (1995-7937) 6.1 (2945-11016) 0.2 (0-200) 0.2 (51-578) 0 (0-16) 0 12 (4-47)

N

Adult

76 76 76 76 76 76 76 76 76 76 76 76 76 76 76 75

10.7 (7.2-14.8) 5.2 (4.7-6.1) 14.4 (12.2-16.8) 43 (37-50) 81 (76-88) 27 (25-30) 33.1 (32.1-34.1) 240 (137-347) 0.3 (69-504) 4.3 (2001-6841) 5.2 (3017-9026) 0.4 (0-145) 0.2 (35-436) 0 (0-14) 0.6 (0.1-1.5) 16 (3-60)

aFrom Ihrig et al. (2001) and M. D. Anderson Cancer Center, Department of Veterinary Sciences; samples from clinically normal chimpanzees. Note. WBC, white blood cells; RBC, red blood cells; Hgb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; Seg, segmented neutrophils; Retic, reticulocyte; Sed, sedimentation. Absolute values are given for value ranges for juvenile, adolescent, and adult chimpanzees for monocytes through basophils.

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Table XXXlI Normal Chimpanzee Values: Serum Chemistries

a

Value

N

Infant

N

Juvenile

N

Adolescent

N

Adult

Total bilirubin (mg/dl) ALT (U/liter) AST (U/liter) Alkaline phosphatase (U/liter) Creatine (mg/dl) Glucose (mg/dl) BUN (mg/dl) Total protein (gm/dl) Albumin (gm/dl) Globulin (gm/dl) Cholesterol (mg/dl) Gamma GT (U/liter) Triglycerides (mg/dl) Calcium (mg/dl) Phosphorus (mg/dl) CPK (U/liter) LDH (U/liter) Chloride (mEq/liter) Sodium (mEq/liter) Potassium (mEq/liter) HDL (mg/dl) LDL (mg/dl)

54

0.3 (.10-.60)

61

0.3 (0-.7)

47

0.3 (.04-.76)

76

0.3 (.21-.58)

73 73 73

33 (23-52) 24 (13-34) 600 (396-914)

67 67 67

40 (23-52) 22 (14-32) 508 (357-704)

60 60 60

30 (18-46) 19 (11-31) 375 (145-789)

76 76 76

29 (20-44) 21 (13-46) 85 (54-153)

55 55 73 73 73 73 30 73 29 6 6 6 10 7 15 15 25 25

0.6 (.5-.9) 83 (68-125) 11 (7-20) 6.7 (6.0-7.6) 3.7 (3.2-4.2) 3.1 (2.4-3.7) 262 (178-357) 17 (9-114) 60 (32-135) 10.1 (9.6-11.9) 5.2 (4.7-7.7) 113 (32-386) 427 (216-553) 108 (100-115) 142 (130-146) 4.1 (2.9-6.0) 80 (44-114) 173 (98-242)

62 61 67 67 67 67 26 61 25 4 3 3 6 2 10 10 23 23

0.6 (.5-.8) 87 (65-120) 14 (10-20) 7.2 (6.4-7.8) 3.9 (3.4-4.2) 3.4 (2.7-4.1) 237 (190-285) 6 (1-20) 72 (49-124) 10.2 (8.7-11.7) 5.0 (1.6-6.1) 156 (98-309) 374 (230-435) 106 (104-120) 143 (129-146) 4.1 (3.6-4.7) 66.5 (43-103) 154 (119-195)

50 49 60 60 60 60 27 60 23 9 9 9 14 9 10 10 21 21

0.8 (.6-1.7) 87 (61-115) 14 (9-23) 7.4 (6.8-8.0) 3.9 (3.5-4.4) 3.4 (2.7-4.1) 224 (162-298) 13 (13-24) 74 (40-108) 9.0 (8.8-9.4) 4.0 (1.9-5.3) 139 (78-366) 264 (125-408) 105 (93-119) 143 (95-147) 4.1 (3.6-4.5) 64 (32.4-92.4) 137 (109-174)

76 76 76 76 76 76 60 76 60 34 33 34 40 28 39 39 59 59

0.9 (.8-1.3) 82 (66-108) 12 (9-17) 7.7 (6.7-8.3) 3.7 (3.3-4.1) 4.0 (3.2-4.7) 216 (170-288) 19 (10-35) 97 (56-164) 9.2 (8.1-10.2) 3.0 (1.8-4.3) 313 (80-553) 279 (203-503) 105 (93-115) 142 (136-148) 3.6 (3.2-4.4) 51 (35-82) 147 (106-209)

aFrom Ihrig et al. (2001) and M. D. Anderson Cancer Center, Departmentof Veterinary Sciences; samples from clinically normal chimpanzees. ALT, alanine aminotransferase;AST, aspartate aminotransferase;BUN, blood urea nitrogen; Gamma GT, gamma gutamyltransferase;CPK, creatinine phosphokinase; LDH, lactate dehydrogenase;HDL, high density lipoprotein, LDL, low density lipoprotein. Note.

panzees in the early 1900s were behavioral, they played a unique role in the aerospace research programs of the 1950s. In the 1960s, chimpanzees were used in the field of organ transplant immunology. During the 1970-1980 era, they were important animal models in cognition and language studies. Chimpanzees continue to make significant contributions in these fields of study. From the 1970s to the present, the chimpanzee has been a valuable model, and in some instances, the only model available to investigate human infectious diseases (hepatitis A, B, C; respiratory syncytial virus; and HIV). Vaccine development continues to be a major area of research interest with these diseases. The unique ability of chimpanzees to resist or develop a milder form of some human diseases like hepatitis, HIV, malaria, and Alzheimer's, as well as the question of why chimpanzees rarely develop cancer, has made the chimpanzee an attractive comparative research subject. As sequencing of the entire human genome nears completion, it is probable that the chimpanzee will play a major role in future genomic research. Because the chimpanzee genome is 98% identical to that of humans (NIH, 1994), its genome is an obvious nonhuman primate species to sequence next. The chimpanzee genome holds significant potential for demonstrating unique genetic characteristics that result in biological differences between the

chimpanzee and the human concerning susceptibility to pathogens, behavior, and cognition.

IV.

PRINCIPLES OF COLONY MANAGEMENT

A.

Housing

Between 1965 and 1980, the biomedical research community made a concerted effort to improve the caging guidelines and standards for nonhuman primates to improve their health and management in a cost-effective manner. This was a formidable task considering the nonhuman primates involved had a weight range between 120 gm and 165 kg (Napier and Napier, 1967). A relatively stable era using larger galvanized or stainless steel hanging cages was the result, and it brought improved sanitation, management, and health care to the industry. The 1985 Amendment to the Animal Welfare Act (Public Law 99-198) changed caging perspectives by challenging the research community to develop documents and follow an appropriate plan for environmental enhancement that would promote the psychological well-being of nonhuman primates. Social (group) housing

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was the most effective large-scale strategy for improving the psychological well-being of primates, so a virtual revolution in housing was initiated. This revolution in creative, novel housing designs, strategies, and systems continues today, but it has already changed the entire concept of housing nonhuman primates. Although most of these modifications and improvements occurred in the late 1980s and early 1990s, the 1996 revision of the "Guide for the Care and Use of Laboratory Animals" ("Guide") (ILAR, 1996) effectively summarizes the contemporary vision for housing nonhuman primates. The primary enclosure for nonhuman primates varies depending on the species, age, size, use, and environmental needs. The 1996 "Guide" reinforces traditional primary enclosure considerations of allowing normal physiologic and behavioral needs of the animal (i.e., urination, defecation, normal posture and movements, maintenance of body temperature, and where indicated, reproduction), allowing the animal to remain clean and dry, allowing adequate ventilation, allowing access to food and water, and providing a safe and secure environment. The major new consideration in the 1996 "Guide" was to recommend social interaction and hierarchical development both within and between enclosures in order to improve the animal's psychological well-being. Nonhuman primates may be housed singly, in pairs, or in larger groups. Individual space requirements are given in Table XXXIII. The 1996 "Guide" recommendations differ from current Animal Welfare Regulations in that the "Guide" states that optimal cage measurements should not be based solely on floor space (engineering standards). Some nonhuman primates benefit more from vertical space (volume of primary enclosure), internal structures, and enrichment opportunities within

Table XXXIII Recommended Space for Nonhuman Primates a

Animals Monkeys (including baboons) Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Apes Group 1 Group 2 Group 3

Weight (kg)

Floor area/ animal (ft2)

Height (inches) b

Up to 1 Up to 3 Up to 10 Up to 15 Up to 25 Up to 30 > 30

1.6 3.0 4.3 6.0 8.0 10.0 15.0

20 30 30 32 36 46 46

Up to 20 Up to 35 > 35 c

10.0 15.0 25.0

55 60 84

aFrom ILAR (1996). bFrom cage floor to cage top. CApes over 50 kg are more effectively housed in permanent housing of masonry, concrete, and/or panel structure than in conventional caging.

the space (complexities) than from floor space. According to the "Guide," space allowances should be derived from performance standards, professional judgment, and experience. When selecting cage material and design, the dexterity, strength, and species-typical behavior of the animal must be considered. The materials used should not only meet the needs of the animal but be easy to sanitize and resist the accumulation of dirt, debris, and moisture. Surfaces should be smooth and resistant to rusting, chipping, cracking, and peeling. Ideal materials are galvanized metal, stainless steel, aluminum, and plastics. Less durable material, such as wood, can provide a more comfortable environment (perches, climbing structures, resting areas, nest boxes), but wooden items must be replaced periodically when sanitation becomes ineffective or damage occurs. Most cage-design literature addresses the Old World species. New World species may require more cage height to account for arboreal lifestyles or long tails. Perches and climbing structures are well used by New World species; marmosets, tamarins, and owl monkeys need nesting boxes as part of their housing. When research design or production programs allow, nonhuman primates that have social housing histories or that can be socially rehabilitated should be housed in compatible pairs or groups. Group housing is complex and must be monitored closely to avoid undue risk to the physical health and wellbeing of the animals. Group-housed nonhuman primates may need more or less space per animal than those that are individually housed. Pair or group housing is preferred even when members of the pair or group have slightly less space per animal than when singly housed. For group housing, determination of the total space needed is not necessarily based on the sum of the amounts recommended for individually housed animals (ILAR, 1996). Experimental design and geographical location often dictate the amount of flexibility available for housing options, but when possible, naturalistic environments with sheltered, outdoor housing should be considered. Some research protocols may allow animals to be housed in pairs or small groups, even outdoors, throughout the experiment. Production colonies may be housed in large outdoor enclosures (field cages, corncribs, corrals, runs, primadomes) with great success. Successful outdoor housing necessitates providing an adequate acclimation period for the animals when first placed outdoors in advance of seasonal changes, grouping compatible animals in a species-appropriate social environment, training animals to enter cages or transfer tunnels to accommodate restraint and transport, and providing adequate security to protect the animals from intruders. Other considerations with outdoor housing are environmental enrichment, food and water, pest control, animal accessibility for health care, and frequent observation of animals. Ground surfaces in outdoor facilities may be native soil and grasses, vegetation or rock, gravel, and concrete. All surfaces must be cleaned periodically to maintain acceptable husbandry/sanitation standards. The benefits of elevated grid flooring are historically well established in individual

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cage design, and the same principle should benefit outdoor housing or shelter areas. Poor cage design and construction can result in hazards to the primate occupants. Toxicity through the ingestion of caging material is possible. For example, leaded paints and galvanized steel have been implicated in inadvertent poisonings, along with certain indoor/outdoor plants, trees, and shrubs used to landscape holding areas (Obeck, 1978). The cage design and finish should eliminate sharp edges; protruding screws/bolts; and any ropes, chains, or cables that might pose a risk of strangulation.

B.

Enrichment Programs

Since the 1985 amendments to the Animal Welfare Act, disciplines within the animal research community have struggled to promulgate minimum standards "for a physical environment adequate to promote the psychological well-being of primates" (CFR, 1999). Most successful and compliant enrichment program strategies do not depend upon tightly written engineering or prescriptive specifications to accommodate the variety of nonhuman primates and situations; on the contrary, they have implemented performance-based strategies that rely heavily on professional judgment in interpreting and applying the regulatory recommendations. Personnel involved in developing, implementing, and evaluating an enrichment plan must have specialized knowledge, skills, and experience with nonhuman primates. Species-specific structures, devices, and foodstuffs can be used to create an environment conducive to normal health and expression of species-typical behaviors. Playscapes and structures for climbing stimulate normal physiologic development. Sight barriers such as crates and barrels allow lowerranking animals to escape from aggressive encounters, thus reducing stress and trauma. Sticks, balls, chew toys, and other play items provide diversion and alleviate boredom. Nonhuman primates can forage as in nature if seeds, grains, fruits, vegetables, or other foods are distributed in a variety of substrates (hay, straw, AstroTurf), or are presented in a puzzle feeder. While numerous scientific publications have been designed to generate data for assessing enrichment strategies on individual species, the 1998 National Research Council publication "The Psychological Well-Being of Nonhuman Primates" (ILAR, 1998) is an excellent overview of the principles and recommendations concerning a quality nonhuman primate enrichment program. The publication committee has effectively evaluated the environmental variables that are most influential in affecting the well-being of nonhuman primates, evaluated the behavioral and physiological measures that are objective indices of the effects of these environmental variables, produced recommendations and procedures for use by institutions in developing plans consistent with federal law, and suggested priorities for future research into the psychological well-being of nonhuman primates. A plan to enhance the psychological well-being of the nonhu-

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man primate has some essential elements. As stated in the 1998 NRC document, the plan must address the social needs of the primate and provide some form of environmental enrichment, that is, opportunities for the expression of species-typical activities. Every plan should consider the following elements: Appropriate social companionship Opportunities to engage in behavior related to foraging, exploration, and other activities appropriate to the species, age, sex, and condition of the animal Housing that permits suitable postural and locomotor expression Interactions with personnel that are generally positive and not a source of unnecessary stress Freedom from unnecessary pain and distress Once a plan is developed and implemented, an assessment of the animal's response should be based on the following criteria: The animal's ability to cope effectively with day-to-day changes in its social and physical environment The animal's ability to engage in species-typical activities The absence of maladaptive or pathological behavior that results in self-injury or other undesirable consequences The presence of a balanced temperament (appropriate balance of aggression and passivity) and absence of chronic signs of distress, as indexed by the presence of affiliative versus distress vocalizations, facial expressions, postures, and physiological responses (e.g., labored breathing, excessive cardiac response, and abnormal hormonal concentrations) Although the aforementioned essentials and criteria for assessment should be considered in each plan developed to enhance the psychological well-being of primates, there are other considerations and conditions in the research setting that must be addressed when developing a plan. The Institutional Animal Care and Use Committee, the Institutional Biosafety Committee, the Personnel Health and Safety Program specialists, the principal investigator, and the veterinarians must all be involved in risk assessments when the experiments involve infectious diseases, atypical rearing, physical restraint, minimally invasive procedures, surgery, multiple uses, conditions involving pain, and studies involving substance abuse or aggression. Even under these circumstances, creative and novel approaches can address some of the nonhuman primate needs for psychological well-being. The 1998 NRC publication also provides specific comments for each taxonomic group (i.e., New World monkeys, Old World monkeys, apes) that supplement the limitations of this chapter. Within these taxonomic groups are general information and recommendations concerning housing, nutrition, social behavior, reproduction and development, cognition, personnel, and veterinary care that should be considered when developing a plan for the psychological enrichment of each species.

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C.

Restraint Techniques

Safe and effective restraint minimizes personnel risk, reduces animal distress, and improves the quality of research data by avoiding the altered physiological parameters prompted by stress-associated increases in cortisone, growth hormone, and glucagon (Brady, 2000). Proper restraint, whether by physical or chemical methods, requires well-trained personnel and wellequipped facilities. The appropriate use of personal protective equipment is essential (Table XXXIV), as is the safe and effective use of restraint devices and equipment. The 1997 death of a regional primate center research technician after an accidental, ocular-splash exposure to cercopithecine herpesvirus 1 (CHV1, B virus) and an increase in research protocols that involve the use of zoonotic pathogens like HIV, SIV, SHIV, hepatitis B, and hepatitis C have heightened the research community's awareness of the risks associated with nonhuman primates (CDCP, 1998).

Table XXXIV Personal ProtectiveEquipmenta Laboratory clothing Laboratory coats, smocks, gowns, total body suits, coveralls, two-piece scrub suits Head coverings Not required except in containment areas requiring a complete clothing change Simple cap, hoods, bouffant cap Shoes and shoe covers Sandals not allowed in biohazard areas For BSL 2 b and BSL 3 areas, a change from street shoes is advised; shoe covers can be used when a complete change of clothes and dedicated shoes are not required Butyl rubber, neoprene, or PVC boots are advised in animal rooms when encountering large amounts of water Gloves Rubber, neoprene, latex, nitrile, PVC, polyvinyl alcohol, surgical Should extend to/beyond wrist Gauntlet-type leather gloves, Kevlar, or stainless steel liners for handling nonhuman primates Respiratory protection Single use, paper "dust" mask can be worn in clean animal rooms; not for use where infectious aerosols present; allowed for necropsies of clean animals Areas where infectious aerosols present, use half-face or full-face respirators with HEPA filters recommended Particulate respirators allowed for BSL 3 areas Eye or face protection Areas of respirable aerosols or droplets, use full-face respirators or halfface respirators plus splash goggles Areas of Herpesvirus simiae (CHV- 1, herpes B virus), use safety shields or face shields plus splash goggles Areas where CHV-1 not a concern, safety shields or face shields sufficient aFrom Kuehne et al. (1995). bBiosafety level.

1. Physical Restraint

Numerous restraint devices, types of equipment, and strategies are available for safe and effective physical restraint of nonhuman primates. Knowledge of species-typical behaviors and dislikes can be leveraged to make the restraint procedures less hazardous to personnel and less traumatic to the animal. Positive-reinforcement training techniques, originally developed and implemented with marine mammals (Laule, 1993), are being successfully used with some nonhuman primates. Effective positive-reinforcement training should significantly reduce the amount of physical and chemical restraint necessary in the future (Schapiro, 2000). Restraint methodology selection will be influenced by the nonhuman primate species, size, gender, temperament, strength, level of debilitation, and reproductive status (Kennett, 1996). a.

Restraint Cages

Restraint cages are the most commonly used and effective method of restraint of the Old World species and chimpanzees. With minimal training, technicians can operate these devices and greatly reduce the risk of receiving bites or scratches. There are a variety of sizes of restraint back cages that can be used on virtually all the nonhuman primates, including the New World species (Klein and Murray, 1995). Restraint cages allow brief procedures (e.g., reading tuberculin tests, treating wounds, and administrating vaccines, medication, and systemic anesthetics) to be accomplished safely. Nevertheless, the type of procedures that can be performed using a restraint back cage is limited. Nasogastric intubation or other protocols that entail medical risk or require too much time should not be attempted using a restraint cage. b.

Restraint Gloves

Well-trained personnel wearing heavy leather or welder'stype gloves can effectively extract from a cage and restrain small nonhuman primates. Some gloves have Kevlar and/or stainless steel liners that increase personnel safety. It is important that these gloves extend above the elbows. The initial use of gloves can be traumatic for the animal; hence, there should be efforts to precondition the animal to the gloves. Generally, hand capture with gloves can be performed on nonhuman primates that are not larger than a young rhesus monkey. Adult male rhesus monkeys or animals of similar size should not be restrained with gloves only. Examples of procedures that can be performed on nonhuman primates restrained with gloves include injections, blood collection, wound treatment, subcutaneous fluid administration, and tuberculin testing (Sauceda and Schmidt, 2000). When using gloved restraint, nonhuman primates weighing more than 1 kg should have their arms pinned behind their back for added security (Martin, 1986).

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Restraint Nets

Nets are used when personnel need to maintain a safe distance from the nonhuman primate being captured. Once the animal has been successfully netted, it can be hand-restrained with gloves or given an anesthetic. Netting is very useful when capturing escaped animals or retrieving individual animals that are housed in social groups. Nets are typically used to capture all the New World species and Old World monkeys up to 3.5 kg (Klein and Murray, 1995).

d.

Pole-and-Collar Restraint

A pole-and-collar method was developed for restraining the larger nonhuman primates. The animal is fitted with a metal or plastic collar that has a metal ring. The pole has a springactivated clip that can be passed through the cage front and connected to the collar at the ring. The nonhuman primate can then be removed from the primary enclosure, restrained at a safe distance from the operator, and allowed to walk to a primate restraint chair for positioning. The advantages of the pole and collar include a minimal amount of training for the nonhuman primate and the personnel, a reduction in opportunity for personnel injury, and once the animal is acclimated, a decreased level of stress for the nonhuman primate (Kennett, 1996). A variation of the pole-and-collar method is the collar-andchain method. This system utilizes a collar, snaphook chain, and a restraint chair. The flexibility of the chain allows easier capture and maneuverability of the animal. Once the animal is captured, the chain serves as a tether system, and the nonhuman primate can be led to a restraint chair. The disadvantage of this method is a greater risk of injury to personnel because there is less control of the animal (Sauceda and Schmidt, 2000). e.

Tether and Vest Restraint

Adapted for use in a variety of New Wodd and Old World monkeys, this restraint method allows continuous physiologic monitoring, biological sampling, and infusion of drugs without the multiple restraint episodes typically associated with longterm studies. The tether device consists of a nylon-mesh vest or jacket and a flexible tether tube, with a swivel assembly connected to the back of the cage (Adams et al., 1988). Cables and catheters for monitoring, collecting, and/or infusing are contained within the stainless steel tether. The swivel assembly allows the animal freedom of movement within the cage, and sampling can take place without physical or chemical restraint (Coelho and Carey, 1990). Advanced designs for baboons have incorporated cardiovascular monitors housed within a backpack that is fitted into the jacket. This modification provides for constant blood-pressure and heart-rate monitoring. This tethering system was designed to allow use with modular caging, where four animals can share one tether in a fourplex cage, thereby al-

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lowing freedom of movement and socialization (Coelho and Carey, 1990). The advantages of the tether and vest include having constant monitoring and sampling with no chemical restraint, less animal stress, less animal handling, less risk of injury to caregivers, and no secondary medical complications associated with long-term chair restraint (McNamee et al., 1984). The disadvantages include the increase in personnel and resources needed to develop and maintain the system throughout the duration of the experiment (Klein and Murray, 1995).

2.

Chemical Restraint

Delivering chemical restraint for nonhuman primates can be accomplished through a variety of device.s. Depending on the size of the animal and its primary enclosure, capture rifles or pistols, blowpipes, pole syringes, or hand injection can be used. For distances greater than 20 meters, CO2-powered rifles are typically used, while CO2-powered pistols suffice for distances less than 20 meters (Kreeger, 1999). When CO2-powered devices are used, the risk of animal injury increases as its size and distance from the device decreases. Bone fractures and internal organ injury can result from using too powerful a restraint device at too short a distance. Blowpipes work well at ranges up to 15 meters with far less risk of injury than when the CO2 or compressed-air devices are used (Klein and Murray, 1995). Pole syringes can be used effectively at distances up to 2 meters. Hand injection is used when restraint cages are available. Acepromazine or other tranquilizers can be used orally when they are disguised in food or juices; however, time of onset and duration of action vary greatly with orally administered drugs. In general, caution should be taken when tranquilizers or sedatives are used in nonhuman primates because, while the drug may have a tranquilizing effect, it may not adequately suppress the animal's stress-arousal response. As a consequence, general anesthetics are more commonly used. The most commonly used drug for restraint of the nonhuman primate is the dissociative anesthetic, ketamine hydrochloride. Ten (10) mg/kg body weight is considered the standard dose for Old World monkeys; higher dosages of 20-25 mg/kg are required for New World species. Combinations of ketamine and acepromazine, xylazine, or diazepam have been used successfully. A ketamine-xylazine combination can be used in a 3:1 volume ratio (3 U ketamine: 1 U xylazine) when the ketamine concentration is 100 mg/ml and the xylazine concentration is 20 mg/ml. Administered intramuscularly, this combination provides good anesthesia and analgesia at a dose of 0.1-0.2 ml/kg body weight (Klein and Murray, 1995). Telazol (tiletamine hydrochloride/zolazepam hydrochloride) has been used successfully for restraint, anesthesia, and procedures of short duration in a variety of nonhuman primates. The key ingredients (tiletamine, zolazepam) are chemically related to ketamine and diazepam, respectively (Plumb, 1995). An intramuscular injection of

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2 - 5 mg/kg body weight provides immobilization and anesthesia for 3 0 - 4 5 min in chimpanzees and macaques. For procedures of greater than 45 min duration, inhalation anesthesia is indicated. Halothane remains a useful inhalation anesthetic in veterinary medicine due to its being safe, potent, noninflammable, and inexpensive. Isoflurane is a similar agent, except it is less potent than halothanel Also, isoflurane has less catecholamine-sensitizing and cardiodepressive properties, making it more safe for hepatic and renal disease patients (Plumb, 1995).

D.

Biosafety

Nonhuman primates pose unique zoonotic risks. Their care and use can present serious hazards to personnel that work directly with them, their tissues, or body fluids. Cercopithecine herpesvirus 1 (CHV-1, B virus), simian immunodeficiency virus (SIV, a retrovirus closely related to HIV-1 and HIV-2), tuberculosis, and bacterial gastroenteritis caused by Shigella, Salmonella, and Campylobacter spp. are some of the more serious diseases that can be transmitted to personnel if biosafety precautions are not practiced. Nonhuman primates experimentally infected with human disease agents can also transmit disease to personnel if appropriate precautions are not followed (Adams et al., 1995). Transmission of natural or experimental infection is usually the result of carelessness or human error (Harding and Liberman, 1995). For a more complete list of zoonotic diseases, their mode of transmission, and the recommended biosafety precautions, the reader is referred to Table XXXV. According to the Centers for Disease Control and Prevention (CDCP), institutions that house experimental nonhuman primates are obligated to provide their workers established practices to ensure that appropriate levels of environmental quality and safety are maintained (CDCP/NIH, 1999). This obligation is driven by a series of federal regulations created for worker safety. Starting in the mid-1980s, the federal standard known as Hazard Communication ("Worker Right to Know") was developed by the Occupational Safety and Health Administration (OSHA) to create a work environment where hazards found in the specific work area are identified, their capabilities for related health problems are recognized, and the workers are given complete access to their respective laboratory results and medical records. This was followed in 1991 by the implementation by OSHA of 29 CFR Part 1910.1030, the Occupational Exposure to Bloodborne Pathogens: Final Rule, which states that blood, body fluids, and tissues infected with human disease agents must be handled in compliance with OSHA standards. Therefore, work practices associated with experimentally infected nonhuman primates must comply. An effective biosafety program implements preventive measures that counteract risks associated with identified infectious agents within the environment. To determine these preventive measures, a risk assessment of the infectious agent must be per-

Table XXXV Zoonotic Diseases

Disease Viral Marburg Ebola Yellow fever Dengue CHV-1 (Herpesvirus simiae, herpes B) Hepatitis A Hepatitis B Hepatitis C Hepatitis D Hepatitis E Systemic T lymphotrophic virus 1

Transmission a

Aerosol, droplet, percutaneous Aerosol, droplet, percutaneous Arthropod vector, Aedes spp. mosquitoes, percutaneous Arthropod vector, Aedes spp. mosquitoes, percutaneous Aerosol, droplet, percutaneous Ingestion Droplet, percutaneous Droplet, percutaneous Droplet, percutaneous Ingestion Droplet, percutaneous

Simian immunodeficiency virus (SIV)

Droplet, percutaneous

HIV-1, HIV-2 Monkeypox Measles Influenza Poliovirus Rubella Rabies

Droplet, percutaneous Percutaneous Aerosol Aerosol Percutaneous, ingestion Aerosol Droplet, percutaneous

Prions Spongiform encephalopathy Bacterial Tuberculosis Mycobacterium spp. Campylobacteriosis Campylobacter spp. Shigellosis Shigella spp. Salmonellosis Salmonella spp. Yersinia enterocolitica Parasitic Nematodes Strongyloides spp. Trichuris spp. Cestodes Trematodes Schistosoma spp. Malaria Toxoplasmosis Trypanosomiasis

Recommended precautions b

BSL 4c BSL 4 BSL 3 BSL 3 BSL 2 BSL 2 BSL 2 BSL 2 BSL 2 BSL 2 BSL 2 with special practices and containment equipment BSL 2 with special practices and containment equipment BSL 2 BSL 2 Immunization Immunization BSL 2 Immunization BSL 2, immunization

Percutaneous, aerosol

BSL 2

Aerosol, percutaneous

Ingestion

BSL 3 if known BSL 2 during quarantine BSL 2

Ingestion, percutaneous

BSL 2

Ingestion, percutaneous

BSL 2

Ingestion

BSL 2

Ingestion Ingestion Ingestion Contact

BSL2 BSL 2 BSL 2 BSL 2

Arthropod vector, percutaneous Ingestion Triatomid bug bite, percutaneous, aerosol, droplet

BSL 2 BSL 2 BSL 2

16. NONHUMAN P

Amebiasis Entamoeba histolytica Balantidiasis Balantidium coli Giardia spp. Cryptosporidiosis Miscellaneous Leptospirosis Leptospira spp. Rickettsia Scrub typhus, Q fever Mycoses Nocardia spp., Aspergillus spp., ringworm, thrush ,,

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Disease

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Recommended precautions b BSL 2

Ingestion Ingestion Ingestion Ingestion

BSL 2 BSL 2 BSL 2 BSL 2

Ingestion, droplet, percutaneous Aerosol, droplet, percutaneous

BSL 2

Contact, aerosol

BSL 2

BSL 2

,, ,,

aFrom CDCP (1999). bFrom CDCP (1995). CBiosafety level.

formed. This assessment addresses such specifics as the pathogenicity of the agent, its infectious dose, the route of transmission, the availability of an effective prophylactic regime, and the experience of the at-risk personnel. Once the assessment is complete, the appropriate biosafety level can be assigned to limit the personnel and environmental exposure level. These potential exposures can then be reduced or eliminated through appropriate containment of the infectious materials. Primary containment is the protection of personnel and the environment from infectious agents through good laboratory practices and safety equipment. These practices are designed to reduce or eliminate exposures and are typically referenced in the facility's biosafety manual (CDCP/NIH, 1999). An example of primary containment would be a procedure to follow after a rhesus monkey bite or scratch. Table XXXVI outlines a protocol designed to reduce the likelihood of transmission of CHV-1. Primary containment safety equipment includes biological safety cabinets, enclosed containers, and personal protective equipment. Gloves, cover clothing, boots, face masks, and shields may have to serve as a primary barrier between personnel and the infectious agent when it is impractical to perform the job in the aforementioned enclosures (CDCP/NIH, 1999). Recommendations concerning a variety of personal protective equipment for working with nonhuman primates are listed in Table XXXIV. Secondary containment is protection of the environment outside the laboratory and is accomplished through a combination of facility design and operational procedures (CDCP/NIH, 1999). These secondary barriers are based on the risk of transmission of the infectious agents. If transmission is through contact exposure with the agent or through contaminated work areas, secondary barriers may involve complete separation of the work area from public and hand-washing facilities. If aerosol

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Table XXXVI Standard Operating Procedures for Rhesus Monkey Bite and Scratch Exposures a 1. Initial wash of wound with Septisol| and water 2. Continuous soaking of wound with fresh 10% bleach solution for 15 min 3. Notification of supervisor, completion of accident report form, recording in log 4. Treatment by physician as recommended by Director, Employee Health 5. Viral cultures of employee wound and macaque (conjunctiva, genitalia, buccal area) 6. Baseline serology for CHV-1 on employee and macaque 7. Supervisor will record the clinical status of the affected person at weekly intervals for one month; patient is queried as to the development of any skin lesions or neurologic symptoms such as itching, pain, or numbness near the wound site; in addition, any acute onset of illness, particularly accompanied by fever and/or headache, reported immediately to the section veterinarian 8. Followup serology on employee and the primate involved at 14-21 days postexposure a From M. D. Anderson Cancer Center, Department of Veterinary Sciences. Note. Septisol| Steris Corporation, St. Louis, MO 63110.

transmission is a possibility, higher levels of containment must be achieved by utilizing both primary and multiple secondary barriers. This may necessitate separate buildings, specialized ventilation systems, and controlled access to these areas (CDCP/ NIH, 1999). Based on a comprehensive assessment of the hazards, an effective biosafety program can prevent personnel exposures, infections, and other complications through a combination of safety equipment, facility design, and strict adherence to biosafety procedures and practices (Richardson, 1995). These combinations are used to achieve recommended biosafety levels that represent conditions under which the agent can be handled safely. Table XXXVII contains a summary of recommended biosafety levels and the associated animal biosafety levels for activities in which experimentally or naturally infected nonhuman primates are used.

E.

Sanitation

To maintain a safe and healthy environment for captive nonhuman primates, a comprehensive sanitation program must be implemented. Consideration is given to the organisms that can be deleterious, the materials and structures that must be sanitized, the products and methods used for sanitizing, and the frequency of sanitizing procedures. As stated in the 1996 "Guide," effective sanitation is composed of two elements, cleaning and disinfecting. Cleaning is defined as the removal of dirt and waste products, while disinfection is the reduction or elimination of pathogenic microorganisms. Providing a sanitary environment can be a challenge when nonhuman primates are group-housed. Solid floors in primary

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BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE Table XXXVII

Recommended Biosafety Levels (BSL) for Activities in Which Experimentallyor Naturally InfectedVertebrateAnimals Are Useda BSL

Agents Not known to consistently cause disease in healthy human adults Associated with human disease. Hazard: percutaneous exposure, ingestion, mucous membrane exposure

Indigenous or exotic agents with potential for aerosol transmission; disease may have serious health effects

Practices

Safety equipment iprimary barriers)

Standard animal care and As required for normal care of managementpractices, including each species appropriate medical surveillance programs ABSLb-1 practice plus ABSL-1 equipmentplus Limited access Primary barriers: containment Biohazard warning signs equipment appropriate for animal Sharps precautions species; PPEsC:laboratory Biosafety manual coats, gloves,face, and Decontamination of all infectious respiratory protection as needed wastes and of animal cages prior to washing ABSL-2 practices plus Controlled access Decontamination of clothing before laundering Cages decontaminatedbefore bedding removed Disinfectant footbath as needed

Facilities (secondarybarriers) Standard animal facility No recirculation of exhaust air Directional air flow recommended Hand-washing sink recommended ABSL- 1 facility plus Autoclave available Hand-washing sink available in animal room Mechanical cage washer used

ABSL-2 equipmentplus ABSL-2 facility plus Containment equipmentfor housing Physical separation from access animals and cage dumping activities corridors Class I or II BSCsd available Self-closing, double-dooraccess for manipulativeprocedures Sealed penetrations (inoculation, necropsy) that may Sealed windows create infectious aerosols. PPEs: Autoclave available in facility appropriate respiratoryprotection

Dangerous/exotic agents that ABSL-3practices plus ABSL-3 equipmentplus pose high risk of lifeEntrance through change room Maximum containmentequipment threatening disease; aerosol where personal clothing is (i.e., Class III BSC or partial transmission, or related removed and laboratory clothing containment equipment agents with unknown risk is put on; showeron exiting in combination with full-body, of transmission All wastes are decontaminated air-supplied, positive-pressure before removalfrom facility personnel suit) used for all procedures and activities i

ABSL-3 facility plus Separate building or isolated zone Dedicated supply and exhaust, vacuum, and decontamination systems Other requirementsoutlined in text

l llll

From CDCP (1999). bAnimal biosafety level; for a more detailed explanation of these levels, see CDCP (1999), pp. 53-74. CPersonal protectiveequipment. dBiosafety cabinet.

a

enclosures can increase the frequency of illnesses caused by fec a l - o r a l transmission. Slatted or grid floors are preferable, since they allow waste products to fall away from the animal's environment, much like a single-cage grid floor. With decreased fecal contamination, the chance of fecal-oral transmission of pathogenic organisms between animals is significantly reduced, even though food items are eaten from the cage floor. Effective sanitation programs focus on a variety of target organisms. Parasites that can be harmful to nonhuman primates include arthropods, protozoa (particularly amebas and coccidia), and helminths (nematodes, cestodes, and trematodes). Bacteria causing significant gastrointestinal morbidity and mortality include Salmonella spp., Campylobacter spp., and Shigella spp. Transmissible viral agents that are of particular concern because they can cause gastrointestinal disease include rotaviruses, hepatitis A virus, and hepatitis E virus. Except for hepatitis E virus, these viruses can occur as natural infections. Experimental inoculation of hepatitis E into owl monkeys,

cynomolgus monkeys, rhesus monkeys, and chimpanzees has shown that they are susceptible and that natural infection is possible (Mansfield and King, 1998). Effective cleaning and disinfecting of both primary and secondary enclosures can be accomplished with water, detergents, bleach, phenolics, and quaternary ammonia compounds. The typical regime would begin with flushing of gross debris with water, followed by disinfecting with products that are tuberculocidal, bactericidal, and virucidal. Cleaning and disinfecting should be performed daily on primary enclosures, including perches, shelves, and enrichment devices. In a hospital setting, it is recommended that the primary enclosure be cleaned and disinfected twice daily (ILAR, 1996). In general, daily disinfection should be supplemented with biweekly sanitation of those primary enclosures and their associated structures, such as cage racks and cage pans. Food and water receptacles should be sanitized either by live steam or 180~ water as found in a mechanical cage washer (CFR, 1999).

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Primary enclosures with dirt floors or absorbent bedding should be spot-cleaned frequently enough to ensure that animals can move about without contacting their excrement (CFR, 1999). Cleaning and disinfection of animal rooms, treatment rooms, storage areas, cage-washing facilities, and corridors should be accomplished at a frequency that is consistent with their use and level of contamination (ILAR, 1996). F.

Environmental Controls

1. Temperature

Captive nonhuman primates should be protected from temperature extremes of less than 45~ and greater than 85~ for more than 4 consecutive hours. Controlled environmental temperature settings should be maintained within a range of 64 ~ 84~ (ILAR, 1996). Temperatures maintained within a facility will depend on a number of criteria: species, age, size, number, and health status (Southers and Ford, 1995). Sudden, prolonged temperature changes should be avoided. Controlled environments should provide nonhuman primate holding rooms with nonrecirculated ventilation at a rate of 10-15 air changes per hr (ILAR, 1996). To avoid temperature-related stress and discomfort, nonhuman primates must be acclimated to the expected seasonal temperature, humidity, and climatic conditions when housed outdoors (CFR, 1999). The deleterious effects of high temperature extremes can be lessened by providing housing areas with shade, misters, wading pools, or other devices and strategies. Cold temperature extremes can be avoided by having secondary enclosures with supplemental heaters, windbreaks, and alternative bedding for the extreme cold. 2.

or gray filters over the light fixtures during their active (dark) phase (S. Gibson, personal communication, 2000).

G.

Effective record-keeping systems are essential in order to adequately manage the well-being of nonhuman primates. A detailed historical record of the animal's prior experimental use and health is invaluable. It is not uncommon for nonhuman primates to have life spans in excess of 20 years and hence to be used in multiple research projects. The permanent animal record should contain relevant information pertaining to the animal's genetic, clinical, surgical, behavioral, reproductive, and research histories (ILAR, 1996). A model record is illustrated in Table XXXVIII. Individual animal records should document the nonhuman primate's permanent identification marking. Typically this involves tattooing a number or code to the chest or inner thigh, a simple technique that remains one of the most common methods of permanent animal identification (Dyke, 1995). Collars with attached identification tags are frequently used with baboons and squirrel monkeys. Subcutaneously placed microtransponders that can contain permanent identification, as well as other information, and be read by a telemetry transceiver are available for use in nonhuman primates. Although the effective range of the reader is limited and the cost for the equipment is high, implantable transponders will become the future permanent identification method of choice for nonhuman primates (Dyke, 1995).

Table XXXVIII

Humidity

Records ,

For most species of nonhuman primates, the recommended range for relative humidity is 30-70% (ILAR, 1996). Some members of the New World group, such as tamarins and marmosets, require 50% humidity (Southers and Ford, 1995). 3.

,

Information to include 1. Animal identification 2. Birth date 3. Acquisition date Source 4. Sire, dam identification Virology testing, if pertinent 5. Vaccination history 6. Weight history 7. Clinical history Hematology Tuberculin testing Virology testing, if pertinent Genetic testing 8. Treatment/morbidity history 9. Surgical history 10. Mating/reproductive history 11. Behavioral history 12. Research history 13. Necropsy results

Lighting

The Animal Welfare Regulations require lighting (natural or artificial) of indoor facilities to be uniformly diffused throughout the enclosure. This allows for routine inspection of the primates, maintenance of acceptable husbandry standards, maintenance of physiologic and neuroendocrinologic stimulation, and assurance of personnel safety. For most primate species, a 12 hr light-12 hr dark diurnal cycle is sufficient (ILAR, 1996) and is assured when regulated by a time-controlled lighting system. For nocturnal species such as Aotus spp., reversal of the above cycle is recommended; diminished illumination to perform observations and husbandry duties can be provided by placing red

Records

,

,

,,

,,,,

,,

.

.

.

.

.

.

.

,

,

,,

i

,

BRUCE J. BERNACKY,SUSANV. GIBSON, MICHALE E. KEELING,AND CHRISTIANR. ABEE

724

V.

MEDICAL MANAGEMENT

Table XXXIX

Tuberculin TestReaction Grades: IntradermalIntrapalpebralTest A.

Preventive Medicine

Laboratory animal veterinarians have the advantage of being able to employ all aspects of a preventive medicine program. The value of preventive medicine in maintaining healthy nonhuman primates far exceeds the benefits of the most sophisticated diagnostic laboratory capability or the most astute laboratory animal diagnostician. A comprehensive preventive medicine program not only involves preemptive immunizations, diagnostic capabilities, and prophylactic drugs, but should permeate the entire health-care and maintenance program. It includes good nutrition, parasite control, facility and primary enclosure design, quarantine and isolation policies, traffic patterns, experimental and social histories, sanitation, vermin control, and awareness of zoonoses. 1.

Quarantine

All newly acquired primates should undergo a quarantine period appropriate to the species. The quarantine program provides segregation of new animals for the time necessary to acclimate animals to their new environment and carry out diagnostic procedures for the detection of pathogens. Quarantined animals harboring pathogenic agents may be treated without exposing other susceptible animals within the facility. The quarantine period should be a minimum of 30 days, ideally 90 days. Selecting the appropriate duration in quarantine depends on the source of the animal(s), facilities available, type of research, institutional policies, and the value of the resident colony. In addition, newly imported primates must be quarantined for a minimum of 31 days at a CDCP-registered primate import facility (Southers and Ford, 1995). Quarantine facility location should allow complete separation from the resident colony; ideally, quarantine should be performed as a separate program. Supplies or equipment should be decontaminated before being transferred from the quarantine area to a "clean area." Personnel traffic patterns should always move from clean to dirty. If staff must move from the quarantine to clean areas, they should shower and put on new clothing before entering the resident colony. On arrival, nonhuman primates should be allowed a 72 hr acclimation period. After the acclimation period, a veterinarian should perform an entry physical examination. This should include individual record formation, hematology, serology, weight, anthelmintics, fecal exam, vaccinations, thoracic radiographs (when appropriate), and a tuberculin test. An intradermal old mammalian tuberculin test should be repeated every 2 weeks in alternating eyelids for the duration of the quarantine period. Each tuberculin test should be evaluated at 24, 48, and 72 hr. Any suspicious tuberculin reaction should be repeated in

Reaction grade

Description of changes No reaction observed Bruise; extravasationof blood in eyelidfrom injection Varying degrees of erythemaof palpebrum without swelling Varying degrees of erythemaof palpebrum with minimum swelling or slight swellingwithout erythema Obvious swellingof palpebrum with drooping of eyelid with varyingdegrees of erythema Swelling and/or necrosis with eyelidclosed

7 days using the opposite eyelid or a shaved region near the umbilicus area. Table XXXIX lists tuberculin test reaction grades. If one animal in the group is positive, all animals in the group must be considered exposed, and the testing and quarantine procedures extended (Martin; 1986). The animal(s) should be observed daily throughout the quarantine period. Key clinical parameters to evaluate include activity, appetite, and excreta. To assure continuity, the same individual should perform these observations as much as possible. Once the quarantine period has been completed, the veterinarian should perform an "exit physical." The veterinarian can release the animal(s) from quarantine or require an extension of the quarantine period. 2.

Immunizations

Historically, various active and passive immunization agents and procedures have been used with nonhuman primates (Southers and Ford, 1995). The decision whether to administer conventional vaccines will depend on a risk-benefit assessment for each situation. While deleterious effects are uncommon, vaccination efficacy is rarely based on scientific data from nonhuman primates. Sometimes immunizations are contraindicated because of research design. Immunizations may serve only as cheap insurance to help protect a valuable research animal. As the demand for high-quality nonhuman primates increases, some vaccine use seems prudent. Tetanus, rabies, measles, and polio vaccines have been common and well documented in nonhuman primates; these may be indicated if animals are housed in large production colonies or outdoor facilities. The results of a recent poll concerning vaccination strategies of primate facilities that house the nonhuman primate species highlighted in this chapter are summarized in Table XL (B. Bernacky, personal communication, 2000). Decisions about the frequency of boosting vaccines must be based on human protective titer literature and random titer sampiing of the nonhuman primate colony. Even though human polio vaccine is still routinely used in chimpanzees, polio has essentially been eradicated; the continued use of the vaccine is

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16. NONHUMAN PRIMATES 1. History update: as changes warrant, in particular reproductive parameters, cage location changes, participation in research, and

Table XL

Vaccination Recommendations

hospitalizations. 2. Weightrecorded: allowsdetection of subtle changes over time; the magnitude of weightloss may indicate a serious disorder. 3. Systemsexamined: to include cardiac, respiratory,ophthalmic,otolaryngeal, dermatologic,gynecologic,orthopedic, lymphatic, and digestive (dental, gastrointestinal). 4. Hematology:shouldinclude a completeblood count, serum chemistries and serologyfor CHV-1, and retroviral surveillancefor Old World monkeys. 5. Tuberculintest: 0.1 ml of full-strength, old mammaliantuberculin in a 25- to 27-gauge ~/2-inchneedle is injected intradermallyinto the upper eyelid. The test is read at 24, 48, and 72 hr; Table XXXIX contains definitions of reaction grades. 6. Fecal examination: shouldinclude testing for nematodes, protozoa and sporozoan parasites, and cultures for Campylobacter spp., Shigella spp., and Salmonella spp. 7. Vaccines:as indicated for the species and exposurehistory (Section V,A,2).

Age at initial immunization

Vaccine

(years)

Booster

Chimpanzee

Polio, MLV a Rabies, killed Tetanus toxoid b

1 2 1

None q3 years q5 years

Macaque

Measles, MLV Rabies, killed Tetanus toxoid

1 1 1

None q3 years q5 years

Baboon

Tetanus toxoid

1

q5 years

Species

Marmoset

Squirrel monkey Owl monkey Modified live virus. bRecommendedfor animals housed outdoors. a

therefore questionable. The effectiveness of yellow fever vaccines in nonhuman primates is well documented; nevertheless, the limited number of nonhuman primates originating from areas of endemic yellow fever does not warrant its routine use. The search for effective vaccines against enteric and pneumococcal diseases in humans is continuing, but at this time these vaccines appear to offer little advantage in managing nonhuman primates. Even the pneumococcal vaccine currently marketed for human use seems to be of questionable effectiveness in the chimpanzee (Jones et al., 1984). Intensive efforts to develop vaccines effective against herpesviruses and retroviruses have been unsuccessful so far. Should these types of vaccines become available, they could play an important role in protecting production colonies of nonhuman primates and preventing significant zoonoses. At this time, specific pathogen-free production colonies must depend on test-and-cull strategies. Although this technique has significantly reduced the zoonotic risk of CHV-1, it is not absolute. All management and handling techniques must be designed to protect against the remote possibility of latent CHV-1 transmission.

8. Radiology, ultrasonography: as needed. 9. Treatments: as needed. A formulary is given in Table XLI.

4.

The significant mortality and zoonotic potential of tuberculosis in nonhuman primates, albeit rare, demands aggressive preventive measures (Motzel et al., 1999; Hill et al., 2000). Immunization techniques (e.g., BCG vaccination) are not practical and have many disadvantages in the United States. Presently, the most effective means of preventing tuberculosis in nonhuman primates entails consistent tuberculin testing, leading to the identification and elimination of tuberculin reactors. The alarming rise in drug-resistant mycobacteria presents an additional serious consideration in diagnosing and treating nonhuman primates for tuberculosis. Currently, the greatest risk to this effective method of detection and control of tuberculosis in nonhuman primates is the availability of old mammalian tuberculin. The very specialized, limited demand for this diagnostic product and its rigid manufacturing specifications make it impractical to manufacture in a cost-effective way. To date, its importance to the research community is appreciated, and its production is continuing. 5.

3.

Surveillance

Frequent animal observations by caretakers, technicians, and veterinarians are a key element of an effective preventive medicine program. Serious illness can go undetected in some nonhuman primates because of their stoic nature. Animal care staff must be trained to recognize subtle physiologic and behavioral changes associated with early onset of disease. Routine physical exams and supporting laboratory work should be performed annually or more frequently based on the health status of the colony. The following elements are essential to a thorough physical examination.

Tuberculosis Testing

Nutrition

A well-nourished nonhuman primate can more effectively resist infectious diseases and functional disorders. The quality of available commercial diets makes taking advantage of the preventive medical benefits of balanced nutrition relatively easy. It is becoming apparent that modification of the traditional commercial nonhuman primate diets, with respect to increasing fiber and reducing fat and protein, can bring benefits to the general health of nonhuman primates. During this era where food enrichment plays a dominant role, enrichment strategies should be tempered with a concern for the animal's nutritional health. Paramount to adequate nutrition in the laboratory setting for

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BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Table XLI Formulary Compound Antibiotics Amoxicillin Ampicillin Baytril Cephalexin Cephapirin sodium Chloramphenicol Doxycycline Erythromycin Gentamicin Metronidazole Penicillin G benzathine Penicillin G potassium Penicillin G procaine Tetracycline Trimethoprimsulfamethoxazole Analgesics Acetylsalicylic acid Acetaminophen Buprenorphine Butorphanol tartrate Flunixin meghemine Naproxen Anesthetics Acepromazine maleate Diazepam Ketamine hydrochloride Midazolam hydrochloride Thiopental sodium Tiletamine hydrochloride Xylazine Halothane w/50% 02, 50% N2 Isoflurane Miscellaneous Atropine Dexamethasone Doxapram Epinephrine Fenbendazole Furosemide Ivermectin Oxytocin Stanozolol Pentobarbital

Macaque a

Chimpanzee a

Baboon b

20 - 40 mg/kg 25 mg/kg 2.5-10 mg/kg 25-50 mg/kg/day

250-500 mg/day 500mg 2.5-10 mg/kg 250 mg

20 mg/kg 25 mg/kg 5.0mg/kg 25 mg/kg

25 mg/kg 5 mg/kg 30-80 mg/kg/day 3-5 mg/kg 25-50 mg/kg 30-60 kU/kg

50 mg/kg/day

25 mg/kg 2.5 mg/kg 50 mg/kg 3 mg/kg 50mg/kg 30 kU/kg

20-40 kU 25 - 50 mg/kg/day

600-1000 kU

10-20 mg/kg 5-10 mg/kg 0.01 mg/kg 0.05- 0.10 mg/kg 0.5- 4 mg/kg 10mg/kg

325-650mg 200- 400 mg 0.01 mg/kg

25-50 mg/kg/day 3-5 mg/kg 250mg 900-1200 kU/kg

25 mg/kg 20 mg/kg

20mg/kg 50 mg/kg

1-2 mg/kg 15 kU 20 kU/kg 15 kU 15 mg/kg 24 mg/kg

10.0mg/kg 0.01 mg/kg

0.01 mg/kg

0.50mg/kg 500 mg initial; then 250mg

0.1-1.0 mg/kg 1.0-5.0 mg 5-10.0 mg/kg

1.0-20.0 mg 10.0 mg/kg

5.0 mg 10.0mg/kg

22- 25 mg/kg 3 - 6 mg/kg 0.25 -0.5 mg/kg 1- 4% induction, 0.5-2.0% maintenance 1-2% induction, 0.5-1.5% maintenance

3-5 mg/kg 0.5 - 1.0 mg/kg 1- 4% induction, 0.5-2.0% maintenance 1-2% induction, 0.5-1.5% maintenance

1- 4% induction, 0.5-2.0% maintenance 1-2% induction, 0.5-1.5% maintenance

0.05 mg/kg 0.5 - 5 mg/day 2 mg/kg 0.1-0.5 mg 50mg/kg • 3days 1-2 mg/kg 200 ~g/kg 5-30 U 10-50 mg/week 0.2 mg/kg

New World monkey c

0.02-0.2 mg/kg 5 - 15 mg/day 2 mg/kg 0.2-1.0 mg 50mg/kg • 3days 1-2 mg/kg 200 Ixg/kg 5-30 U 0.2 mg/kg

aFrom M. D. Anderson Cancer Center, Department of Veterinary Sciences. bFrom Michelle Leland, Southwest Foundation. r Abee (1985) and Brady (2000).

20- 40mg/kg, SC 0.225 mg 15 - 20 mg/kg

4 - 8 mg/kg

Dosage

BID-QID BID BID BID-QID QID; IM, IV BID-QID; PO BID BID BID-TID; IM BID-TID EOD; IM, SC IV, IM, SC SID; SC BID BID; PO

TID-QID; PO TID; PO BID; IM, SC QID; IM SID; IM BID-TID

IM; PO IM Prn; IV IV IM, IV IM, IV SC

2 - 4 % induction, 1-3% maintenance

0.05 mg/kg 0.5 mg/kg 2 mg/kg 0.1 mg 1 mg/kg 200 txg/kg 8U

IM IM, Day IV IM PO PO, IM PO, SC Prn, IM, SC IM; • weeks IV; euthanasia

16. NONHUMANPRIMATES nonhuman primates is assuring the availability of sufficient amounts of food for all animals when animals are paired or housed in social groups. Food-delivery techniques are thus additional considerations in maintaining good nutritional health. 6.

Occupational Health Program

The potential exchange of diseases between humans and nonhuman primates requires that those managing the care of nonhuman primates develop, implement, and sustain a comprehensive personnel health program designed to meet the needs of their employees. In addition, the institution should have a standard operating procedure following suspected exposure to a zoonotic agent. An example protocol is depicted in Table XXXVI. Although policies vary between situations and facilities, there are certain basic elements that should be considered in any risk analysis. The 1997 NRC publication "Occupational Health and Safety in the Care and Use of Research Animals" is an excellent guide for developing an effective program. A program must have oversight by occupational health specialists to protect personnel, along with a laboratory animal veterinary specialist who can provide input to the program regarding potential zoonotic agents. The veterinarian should also provide expertise regarding protection of nonhuman primates from human pathogens that could be transmitted to animals by the public, contract workers, students, caregivers, and investigators.

B.

Clinical Techniques

With today's pediatric care devices, instrumentation, and biotechnology, there are few limitations to collecting biological samples, administering therapy, and performing special techniques on nonhuman primates. Based on body size, the New World monkeys are at times more challenging, but most investigative and animal care needs can be met if resources (financial, personnel, equipment) are available. Such constraints are more rare with Old World monkeys and chimpanzees. The techniques described in this section are applicable for delivering highquality veterinary care to the nonhuman primate as well as collecting data for biomedical research programs. Effective use of these techniques significantly reduces the need to euthanatize the animals during a study, thus reducing the number of nonhuman primates required in a given study. The list of zoonotic pathogens continues to grow as more is learned about the simian immunodeficiency viruses (SIV), simian retroviruses (SRV), foamy viruses, ectopic Ebola virus, and recombinant agents like simian-human immunodeficiency virus (SHIV). (See Chapter 25.) Considering this level of risk, effective restraint and chemical immobilization are paramount to preventing human exposure. Drug choices and regimens are given in Table XLI.

727

Certain special techniques are beyond the scope of this chapter but are frequently used when working with nonhuman primates. These techniques may involve the use of Alzet osmotic pumps (ALZA Corporation, Palo Alto, California), Ommaya reservoirs (Integra NeuroSciences, Plainsboro, New Jersey), and sophisticated imaging techniques such as computer assisted tomography (CT), magnetic resonance imaging (MRI), and position emission tomography (PET). The techniques addressed in this section are intended to aid the clinical veterinarian in the routine collection of body fluids or tissue and the effective delivery of therapy. Techniques not addressed here have been documented elsewhere in the human and veterinary literature (Dysko and Hoskins, 1995; Levin, 1995). I.

Fluid Collection a.

Blood

The most common site for venipuncture in the nonhuman primate is the femoral vein. In smaller species and infants, the venipuncture is "blind," aided by the anatomy of the femoral triangle. In larger species or adults, the femoral artery and adjacent vein can be palpated. In adult macaques and chimpanzees, blood can also be collected from the saphenous and cephalic veins. The latter, however, are more commonly used for fluid administration. Venipuncture sites other than the femoral triangle in New World species include the saphenous veins and the bilateral tail veins (Abee, 1985). The jugular vein represents an alternative site for venipuncture in all species. Various veterinary and human rule-of-thumb formulas for calculating a safe, maximum blood draw have been used successfully in nonhuman primates; one is considered to be 15% of the total blood volume to be withdrawn no more frequently than biweekly. McGuill and Rowan (1989) recommend the 10%-10% rule, which states that the maximal blood sample size is 10% of a blood volume estimated to be 10% of the body weight. When all blood components are not required, blood samples can exceed the calculated limits if the plasma or red cells that were collected are returned to the animal after separation, along with appropriate fluid replacement. For macaques, a 20- to 23-gauge needle on a 10 ml syringe works well for the typical sites of blood collection. For chimpanzees, an 18- to 20-gauge needle on a 20 to 30 ml syringe can be used. In squirrel monkeys, the femoral vein is the site of choice, with a .75- to 1.0-inch, 21- to 23-gauge needle; while marmosets should be sampled using the femoral vein, with a 25to 26-gauge needle and a 1.0 ml syringe. Butterfly needles are commonly used with infants and smaller species because the needle and wing lie flat and a successful puncture can be detected by gravity flow (Dysko and Hoskins, 1995). Some vacuum-type blood collection systems, such as the Sarstedt SMonovette Blood Collection System (Newton, North Carolina), are especially safe for personnel to use in larger primates,

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BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

namely macaques, baboons, and chimpanzees. However, they are not recommended for smaller animals because the smalldiameter vessels may collapse. In the research setting, nonhuman primates can be trained to allow voluntary hypodermic injection, urine collection, and venipuncture. This collection technique is excellent for pharmacokinetic studies requiring repeated small blood samples without the complications of anesthetic drugs or restraint distress. Specialized equipment or cage modifications may be necessary to permit voluntary exteriorization of a limb and assure personnel safety (Schapiro, 2000). b.

Urine

For routine urinalysis, urine can be collected from clean waste pans or via the free-catch method. The major disadvantage of this technique is bacterial contamination. Several methods have been described as modifications to this technique (Dysko and Hoskins, 1995). Catheterization of the urinary tract in both males and females can be done in macaques, baboons, chimpanzees, and squirrel monkeys. Bahnson et al. (1988) describes catheterizing cynomolgus monkeys with 7.0 to 9.0 French catheters while rhesus monkeys can be collected with 3.5 to 4.0 French catheters. A 3.0 French tomcat catheter may also be used for urethral catheterization of squirrel monkeys. Aseptic catheterization of the urinary tract of both the male and female nonhuman primate is possible, but this usually requires physical or chemical restraint and experience overcoming anatomical differences. Cystocentesis is the preferredmethod for sterile urine collection. Cystocentesis by puncture of the surgically prepared suprapubic site using a 1.5- to 2.0-inch, 20- to 22gauge needle is effective but may be difficult without experience (Keeling and Wolfe, 1975). Cystocentesis in the chimpanzee can be accomplished using a 2.0- to 3.0-inch, 20-gauge spinal needle. c.

to 2.0-inch, 22-gauge needle, while in the chimpanzee a 2.0- to 3.0-inch, 20-gauge spinal needle is used. Geretschlager et al. (1987) and Lipman et al. (1988) describe techniques for marmosets and cynomolgus monkeys, respectively. ii. Lumbar. Collection of CSF is most often accomplished by lumbar puncture in an anesthetized animal. A slight flexion of the spine will facilitate the widening of the intervertebral space. A horizontal line between both iliac crests will bisect the intervertebral space that should be entered. Based upon the animal size, an 18- to 20-gauge spinal needle can be used (Dysko and Hoskins, 1995). If there is difficulty entering the vertebral space at the transverse plane of the iliac crests, either of the two spaces above can serve as entry sites. In smaller animals, once the needle is in place, a slight rotation of the needle while applying pressure to the carotid arteries will sometimes establish CSF flow. 2.

Tissue Collection

a.

Skin Scraping

This diagnostic procedure involves abrading the skin with a scalpel blade and observing the debris microscopically to detect ectoparasites. Nonhuman primates are susceptible to skin parasites such as mites (Sarcoptes and Demodex spp.), fungal dermatophytes (Microsporum and Trichophyton spp.), and lice (Johnson-Delaney, 1994). An adequate skin scraping can confirm and differentiate ectoparasites. Scrapings should be collected from undisturbed and untreated areas (Bistner and Ford, 1995). For Demodex spp., the skin should be pinched prior to scraping, and for Sarcoptes spp., large areas should be sampled. Dermatophyte scrapings can be used to inoculate culture media or a Wood's light can be used to detect microsporum fluorescence. The hair shafts collected from such scrapings can be examined for adult lice or eggs (Georgi, 1980).

Cerebrospinal Fluid

Cerebrospinal fluid (CSF) can be collected from the cisterna magna or the lumbar area. When the subarachnoid space is to be entered, the animal must be anesthetized and the area surgically prepared. Smith and Lackner (1993) detailed comparisons of fluid from the lumbar region to that of the cisterna magna, finding lumbar fluid to have higher concentrations of total protein, albumin, and IgG and lower concentrations of glucose and potassium. The report states that similar findings for total protein are seen in humans (Dysko and Hoskins, 1995). i. Cisterna magna. Similar to CSF collection in the canine, CSF in the nonhuman primate should be collected at the junction of a line that bisects the cranial wings of the atlas and a line from the external occipital protuberance (Bistner and Ford, 1995). In macaques, the procedure can be performed with a 1.5-

b.

Skin Biopsy

The surgical removal of a small section of skin may be needed when a differential diagnosis requires histopathology or impression smears. These samples can demonstrate the presence of bacterial, fungal, and parasitic organisms, and identify neoplastic or immune-mediated skin disease (Crow and Walshaw, 1987). i. Cutaneous punch biopsy. The punch biopsy is a quick method of sampling a small core of skin for histopathological evaluation. The biopsy samples should include both diseased and normal tissue, and the site should be surgically prepared. Punch biopsies offer a range of sizes, but the typical diameters are 4 - 8 mm (Dysko and Hoskins, 1995). Skin sutures may be needed to close the biopsy site.

16. NONHUMAN.PRIMATES ii. Cutaneous wedge biopsy. When a large full-thickness skin biopsy is required, the punch biopsy is inappropriate. An excisional biopsy is used to collect the entire lesion. If only a portion of the lesion is biopsied, an incisional biopsy is utilized (Bistner and Ford, 1995). With either technique, different stages of the lesion should be sampled with normal tissue; traumatized or crusted lesions should be avoided (Crow and Walshaw, 1987). The biopsy site should be closed with sutures. When skin biopsies are collected, multiple samples will increase the likelihood of obtaining a diagnostic sample. These biopsies should be performed under general anesthesia. c.

Lymph Node Biopsy

Lymph node biopsies are indicated when there is lymph node enlargement, generalized lymphadenopathy, or suspicion of tumor metastasis (Dysko and Hoskins, 1995). Lymph node samples can be collected by needle aspiration, punch biopsy, and excisional biopsy. The most accessible lymph nodes in nonhuman primates are the axillary and superficial inguinal nodes. During exploratory surgery, excisional biopsies are most frequently done on mesenteric and iliac lymph nodes (Boothe, 1990). d.

Bone Marrow

Needle aspiration is the most common technique used to collect bone marrow samples from nonhuman primates; the preferred site is the iliac crest. While other sites can be used (trochanter of femur, tibial tuberosity, greater tubercle of the proximal humerus, sternum, rib, and ischial tuberosity), the iliac crest is usually an active site of marrow production (Perman et al., 1974). In smaller species (marmosets, owl monkeys, and squirrel monkeys), the size of the marrow cavity may be small, and minimal aspiration will rupture vasculature in the space, resuiting in a peripheral blood sample rather than a sample of bone marrow. 3.

Radiology

Diagnostic radiography has proven to be a beneficial diagnostic tool in nonhuman primate medicine. Human pediatric- or adult-technique charts work well for nonhuman primates. Thoracic pathology is common and should be one of the diagnostic procedures performed when tuberculosis is a consideration. To be of greatest diagnostic value, nonhuman primate thoracic radiographs should be made with the animal in an upright posture. This positioning will necessitate a vertical cassette holder. Abdominal radiographs in the nonhuman primate are significantly enhanced by pneumoperitonography (Morgan et al., 1975). This can be combined with contrast media to increase diagnostic effectiveness. It is a relatively simple and safe technique. Carbon dioxide is instilled into the peritoneal cavity un-

729

til there is moderate distension. Various radiographic views (anterior/posterior, posterior/anterior, and lateral) can capitalize on nonsupported, gravitational positioning of the organs. The carbon dioxide will be absorbed in 1-2 hr. 4.

Ultrasonography

Many clinical and diagnostic procedures are facilitated by ultrasound. Fine-needle aspiration of organs and joints, as well as cavity and abscess drainage and centesis of abdominal, bladder, and thoracic regions, can be assisted with ultrasound guidance. Organs like the liver, kidney, and prostate are within reach for ultrasound-guided biopsies. Real-time ultrasonography can assess the form and function of the cardiovascular system. Ultrasonography of the reproductive system can detect prostate abnormalities, follicular and luteal changes, sex of a fetus, and gestational age (Blevins et al., 1998). Morphological characteristics detected by fetal ultrasound (crown-rump length, biparietal diameter, head circumference, and femur length) have been determined for the rhesus monkey, baboon, and chimpanzee. Longitudinal growth curves established for these species can be used to predict estimated parturition dates (Nyland et al., 1984; Herring et al., 1991; Lee et al., 1991). Preparation of the animal should include full sedation, and the region of interest should be clipped, shaved, and wiped with alcohol or soapy water. Acoustic gel should be used as the interface between the animal's skin and the transducer. A 3.0 to 7.5 MHz transducer is adequate for most areas to be scanned, although most ocular ultrasonography requires a 10.0 MHz scan head (Blevins et al., 1998). 5.

Other Techniques a.

Intravenous Administration~Collection

Indwelling catheter placement is a frequently employed technique in nonhuman primates for extended intravenous fluiddelivery therapy and investigative data collection. A conventional surgical cut-down procedure is usually the most effective way to place the indwelling catheter. For the chimpanzee, baboon, and macaque, the cephalic or saphenous veins can be employed. Numerous veterinary and human catheter systems can be used in combination with limb immobilization by splint and bandage or lightweight casting material. More sophisticated systems can be used by employing a jacket with tether or backpack equipment. b.

Intraosseous Administration

Intraosseous infusion of fluids may be used in severely debilitated, hypotensive animals when venous access is limited. The most accessible site in the owl monkey is the trochanteric fossa of the femur (Baer, 1994). The site is prepared aseptically, and

BRUCE J. BERNACKY,SUSANV. GIBSON, MICHALE E. KEELING,AND CHRISTIANR. ABEE

730

the skin and periosteum are anesthetized with 1% lidocaine. A 20-gauge needle is introduced aseptically into the medullary cavity through the trochanteric fossa. The needle may be secured by suturing a tape butterfly, which incorporates the hub of the needle to the skin. Fluid is administered through a standard infusion set; flow rate is limited because the bone marrow cavity cannot expand to accept increased volumes. c.

Nasogastric Intubation

Nasogastric intubation is commonly practiced. Nasogastric intubation (5 to 8 French nasogastric tube) can be a rapid and effective means of delivering oral fluids and medication. The procedure is vital in relieving acute gastric dilation (bloat), and culture of gastric lavage has been used to confirm mycobacterial infection (Baron et al., 1994). Adult human nasogastric tubes can be used in adult chimpanzees. d.

Rectal Prolapse Repair

Nonhuman primates of any age or sex can experience rectal prolapse, but it is most frequently diagnosed in young animals. Typically, rectal prolapse results from straining and severe diarrhea associated with enteritis or colitis. Other causes include social or management-related stress, rectal foreign bodies, lacerations, neoplasia of the rectum or distal colon, dystocia, urolithiasis, and prostatitis (Merck, 1991; Brown and Swenson, 1995). The prolapse may be partial or complete. Partial prolapse involves only the rectal mucosa and appears as a red, swollen, doughnut-shaped mass. Complete prolapse appears as a cylindrically shaped, edematous mass involving all layers of the rectal wall (Sherding, 1994). Successful treatment involves reduction of the prolapse and identification of the underlying cause. Partial prolapses may resolve spontaneously or may require manual reduction using lubricants and hypertonic sugar compresses to reduce the associated edema. If manual reduction is not possible, an enema of lidocaine gel may be helpful. The gel aids in extending the tissue and relieves the urge to strain (Brown and Swenson, 1995). A perianal purse-string suture may be needed if straining is expected to occur while the primary cause is being treated. The suture should remain in place for 5 - 7 days. If the viability of prolapsed tissue is questionable, then rectal resection and anastomosis should be performed. When the tissue is healthy but manual reduction fails after repeated attempts that include purse-string placement, a celiotomy and colopexy are advised (Merck, 1991). e.

Dental Procedures

The canine teeth of male Old World monkeys are long, sharp, and capable of inflicting considerable trauma to cagemates and personnel. To reduce the risk and severity of bite wounds, it is

often necessary that the canine teeth be modified. In the past, canine teeth were extracted, but this procedure has fallen out of favor due to the resulting migration of adjacent teeth and malocclusion (Brown and Swenson, 1995). Bass and Stark (1980) reported a technique for blunting canine teeth that involved shortening them tO the same height as the adjacent premolars. The exposed root canal pulp is cleared and the cavity filled with amalgam. This technique causes less trauma to the animal, leaves no extraction wound, prevents tooth migration and malocclusion, and is easy to perform. Coman et al. (1998) described a pulpotomy technique from human dentistry. Materials needed are calcium hydroxide as the pulpal capping agent, varnish as a barrier layer and sealer for the pulp and dentinal tubules, and amalgam as the restorative material. The method promotes the maintenance of a viable tooth through a fast (45 min/monkey), less invasive procedure that results in little patient discomfort and no sequelae of tooth migration. Schofield et al. (1991) described a technique, termed root banking, adapted from human dentistry. This technique involves amputating the canine tooth below the level of the alveolar bone crest and covering the exposed pulp with a mucoperiosteal gingival flap. A vital tooth root is preserved, buried in alveolar bone, and covered with normal mucosa. Periapical abscess formation, a potential complication of the root canal procedure, does not occur with root banking (Brown and Swenson, 1995).

VI.

A. 1.

DISEASES

Bacterial Diseases

Enteric

Enteric disease of bacterial etiology is a primary cause of morbidity and mortality in Old World monkeys but is less frequently encountered in New World species. Determining the etiology of a bacterial diarrhea may be quite difficult. Commonly, two or more potentially pathogenic bacteria may be isolated from an individual with diarrhea. The clinician must ascertain the primary pathogen and treat appropriately. A summary of bacterial enteric diseases by isolate and primate species is presented in Table XLII. a.

Shigellosis

Shigella spp. are among the most common enteric pathogens recovered from captive nonhuman primates, occurring in a variety of species such as marmosets, tamarins, macaques, baboons, and apes. Shigella flexneri is the most frequent isolate, including serotypes l a, 2a, 3, 4, 5, 6, and 15 (Russell and DeTolla, 1993), with S. sonnei and S. boydii occurring less

16. NONHUMAN PRIMATES

731

Table XLII Enteric Bacterial Infections Agent Shigella spp.

Campylobacter spp.

Escherichia coli

Primate

Clinical signs

Macaque

Diarrhea _ blood +_ mucus _ mucosal fragments Depression, hunched posture

Marmoset Tamarin

Lethargy, dehydration, depression Pale mucous membranes Blood in feces

Macaque

Mucohemorrhagic diarrhea

Baboon

Chronic diarrhea

Marmoset

Watery or mucohemorrhagic diarrhea

Chimpanzee

Mild-severe, watery diarrhea; or dysentery

Squirrel monkey

Infant death

Owl monkey

Profuse, foul-smelling, blood-tinged diarrhea Dehydration, depression

Tamarin

Watery diarrhea

Salmonella spp.

Macaque

Diarrhea, pyrexia Infants nwatery diarrhea ___ blood _ mucus

Yersinia spp.

Macaque

Diarrhea, vomition, blood-stained feces Depression, abdominal pain Dehydration Abortions/stillbirths Weak, inactive, enlarged cervical lymph nodes Weakness, depression, diarrhea, abdominal distension, splenomegaly

Squirrel monkey Owl monkey

Helicobacter s p p .

Lawsonia intracellularis

Macaque

Clinical pathology Leukocytosis with left shift

Chronic diarrhea

Macaque

Mild or transient diarrhea, abdominal distension Death, no clinical signs

Enrofloxacin 5 mg/kg ~b or multidrug therapy c Rehydrate Neomycind in water for 10 days Quarantine

Hyponatremia, hypochloremia acidosis

Usually self-limiting Rehydrate

Normal WBC to leukocytosis with left shift

Erythromycin 25 mg/day e

Ampicillin, Gentamicin, or trimethoprimsulfamethoxazole/ Intestinal protectant Rehydrate Usually multidrug resistant; carrier state can develop Leukocytosis Hyponatremia, hypochloremia prerenal azotemia hyperfibrinogenemia

Usually unsuccessful Chloramphenicol 100 mg/kg, divided doses TID g Rehydrate

Neutrophilia with left shift

Trimethoprim-sulfamethoxazole or gentamicin I Rehydrate Oral intestinal protectant

Gastric: anorexia, vomiting Intestinal: chronic diarrhea

Cotton-top tamarin

Treatment

Triple antibiotic therapy: amoxicillin, metranidazole, bismuth (other combinations also approved for humans) Anemia

aFrom Line et al. (1992). bFrom Banish et al. (1993b). CFrom Olson et al. (1986); therapy consisted of oral trimethoprim-sulfamethoxazole, erythromycin, and tetracycline. dFrom Cooper and Needham (1976). "From Pinheiro et al. (1993). fFrom Weller (1994); drug doses not provided in text. gFrom Rosenberg et al. (1980).

732

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

frequently. Endemic infections within colonies are maintained by asymptomatic carriers (Banish et aL, 1993b; Russell and DeTolla, 1993). Infection is spread by the fecal-oral route among primates within the same group, by movement of primates between groups, and through importation of new primates into the colony. Infection and disease with subsequent antibody production do not provide immunity, and animals may be chronically reinfected (Banish et al., 1993a). Overt disease, manifested by diarrhea or dysentery, may not occur in endemically infected animals without a precipitating stressful event, such as social group disruption or formation or transport to a new facility. Clinical signs of shigellosis vary in Old World monkeys. Dysentery is characterized by foul-smelling, liquid stool containing mucus, frank blood, and/or mucosal fragments. Affected monkeys are weak and moderately to severely dehydrated, and they require prompt medical treatment to correct life-threatening fluid and electrolyte imbalances. A more common form of shigellosis is a subacute to chronic diarrhea with liquid to semisoft stool. Diarrhea is intermittent to episodic; occasionally animals will have firm, mucus-laden feces with streaks of fresh blood. These animals do not appear clinically ill, and episodes frequently resolve spontaneously. However, they serve as sources of infection for other individuals within the colony. Gingivitis, abortion, and air sac infection are nonenteric forms of shigella infections that may occur in rhesus monkeys (McClure et al., 1976). Diarrhea is not a primary clinical sign of shigellosis in marmosets and tamarins. Instead, affected animals are lethargic, depressed, and dehydrated with dried blood found around the anus (Cooper and Needham, 1976). Lesions of enteric shigellosis occur primarily in the cecum and colon. The colonic mucosa is usually covered with a fibrinopurulent exudate, and the intestinal wall is edematous and hemorrhagic with focal areas of ulceration. Luminal contents vary from fluid mucus with fibrin and cellular debris to frank hemorrhage and mucus in the lumen (Lindsey et al., 1971; Mulder, 1971). Intussusception of the small intestine, rectal prolapse, splenomegaly, and mesenteric lymphadenopathy may occur. Gross lesions in marmosets and tamarins are confined to the cecum and colon (Cooper and Needham, 1976) and consist of multifocal ulceration and petechiation in the mucosa, with blood and fluid feces found throughout the large bowel. Treatment for shigellosis should include antibiotic therapy based on sensitivity testing and aggressive correction of deficits in hydration, acid-base balance, and electrolytes. Empirical antibiotic therapy may be necessary in acute cases, based on colony history. Shigella spp. rapidly develop antibiotic resistance, and many species have multiple drug resistance. Antibiotics that have been reported to be successful in treating shigella infections include enrofloxacin, 5 mg/kg body weight once a day (Line et al., 1992; Banish et al., 1993b), and combination therapy with oral trimethoprim-sulfamethoxazole, erythromycin, and tetracycline (Olson et al., 1986). Shigella spp. were eliminated from a colony of callitrichids through quarantine

procedures and administration of neomycin in the drinking water for 10 days (Cooper and Needham, 1976). Shigellosis is a zoonotic disease and has been transmitted from monkeys to children, pet owners, animal caretakers, and research technicians (Mulder, 1971; Fox, 1975; Tribe and Fleming, 1983; Kennedy et al,, 1993). Severe dysentery resulted from infection, and in one instance death occurred (Fox, 1975). b.

Campylobacteriosis

Campylobacterjejuni and C. coli are the most frequent fecal bacterial isolates from subclinical and clinically affected nonhuman primates (Tribe et al., 1979; McClure et al., 1986; Russell et al., 1987). Infection usually occurs by the fecal-oral route. Prevalence of infection varies greatly, with some colonies reporting a 100% infection rate (Russell et aL, 1988) and with isolates from clinically normal monkeys composing up to 81% of total isolations (Taylor et al., 1989). Campylobacter jejuni infection in macaques is probably the result of exposure to contaminated water and foodstuffs after capture (Morton et al., 1983). Prevalence of infection in macaques increases with time in captivity. In contrast, recently trapped tamarins had a higher prevalence of infection with C. coli initially than after they had been housed in the laboratory for 1 year (Gozalo et aL, 1991). Disease due to campylobacter infection usually presents as watery diarrhea, although mucohemorrhagic diarrhea has also been reported (Paul-Murphy, 1993). Primates with diarrhea and campylobacter infection may have normal white blood counts or leukocytosis with a left shift (Paul-Murphy, 1993). Severe electrolyte abnormalities, including hyponatremia (Na < 132 mEq/liter), hypochloremia (C1 < 93 mEq/liter), acidosis, and a high anion gap, occur in monkeys with campylobacter diarrhea (George and Lerche, 1990). Based on these reported electrolyte imbalances, rehydration and replacement of electrolytes by normonatremic fluids such as lactated Ringer's solution or normal saline should be considered (George and Lerche, 1990). Because many infections are self-limiting and reinfection frequent (Russell et al., 1987), the efficacy of antibiotic treatment for campylobacter diarrhea is debatable and should probably be determined on a case-by-case basis. Erythromycin has been the antibiotic of choice; however, resistant campylobacter strains have been reported (Tribe and Fleming, 1983). c.

Colibacillosis

Isolation of Escherichia coli from a primate with diarrhea can be a diagnostic dilemma (Thomson and Schemer, 1996), as it is a frequent normal fecal isolate. Mucoid and hemolytic colony types and specific serotypes of E. coli have been associated with increased pathogenicity. Young chimpanzees with pathogenic E. coli infection of serotypes O119:B14, O55:B5, and O26:B6 had mild to moderately severe, watery diarrhea for 2 to 10 days (McClure et al., 1972). Small amounts of mucus or blood were

16.

NONHUMANPRIMATES

found occasionally in the stool. Peracute disease developed in some of the chimpanzees, resulting in death within hours. Death with no clinical signs occurred with infections of E. coli serotype O 13 in squirrel monkey infants (Scimeca and Brady, 1990). Tamarins infected with a hemolytic E. coli developed watery diarrhea (Potkay, 1992). Gross lesions in the apes included diffuse mucosal hemorrhage in the gastrointestinal tract, splenomegaly, congested and slightly enlarged mesenteric lymph nodes, pulmonary hemorrhages, and edema (McClure et al., 1976). Gross lesions in the squirrel monkey infants were not diagnostic; invasive colitis and septic meningitis were seen microscopically (Scimeca and Brady, 1990). Tamarins with diarrhea had congestion, edema, and necrosis of the intestinal mucosa (Potkay, 1992). Most recently, enteropathogenic E. coli (EPEC) have been identified in rhesus monkeys infected with simian immunodeficiency virus (Mansfield et al., 2001). Animals with EPEC often had an infection with other opportunistic enteric pathogens. Clinically, infection was associated with persistent diarrhea and wasting; EPEC infection was more frequent in animals dying at < 1 year of age. The EPEC was histologically characterized by the typical attaching and effacing lesions. d.

Salmonellosis

Frequently reported serovars of Salmonella spp. from nonhuman primates have been S. typhimurium, S. choleraesuis, S. anatum, S. stanley, S. derby, and S. oranienburg (Good et al., 1969; McClure, 1980; Potkay, 1992). Salmonella infection usually occurs from fecal-oral transmission. Infection can result from ingestion of contaminated food, water, or fomites; airborne infection is rare. Insects have served as mechanical vectors. Salmonellae can survive and multiply for relatively long periods in the environment, Reports of salmonella infection and disease within established primate colonies are rare. A mild to severe diarrheal disease associated with infection by either S. miami or S. oranienberg took place in chimpanzees coinfected with Shigella spp. and intestinal parasites (Galton et al., 1948). Salmonella organisms were isolated from 6% of enteric cultures from nonhuman primates with diarrhea at a primate research center (McClure, 1980); 21 of the isolates were S. derby recovered primarily from rhesus infants during a nursery outbreak (McClure, 1980). Clinical signs of enteric salmonellosis include watery diarrhea, sometimes with hemorrhage or mucus. Animals are often pyrexic (Paul-Murphy, 1993). Extraintestinal signs include neonatal septicemia, abortion, osteomyelitis, and pyelonephritis (Thurman et al., 1983; Klumpp et aL, 1986; Duncan et al., 1994). Spontaneous, clinical enteric salmonellosis is characterized by edema, hyperemia, and rare mucosal ulceration in the ileum and colon. Enlargement of the spleen and mesenteric lymph nodes may occur. Treatment for salmonella infections requires antibiotic therapy and supportive care to compensate for fluid loss and electrolyte and acid-base imbalances that result from diarrhea. Many isolates have multiple antibiotic resis-

733

tance. Antibiotic therapy should be reserved for animals with severe diarrhea or septicemia to reduce the likelihood that a carrier state may develop. The zoonotic potential of salmonellosis and the difficulty in eliminating the carrier state may necessitate the culling of carrier animals. e.

Yersiniosis

Yersinia enterocolitica and Y. pseudotuberculosis cause fulminating enteric and systemic disease in marmosets, owl monkeys, squirrel monkeys, and Old World monkeys. Diarrhea, vomiting, severe abdominal pain, mild dehydration, and bloodstained feces were reported for macaques infected with Y. pseudotuberculosis (Bronson et al., 1972; MacArthur and Wood, 1983). Leukocytosis due to neutrophilia, hyponatremia, hypochloremia, prerenal azotemia, and moderate hyperfibrinogenemia were reported in affected macaques by Rosenberg et al. (1980). Abortions and stillbirths occurred in some animals. Yersinia-infected squirrel monkeys had nonspecific clinical signs, such as inactivity, weakness, and failure to cling to the dam, and cervical lymph node enlargement (Plesker and Carlos, 1992). Yersinia infections in nonhuman primates usually present with a triad of lesions at necropsy: multifocal hepatic and splenic necrosis or abscess formation, mesenteric lymphadenopathy, and ulcerative enterocolitis (Fig. 12). Squirrel monkeys have an unusual presentation in that cervical lymph nodes are also markedly enlarged (Plesker and Carlos, 1992). Multifocal, purulent, and necrotizing hepatitis, splenitis, lymphadenitis, and ulcerative gastroenterocolitis are the characteristic microscopic

Fig. 12. Hepatic abscesses due to Yersinia enterocolitica infection in a squirrel monkey,Saimiri spp.

734

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

lesions. Frequently, bacterial colonies or large masses of bacteria are seen within the central core of lesions, encased by necrotic debris with a peripheral border of mononuclear cells and neutrophils (Chang et al., 1980). Intestinal lesions range from focal mucosal necrosis with bacterial colonization to fullthickness mucosal necrosis with adherent intestinal contents, cellular debris, and bacteria (McClure et al., 1971; Baggs et al., 1976), with ulcerative lesions overlying gut-associated lymphoid tissue (Bronson et al., 1972). Treatment is usually unsuccessful due to the fulminating nature of the disease. f

Helicobacter spp.

Doenges (1939) probably provided the original description of Helicobacter pylori infection in monkeys when he described short spirochetes in the gastric glands of clinically normal rhesus monkeys. Isolation of H. pylori from stomachs of nonhuman primates, particularly macaques, has been reported by several authors since that time. Presence of H. pylori in the gastric mucosa of monkeys is often accompanied by persistent lymphocytic plasmacytic gastritis (Baskerville and Newell, 1988; Dubois et al., 1994; Reindel et al., 1999). Gastritis may or may not be present in monkeys infected with a related organism, "H. heilmannii" (Dubois et al., 1991). Clinical signs of gastric helicobacter infection may include inappetence and occasional vomiting. A novel Helicobacter sp. has been isolated and characterized from colons of cotton-top tamarins, Saguinas oedipus, with inflammatory bowel disease (Saunders et al., 1999). A specific serological response to this novel Helicobacter spp. was also found in cotton-top tamarins with chronic colitis (Whary et al., 1999). Similarly, novel Helicobacter spp. as well as H. cinaedi have been isolated from inflamed colons of rhesus monkeys (Fox et al., 2001a,b). Chronic, idiopathic, diffuse colitis is a well-recognized clinical and pathological entity in captive rhesus monkeys, characterized histologically by chronic, moderate to severe colitis and typhlitis, with diffuse mononuclear inflammation of lamina propria, reactive lymphoid hyperplasia, and multifocal microabscesses. The exact role of Helicobacter spp. in this disease entity has not been determined. g.

Lawsonia intracellularis

Klein et al. (1999) reported a fatal proliferative enteritis in juvenile, colony-born rhesus monkeys due to infection with Lawsonia intracellularis. Affected monkeys were between 6 and 16 months of age. Clinical histories included depression, mild or transient diarrhea, and abdominal distension, although most animals were found dead or moribund with no clinical history. Moribund animals were hypothermic and anemic, hematocrit = 10. The primary gross lesion was segmental thickening and pallor of the distal 5 cm of ileum. Distortion or loss of villouscrypt architecture, blunting and fusion of villi, and proliferation

of pseudostratified tall columnar epithelial cells with varying degrees of inflammation were seen microscopically. 2.

Respiratory and Nervous System Disease

Bacterial infections of the respiratory system frequently spread to the central nervous system in nonhuman primates and can progress to severe, life-threatening disease. Predisposing factors include recent shipping, overcrowding, and concomitant viral infections. Bacteria associated with respiratory and nervous system infections and the species affected are listed in Table XLIII. a.

Streptococcus pneumoniae

By far the most devastating respiratory pathogen in nonhuman primates is Streptococcus pneumoniae. Streptococcal infections are acquired by aerosol via the upper respiratory tract, middle ear, or mouth (Graczyk et al., 1995). Stress-related factors including capture, transportation, and quarantine (Kaufmann and Quist, 1969a; Fox and Wikse, 1971), viral infection (Brendt et al., 1974; Jones et al., 1984), and decreased immunocompetence in neonates due to waning passive immunity (Graczyk et al., 1995) predispose nonhuman primates to S. pneumoniae infection and disease. In macaques, the disease is usually rapidly progressive; death may occur within hours of the onset of clinical signs (Gilbert et al., 1987). In chimpanzees, clinical signs are initially those of an upper respiratory infection: coughing and a seromucoid to mucopurulent nasal discharge are followed by neurological signs (Keeling and McClure, 1974; Solleveld et al., 1984). Duration of clinical illness can range from 2 to 14 days in chimpanzees. The presence of free or phagocytized, encapsulated, grampositive diplococci in smears of cerebrospinal fluid provides a presumptive diagnosis of pneumococcal meningitis (Keeling and McClure, 1974; Solleveld et al., 1984). Aggressive treatment with antibiotics, intravenous fluids and electrolytes, and diazepam (20-30 mg/day) to modulate seizure activity has been effective in chimpanzees (Solleveld et aL, 1984). Gross lesions are engorgement of the meningeal vasculature, thickening and opacification of the leptomeninges, and purulent exudation over the cortex and/or the ventricles (Fox and Wikse, 1971; Solleveld et al., 1984; Graczyk et al., 1995). Lesions may extend to the spinal cord. In chronic disease, asymmetry of the cerebral hemispheres and severe malacia of the ventral frontal lobes of the brain may occur (Solleveld et al., 1984). Congestion of the lungs, pulmonary edema, acute purulent bronchopneumonia, and gray hepatization of the ventral lung lobes have been reported in macaques, as well as diffuse, suppurative peritonitis with adhesions, suppurative arthritis, and panophthalmitis (Fox and Wikse, 1971; Herman and Fox, 1971). In chimpanzees, lesions have included purulent otitis interna, sinusitis, and tonsillitis (Solleveld et al., 1984).

735

16. NONHUMAN PRIMATES Table XLIII

Bacterial Respiratory and Nervous System Infections Agent

Streptococcus pneumoniae

Primate Chimpanzee

Clinical signs and clinical pathology URI Slight cough, mucopurulent nasal discharge Conjunctivitis Pyrexia Leukocytosis Meningitis

Treatment/prevention Penicillin, 8 x 106U/day and ampicillin 4 gm/day and diazepam 20-30mg/day a or Penicillin 2 • 105U/day and ampicillin 200-400mg and chloramphenico125 mg/kg TID b or Ceftriaxone 50-100mg/kg c

Lethargy, vestibular signs Head holding, lip droop Seizures, dysphagia Macaque

Klebsiella pneumoniae

Squirrel monkey

Panophthalmitis, conjunctivitis Lethargy, incoordination, hypothermia Depressed reflexes Ataxia, muscle tremors, head pressing Nuchal rigidity, nystagmus Leukocytosis with left shift Cough Clear-mucopurulent nasal discharge Rales Pulmonary congestion Anorexia, adipsia Listlessness, reluctance to move Droopy eyelids Leukocytosis due to neutrophilia Swollen throat, abscesses in throat, infant deaths

Callitrichid

Death, no clinical signs

Owl monkey

Death, no clinical signs Anorexia, pyrexia, sneezing, coughing, dyspnea Cervical-mandibular swelling Serous to mucopurulent nasal discharge Facial edema; painful, distended abdomen Mucopurulent nasal discharge Dyspnea during handling Pyrexia, bright and alert Deaths in animals < 1 year old Serous to mucopurulent nasal and ocular discharges Dyspnea, depression, anorexia, weight loss Usually self-limiting Unsteady gait, circling Nystagmus, head tilt Edema of eyelids, serosanguinous discharge from ears Lethargy, anorexia, depression, weight loss Dyspnea Seizures Localized swelling and exudation associated with abscesses Postsurgical air sacculitis; abscess from catheterization

Chimpanzee

Macaque

Bordetella bronchiseptica

Marmoset

Owl monkey

Pasteurella spp.

Squirrel monkey

Owl monkey

Baboon

Autogenous bacterind 2 doses at 1 month interval Autogenous bacterin e Tetracycline, 55 mg/kg body weight y Autogenous bacterin g or Cephalothin, gentamicin Trimethoprim-sulfamethoxazole Amikacin or kanamycinh Oxytetracycline i Autogenous bacterin y

Chloramphenicol, amoxicillin, or tetracycline h

Chloramphenicol Penicillin Ampicillin h

Remove catheter

(continues)

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

736

Table XLIII (Continued)

Agent

Primate

Nocardia spp.

Macaque

Moraxella spp.

Baboon Squirrel monkey Cynomolgus

Clinical signs and clinical pathology Dyspnea, epistaxis Chronic weight loss; abdominaldistension and discomfort Chronic intermittentdiarrhea Lethargy, depression Coma Cutaneous draining tract Subcutaneous nodules Epistaxis, mucohemorrhagicnasal discharge

aFrom Keeling and McClure (1974). bFrom Solleveld et al. (1984). CFromPernikoffand Orkin (1991). dFrom Postal et al. (1988). eFrom Gozalo and Montoya (1992).

Treatment/prevention

Excision Long-acting penicillinj

fFrom Chalmers et al. (1983). gFrom Obaldia (1991). hFrom Weller (1994); drug doses not provided in text. /From Baskerville et al. (1983). iFrom VandeWoudeand Luzarraga (1991).

Microscopically, S. pneumoniae infection is characterized by fibrinopurulent leptomeningitis extending into the cerebral and cerebellar cortices (Fox and Wikse, 1971; Solleveld et aL, 1984) (Fig. 13). Necrotizing vasculitis and thrombosis due to fibrin deposition are common. Lung lesions range from acute serous inflammation with hyperemic congestion to exudative bronchopneumonia (Kaufmann and Quist, 1969a; Fox and Wikse, 1971). b.

Klebsiella pneumoniae

Klebsiella pneumoniae infection can result in pneumonia, meningitis, air sacculitis, septicemia, peritonitis, and enteritis in New and Old World primate species and in apes. Antemortem diagnosis is frequently not possible due to the acute course of the disease. New World monkeys in particular may die from septicemia or peritonitis with no clinical signs (Snyder et al., 1970; Giles et al., 1974; Gonzalo and Montoya, 1991). Young animals are more frequently affected than adults (Campos et al., 1981). A 64% incidence of infection has been reported for recently imported tamarins, and a 2 5 - 2 9 % incidence of klebsiella infection and disease has been reported for some owl monkey colonies (Snyder et al., 1970). Pneumonia and septicemia due to K. pneumoniae caused mortality rates approaching 100% in young Guyanese squirrel monkeys (Moisson and Gysin, 1994). Maternal neglect, trauma, and failure of hand rearing predisposed infants to disease. Klebsiella infection in young chimpanzees has been a primary cause of respiratory disease deaths (Schmidt and Butler, 1971). Fibrinous lobar pneumonia, purulent peritonitis, and mesenteric lymphadenopathy are the primary lesions reported in callitrichids infected with K. pneumoniae (Gozalo and Montoya, 1991, 1992). Enteritis and hepatomegaly were reported in a

Fig. 13. Meningitisresultingfrom Streptococcus pneumoniae infection in a

macaque.

16. NONHUMANPRIMATES colony of common marmosets by Campos et al. (1981). Purulent meningitis, consolidative pneumonia, intestinal hemorrhages, peritonitis, and air sacculitis occur in owl monkeys with K. pneumoniae infections (Gozalo and Montoya, 1991, 1992). Lesions in owl monkeys with acute air sacculitis range from diffuse hyperemia of the air sac rnucosa to thickening with edema of the adventitia (Giles et al., 1974) to purulent exudation extending to the salivary glands, trachea, and larynx. Gross necropsy findings reported in rhesus monkeys were exudative bronchopneumonia and hemopurulent meningitis (Good and May, 1971; Fox and Rohovsky, 1975). Chimpanzees had firm, inflated, red-purple lung lobes with yellow-white foci throughout the parenchyma (Schmidt and Butler, 1971). Microscopically, intense congestion with infiltrates of mononuclear and polymorphonuclear leukocytes occurred in affected organs of common marmosets (Campos et al., 1981). Inflammation and suppuration of the Peyer's patches and cecal lymphoid tissue were observed in owl monkeys with klebsiella septicemia (Gozalo and Montoya, 1991). Acute air sacculitis in owl monkeys was characterized by bacterial thromboembolism of the subepithelial vasculature of the air sac (Giles et al., 1974). Diffuse fibrinopurulent bronchopneumonia, suppurative bronchitis, and pleuritis were reported in rhesus monkeys by Fox and Rohovsky (1975), as well as fibrinopurulent meningitis, vasculitis, and thrombosis of meningeal vessels. Thickening of alveolar septae, congestion, alveolar hemorrhage, and edema with mononuclear and neutrophilic inflammatory cell infiltrates characterized pneumonia in chimpanzees (Schmidt and Buffer, 1971). Multifocal microabscesses and filling of bronchioles with necrotic debris and inflammatory cells were described. Treatment of infections caused by K. pneumoniae has been difficult due to the fulminating course of disease and the high incidence of multiple drug resistance (Fox and Rohovsky, 1975). Vaccination utilizing autogenous bacterins has been effective in preventing klebsiella infection and disease in marmosets, owl monkeys, and squirrel monkeys (Postal et al., 1988; Obaldia, 1991). c.

737 et aL, 1983). The typical microscopic pulmonary lesion was purulent bronchopneumonia with multifocal necrosis of the bronchiolar epithelium and filling of bronchioles with basophilic necrotic cellular debris, polymorphonuclear cells, and macrophages. Treatment of adult common marmosets with intramuscular oxytetracycline for 8 days resolved clinical disease, but it did not eliminate the organism from the nasal passages (Baskerville et al., 1983). Use of an autogenous bacterin in Callithrix jacchus colonies has been reported (Chalmers et al., 1983). d.

Pasteurella multocida

Pasteurella multocida is an opportunistic pathogen of squirrel monkeys and owl monkeys affecting recently shipped animals and animals in poor condition (Greenstein et al., 1965; Benjamin and Lang, 1971; Pinkerton, 1972). Squirrel monkeys presented with unsteady gait, nystagmus, head tilt, and circling. Meningitis, otitis media, lymphadenitis, and myocarditis were diagnosed at necropsy (Greenstein et al., 1965) (Fig. 14). Pneumonia, pleuritis, and meningitis occurred in affected owl monkeys. Necrotizing, fibrinopurulent pneumonia with thrombosis of

Bordetella bronchiseptica

BordeteUa bronchiseptica is carried as a commensal within the nasopharynx of many monkeys. Historically, disease has been associated with recent shipping, quarantine, poor condition, and overcrowding (Seibold et al., 1970; Pinkerton, 1972), although outbreaks have occurred in established colonies of common marmosets in which the initiating factor was unknown (Baskerville et al., 1983; Chalmers et al., 1983). Clinical signs in affected common marmosets included bilateral mucopurulent nasal discharge, dyspnea during handling, and pyrexia. Marmosets usually remained bright and alert and in good body condition. Death occurred in marmosets less than 1 year of age, but adults survived. Bronchopneumonia affecting all lung lobes was the primary gross lesion (Baskerville et al., 1983; Chalmers

Fig. 14. Meningitisdue to PasteureUa multocida infectionin an owl monkey,Aotus spp. Note thickenedmeningesand exudate. (Photographfrom Cynthia Besch-Williford.Courtesyof the ResearchAnimalDiagnosticand InvestigativeLaboratory,Universityof Missouri-Columbia.)

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

738

small blood vessels and severe, acute interstitial pneumonia with multifocal necrosis of alveolar septae were the primary microscopic lesions. Baboons have developed pasteurella infections secondary to surgical procedures, chair restraint, and chronic catheterization (Bronsdon and DiGiacomo, 1993). e.

Nocardia spp.

Nocardia spp. are aerobic actinomycetes found in richly fertilized soil as saprophytes on decaying vegetation. Nocardia asteroides is the most common isolate in nonhuman primates. Infection occurs following contact with skin wounds, inhalation, or ingestion. Clinical signs include dyspnea and epistaxis (McClure et al., 1976), chronic weight loss, abdominal distension and discomfort (Liebenberg and Giddens, 1985), chronic intermittent diarrhea, lethargy or depression (Jones and Wyand, 1966), and coma (A1-Doory et al., 1969). Radiographically, pulmonary lesions of nocardiosis cannot be distinguished from tuberculosis. Gross pulmonary lesions include multinodular to diffuse red to gray areas of consolidation, pulmonary hemorrhage and edema, abscesses, and cavitary lesions. Disseminated nocardiosis with multifocal abscesses in the omentum, mesentery, liver, kidney, and stomach has been reported in macaques (Liebenberg and Giddens, 1985) in addition to hemorrhage and multifocal abscesses in the brain (Sakakibara et al., 1984). Nocardiosis may also present as a draining multinodular cutaneous lesion (Boncyk et al., 1975) or as subcutaneous nodules (Kessler and Brown, 1981). Multifocal to coalescing pyogranulomas containing sulfur granules with large colonies of filamentous bacteria are characteristic. Multinucleate giant cells are found at the lesion periphery. There are no reports of successful treatment of nocardiosis in nonhuman primates.

f.

Moraxella catarrhalis

Moraxella catarrhalis is a gram-negative diplococcus associated with epistaxis and mucohemorrhagic nasal discharge in cynomolgus macaques (VandeWoude and Luzarraga, 1991). Clinical signs may also include sneezing and occasionally, periorbital swelling. White blood cells, erythrocytes, and large diplococcal organisms are seen in nasal cytologic preparations. Treatment with long-acting penicillin is effective (VandeWoude and Luzarraga, 1991).

3.

Tuberculosis and Mycobacteriosis

Tuberculosis is an insidious disease capable of causing high morbidity and mortality within a nonhuman primate colony. Tuberculosis is caused by Mycobacterium tuberculosis, M. bovis, or M. africanum and is usually acquired though human contact. There is no apparent difference in tuberculous disease in nonhuman primates caused by M. tuberculosis or M. bovis (McLaughlin, 1978); distribution and character of the lesions are identical. Kaufmann et al. (1975) estimated that up to 10% of tuberculosis outbreaks in nonhuman primates were caused by M. bovis.

Epizootic tuberculosis in nonhuman primates usually takes place by aerosol transmission, but transmission has occurred by ingestion, direct contact, and contact with fomites, including contaminated thermometers (Riordan, 1949) and a tattooing needle (Allen and Kinard, 1958). Although particular species may be more or less susceptible to disease, all nonhuman primates can develop tuberculosis. Frequently monkeys are found dead with no previous clinical history (Renquist and Whitney, 1978). A summary of clinical signs associated with tuberculosis is presented in Table XLIV.

Table XLIV

Tuberculosis in NonhumanPrimates Agent Mycobacterium tuberculosis, M. bovis

Species Macaque

Baboon Squirrel monkey Owl monkey Callitrichid

Clinical signs Persistent cough Chronic fatigue Exertional dyspnea Anorexia, weight loss Peripheral lymph node enlargement +_ draining tract Cutaneous abscesses Enlarged liver or spleen on palpation Lethargy, emaciation Positive TB test +__clinical signs Weakness, lethargy, paroxysmal cough Serous ocular and nasal discharge Weight loss, dehydration, depression No clinical signs reported

Clinical pathology Anemia, normocytic, normochromic Leukocytosis Lymphopenia Elevated serum globulins Elevated erythrocyte sedimentation rate

16. NONHUMANPRIMATES

Fig. 15.

pneumonia.

739

Pulmonarytuberculosisin a rhesus monkey,Macaca mulatta, infected with Mycobacterium bovis. Note enlarged hilar lymphnodes and tuberculous

Gross lesions of tuberculosis include caseous nodules in the hilar lymph nodes and lung, large cavitary and coalescing lesions within the lung, and tubercles extending into the thoracic pleura (Fig. 15). In advanced disease, there is secondary spread to the spleen, kidney, liver, and various lymph nodes with either multifocal miliary disease or larger nodular foci of caseation. Less frequently, tuberculous nodules are found in the cerebrum (Machotka et al., 1975), spinal column, omentum, uterus and ovary (McLauglin, 1978), peripheral lymph nodes, skin (Lindsey and Melby, 1966), mammary gland, or spinal vertebra (Fox

Fig. 16.

et al., 1974) (Fig. 16). The typical microscopic lesions are unencapsulated granulomas of varying size with a necrotic core. Surrounding the core are a layer of epithelioid macrophages and lesser numbers of neutrophils, with multinucleate Langhans' giant cells at the periphery (Fig. 17). Mineralization is uncommon. Acid-fast bacilli (AFB) can be demonstrated within the lesions both intra- and extracellularly using an acid-fast stain. Diagnosis of tuberculosis antemortem is based on the intradermal tuberculin test. Swelling, erythema, edema, and ptosis are indicative of a suspect or positive reaction (Fig. 18)

Cutaneoustuberculosisin the inguinal lymphnode of a rhesus monkey.

740

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Fig. 17. Langhans'giant cells associatedwith pulmonarytuberculosis in a rhesus monkey,Macaca mulatta.

(Table XXXIX). McLauglin and Marrs (1978) reported no advantage in using M. bovis purified protein derivative (PPD) over mammalian tuberculin in the detection of monkeys infected with M. bovis; in fact, mammalian tuberculin was the superior diagnostic reagent. The tuberculin test is limited in its sensitivity in that animals with early or advanced disease may give

Fig. 18. Positivetuberculin reaction in rhesus monkey.

false-negative reactions. Concomitant disease such as measles may also result in a false-negative reaction due to immunosuppression. Therapy with isoniazid will also invalidate the tuberculin test (Dillehay and Huerkemp, 1990). False positives may result from exposure to Freund's complete adjuvant (Pierce and Dukelow, 1988), trauma due to improper administration of the test, or nonspecific reactivity to the vehicle. Recent difficulties in obtaining old tuberculin further limit the application of this test. Alternative tests such as enzyme-linked immunosorbent assays have been used to detect specific antibodies to mycobacteria. However, the lack of clear-cut differences in titers between uninfected and infected monkeys, and difficulty choosing a universal antigen for the test reagent, have limited the effectiveness of the method (Corcoran and Thoen, 1991). Thoracic radiography may provide confirmation of pulmonary disease but cannot distinguish between tuberculosis and other cavitary diseases of the lung, such as nocardiosis or cryptococcosis. Culture of M. bovis or M. tuberculosis may be difficult, especially early in disease when there are low numbers of mycobacteria present. Detection of infection by screening feces, sputum, or necropsy tissues using specific mycobacterial primers in polymerase chain reaction (PCR) for mycobacterial DNA may provide a more rapid diagnosis of tuberculosis (Brammer et al., 1995). Mycobacterium avium-intracellulare complex, also known as M. avium complex (MAC), consists of pathogenic, saprophytic, nontuberculous mycobacteria that are contracted through exposure to soil, water, and infected tissues. Entry into the body can be through the respiratory, oral, and cutaneous routes. A summary of nonhuman primate mycobacterioses is found in Table XLV. Nonhuman primates are resistant to experimental

16. NONHUMAN P

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Table XLV Mycobacteriosisin NonhumanPrimates Agent/species

Mycobacterium avium-intracellulare Macaques (Coinfection with SIV) M. leprae Chimpanzee M. kansasii Macaques, squirrel monkeys M. gordonae Squirrel monkeys

Clinical signs

Clinical pathology

Intermittent-chronic diarrhea, refractory to treatment Marked weight loss General lymphadenopathy Draining fistula or ulcerated cutaneous lesions Self-mutilation hands and feet Multiple, eroded nodular lesions face and ears Nodules and papules along lateral extremities, scrotum, perineum None, detected by TB test

Anemia, normocytic, normochromic Hypoalbuminemia Hypergammaglobulinemia

None, detected by TB test

None

infection with M. avium-intracellulare; however, there are several reports of naturally acquired disease in immunocompromised macaques with concomitant SIV infection. Disease primarily involved the gastrointestinal tract and reticuloendothelial system (Sesline, 1978; Holmberg et al., 1985), with rare thoracic disease (Sedgwick et al., 1970). There are two reports of cutaneous manifestations (Latt, 1975; Bellinger and Bullock, 1988). Clinical signs of infection ranged from none (Sedgwick et al., 1970) to intermittent or continuous diarrhea refractory to treatment, accompanied by dramatic weight loss and generalized lymphadenopathy (Sesline, 1978). Infected macaques usually survived for 1 year after onset of clinical signs. In some macaques, no gross lesions were associated with M. avium infection (Sedgwick et al., 1970). Those with intestinal illness had mild to severe thickening of the intestinal wall from the distal small intestine through the cecum and colon (Sesline, 1978; Holmberg et al., 1985). Enlargement and edema of mesenteric lymph nodes and splenomegaly were frequently observed. Diffuse granulomatous inflammation of the terminal ileum and proximal colon due to infiltration of large, foamy macrophages into the mucosa and submucosa was characteristic. Numerous AFB were found in the cytoplasm of macrophages in Ziehl-Neelsen-stained intestinal sections. The animal with the focal cutaneous lesion had numerous AFB-containing macrophages, epithelioid cells, and Langhans' giant cells, with granulation tissue (Bellinger and Bullock, 1988). This animal recovered spontaneously after removal of the lesion. Diagnosis of M. avium-intracellulare infection can be difficult. Responses to tuberculin testing, even when using avium tuberculin, are variable (Sedgwick et al., 1970; Holmberg et al., 1985). A positive test using old tuberculin may be transitory erythema at 24 hr postinoculation (Sedgwick et al., 1970). Biopsy of colonic tissue or a suspect cutaneous site is a helpful diagnostic tool in that it can provide tissue for both histopathology and PCR analysis for mycobacterial DNA. Culture of the organism from feces or lesions is time consuming. Treatment is not recommended because this organism has resistance to multiple drugs. Mycobacterium leprae is the etiologic agent of leprosy or

None

None

Hansen's disease. Leprosy is a chronic granulomatous disease affecting the skin and peripheral nerves with lesions preferentially occurring in cooler areas, i.e., the extremities, of the host. Naturally acquired infections with M. leprae have been reported in a sooty mangabey, Cercocebus atys (Meyers et al., 1985), and in chimpanzees (Leininger et al., 1978; Hubbard et al., 1991; Alford et al., 1996). Multiple, eroded, nodular skin lesions of the face and ears occurred in chimpanzees with leprosy (Leininger et al., 1978; Hubbard et al., 1991; Alford et al., 1996). One chimpanzee had a 10-year history of self-mutilation of the hands and feet (Alford et al., 1996). In this individual and another chimpanzee at the same facility, nodular and papular lesions were also found on the lateral surfaces of the arms and legs, scrotum, perineum, and penis. The face and extremities appeared edematous and swollen. Many of the nodular lesions became ulcerated. Multidrug therapy consisting of rifampin (600 mg daily), dapsone (100 mg daily), and clofazimine (100 mg daily) was given for 6 months in combination with topical therapy of sugar and saline irrigating solution applied to deep ulcerated areas (Alford et al., 1996). After 4 years of treatment, biopsy of previously lesioned skin sites revealed no AFB and no remarkable lesions. Dapsone was continued for life. Microscopic lesions observed in biopsy or necropsy specimens revealed epidermal thinning and subepidermal clear zones (Leininger et al., 1978; Meyers et al., 1985; Hubbard et al., 1991). Dermal inflammation was primarily histiocytic, with inflammatory cells localized around neurovascular channels. Large numbers of AFB were observed in histiocytes and lesser numbers within nerves. Tuberculin skin tests were negative, lepromin skin tests were positive, and specific serum antibodies to M. leprae were detected (Meyers et aL, 1985; Hubbard et al., 1991). Mycobacteriosis due to M. kansasii infection has been reported in rhesus monkeys and squirrel monkeys. Unlike indications in M. tuberculosis infection, there was no evidence of animal-to-animal transmission. In a breeding colony of rhesus monkeys, 71 monkeys developed positive tuberculin reactions,

742

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

and 60 monkeys were culture positive for M. kansasii (Valerio et al., 1979). Infection was initially detected by tuberculin test using mammalian tuberculin intradermally in both the eyelid and the abdomen. Minimal ptosis, with mild thickening of the eyelid and little or no hyperemia, was observed. A 1-2 cm area of blanched, thickened skin was found at the abdominal test site. Severity of tuberculin reactions became more pronounced in animals that were retested. Tuberculin testing with specific M. kansasii antigen or PPD was not diagnostically superior to testing with mammalian tuberculin (Valerio et al., 1979). Mycobacterium kansasii infection in squirrel monkeys was also detected by routine tuberculin testing (Brammer et al., 1995). Diagnosis of M. kansasii infection was preliminarily confirmed by use of genus-specific probes for mycobacterial DNA in PCR testing of bronchial lymph nodes. The organism eventually was isolated from 3 of 5 tuberculin reactors. Gross lesions of M. kansasii infection in rhesus monkeys included 1 to 2 pulmonary nodules, some containing yellowgreen caseous material, and tuberculous-type lesions in the mediastinal lymph nodes (Valerio et al., 1979). Some monkeys had miliary lesions in the liver and/or spleen. Squirrel monkeys had enlarged bronchial, cervical, and/or mediastinal lymph nodes (Bremmer et al., 1995). Microscopic lesions were indistinguishable from those of tuberculosis. Tuberculin testing of a colony of squirrel monkeys identified 3 tuberculin reactors (Soave et al., 1981). Two monkeys were euthanatized and necropsied. There were no gross or microscopic lesions, but M. gordonae was isolated from the mediastinal lymph nodes, spleen, and/or liver. 4.

Other Bacterial Infections

Tetanus due to Clostridium tetani has been a significant cause of mortality in free-ranging or outdoor-housed monkeys. Teta-

nus occurred secondary to soil contamination of fight wounds or to parturition in rhesus macaques (DiGiacomo and Missakian, 1972; Rawlins and Kessler, 1982), bite wounds or other skin trauma in squirrel monkeys (Kessler and Brown, 1979), and frostbite lesions in baboons (Goodwin et al., 1987). Clinical signs are summarized in Table XLVI. Tetanus is a clinical diagnosis because culture of the organism is difficult. Tetanus toxoid administered intramuscularly followed by a single booster was protective for rhesus macaques and baboons (Kessler et al., 1988; Goodwin et al., 1987). Immunization with tetanus toxoid is advised for populations at risk. Naturally acquired leptospiral infections have been reported in tamarins, squirrel monkeys, baboons, and chimpanzees (Wilbert and Delorme, 1927; Fear et al., 1968; Perolat et al., 1992; Reid et al., 1993). In squirrel monkeys and baboons, leptospirosis is associated with abortion. Squirrel monkeys may die without clinical signs or present with pyrexia, marked dehydration, and icterus (Perolat et al., 1992). Gross necropsy lesions include jaundice of mucous membranes, subcutaneous tissues, and viscera; hemorrhagic pneumonia; renal necrosis and hemorrhage; and hepatomegaly (Perolat et al., 1992; Reid et al., 1993). Microscopic lesions include pulmonary hemorrhage and edema, multifocal hepatocellular degeneration and bile stasis, interstitial nephritis, tubular epithelial necrosis, and exudative glomerulopathy. Spirochetes can be seen with silver staining of renal tissues. Immunization with an inactivated vaccine was effective in eliminating leptospiral disease in a colony of squirrel monkeys (Perolat et al., 1992). Staphylococcus and Pseudomonas are bacteria genera associated with a variety of disease conditions, usually affecting individual animals. Staphylococci have been recovered from abscesses, ascitic fluid, ocular discharges, and fistulas of clinically ill owl monkeys (Weller, 1994) and from a tamarin with bronchopneumonia (Deinhardt et al., 1967a) (Fig. 19). Staphylococ-

Table XLVI Tetanus (Clostridium tetani) in Primates Species

History

Clinical signs

Treatment

Rhesus

Free-ranging Fight wounds Postpartum

Torpor Unable to prehend food Difficulty swallowing Excessive thirst Progressive stiffness, abduction pectoral limbs Bipedal running and hopping Piloerection Trismus, opisthotonus, status epilepticus

None attempted; some obese animals recovered spontaneously

Baboon

Outdoor housing Frostbite

Moribund Trismus and extensor rigidity

1500 units veterinary tetanus antitoxin Acepromazine 3 - 5 mg QID x 4 weeks Valium 1.5 mg QID x 10 daysa

Squirrel monkey

Outdoor housing Bite wounds, trauma

Slow, deliberate, stiff gait Reluctance to move Trismus, extensor rigidity, and opisthotonus

Treatment unsuccessful

aFrom Goodwin et al. (1987).

16. NONHUMAN PRIMATES

743

Fig. 19.

Staphyloccocalfibrinopurulentpleuritis and pneumoniain a squirrel monkey,Saimiri spp.

cus a u r e u s was isolated from macaques with clinical vaginitis or pyometra (Lang and Benjamin, 1969; Doyle et al., 1991), and

from a retrobulbar abscess in a rhesus infant (Rosenberg and Blouin, 1979). At Yerkes Regional Primate Research Center (Atlanta, GA) S. a u r e u s was the most frequent isolate from external wounds and joint specimens from monkeys with arthritis, and a frequent isolate of blood cultures from clinical cases or

Fig. 20.

necropsies (McClure et al., 1986). Staphylococcal infection is a frequent problem with indwelling catheters; it can cause serious disease as well as reduce catheter life span by half (Taylor and Grady, 1998) (Fig. 20). In this report, 64% of catheter infections in macaques were due to staphylococci, many of which were multiple-antibiotic resistant. P s e u d o m o n a s spp., including P. aeruginosa, have been

Bacterialembolus in the lung due to staphyloccocalinfection in a squirrel monkey,Saimiri spp.

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BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

recovered from monkeys with diarrhea, from external wounds, blood cultures, and air sac infections (McClure, 1980). Pseudomonas spp. were associated with bronchopneumonia, empyema, vegetative endocarditis, pancarditis, and septicemia in Callitrichidae (Deinhardt et al., 1967b; Cicmanec, 1977). Pseudomonas aeruginosa has been isolated from squirrel monkeys with meningitis, pododermatitis, cellulitis (Line et al., 1984; Lausen et al., 1986), and a maxillofacial abscess (Langner et al., 1986), and also from a chimpanzee with suppurative nephritis (Migaki et al., 1979). Treatment in all cases entailed aggressive antibiotic administration. Successful antibiotic regimens included intramuscular gentamicin (1.25 mg/kg every 12 hr), procaine penicillin (50,000 U/kg every 12 hr), and polymyxin B (12,500 IU/kg every 12 hr) (Line et al., 1984; Lausen et al., 1986).

B. 1.

M y c o t i c Diseases

Respiratory

Pneumocystosis has been reported in nonhuman primates with debilitation due to recent importation, bacterial infection, neoplasia, or immunodeficiency associated with retroviral infection (Poelma, 1975; Chandler et al., 1976; Letvin et al., 1983; Kestler et al., 1990; Lowenstein et al., 1992). Pneumocystis carinii also occurred as an endemic infection within a colony of tamarins, Saguinusfuscicollis and S. oedipus (Richter et al., 1978). Clinical signs included progressive weight loss, anorexia, and failure to thrive. Chimpanzees with pneumocystosis and erythroleukemia developed anorexia, pyrexia, dyspnea, cyanosis, and pneumonia (Chandler et al., 1976). Thoracic radiographs revealed extensive infiltrates in the lung lobes. Treatment was unsuccessful. Gross lesions attributable to pneumocystosis include partial lung collapse; firm, rubbery lungs with multiple 1-2 mm gray nodules throughout the lungs; and multifocal or diffuse consolidation with subpleural hemorrhages (Chandler et al., 1976; Richter et al., 1978). Pneumocystosis is characterized by multiple foci in which alveoli are filled with granular, eosinophilic foamy material (Chandler et al., 1976). Interstitial pneumonia with a mononuclear cell infiltrate occurred in chimpanzees and tamarins. Silver or periodic acid-Schiff stains revealed cystic pneumocystis organisms within a honeycomb matrix. Organisms were dark brown to black, round, ovoid, or cup-shaped cysts, 4 - 6 ~tm in diameter. Diagnosis of P. carinii infection is usually based on characteristic histologic lesions. Pneumocystosis should be considered in an immunocompromised animal with dyspnea or other indication of pulmonary disease. Trimethoprim-sulfamethoxazole, dapsone, and aerosolized pentamidine have been effective in prophylaxis and treatment of pneumocystis infections in humans.

2.

Superficial

Dermatophytosis or ringworm occurs rarely in nonhuman primates and is caused by organisms of the genera Microsporum and Trichophyton. Lesions include baldness, typical circular or ring-shaped lesions, generalized scaliness, and patchy hair loss to generalized alopecia (A1-Doory, 1972). Microsporum canis has been isolated from New World monkeys (Kaplan et al., 1957), a rhesus monkey (Baker et al., 1971), and a chimpanzee (Klokke and deVries, 1963). Trichophyton mentagrophytes has been recovered from rhesus monkeys (Hauck and Klehr, 1977) and capuchins (Bagnell and Grunberg, 1972). An epizootic of ringworm caused by T. violaceum occurred in 60 baboons housed in the same cage in Sukhumi (Voronin et al., 1948). The source of infection was believed to be dogs previously held in the cage.

3.

Systemic

a.

Coccidioidomycosis

Infections with Coccidiodes immitis have been reported in baboons, macaques, and apes (Migaki, 1986). Clinical signs associated with respiratory disease included nasal discharge, cough, and dyspnea. Vertebral C. immitis infection in a baboon resulted in lameness and reluctance to move (Rosenberg et al., 1984); and in a rhesus monkey, in altered gait leading to paralysis (Castleman et al., 1980). In all reports, affected monkeys and apes had a history of outdoor housing in California or Texas. Radiography can reveal severe pulmonary disease with multiple cavitary lesions or lysis of vertebral bone. Serologic testing for specific antibody by complement fixation or tube precipitin test, or skin testing, may be helpful in reaching a diagnosis. Coccidioides immitis infections usually result in disseminated disease, with lesions most frequently found in the lung and vertebrae (Migaki, 1986). Firm white nodules, partial or complete collapse of a lung lobe, pleural adhesions, and cavitations are common lesions (Breznock et al., 1975; Castleman et al., 1980; Bellini et al., 1991). The hilar lymph nodes may be enlarged. Paravertebral masses or abscesses with invasion into the vertebrae and spinal cord were reported for monkeys with lameness or paralysis. Microscopic lesions are typically pyogranulomas bordered by multinucleate giant cells. Lesions contain thickwalled spherules 10-60 ~tm in diameter filled with 2 - 4 ~tm round endospores. b.

Histoplasmosis

Spontaneous histoplasmosis due to Histoplasma capsulatum var. capsulatum has been reported in a squirrel monkey with granulomatous pneumonia, hepatitis, and splenitis (Bergeland et al., 1970), an owl monkey (Weller et al., 1990), and a rhesus monkey infected with SIV (Baskin, 1991). The owl monkey presented with weight loss and splenomegaly and had neu-

16. NONHUMANPRIMATES tropenia, eosinophilia, hypercalcemia, hypercholesterolemia, and hypophosphatemia. The rhesus monkey experienced diarrhea, weight loss, anorexia, and dyspnea. Marked splenomegaly and enlargement of the mesenteric lymph nodes were found at necropsy (Baskin, 1991). In the rhesus monkey, there was diffuse histiocytosis of many organs, including intestines, mesenteric and peripheral lymph nodes, spleen, liver, adrenal gland, and bone marrow. Disseminated microgranulomas with multinucleated Langhans'-type giant cells were seen microscopically in affected abdominal viscera and lymph nodes of the owl and squirrel monkeys. Small yeasts, 2 - 4 gm in diameter, with a thin cell wall and a central basophilic structure, were found in the cytoplasm of macrophages or giant cells. Histoplasma capsulatum var. duboisii is the etiology of African or large-form histoplasmosis. The agent is indigenous to Africa, but infection has been spread among feral and laboratory-born baboons in Texas (Butler and Hubbard, 1991). Direct contact with infected baboons through grooming or licking of skin lesions appeared to be the most likely mode of transmission in this epizootic. Lesions were usually confined to the skin, especially surfaces that contacted the ground, such as hands, buttocks, and tail, but also occurred on the face, ears, and scrotum. Papules, pustules 5-10 mm in diameter, or ulcerative granulomas 1-2 cm in diameter were common (Butler et al., 1988; Butler and Hubbard, 1991). Radiography may reveal osteolytic lesions in the skull, digits, and vertebrae underlying affected areas of skin. Numerous discrete, elevated, and ulcerated skin lesions on the face, ears, hands, feet, tail and/or perineum, and rarely, the torso, were found at necropsy (Butler and Hubbard, 1991), with enlargement of superficial and retroperitoneal lymph nodes, particularly those draining areas with skin le-

745

sions. Extension of cutaneous lesions to underlying bone resuited in osteolysis (Walker and Spooner, 1960; Butler et al., 1988; Butler and Hubbard, 1991). Pyogranulomatous inflammation of the dermis and subcutaneous tissues with marked histiocytic infiltrates, large numbers of both Langhans' and foreign body-type multinucleated giant cells, and neutrophils was reported by Butler and Hubbard (1991). Numerous intracellular and extracellular, 8-15 am, uninucleate yeast cells were found throughout the lesions. Diagnosis of H. capsulatum var. duboisii can be made based on the characteristic appearance of the organism and resultant tissue inflammation (Butler and Hubbard, 1991). Antifungal therapy has been ineffective. Surgical excision of lesions was effective in eliminating infection from 13 baboons. c.

Candidiasis

Candidiasis is caused by yeast of the genus Candida, usually C. albicans, a normal saprophytic inhabitant of the mucous membranes of the alimentary and genital tracts and skin of nonhuman primates (Migaki et al., 1982). Predisposing factors for clinical candidiasis include antibiotic therapy, recent importation, age, concomitant mycobacterial infection, retroviral infection, routine oral gavage, or parasitism (Migaki et al., 1982; Tucker, 1984; Lowenstein et al., 1992). Anorexia, dysphagia, open-mouth breathing due to ulcers of the hard palate, dehydration, and diarrhea are associated with candidiasis (Kaufmann 1969; Kaufmann and Quist, 1969b; Wikse et al., 1970; Weller, 1994). Onychomycosis, with shortening, erosion, and deformation of the nails, and balanitis have also been associated with candidiasis (Kerber et al., 196.8; Wikse et al., 1970).

Fig. 21. Candidiasisof the esophaguswith pseudomembraneformationand large numbersof candidal pseudohyphaeand blastospores.

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BRUCE J. BERNACKY, SUSAN V. GIBSON, M I C H A L E E. KEELING, AND CHRISTIAN R. ABEE

Pseudomembrane formation results from candidal overgrowth. White or creamy plaques are found on the tongue, buccal cavity, esophagus, and intestine. Large clusters of pseudohyphae and blastospores, 3 - 5 pm in diameter, are seen in the superficial portion of the epithelium of mucous membranes (Migaki et al., 1982). Pseudomembranes are composed of degenerate and sloughed epithelial cells, neutrophilic infiltrates, and numerous yeasts (Fig. 21). Necrosis and ulceration result from deep invasion of the epithelium and lamina propria. Oral nystatin suspension is an effective treatment for lesions of the oral cavity or digestive system. d.

Cryptococcosis

Cryptococcosis is a systemic infection of humans and animals caused by a yeastlike fungus, Cryptococcus neoformans. Transmission usually occurs by inhalation of spores but also as a result of direct contact. Cryptococcosis has been described in New World and Old World monkeys. Clinical signs vary, as infection is usually disseminated. Central nervous system signs include depression (Miller and Boever, 1983), seizures, and blindness (Sly et al., 1977). A squirrel monkey developed a deforming mass of the lower jaw, significant weight loss, and leukopenia with lymphopenia and anemia (Roussilhon et al.,

1987). There are two general forms of gross lesions: a gelatinous mass loosely organized with no defined capsule, or a solid, granulomatous mass. Emaciation, splenomegaly, granulomatous pneumonia, and lymphadenopathy involving the hilar or peripheral lymph nodes are common features (Sly et al., 1977; Roussilhon et al., 1987). Histologically, large, irregularly sized yeast cells with an abundant polysaccharide capsule occur singly, in small aggregates, or in large masses. The degree and type of inflammation range from none to acute or granulomatous inflammation. Cytologic evaluation of cutaneous masses or spinal fluid can provide rapid diagnosis of cryptococcal infection. India-ink preparations allow visualization of the organism against a black background but are not diagnostic unless budding is observed. C. 1.

Viral Diseases

DNA Viruses

a.

Herpesviruses

Viruses in the subfamily Alphaherpesvirinae commonly cause subclinical infection in the natural host. Viral infections are persistent and latent. Clinical disease when present is usually

Table X L V I I Alphaherpesviruses: Disease in Host Species Host/reservoir

Synonyms

Lesions in host species Common, usually subclinical, latent infection Virus shed in oral or genital secretions Transmitted by biting, sexual behavior, fomites Disease usually self-limiting with vesicles and ulcers of the oral and genital mucosa, resolved in 10 -14 days Rare systemic infections are fatal Multifocal, necrotizing hepatitis a Hemorrhagic interstitial pneumonia b Intranuclear inclusion bodies in endothelial cells Common subclinical, latent infection in baboons Virus shed in genital and oral secretions Venereal transmission c Small vesicles or pustules on genital or less commonly oral mucosa with primary infection or recrudesence Severe genital lesions may occur c Neutralizing antibodies found in stump-tailed macaques with no history of clinical disease d Usually no clinical disease, latent infection, shed in saliva Ulcerative stomatitis e Common, symptomatic infection with latency HSV-1 oral lesions and/or encephalitis HSV-2 sexually transmitted, genital lesions in adults and disseminated disease in children

Cercopithecine herpesvirus 1

Macaque

Herpes B, Herpes simiae, B virus

Cercopithecine herpesvirus 2

Baboon African green monkey

Herpes papionus SA 8

Cercopithecine herpesvirus 6, 7, 9 Saimirine herpesvirus 1

Unknown Stump-tailed macaque? Squirrel monkey

Human herpesvirus 1, 2

Human

Simian varicellovirus Simian varicella Herpes tamarinus Herpes T Herpes platyrrhinae Herpes hominus Herpes simplex

,,

aFrom Simon et al. (1993). bFrom Espana (1973).

~From Levin et al. (1988). dFrom Mansfield and King (1998).

eFrom King et al. (1967).

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7

Ulcerationof the tonguedue to Herpesvirus saimiri 1 infectionin a youngBoliviansquirrel monkey,Saimiri boliviensis boliviensis.

self-limiting and consists of oral or genital vesicles that resolve over time (Table XLVII; Fig. 22). Systemic, fatal disease occurs on rare occasion in the natural host. Disease is much more severe when an alphaherpesvirus infects an aberrant host; often it is fulminating, systemic, and fatal. An exception to this is infection of apes with human herpesviruses 1, 2, or 3, which are usually self-limiting (McClure and Keeling, 1971; Mansfield and King, 1998). Clinical signs and lesions of alphaherpesvirus infection in aberrant primate hosts are found in Table XLVIII. Cercopithecine herpesvirus 1 (CHV-1), also known as herpesvirus simiae or B virus, causes a persistent, subclinical latent infection in the natural host species, macaques; and a disseminated viral infection in humans, resulting in ascending paralysis, encephalitis, and death in about 70% of cases (Weigler, 1992). An increased number of human cases have been reported since the late 1980s (Holmes et al., 1990). Contact with monkey saliva, tissues, or tissue fluids is the most common route of transmission; person-to-person transmission has been reported in one instance. In 1997, death of a technician resulted from an ocular splash of material from a rhesus monkey (CDCP, 1998). Because of the number of human cases and the high fatality rate of this zoonosis, guidelines for protective clothing and procedures for handling macaques have been published (CDCP, 1987, 1998; Holmes et al., 1995). In addition, specific postexposure procedures for wound cleansing, and collection and testing of samples from both human and macaque for virus isolation, PCR for viral DNA, and viral antibody testing have been recommended (Table XXXVI). Specific-pathogen-flee colonies of macaques have been developed and are being expanded to provide monkeys that are free of CHV-1. Cercopithecine herpesviruses 6, 7, and 9 compose the group of viruses known as simian varicella. The reservoir host has not

been identified; however, serum neutralizing antibodies have been detected in stump-tailed macaques with no history of clinical disease (Mansfield and King, 1998). The disease in aberrant hosts, including African and Asian nonhuman primate species, is characterized by the development of a vesicular dermatitis progressing to death within 48 hr (Fig. 23). Disseminated infection affects the lungs, liver, and gastrointestinal tract (Table XLVIII; Fig. 24). Cytomegaloviruses (CMV) or betaherpesviruses commonly occur in humans and nonhuman primate species but rarely cause overt disease. These viruses have a narrow host range, although interspecies transmission is documented (Swack and Hsuing, 1982). Virus is transmitted horizontally in blood, saliva, milk, urine, and semen (Asher et al., 1974). Clinical disease is associated with intrauterine infection or immunosuppression. In macaques infected with SIV, CMV reactivation results in necrotizing encephalitis, enteritis, lymphadenitis, and/or interstitial pneumonia (Baskin, 1987). Gammaherpesviruses are oncogenic. Experimental or naturally acquired infection in aberrant primate hosts results in lymphoma or lymphocytic leukemia (Table XLIX). Malignant lymphoma has been reported in baboons and macaques infected with Epstein-Barr virus-like (EBV-like) viruses (Deinhardt et al., 1978; Rangan et al., 1995). Coinfections with EBV, or EBV-like viruses, and SIV in macaques have been associated with oral lesions resembling hairy leukoplakia and lymphoma (Feichtinger et al., 1992; Baskin et al., 1995). b.

Poxviruses

Poxvirus infections in nonhuman primates are usually nonfatal. Clinical presentation includes the development of a

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

748

Table X L V I I I Alphaherpesviruses: Disease in Aberrant Host Species Species aberrantly infected

Virus

Disease and lesions Vesicular dermatitis _ pruritis at inoculation site 3-24 days postexposure Lymphangitis and lymphadenopathy "Flulike" symptoms: fever, conjunctivitis, paresthesia, muscle weakness N6urologic symptoms, coma, death Necrotizing encephalitis

Cercopithecine herpesvirus 1

Human a

Cercopithecine herpesvirus 2

None reported

Cercopithecine herpesvirus 6, 7, 9

African green monkey b Patas monkey Macaque

Disseminated vesicular exanthema; death in 48 hr High morbidity and mortality; in some cases associated with recent importation Latent infection with reactivation demonstrated in African green monkeys c Disseminated infection with necrotizing lesions in lung, liver, and gastrointestinal tract Cowdry type A intranuclear inclusion bodies (Fig. 24)

Saimirine herpesvirus 1

Owl monkey d Marmoset, tamarin

Inadvertant exposure to host species Vesicular oral, labial, and/or dermal lesions 7-10 days postinfection Pruritis, anorexia, depression; with death in 24 - 48 hr; high morbidity and mortality Necrosis of epidermis, oral, and gastrointestinal mucosa; multifocal hepatic necrosis Cowdry type A intranuclear inclusion bodies; multinucleate giant cells in epidermis Modified live vaccine can cause outbreaks of disease*

Human herpesvirus 1, 2

Ape

Mild, usually self-limiting oral vesicular disease; fatal meningoencephalitis reported in gibbons y

Owl monkey g Tamarin (experimental) h

Fatal, disseminated infection similar to that caused by saimirine herpesvirus 1, more severe facial lesions, with blepharitis and stomatitis; encephalitis more common Distinguish by virus isolation and identification Modified live vaccine developed and protective for owl monkeys i

Ape j

Mild, self-limiting vesicular dermatitis

Human herpesvirus 3 (may be simian in origin)

/'From Smith et al. (1969). gFrom Melendez et al. (1970). hFrom Felsberg et al. (1972). /From Daniel et al. (1978). JFrom McClure and Keeling (1971).

aFrom McChesney et al. (1989) and Weigler (1992). bFrom Clarkson et al. (1967), Felsenfeld and Schmidt (1975) and Wenner et aL (1977). CFrom Soike et al. (1984). dFrom Hunt and Melendez (1966). e From Asher et al. (1974).

Table XLIX Gammaherpesviruses Virus Saimirine herpesvirus 2

Synonym Herpesvirus saimiri 2

Natural host

None

Squirrel monkey Owl monkey Callitrichids Howler monkey Spider monkey

Ateline herpesvirus 2

Herpesvirus ateles

Herpesvirus 4

Lymphoma or lymphocytic leukemia (experimental) a Naturally acquired disease rare in callitrichids T-cell origin Large, pleomorphic, histiocytic cells or well-differentiated lymphocytes None

Spider monkey Owl monkey Callitrichids

Epstein-Barr virus (EBV)

Disease

Aberrant host

Lymphoma or leukemia (experimental) Asymptomatic or infectious mononucleosis Fever, pharyngitis, lymphadenopathy Circulating atypical lymphocytes

Human

New World monkeys

Large cell lymphoma (experimental) b

16. NONHUMAN PRIMATES

749

Table XLIX (Continued)

Virus EBV-like viruses

Synonym Chimpanzee herpes Orangutan herpes Gorilla herpes Rhesus leukocyte-associated herpes Herpes papionis

African green monkey EBV-like virus

Natural host

Aberranthost

Disease

Apes

None

Rhesus Baboon Vervet

Subclinical infection common Malignant lymphoma in baboons and macaquesc Coinfection with SIV: lymphoma in macaques or lesions resembling oral hairy leukoplakiad Callitrichids

aFrom Melendez et al. (1970). bFrom Cameron et al. (1987) and Neidobitek et al. (1994). CFrom Deinhardt et al. (1978) and Rangan et al. (1995).

MalignantB-cell lymphoma (experimental)e

dFrom Baskin et al. (1995) and Feichtinger et al. (1992). eFrom Miller et aL (1977).

papular rash progressing to umbilicated pox lesions as seen in monkeypox, tanapox, and marmoset poxvirus infections; development of small to large skin masses as occurs in Yaba virus infection; and small, raised lesions on the face as seen with molluscum contagiosum (Table L). Primate poxviruses can be

infectious for humans. Zoonotic disease is usually self-limiting; however, m o n k e y p o x infection has caused fatalities in children. H u m a n - t o - h u m a n transmission is rare.

Fig. 23. Vesicleformation in the epidermis due to simian varicella infection (Cercopithecine herpesviruses 6, 7, and 9).

Fig. 24. Intranuclear inclusion bodies and hepatic necrosis due to simian varicella infection (Cercopithecine herpesviruses 6, 7, and 9).

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

750

Table L Poxviruses Virus

Host (naturally acquired disease)

Disease/lesions

African green monkeysa Macaques b Apes Captive New World monkeys

High prevalence of antibodies; no clinical disease Spread by aerosol, direct contact, and biting insects Viremia 3 - 4 days postinfection (pi); fever, anxiety, aggression Papules progress to vesicular rash 6 - 7 days pi with umbilication to classic pock lesion; buttocks, hands, and feet Dissemination to lung, spleen, and mucous membranes Disease not always fatal, recovered monkeys immune Hemorrhagic necrosis of lungs Intracytoplasmic inclusion bodies

Humans c

Sporadic outbreaks in Africa; human-to-human transmission rare Fatigue, fever, headache, back pain, lymphadenopathy Vesiculopapular rash over face and body 10-15% case fatality rate Vaccination against smallpox provides immunity

Macaques, baboons `/

Transmission unknown, suspect arthropod vector Subcutaneous masses of varying size on feet, hands, and face Oral masses in baboons Large masses may ulcerate; masses regress in 6 weeks Nonencapsulated masses of large pleomorphic histiocytes ___large cytoplasmic inclusion bodies

Humans

Pseudotumors <- 2 cm on hands and feet Lymphadenopathy, fever Lesions regress in weeks

Humans e

Benign cutaneous skin infection in East Africa Fever for 2-3 days with headache, backache, and prostration 1-2 small papules on face, extremities, or trunk; may umbilicate

Macaques f

Small red papules develop 4 - 5 days pi on face, thorax, petineum Progress to 1 cm raised foci 14 days pi Papules become umbilicated, have red margins, may ulcerate Resolve in 3 - 8 weeks Epidermal proliferation and ballooning degeneration Intracytoplasmic and intranuclear inclusion bodies

Marmoset poxvirus

Callithrix j a c c h u s g

Papulovesicular disease lasting 4 - 6 weeks Not disseminated, not fatal

Molluscum contagiosum

Humans

Mildly contagious, chronic skin disease Multiple small skin tumors filled with waxy material Large central acanthocytes have cytoplasmic inclusion bodies

Chimpanzees h

Small firm lesions with waxlike contents on face and inguinal area Large intracytoplasmic inclusion bodies in lesions

Monkeypox

Yaba virus

Tanapox

aFrom Arita et al. (1972). bFrom Mutumbo et al. (1983). CFrom McConnell et al. (1968) and Arita et al. (1985). dFrom Downie (1972) and Bruestle et al. (1981). eFrom Jezek (1985) and McNulty (1995). fFrom Downie and Espana (1972). gFrom Gough et al. (1982). hFrom Douglas et al. (1967).

16. NONHUMANPRIMATES

c.

Other DNA Viruses

Adenoviruses have been isolated from New World and Old World monkey species and apes, both from clinically affected and healthy animals. Subclinical infection is common in nonhuman primates. Clinical disease may be enteric or respiratory depending on viral tropism. Animals with enteric disease may develop diarrhea, which resolves in 10-14 days. Virus can be shed several weeks after clinical recovery. Adenovirus-induced necrotizing pancreatitis has been reported in immunocompromised macaques with natural or experimental retroviral infections (Chandler and McClure, 1982; Martin et al., 1991). Diarrhea and death occurred in these monkeys. Sneezing, coughing, and rapid respiration occur in clinical adenovirus respiratory tract infections. Dyspnea and cyanosis may develop in severe infections. Adults with respiratory disease usually recover in 710 days. Mortality is low except in neonates with secondary bacterial infections that contribute to mortality. Polyomavirus macacae, more commonly known as simian virus 40 (SV40), is a common latent viral infection of Asian macaques. The virus was originally isolated from rhesus or cynomolgus kidney cell cultures used for production of polio vaccine. Simian virus 40 transforms cells in vitro and is oncogenic when inoculated into hamsters. Clinical disease in macaques is rare and results from reactivation of latent infection, usually due to immunosuppression associated with SIV infection (Horvath et al., 1992). Lesions include demyelination in the cerebral white matter and subependymal areas, chronic tubulointerstitial nephritis with hypertrophy/hyperplasia of collecting tubule epithelium, and proliferative interstitial pneumonia (Mansfield and King, 1998). Intranuclear inclusion bodies are found in affected areas. Simian virus 40 has been isolated from human neoplasms and from human cases of progressive multifocal leukoencephalopathy, but a causal relationship has not been established (Melnick and Butal, 1988). Papillomavirus infections and associated dermal or oral lesions have been diagnosed in macaques and chimpanzees. In chimpanzees, focal epithelial hyperplasia characterized by the development of multiple, sessile, well-circumscribed proliferative structures in the oral mucosa is associated with papillomavirus infection. These masses are small (-----0.5 cm) and may persist for extended periods and then undergo spontaneous regression (Glad and Nesland, 1995). Three distinct parvoviruses of the erythrovirus group have been isolated from cynomolgus, rhesus, and pig-tailed macaques with anemia (O'Sullivan et al., 1994, 1996; Green et al., 2000). Severe normocytic, normochromic anemia was reported for cynomolgus monkeys with parvovirus infection and concomitant SRV infection, and for cynomolgus monkeys with parvovirus infection in the high-dose test group of a drug safety study (O'Sullivan et al., 1996). Experimental infection of cynomolgus monkeys with the parvovirus isolate resulted in mild anemia with destruction of erythroid cells in the bone marrow during peak viremia (O' Sullivan et al., 1997).

751 2.

RNA Viruses

a.

Viruses Causing Hemorrhagic Fevers

Simian hemorrhagic fever is a highly contagious, fatal viral disease of macaques caused by an arterivirus. Outbreaks have occurred in macaque colonies in the United States and Europe following exposure to infected blood or tissue from reservoir host species, usually the patas monkey (Erythrocebus patas). Infected macaques develop a bleeding diathesis that progresses to death. A lesion that distinguishes simian hemorrhagic fever from other hemorrhagic fevers in macaques is hemorrhagic necrosis of the proximal duodenum. Clinical signs and pathology are summarized in Table LI. Nonhuman primates serve as reservoir hosts for flaviviruses that can cause a hemorrhagic fever syndrome in human populations. These diseases may be spread by arthropod vectors or be directly transmitted between mammalian hosts. Disease in the nonhuman primate host may be subclinical, or the host may develop fatal hemorrhagic disease (Table LII). Two distinct groups of filoviruses are associated with hemorrhagic fever in humans or nonhuman primates (Table LIII). In 1967, Marburg virus caused an outbreak of hemorrhagic fever in laboratory workers handling cell cultures from African green monkeys. Since then, small outbreaks of disease associated with this virus have been reported in Africa. Ebola virus is associated with hemorrhagic fever in humans and macaques. Two strains of Ebola, Zaire and Sudan, have caused outbreaks in humans with mortality rates approaching 80%. No reservoir host has been identified. Ebola-Reston was identified in newly imported cynomolgus macaques with high mortality due to hemorrhagic fever in 1989-1990. The monkeys were coinfected with simian hemorrhagic fever virus (SHFV) (Dalgard et al., 1992). Subsequent outbreaks of disease due to Ebola-Reston occurred in cynomolgus monkeys in 1992 and 1996 (Rollin et al., 1999). Transmission occurred among macaques by direct contact, fomites, and aerosolization. Clinical signs included anorexia, lethargy, and death without premonitory clinical signs. In experimental disease, clinical signs included anorexia, lethargy, hypothermia, occasional nasal discharge, splenomegaly, facial petechia, and severe subcutaneous hemorrhages at venipuncture sites (Jahrling et al., 1996). Disease progressed rapidly to cardiovascular collapse, severe depression, and coma. Elevated liver enzymes and lactate dehydrogenase were seen in the first outbreak. Gross lesions are similar to those reported for SHFV. Multifocal hepatocellular necrosis, multifocal necrosis within the zona glomerulosa of the adrenal gland, and mild interstitial pneumonia are microscopic lesions that distinguish Ebola-Reston from SHFV (Dalgard et al., 1992). Large eosinophilic or amphophilic intracytoplasmic inclusion bodies may be found in the liver and adrenal gland. No Ebola-like disease has been reported in animal handlers or other personnel exposed to infected macaques or their tissues;

752

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Table LI Simian Hemorrhagic Fever Host

Disease

Reservoir: patas, African green monkey, baboon

Usually subclinical, may be persistently virernic a

Aberrant: Asian macaque

Fulminant, fatal hemorrhagic disease Initial spread by contact with blood/tissue of reservoir species Highly contagious among macaques once established Aerosol, direct contact, or fomite transmissionb Fever, bleeding diathesis including ecchymoses and petechia Epistaxis, hematuria, melena Depression, photophobia, cyanosis, death c Hemorrhage and necrosis of proximal duodenum Random hemorrhage GI tract, liver, kidney, lung, subcutis Splenomegaly Lesions consistent with D!C, fibrin thrombi in glomeruli Necrosis of lymphoid tissue; cortical thymic necrosis d

From Gravell et al. (1980). bFrom Renquist (1990). Note. DIC, disseminated intravascular coagution. a

cFrom Palmer et al. (1968). dFrom Zack (1993).

Table LII Flaviviruses Virus Yellow fever

Host

Vector

African monkeys New World monkeys

Mosquito

Langurs Bonnet macaques

Ixodid tick

Macaques Langurs Humansb

Mosquito

Hugh-Jones et al. (1995). bFrom Rigau-Perez et al. (1998) and George and Lam (1997).

a

Pancytopenia, fever, bradyca~ia Epistaxis and gastrointestinal hemorrhage Mu.ltifocal hepatocellular necrosis Hemorrhage adrenal, brain, kidney, lung Nonsuppurative encephalomyelitis Fever, chills, lower back and leg pain, headache, insomnia, anorexia Stiff neck, tremors, confusion Fever lasts 1-1 ~ weeks Epistaxis, gastrointestinal hemorrhage (fatalities)

Humansa

Dengue

:Subclinical, short-lived viremia Severe, epizootic, fatal Icterus, multiple hemorrhages Multifocal hepatocellular necrosis Hepatic fatty degeneration Councilman and Torres b ~ e s High fever, chilis, headache, backache, myalgia prostration, nausea, vomiting Epistaxis, oral bleeding, hematemesis Jaundice Death 3-7 days postinfection Case fatality rate 5-50%

Humansa

Kyasanur Forest disease

Disease

No clinical disease High incidence of antibody titers Fever, headache, myalgia, rash, nausea, vomiting Dengue hemorrhagic fever Increased vascular permeability Disseminated intravascular coagulation Thrombocytopenia Shock Fulminant hepatitis with encephalopathy

16. NONHUMANPRIMATES

753 Table LIII

Filoviruses i

Virus

Strain

Marburg

Ebola

Zaire

Reservoirhost

Disease in human

African green monkey

Fever, headache, muscle and joint aches, vomitingand diarrhea Maculopapularrash; gastrointestinalbleeding and epistaxis Hemorrhagic feverin technicians handling cell cultures Secondary and tertiary infections with human contacts 28% fatalities in primarycases; no disease in monkeys Headache, fever, myalgia,and nausea Maculopapularrash, sore throat, vomiting, and diarrhea Epistaxis, melena, hematemesis,bloodydiarrhea Hemorrhagic fever in humanswith 86% mortality Spread to close contacts and medical personnel Lowermortalityrate in contact cases suggestsviral attenuation Denguelike disease followingchimpanzeenecropsy No clinical signs of infection in animal handlers Handlers developedneutralizing antibodies indicating infection

Unknown

Sudan

Cote d'Ivoire Ebola-Reston

Chimpanzee Macaque?

however, detectable antibodies have been found in a small number of animal handlers, indicating that human infection with Ebola-Reston does occur (Miranda et al., 1999). Following the initial diagnosis of Ebola-Reston in 1990, CDCP quarantine requirements for imported nonhuman primates became more stringent. Quarantine of imported primates was and is limited to laboratories with biosafety level 3 containment facilities. Disease-control measures emphasize protection of employees from exposure, prevention of spread among animals, and testing of all animals, in particular those that become ill or die during quarantine (DeMarcus et al., 1999). b.

Paramyxoviruses

Measles virus infection has been common in nonhuman primates. Serologic testing of primate populations indicates that infection may be subclinical in macaques (Kalter and Heberling. 1990), but severe disease may also occur (Willy et al., 1999). Clinical signs and disease progression are detailed in Table LIV (see Figs. 25 and 26). In addition to the typical skin lesions, clinical disease may be respiratory, gastrointestinal (GI), or neurologic; abortions have also been reported (Renne et al., 1973; Steele et al., 1982, Roberts et al., 1988). Measles infection induces immunosuppression in the host (McChesney et al., 1989), increasing susceptibility to secondary bacterial infections. Measles infection in monkeys is usually acquired through contact with infected humans. Prevention of disease may be accomplished through screening of personnel to ensure adequate vaccination or measles infection history and/or vaccination of susceptible primate populations (Willy et al., 1999). Vaccination using modified-live measles vaccine has been shown to be safe and effective in some species of New World or Old World nonhuman primates (Albrecht et al., 1980; Willy et al., 1999). Vaccination using canine distemper/measles virus vaccine has

been reported to induce a measurable antibody response in rhesus monkeys (Staley et al., 1995). However, measles vaccination with modified-live products can cause immunosuppression that interferes with intradermal skin testing for tuberculosis; vaccinated monkeys may require up to 4 weeks to return to normal (Staley et al., 1995). Serologic surveys of wild and captive monkey and ape populations indicate that parainfluenza virus infections occur frequently. Transmission is by aerosol or direct contact with infected secretions. Clinical disease is usually self-limiting and ranges from mild upper respiratory disease to pneumonia. Morbidity within a primate population is usually high, with low mortality. Mortality is associated with secondary bacterial infection. The death of a juvenile chimpanzee has been attributed to respiratory syncytial virus infection (Clarke et al., 1994). c.

Retroviruses

Simian T-cell leukemia virus (STLV-1) is a type C retrovirus associated with lymphoproliferative disease in Old World monkeys and apes (Lee et al., 1985; McCarthy et al., 1990; TrainaDorge et al., 1992). Infection is usually subclinical with a high rate of infection in feral and captive baboons, African green monkeys, and macaques. In baboons, leukemia/lymphoma associated with STLV-1 resembles adult T-cell leukemia/lymphoma in humans. Clinical signs include anorexia, depression, lymph node enlargement, and hepatosplenomegaly. Overt leukemia occurs in greater than 50% of cases, and multilobulated, neoplastic lymphocytes occasionally may be found in peripheral blood smears (McCarthy et al., 1990). Simian retroviruses type D (SRV/D), particularly SRV/D-1 and SRV/D-2, are the primary cause of viral-induced immunodeficiency in captive macaque species. The type D virus is endemic in several Asian macaque species, and prevalence is high

754

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE Table LIV

Paramyxoviruses Genus Morbillivirus

Paramyxovirus

Virus Measles virus

Species

Disease

Old World monkeys New World monkeys

6- to 10-day incubation Maculopapular rash, facial hyperemia, cough, conjunctivitis, coryza, epistaxisb May develop pneumonia Gastrointestinal signs may predominatec Secondary bacterial infections common Syncytia in skin, lung, and lymphoid tissues (Fig. 25) __+intracytoplasmic(IC) and intranuclear (IN) inclusion bodies

Marmosets

Gastrointestinal disease, usually no rashd High mortality Necrotizing gastroenteritis Syncytia in gastrointestinalepithelium (Fig. 26) ___IC and/or IN inclusion bodies Anorexia, dehydration, diarrheae 10-100% mortality Necrotizing typhlocolitis Syncytia in crypt epithelium, pancreas, kidney, liver, and bile duct IC inclusion bodies

Paramyxovirus saguinus

Callitrichids

Parainfluenza type 1 (Sendai virus)

Marmosets

a

Persistent sneezing, tachypnea, dyspnea Ocular and nasal discharge Depression, anorexia, piloerection High morbidity (50%), low mortalityf Congestion and/or consolidation of lungs Interstitial pneumoniag

Pneumovirus

Parainfluenza type 3 (simian agent 10)

Patas, gibbons Chimpanzees

Upper respiratory tract infection Predisposed to streptococcalinfectionh

Respiratory syncytial virus

Chimpanzees

Upper respiratory tract infection Rhinorrhea, sneezing, coughing, fever Fatality in juvenile chimpanzee/

From Hall et al. ( 1971). ~ Willy et al. (1999). cFrom Roberts et al. (1988). dFrom Albrecht et al. (1980). eFrom Fraser et al. (1978). a

in captive macaque colonies. The most likely mode of transmission is through inoculation with blood or saliva by biting (Lerche e t al., 1986); however, vertical transmission and transmission from the dam to infant during the perinatal or postnatal period has been documented (Tsai e t al., 1990). Several clinical syndromes can occur with SRV/D infection: (1) persistent carrier state with no clinical disease with or without an antibody response, (2) severe immunodeficiency and viremia with or without an antibody response, (3) clearance of infection with an appropriate antibody response, (4) retroperitoneal or subcutaneous fibromatosis, or (5) persistent lymphadenopathy (Lowenstein, 1993). A case definition of simian AIDS induced by SRV/D is presented in Table LV. Immunosuppressed animals are particularly susceptible to pyogenic bacterial infections,

fFrom Flecknell et al. (1983). gFrom Sutherland et aL (1986). hFrom Churchhill (1963), Martin and Kaye (1983), and Jones et al. (1984). /From Clarke et al. (1994).

disseminated CMV, candidiasis, intestinal cryptosporidiosis, and noma or cancrum oris (Lowenstein, 1993). Proliferative lesions vary with virus serotype. Retroperitoneal fibromatosis is most frequently associated with SRV/D-2 infection and can present as a multinodular to coalescent mass originating from the ileocecal junction and involving the root of the mesentery, mesenteric lymph nodes, and gastrointestinal tract. Lesions range from small plaques or nodules spread across mesothelial surfaces to encasement of the gastrointestinal tract in a large fibrotic mass. Subcutaneous fibromatosis is more often associated with SRV/D-1 infection and is characterized by multiple nodules in the subcutis and oral cavity (Tsai e t al., 1985). Microscopically, proliferative lesions are highly vascular with

755

16. NONHUMAN PRIMATES

Fig. 25. Measles virus syncytia formation in lung parenchyma of a tamarin, Saguinus mystax.

Fig. 26. Measles virus syncytia in small intestinal epithelium of a tamarin, Saguinus mystax.

756

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Table LV Case Definition of Simian Type D Retrovirus-Induced Simian AIDSa Generalized lymphadenopathy and/or splenomegaly accompanied by at least four of the following clinical and laboratory findings: Weight loss (> 10%) Fever (> 103~ Persistent refractory diarrhea Opportunistic infections Noma (cancrum oris) Retroperitoneal fibromatosis Hematologic abnormalities Anemia (PCV < 30%) Neutropenia (< 1700) Lymphopenia (< 1600) Thrombocytopenia (< 50,000) Pancytopenia Bone marrow hyperplasia Characteristic lymph node lesions aFrom Lackner (1988).

intersecting fascicles of spindle-shaped cells that infiltrate along serosal surfaces and encompass normal abdominal structures, usually accompanied by lymphoplasmacytic inflammation (Mansfield and King, 1998). In severely immunosuppressed animals, marked lymphoid depletion with effacement of normal architecture occurs in the spleen, thymus, and lymph nodes. Histiocytes replace depleted plasma cells and lymphocytes in the paracortex, and follicles contain hyalinized arterioles (Osborn et al., 1984). Establishment of SRV/D-free colonies by identification of SRV/D-negative animals is difficult because serologic testing is inadequate. Apparently healthy, seronegative monkeys with persistent infections can only be identified by virus isolation or PCR for viral RNA. A successful protocol combining viral isolation and antibody screening has been described by Lerche et al. (1994). Simian immunodeficiency viruses (SIVs) are lentiviruses closely related to human immunodeficiency viruses HIV-1 and HIV-2. Serologic studies have revealed that seropositive animals are frequently found among wild and captive African monkey populations, while seropositive animals are infrequently found in captive, but not wild Asian monkey (macaque) populations. Simian immunodeficiency virus isolates are identified by subscript to indicate the species of origin, i.e., SIVmac was isolated from a rhesus monkey and SIVcpz from a chimpanzee. Simian immunodeficiency viruses in African species are of relatively low pathogenicity, sexual transmission is most likely (Phillips-Conroy et al., 1994), and transmission from dam to infant has also been proposed (Fultz et al., 1990). In macaque species, SIVs produce devastating AIDs-like disease; horizontal transmission by biting or being bitten, sexual transmission, and dam-to-infant transmission have been demonstrated in natural or experimental infections. Common lesions of uncomplicated

SIV infection in macaques are listed in Table LVI. Opportunistic infections occur with Mycobacterium avium complex, CMV, adenovirus, papovavirus, Pneumocystis carinii, Cryptosporidium spp., Cryptococcus neoformans, toxoplasmosis, and candidiasis. Development of malignant lymphoma among SIVinfected macaque populations has been described (Lowenstine et al., 1992). Unlike SRV infection, seronegative, viral-positive SIV infection in macaques is rare. Serologic testing of macaque colonies and removal of reactors is an effective way to eliminate infection from the population (Lowenstine et al., 1986). Asian monkeys should not have direct contact with African species or their tissues.

d.

Other RNA Viruses

Rabies has been reported in tamarins, squirrel monkeys, macaques, and chimpanzees (Richardson and Humphrey, 1971; Fiennes, 1972). Although current housing practices minimize the possibility of contact with carrier species, possible rabies exposure should be considered when primates are housed outdoors in rabies endemic areas. Reported clinical cases in nonhuman primates are extremely rare. Clinical signs include irritability, self-mutilation, and paralysis of pharyngeal and pelvic

Table LVI Lesions Associated with SIV Infection in Macaques a System

Comment

Lymphoid

Follicular hyperplasia Follicular involution ___expanded paracortical regions Depletion of follicular and paracortical regions Granulomatous (giant cell) lymphadenitis Generalized lymphoproliferative syndrome

Nervous

Nonsuppurative histiocytic meningoencephalitis Multifocal perivascular aggregates of giant cells and histiocytes

Gastrointestinal

Enteropathy; disseminated giant cell disease involving the lamina propria

Cardiopulmonary

Arteriopathy with medial and intimal proliferation of pulmonary arteries Thrombosis, hemorrhage, consolidation, and infarction of lung

Skin

Viral exanthema in experimental infections Perivascular lymphocytic dermatitis

Respiratory

Giant cell interstitial pneumonia

aAdapted from Mansfield and King (1998).

16. NONHUMANPRIMATES muscles. Nonhuman primates may be vaccinated with killed vaccine; however, the efficacy of the vaccine is unknown. Use of attenuated rabies vaccine is contraindicated as vaccine-induced disease is believed to have occurred in New World species (McClure et al., 1972). Montali et al. (1993) reported on the development of a rapidly progressive viral hepatitis in callitrichids in zoological collections due to infection with lymphocytic choriomeningitis virus (LCMV). Infection has been shown to occur following feeding of neonatal mice infected with LCMV and is postulated to occur from consumption of naturally infected wild mice (Montali et al., 1993). Horizontal transmission between callitrichids does not occur; however, vertical transmission has been demonstrated. Clinical signs include anorexia, dyspnea, lethargy and weakness, ataxia or incoordination, and in some instances, seizures (Montali et al., 1993). Affected animals may be jaundiced. Elevated liver enzymes, bilirubin, and alkaline phosphatase levels have been reported. Mortality can be high although serologic evidence of infection has been demonstrated in animals without history of clinical disease (Potkay, 1992). Mortality is related to the amount of infected mouse tissue consumed. Gross lesions include hepatomegaly, splenomegaly, jaundice, subcutaneous and intramuscular hemorrhages, and pleural or pericardial effusions (Montali et al., 1993). Liver lesions include multifocal hepatocellular necrosis with lymphocytic and neutrophilic infiltrates. Acidophilic apoptotic hepatocytes are found within sinusoids and Kupffer's cells (Montali et al., 1993). Necrosis of abdominal lymph nodes, adrenal glands, spleen, and gastrointestinal tract may be seen. Lymphocytic choriomeningitis virus is a zoonotic disease, and veterinarians in contact with infected marmosets developed antibody titers to LCMV (Montali et al., 1995). Naturally occurring cases of hepatitis A virus have been described in chimpanzees, owl monkeys, African green monkeys, and cynomolgus monkeys. Serologic testing indicates that infection occurs in both wild and captive nonhuman primate populations, including many New and Old World species. Transmission is fecal-oral. Infection is usually self-limiting with no clinical disease. Elevated serum alanine aminotransferase and aspartate aminotransferase, 2-10 times above normal levels, with mild elevations of bilirubin are characteristic (Mansfield and King, 1998). Microscopic lesions include focal hepatocellular necrosis with nonsuppurative inflammatory infiltrates in the portal areas. Bile duct hyperplasia and necrosis of bile duct epithelium has been described in chimpanzees. Brack (1987) reported cases of human infection with hepatitis A virus contracted from nonhuman primates, particularly from chimpanzees. Fatal infection with encephalomyocarditis viruses has been reported in owl monkeys, squirrel monkeys, baboons, rhesus macaques, and chimpanzees (Gainer, 1967; Blanchard et al., 1987; Hubbard et al., 1992; Baskin, 1993). Wild mice and rats are the reservoir hosts for this group of viruses, and fecal con-

757

tamination of feed, water, or enclosures has been postulated as the source of infection in nonhuman primates. Death with no premonitory clinical signs is usual in naturally infected monkeys. Pericardial effusion, white-tan mottling of the myocardium, and pulmonary congestion may be observed at necropsy. Nonsuppurative necrotizing myocarditis is the most important microscopic lesion. Placental infection and subsequent abortion can occur (Hubbard et al., 1992). Elimination of feral rodents and cleaning of facilities are essential for prevention and control. Naturally occurring infections with poliovirus have been reported for the great apes and rhesus monkeys. Infection may cause no clinical disease, or infected animals may develop paresis and paraplegia and then die. Lesions are located in the gray matter of the central nervous system and include perivascular inflammatory cell aggregates and meningeal infiltrates with neuronal necrosis and glial nodules. Vaccination of great apes with oral trivalent polio vaccine is recommended. Vaccination has been used effectively in wild chimpanzee populations to prevent disease (Morbeck et al., 1990).

D.

Parasitic Diseases

Nonhuman primates, particularly feral animals, may be infested with a variety of parasites. Many of these infections are incidental or subclinical in nature and will not be presented in detail. Tables LVII-LXIV list parasites that produce clinical disease in nonhuman primates; disease, pathology, diagnosis, and treatment are briefly described. Figures 27-42 illustrate ova, adult parasites, or gross or microscopic lesions.

E.

Nutritional Diseases

Some nutritional diseases of nonhuman primates are summarized in Table LXV (see next page). Most are due to vitamin deficiencies, with one instance of vitamin overfeeding. Simian bone disease, also known as nutritional hyperparathyroidism due to insufficient calcium or improper calcium-phosphorus ratio in the diet, is included although its occurrence is now rare with the use of commercially formulated feeds. Errors can and have occurred during feed manufacture, and a specific batch of a commercial diet may be misformulated (Ratterree et al., 1990; Eisele et al., 1992). Table LXV is not inclusive of nutritional diseases but provides a synopsis of those commonly reported. References in the table footnotes can be used to find more detail on these conditions. In general, young and rapidly growing animals are more likely to develop clinical disease due to nutritional deficiencies. Clinical signs of nutritional disorders may overlap or be sufficiently nonspecific to preclude diagnosis, as in vitamin C and D deficiencies. Radiography to determine specific lesion site and

cr

Table LVII

Enteric Protozoa Parasite

Affected species a

Location

Clinical disease/pathology

Diagnosis

Treatment

Flagellates

Giarclia spp.

Small intestine OWM Apes

Frequently subclinical Diarrhea and vomiting ___steatorrhea

Saline wet mount, fresh feces Fecal concentration for cysts

Metronidazole b,c 3 0 - 5 0 m g / k g BID X 5-10 days Furozolidine ~

1.5 mg/kg X 7 days (marmosets)

Trichomonas spp.

NWM OWM

Intestine Stomach, pelvic cavity (invasive disease in rhesus)

Diarrhea reported for callitrichids

Saline wet mount, fresh feces Rectal swab and culture

Metronidazole a 17.5-25mg/kg BID • 10 days

NWM OWM Apes

Colon, cecum

Varies, more severe in young monkeys and NWM Anorexia, vomiting Severe diarrhea _ hemorrhage Necroulcerative colitis Flask-shaped ulcers Amebic abscesses in liver, lungs, or central nervous system

Saline wet mount Iodine, trichrome, or Giemsa stain Trophozoite 2 0 - 3 0 Ixm diameter Organism in lesioned organs Giemsa, trichrome, PAS stains

Metronidazole c

Amoebas

Entamoeba histolytica

30mg/kg TID X 5 - 1 0 days or in combination with Diiodohydroxyquin c

3 0 - 4 0 mg/kg TID Tetracycline r

2 5 - 5 0 m g / k g 5 - 1 0 days Chloroquin c

5 mg/kg x 14 days Chloramphenicol e

50-100 mg/kg BID Paromomycin f

12.5-15 mg/kg BID x 5-10 days Coccidians

Isospora spp.

Callitrichids

Intestine

Diarrhea

Fecal flotation Saline wet mount

Sulfamethoxine f

50 m.g/kg/day 1, then 25 mg/kg/day

(continues)

Table LVII (Continued) Parasite Cryptosporidium

spp.

Affected species a

Location

Clinical disease/pathology

Diagnosis

Treatment

Prosimians NWM OWM

Intestine Bile duct, pancreatic duct, conjunctiva, trachea, bronchioles in immunosuppressed animals

Intractable diarrhea Profuse, watery diarrhea Depression, weight loss Hypothermia, anorexia Dehydration Fluid and gas distension of intestine Mesenteric LN enlargement Blunting and fusion of villi Villous atrophy (Fig. 27) Increased mitotic index in crypts Hyperplasia of biliary and pancreatic duct epithelium; periductal fibrosis

Stain fecal smears or concentrates Flotation Direct or indirect FAS Stool antigen detection assay Formalin-fixed feces Histology 4 - 5 p~m oocysts on brush border of enterocytes (Fig. 28)

Supportive care Replace fluid and electrolytes Antidiarrheals Antibiotics

NWM

Cecum, colon

Usually nonpathogenic Can cause severe, ulcerative colitis in apes Anorexia, weight loss, weakness, lethargy Watery diarrhea, tenesmus, rectal prolapse

Large ciliated ovoid organisms 30-150 X 25-120 Ixm

M e t r o n i d a z o l e c,g

Ciliates

Balantidium coli

OWM Apes

aNWM, New World monkeys; OWM, Old World monkeys. bFrom Peisert et al. (1983). CFrom Lehner (1984). dFrom Brady et al. (1988). eFrom Renquist and Whitney (1987). IFrom Wolff (1993). gFrom Swenson et al. (1979).

35-50 mg/kg/day divided doses TID Tetracycline g

40 mg/kg PO divided dose TID Diiodohydroxyquin

8

40 mg/kg PO divided doses TID x 14-21 days

760

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Fig. 27.

Fig. 28.

Fusion of villi due to cryptosporidial infestation.

Cryptosporidium organisms adherent to enterocytes.

Table LVIII Hemoprotozoa Parasite

Trypanosoma cruzi

Host a NWM OWM Apes

Location

Clinical disease/pathology

Blood (trypomastigote)

Subclinical Anemia, hepatosplenomegaly Lymphadenitis Generalized edema Anorexia, depression, weight loss Right bundle branch block-ECG

Skeletal and cardiac muscle, reticuloendothelial system (amastigote)

Myocarditis Pseudocysts within myocardial fibers contain 1.5- 4.0 p~m round-to-oval organisms

Treatment

Diagnosis Smear, blood or body fluid, thick and thin

None

Serology EIA or CF Histology

Chloroquine c 2.5-5 mg/kg IM X 4 - 7 days followed by Primaquine 0.75 mg/kg P O x 14 days

Plasmodium brazilianum

NWM

Erythrocytes

Usually persistent low parasitemia Can cause severe, fatal disease Anemia, cyclic pyrexia Hepatosplenomegaly Depression, death

P. knowlesi, cynomolgi, fieldi, fragile, inui

Macaques Leaf monkeys

Erythrocytes

Usually subclinical unless splenectomized or immunosuppressed Anorexia, fever, weakness, splenomegaly

As above

P. pitheci, rodhairi, reichenowi, schwetzi

Great apes

Erythrocytes

Usually subclinical parasitemia Some strains cross-infective with humans

As above

P. hylobati, youngi, eylesi

Gibbons

Erythrocytes

Fever with parasitemia, pathogenic

As above

Hepatocystis spp.

OWM Apes

Blood, liver

Subclinical infection Numerous, random, gray-white foci on surface of liver (merocyst) Eosinophilic granulomatous reaction to ruptured cysts Focal fibrosis when healed

Thick or thin blood smears Typical hepatic lesions

Thick blood smear Serology (FA)

None Vector control

Histology

Babesia pitheci

NWM OWM

Erythrocytes

Severe anemia, death with splenectomy Mild disease in intact monkeys

Blood smear Pyriform 2 - 6 ~tm organisms

None

Entopolypoides macacai

OWM Apes

Erythrocytes

Usually subclinical Fever, anemia, monocytosis Hemolytic anemia and icterus following splenectomy or immunosuppression

Blood smear

None

aNWM, New World monkeys; OWM, Old World monkeys. bEIA, enzyme-linked immunoassay; CF, complement fixation. CFrom Lehner (1984).

762

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Table LIX Disseminated Protozoal Infestations Parasite

Toxoplasma gondii

Host a

Location

Clinical disease/pathology

Prosimians NWM OWM Apes

Lymph nodes, liver, lung, spleen, intestine, brain, heart

Death, no clinical signs Anorexia, listlessness, weakness Depression, somnolence Emesis, diarrhea Oculonasal discharges Dyspnea, tachypnea Neurologic signs Circling, head holding, head hitting, incoordination, paresis, convulsions Hepatosplenomegaly Ulcerative enteritis Lymphadenopathy Pulmonary edema

Encephalitozoon cuniculi

Squirrel monkeys

Brain, kidney, lung, adrenal glands, liver, placenta

None or nonspecific, death Granulomatous inflammation (Fig. 30)

Diagnosis Serology (CF, IFA, HA1)b Impression smears of spleen, lung, lymph nodes

Treatment No treatment reported for nonhuman primates

Pediatric treatment regimen (humans) c Histology, Gram-, PAS- 4 - 8 I~m Sulfadiazine banana-shaped tachyzooites in 100 mg/kg/day; in divided 60 ~m cyst (Fig. 29) doses BID x 1 month Animal inoculation Pyrimetharnine Load with 1 mg/kg/day x 2 - 4 days; then 1 mg/kg/day Folio acid 1 mg/day to prevent bone marrow depression

2.5 X 1.5 lxm oval organisms in 60-120 I~m pseudocysts in tissues (Fig. 31) Gram+, PAS + Serology IFA, ELISA Organisms in urine

None

NWM, New World monkeys; OWM, Old World monkeys. bCF, complement fixation; IFA, indirect fluorescent antibody assay; and HAI, hemagglutination inhibition test. CFrom Lehner (1984).

Table LX Acanthocephalans

Parasite

Host a

Prosthenorchis elegans, P. spirula

NWM

Location Ileum, cecum, colon

Prosimians

Clinical disease/pathology

Diagnosis

Anorexia, dehydration Abdominal distension Diarrhea Debilitation Weight loss Death

Fecal smears Formalin ether sedimentation (standard flotation not effective) Flexible fiberoptic proctoscopy Palpation of abdominal masses Necropsy (Fig. 32)

Treatment Carbon tetrachloride 0.5 ml/kg PO once

(Efficiency and long-term safety of this treatment is not established)b

aNVC'M, New World monkeys. bFrom Toft and Eberhard (1998).

Table LXI Scabies Parasite

Sarcoptes scablei

Hosta

Location

Clinical disease/pathology

Diagnosis

Treatment

OWM

Skin

Severe pruritis Anorexia, weight loss, weakness Tremors Alopecia, scaling and thickening of skin Self-mutilation Bacterial dermatitis Death

Deep skin scrapings for parasites and eggs (Fig. 33)

Ivermectin 200mcg/kg; repeat in 3 weeks b

Apes

Old World monkeys. bFrom Toft and Eberhard (1998). a OWM,

16. NONHUMAN PRIMATES

763

Fig. 29. Hepatocellular necrosis due to Toxoplasma gondii infestation. Note toxoplasma tachyzoites within hepatocytes.

Fig. 30. Microgranuloma in the brain of an infant squirrel monkey, Saimiri spp., associated with Encephalitozoon spp.

763

764

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Fig. 31. Encephalitozoon spp. pseudocyst, squirrel monkey cerebellum.

Fig. 32.

Thorny-headed worms, Prosthenorchis elegans, attached to the ileal mucosa of a tamarin, Saguinus mystax.

Table

LXII

Nematodes Parasite

Strongyloides cebus

Host a NWM

Location Intestine

Clinical disease/pathology

Fecal flotation, larvae, or larvated ova Necropsy, histology (Fig. 34, 35, and 36)

Ivermeetin b 200 mcg/kg IM

Diarrhea _ hemorrhage _ mucus Dermatitis, urticaria Vomiting, dehydration Debilitation, emaciation Cough, dyspnea Fatalities in apes, patas Enterocolitisi catarrhal, hemorrhagic, or necrotizing Peritonitis Pulmonary hemorrhage

Fecal flotation, larvae, or larvated ova Necropsy, histology

Ivermectin b

Usually subclinical Death in spider monkey due to overwhelming infestation

Observation of adults at anus Perianal tape test or swab (Fig. 37) Fecal flotation Ellipsoid, asymmetric ova

Thiabendazole e

May be subclinical Anal pruritis and irritation Self-mutilation Aggressiveness Fatalities in chimpanzees Ulcerative colitis, peritonitis, lymphadenitis

Observation of adults at anus Perianal tape test or swab Fecal flotation Ellipsoid, asymmetric ova

Mebendazole d

Weight loss, diarrhea Unthrifty Subserosal nodules 2 - 4 mm in colon and mesentery

Fecal flotation, hookworm like ova Identify larvae following stool culture (Fig. 38)

Thiabendazole a

Usually none, eosinophilia Fatality in a woolly monkey

S. fulleborni, S. stercoralis

Trypanoxyuris sp.

OWM Apes

NWM

Intestine Filariform larvae in lungs and other parenchymous organs

Cecum, colon

Enterobius ve rmicularis, E. anthropopitheci

OWM Apes Chimpanzees

Cecum, colon

Oesophagostomum spp.

OWM Apes

Cecum, colon

Treatment

Diagnosis

Thiabendazole c

50-100mg/kg PO for 1, 2, or 5 days 200 mcg/kg IM, PO Thiabendazole c

5 0 - 1 0 0 mg/kg PO for 1, 2, or 5 days Mebendazole d

22 mg/kg/day PO or SQ x 2 - 3 days Levamisole d 10mg/kg PO or SQ • 2 - 3 days Pyrantel pamoate d

11 mg/kg PO once 5 0 - 1 0 0 mg/kg PO

100mg/kg PO adult ape 10mg/kg PO infant or smaller species P y r a n t e l pamoate d 11 mg/kg PO once

2 5 - 1 0 0 m g / k g PO for 1-2 days Levamisole d 10mg/kg PO or SQ once Mebendazole d

40 mg/kg PO, divided dose TID X 3 - 5 days Repeat all treatments at 10 -14 days

Molineus elegans, M. torulosis

NWM

Pylorus, duodenum Pancreatic ducts

M. elegans usually subclinical Ulcerative enteritis _ hemorrhage with M. torulosis Serosal nodules in duodenum Chronic pancreatitis

Fecal flotation Necropsy

None

(continues)

Table LXH (Continued)

Parasite

Hosta

Location

OWM Apes

Intestine

Trichospirura leptosoma

NWM

Pterygodermatites nycticebi, P. alphi

Prosimians NWM Gibbons

Ascaris lumbricoides

Trichuris spp.

Anatrichosoma cynomolgi

Clinical disease/pathology

Treatment

Usually subclinical Deaths associated with intestinal blockage due to heavy parasitism and migration to liver, bile ducts

Fecal flotation, roundworm ova (Fig. 39)

Mebendazole f

Pancreatic ducts

Weight loss, wasting disease Acute/chronic pancreatitis Jaundice due to bile duct obstruction

Fecal flotation Thick-shelled, larvated ova

None

Intestine

Watery diarrhea, anorexia, weakness (tamarins) Anemia, leukopenia, hypoproteinemia Pseudomembranous necrotizing enteritis

Fecal flotation, spirurid ova Adults, larvae in feces Necropsy, histology

Ivermectin g

Anorexia, mucoid or watery diarrhea, and occasionally death may occur with heavy infestations

Fecal flotation Bipolar operculated ova (Fig. 40)

Subclinical nasal infestation or mild serous discharge Pruritis of hands and feet Vesicle/pustules in skin Regional lymph node enlargement

Nasal or epidermal swabs or s c r a p i n g s i ova (Fig. 41)

NWM OWM Apes

Cecum, colon

NWM OWM Apes

Nasal mucosa Secondary infestation of hands and feet (creeping eruption)

New World monkeys; OWM, Old World monkeys. bFrom Brack and Rietschel (1986) and Battles et al. (1988). CFrom Bingham and Rabstein (1964), Flynn (1973), Swenson et al. (1979), Lehner (1984), and Abee (1985). dFrom Swenson et al. (1979). eFrom Lehner (1984). fFrom Toft and Eberhard (1998). gFrom Blampied et al. (1983). hFrom Welshman (1985). /From Kumar et al. (1978). JFrom Harwell and Dalgard (1979). a NWM,

Diagnosis

22 mg/kg PO • 3 days Pyrantel p a m o a t e f 11 mg/kg PO once, repeat in 10-14 days

0.5 mcg/kg SQ x 3 days predilute in sterile water for smaller primates (marmosets) Mebendazole g

40mg/kg x 3 days Mebendazole d

40mg/kg PO BID x 5 days Dichlorvos d 10mg/kg PO SID, 1-2 days Levamisole h 7.5 mg/kg SQ x 2 at 2-week interval F l u b e n d a z o l e (5 %)i 27-50mg/kg BID x 5 days (baboons) Fenbendazole /

10-25 mg/kg PO SID x 3-10 days

767

16. NONHUMAN PRIMATES

Fig. 33.

Sarcoptes scabiei mite from a rhesus monkey, Macaca mulatta.

Fig. 34.

Larvated Strongyloides egg, fecal flotation.

768

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Fig, 35.

Fig. 36.

Strongyloides cebus larva, fecal flotation.

Strongyloides cebus, adult parasite in the small intestine of a squirrel monkey, Saimiri spp.

769

16. N O N H U M A N PRIMATES

Fig. 37.

Pinworm eggs on a perianal tape test from an owl monkey, Aotus spp.

Fig. 38.

Oesophagostomum sp. or nodular worm in intestine of rhesus monkey.

770

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Fig. 39.

Fig. 40.

Egg ofAscaris lumbricoides, fecal flotation from a chimpanzee.

Trichuris trichiura egg, fecal flotation from a baboon, Papio spp., iodine stain.

771

16. NONHUMAN PRIMATES

Fig. 41.

Fig. 42.

Anatrichosoma cynomolgi egg, fecal flotation.

Cysticercus in the brain of a stump-tailed macaque, Macaca nemestrina.

772

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE Table L X I I I

Cestodes Parasite

Location

Host a

Clinical disease/pathology

Diagnosis

Treatment

All

Small intestine

Abdominal pain, tucked abdomen, crouching Anorexia, vomiting Catarrhal enteritis Abscessed mesenteric lymph node

Fecal flotation Ova Proglottids in feces Necropsy

Niclosamide b 100 mg/kg PO once Bunamidine b 25-100 mg/kg PO once Praziquantel c 0.1 mg/kg IM

Cysticercosis Taenidae

All

Abdominal or thoracic cavities Muscle Subcutaneous tissues Central nervous system (CNS)

Clinical signs dependent on number and location of cysticerci

Necropsy (Fig. 42)

None

Coenurosis Multiceps spp.

Prosimians OWM

Subcutaneous tissue Peritoneal cavity Brain Liver

Dependent on number and location of coenuri Usually subclinical except in CNS

Radiography Palpation of mass in SQ tissues

None

Hydatidosis Echinoccocus granulosus

All

Abdominal or thoracic cavities Liver Lungs Retrobulbar Subcutaneous tissue

Usually subclinical, may mimic neoplasic disease Abdominal distension Exophthalmia Localized subcutaneous mass Anaphylactic shock, death following cyst rupture in lungs

Radiography Ultrasound Serology: intradermal skin test HA d

None

Hymenolepis nana

Larval Diseases

All, all nonhuman primates; OWM, Old World monkeys. bFrom Swenson et al. (1979). cFrom Welshman (1985). dHA, hemagglutination assay. a

Table L X I V

Trematodes Parasite

Host a

Location

Clinical disease/pathology

Treatment

Diagnosis

Gastrodiscoides hominis

OWM

Cecum, colon

Mucoid diarrhea Mild chronic colitis

Ova in feces

None

Watsonius spp.

OWM

Intestine

Diarrhea, severe enteritis Death

Ova in feces

None

Paragonimus westermanii

OWM

Lung Ectopic sites include brain and liver

Cough, wheezing Blood in sputum Moist rales Progressive weight loss

Ova in feces Necropsy

None

Schistosoma mansoni, S. haematobium, S. matheei

NWM OWM Apes

Mesenteric veins (S. mansoni and S. matheei) Portal veins (S. haematobium)

Usually subclinical Fever, hemorrhagic diarrhea Hematuria Ascites

Ova in feces or urine Necropsy, adults in veins

Praziquantal (56.8 mg/ml) b 0.2 cm3/kg if < 1 kg body weight; 0.1 cm3/kg if > 2 kg body weight

a NWM, New World monkeys; OWM, Old World monkeys. bFrom Toft and Eberhard (1998).

Table L X V Nutritional Diseases Condition

Common name

Species affected

Clinical signs

Hypovitaminosis A

Rhesus monkeys a

Abortion

Hypervitaminosis A

Callitrichids b

Musculoskeletal lameness, paresis cachexia, debilitation, alopecia

Rhesus monkeys c (experimental)

Visual impairment Spastic paralysis of hindlimbs Decreased range of motion in knees Painful, swollen joints Contracted tendons Hand walking Megaloblastic anemia

Hypovitaminosis B 12

May contribute to "cage paralysis"

Chimpanzees Hypovitaminosis C

Scurvy

All primates (young animals most affected)

Squirrel monkeys

Reluctance to move Joint pain and tenderness Lameness, abnormal locomotion Gingival swelling, hyperemia, petechia Bruising Microcytic anemia Acute cephalohematoma

Lesions

Treatment Correct diet

Spinal hyperostosis and spinal ankylosis

Correct diet Irreversible

Subperiosteal hemorrhage Epiphyseal fracture Periodontal bone resorption Long bone fractures Periosteal elevation long bones

25 mg/kg ascorbic acid IM BID X 5 days e

Periosteal elevation skull (Fig. 43)

50mg ascorbic acid IM single dose f or 10mg/kg body weight/day g

250 mg ascorbic acid IM x 2 days and oral 30-100 mg/kg/day d

Hypovitaminosis D 2

Rickets

Old World monkeys

Failure to grow Wrists and knee enlargement Bowing of long bones

Cupping of epiphyses Decreased bone density

Provide D2 in diet Ultraviolet light

Hypovitaminosis D 3

Rickets

New World monkeys (young animals)

Growth retardation Impaired ambulation Fractured long bones Masticatory weakness, difficulty chewing Inanition, death

Metaphyseal cupping and fraying of femur and tibia

1 IU/gm diet D 3 preventive h Provide source of ultraviolet B radiation /

(continues)

Table LXV ( C o n t i n u e d )

Condition

Common name

Folic acid deficiency

Species affected Monkeys

Squirrel monkeys Hypovitaminosis E

Calcium deficiency

Nutritional cardiomyopathy

Baboons l

Vitamin E-responsive anemia

Owl monkeys m (gray-necked)

Contributes to marmoset wasting syndrome

Tamarins n

Simian bone disease; osteomalacia

Prosimians and simians ~

aFrom O'Toole et al. (1974). bFrom Demontoy et al. (1979). CFrom Kark et al. (1974). Agamanolis et al. (1976). aFrom Eisele et al. (1992). eFrom Ratterree et al. (1990). fFrom Kessler (1970). gFrom Lehner et al. (1968). hFrom Lehner et al. (1967). i From Gacad et al. (1992). J From Wixson and Griffith (1986). kFrom Rasmussen et al. (1980). lFrom Liu et al. (1984). mFrom Sehgal et al. (1980) and Weller (1994). "From Baskin et al. (1983). ~ Krook and Barrett (1962) and Snyder et al. (1980).

Clinical signs

Lesions

Megaloblastic anemia j Weight loss, petechia Anorexia, gingivitis, dehydration Diarrhea Alopecia, scaly dermatitis Megaloblastic anemia of pregnancy k Death, no clinical signs Heart failure Dyspnea Weakness, pallor, heart murmur Icterus Hemolytic anemia, hematocrit ~< 15 Weight loss, muscle atrophy Anemia Decreased locomotion Impaired mobility

Treatment

109 lxg folic acid/day preventive White areas of myocardium Acute myocytolysis, fibrosis

Vitamin E

Splenomegaly

Vitamin E and selenium, 02, supportive therapy, transfusion (rarely) Not responsive to treatment but vitamin E was preventive

Myositis, steatitis

Kyphosis Bowing, fractured long bones Thickened jaw Dental displacement

Correct calcium: phosphorus in diet

16. NONHUMANPRIMATES appearance, diet analysis to determine if there is an adequate amount of the nutrient in question, necropsy and histologic diagnoses, and response to treatment can contribute to making a specific diagnosis. Management practices, feeding varied diets of multiple foods, and lack of knowledge of nutritional requirements for a specific animal may interact and result in a nutritional deficiency. (Fig. 43). An example is wasting marmoset syndrome. Affected callitrichids have chronic diarrhea, colitis, and in some cases, hemolytic anemia. Animals experience marked weight loss and muscle atrophy and develop alopecia. Both managerial and dietary factors probably contribute to this syndrome. The practice of feeding groups of marmosets or tamarins on the cage floor or at one feed site has been considered contributory to the development of wasting marmoset syndrome (Tardif and Richter, 1981). The dominant callitrichid may guard the food station and prevent or limit access to other group members. A single commercial diet has not proven to be adequate for callitrichids, so many laboratories feed a varied diet that may or may not be nu-

Fig. 43. Hyperostosisof a squirrel monkeyskull subsequentto cephalohematoma formationfrom vitaminC deficiency.

775

tritionally adequate. Primary nutritional vitamin E deficiency and protein deficiency due to chronic diarrhea have been proposed as etiologies for this syndrome (Baskin et al., 1983; Tucker, 1984). Other investigators have suggested that the etiology of this syndrome is inadequate calories per gram of diet, combined with a high daily gross energy requirement for callitrichids (Barnard et al., 1988).

F. 1.

Miscellaneous Disorders

Trauma

Trauma is a common medical problem requiring treatment in pair or group-housed primates. Aggression between animals can cause severe wounding or death. Trauma frequently involves injury to tissues beneath the skin; associated tissue damage may require treatment for shock, sepsis, or multiple organ failure. Severely wounded animals require vigorous treatment, including intravenous fluid and electrolyte therapy. Crush injuries to the skin are commonly associated with bite wounds. The extent of injury can easily be underestimated because there are no visible lacerations and external hemorrhage may be minimal or nonexistent. Laboratory values may be within normal limits. Several days later injured areas have developed ischemic necrosis characterized by dry, blackened, devitalized skin. Underlying tissues may be devitalized and require debridement. Secondary infection is common. In severe cases, damage to underlying tissues can result in myoglobinuria, myoglobin casts in the kidney, and resultant fatal acute renal disease. Accurate early diagnosis of crush injury and aggressive fluid therapy can ameliorate these sequelae. Another cause of traumatic injury in macaques is self-mutilation or self-injurious behavior (SIB). In its mildest form, SIB is expressed as hair pulling; head banging and self-biting are the more severe manifestations of SIB. From 5 to 12% of individually housed rhesus macaques have been reported to engage in SIB (Bayne et al., 1995; Novak et al., 1998). Wounds usually consist of bites or slashes to skin and muscle. Episodes of selfbiting behavior are more frequent than actual wounding. Attempts to decrease the occurrence of SIB through environmental enrichment with puzzle feeders or other manipulanda have been unsuccessful (Novak et al., 1998). Cutting or blunting of the canine teeth decreases wounding but does not stop selfbiting behavior (Bayne et al., 1995). Tail injuries due to tail chewing are a major problem in grouphoused squirrel monkeys. Occasionally 1 or 2 animals within a group will bite the tails of other members. Once injured, infection or irritation of the tail can lead to self-mutilation of the existing wound (Abee, 1985). Blood supply to the tail is poor, and gangrene or infection are frequent sequelae. Tail injuries may be self-inflicted; squirrel monkeys with abscessed teeth or other

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dental problems may gnaw the tail in order to alleviate the primary source of pain (Abee, 1985). Treatment of severely injured or infected tails is partial amputation of the tail to remove the damaged segment.

2.

Hypoglycemia

Squirrel monkeys, marmosets, and owl monkeys may develop hypoglycemia as a primary disorder. Hypoglycemia occurs more commonly in young animals, particularly infants, but can also be seen in older, debilitated animals. Predisposing factors include higher basal metabolic rates, lower percentage of body adipose tissue, limited glycogen reserves, limited gluconeogenic enzymes, and limited ability to utilize ketones or fatty acids (Abee, 1985; Baer, 1994). Separation from the social group, accidental feed deprivation, complications during weaning, prolonged research procedures, fasting, or anorexia due to an underlying disease condition can place monkeys at risk for hypoglycemia. Severity of clinical signs is related to the degree of hypoglycemia. Clinical signs include weakness, lethargy, disorientation, seizures, or unconsciousness (Baer, 1994). Glucose meters developed for human diabetics are useful in diagnosis as they require only one to two drops of blood to make a fast and accurate determination of blood glucose levels. Squirrel monkeys with blood glucose levels less than 40 mg/dl and owl monkeys with glucose levels less than 50 mg/dl are considered hypoglycemic. Conscious hypoglycemic animals can be given oral glucose, sucrose solutions, or fruit juice. Alternatively, warmed 5% dextrose solutions may be administered intravenously at 5 - 8 ml/100 gm body weight (Abee, 1985; Baer, 1994). Use of concentrated dextrose solutions intravenously in New World monkeys is contraindicated (Brady, 2000). Treatment of unconscious squirrel monkeys with 20% dextrose orally by stomach tube at approximately 1 ml/100 gm body weight is effective (Brady, 2000). Hypoglycemia is not likely to be a primary disease condition in larger, Old World monkeys. Treatment with an intravenous bolus of 10 ml of 50% dextrose is recommended for hypoglycemic animals weighing > 5 kg (Rosenberg, 1995). If peripheral circulation and hydration are poor, then 1020% dextrose solution can be administered intravenously. Solutions may be given orally if the monkey has a good swallowing reflex. 3.

Hypothermia and Hyperthermia

Primates housed outdoors are susceptible to hypothermia and hyperthermia associated with extremes in the weather. Particular care should be taken in the introduction of animals to outdoor housing; acclimatization should take place when temperatures are not extreme. Shelter and supplemental heat are required in areas where temperatures drop below freezing. Simi-

larly, shade and shelter are necessary to provide relief during the summer. In areas with more severe summers, water misters may need to be provided to help keep animals cool. Temperature extremes may also occur in indoor housing due to a failure of environmental systems. Hypothermia is frequently encountered in neonatal monkeys that have been separated from or rejected by the dam. These animals are also likely to be hypoglycemic. Moribund infants may appear lifeless when discovered; vigorous toweling, gradual warming, and correction of hypoglycemia, if present, can revitalize them. Treatment for hypothermia is best accomplished by placing the animal in a lukewarm water bath and monitoring body temperature every 4 - 5 min. Alternative methods of warming include use of recirculating water pads, warm-air blankets, warmed fluid enemas, and warmed intravenous fluids, but they do not warm the animal as rapidly. Electric heating pads and heat lamps are contraindicated as extreme temperatures and subsequent burns or overheating of the animal can result. Treatment for hyperthermia is also best accomplished by a cool- to room-temperature water bath with frequent monitoring of body temperature. 4.

Acute Gastric Dilatation

Acute gastric dilatation or bloat in nonhuman primates has occurred following overeating and drinking, following alteration of gastric flora from antimicrobial therapy, or following anesthesia, transportation, or other change in routine (Soave, 1978; Stein et al., 1981). Bloat has been associated with gastric proliferation of Clostridium perfringens (Bennett et al., 1980; Stein et al., 1981), although a definitive causal relationship has not been proven. In one review, Bennett et al. (1980) reported that none of the monkeys involved had had a disruption in schedule nor had they been recently anesthetized or tranquilized. Clostridium perfringens was isolated from gastric contents of 21 of the 24 monkeys and from monkey diet biscuits fed to the animals. Individual monkeys may have a predisposition for developing acute gastric dilatation and experience multiple episodes (Soave, 1978). Early clinical signs include discomfort, as indicated by frequent grimacing and reduction in activity (Soave, 1978). As the disease progresses, monkeys may crouch or lie prone in the cage (Newton et al., 1971; Soave, 1978). Marked abdominal distension, shallow labored respiration, and coma occur terminally. Frequently monkeys are found dead with no clinical signs. Acute gastric dilatation is a medical emergency and must be treated promptly. Soave (1978) reported the following procedures for treatment of bloat in macaques: sedation with ketamine hydrochloride (10-15 mg/kg body weight IM); gastric intubation to relieve intragastric pressure; administration of an agent to control gas formation; oral administration of ampicillin

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REFERENCES

Fig. 44. Swelling of the sinus due to an abscessed canine tooth in an adult male Bolivian squirrel monkey, Saimiri boliviensis boliviensis. Sinus has been olSened to facilitate drainage following extraction of the tooth.

(30,000 I U / k g ) ; intravenous administration of lactated Ringer's solution ( 2 0 - 3 0 m l / k g ) ; and cortisone (1 m g / k g ) administered IV or I M to counter shock. Gas and fluid should be r e m o v e d slowly to m i n i m i z e vascular collapse following release of gastric pressure.

5.

Dental Abscesses

Severe wear and abscessation of teeth are c o m m o n in adult squirrel m o n k e y s and also occur in other species of nonh u m a n primates. A b s c e s s e s of the u p p e r canine teeth present as swellings b e n e a t h the eye and will rupture if left untreated (Fig. 44). A b s c e s s a t i o n of m o l a r teeth in squirrel m o n k e y s usually extends into the infraorbital region of the eye and can lead to e x o p h t h a l m o s and blindness (Abee, 1985). E n u c l e a t i o n of the eye m a y be required.

ACKNOWLEDGMENTS

We a c k n o w l e d g e the following p e o p l e for their assistance and support during the p r e p a r a t i o n of this d o c u m e n t : Ms. L i n d a Karnstadt, Ms. R e g i n a M c C r e e r y , Ms. L a u r a Zapalac, Ms. Susan L a m b e t h , Dr. David Elmore, Dr. Pat Frost, Dr. G e n e Hubbard, Dr. M e l a n i e Ihrig, Dr. D. R i c k Lee, Dr. M i c h e l l e Leland, Dr. Steve Schapiro, and Dr. Jim Weed.

Abee, C. R. (1985). Medical care and management of the squirrel monkey. In "Handbook of Squirrel Monkey Research" (L. A. Rosenblum and C. L. Coe, eds.), pp. 447-487. Plenum Press, New York. Abee, C. R. (2000). Squirrel monkey (Saimiri spp.) research and resources. ILAR J. 41, 2-9. Abegglen, J. J. (1984). "On Socialization in Hamadryas Baboons." Associated Univ. Press, Cranbury, New Jersey. Adams, M. R., Kaplan, J. R., Manuck, S. B., Uberseder, B., and Larkin, K. T. (1988). Persistent sympathetic nervous system arousal associated with tethering in cynomolgus macaques. Lab. Anim. Sci. 38, 279-281. Adams, S. R., Muchmore, E., and Richardson, J. H. (1995). Biosafety. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 377-420. Academic Press, San Diego. Agamanolis, D. P., Chester, E. M., Victor, M., Kark, J., Hines, J. D., and Harris, J. W. (1976). Neuropathy of experimental vitamin B12 deficiency in monkeys. Neurology 26, 906-914. Aikawa, M., Broderson, J. R., Igarashi, I., Jacobs, G., Pappaioanou, M., Collins, W. E., and Campbell, C. C. (1988). "An Atlas of Renal Disease in Aotus Monkeys with Experimental Plasmodial Infection." American Institute of Biological Services, Arlington, Virginia. Aksel, S., Diamond, E. J., Hazelton, J., Wiebe, R. H., and Abee, C. R. (1985). Progesterone as a predictor of cyclicity in Bolivian squirrel monkeys (Saimiri sciureus) during the breeding season. Lab. Anim. Sci. 35, 54-57. Albrecht, P., Lorenz, D., Klutch, M. J., Vickers, J. H., and Ennis, E A. (1980). Fatal measles infection in marmosets: Pathogenesis and prophylaxis. Infect. Immun. 27, 969-978. A1-Doory, Y. (1972). Fungal and bacterial diseases. In "Pathology of Simian Primates" (R. N. T-W. Fiennes, ed.), pp. 206-241. Karger, Basel and New York. A1-Doory, Y., Pinkerton, M. E., and Vice, T. E. (1969). Pulmonary nocardiosis in a vervet monkey. J. Am. Vet. Med. Assoc. 155, 1179-1180. Alexander, S., Yeoman, R., Williams, L., Aksel, S., and Abee, C. R. (1991). Confirmation of ovulation and characterization of LH and progesterone secretory patterns in cycling, iso-sexually housed Bolivian squirrel monkeys (Saimiri boliviensis boliviensis). Am. J. Primatol. 23, 55-60. Alford, P. L., Lee, D. R., Binhazim, A. A., Hubbard, G. B., and Matherne, C. M. (1996). Naturally acquired leprosy in two wild-born chimpanzees. Lab. Anim. Sci. 46, 341-346. Allen, A. M., and Kinard, R. E (1958). Primary cutaneous inoculation of tuberculosis in Macaca mulatta monkeys. Am. J. Pathol. 34, 337-345. Altmann, J., Altmann, S. A., Haufsfater, G., and McCuskey, S. (1977). Life histories of yellow baboons: Physical development, reproductive parameters, and infant mortality. Primates 18, 315-330. Anderson, C. O., and Mason, W. A. (1977). Hormones and social behavior of squirrel monkeys (Saimiri sciureus). 1. Effect of endocrine status of females on behavior within heterosexual pairs. Horm. Behav. 8, 100-106. Anderson, E. T., Lewis, J. P., Passovoy, M., and Trobaugh, F. E., Jr. (1967). Marmosets as laboratory animals. II. The hematology of laboratory kept marmosets. Lab. Anim. Care 17, 30-40. Aquino, R., and Encarnacfon, E (1986). Population structure of Aotus nancymae (Cebidae: Primates) in Peruvian Amazon lowland forest. Am. J. Primatol. 11, 1-7 . . . . . / Aquino, R., Puertas, E, andEncarnacfon, E (1990). Supplemental notes on population parameters of northeastern Peruvian night monkeys, genus Aotus (Cebidae). Am. J. Primatol. 21, 215-221. Ariga, S., Dukelow, W. R., Emley, G. S., and Hutchinson R. R. (1978). Possible errors in identification of squirrel monkeys (Saimiri sciureus) from different South American points of export. J. Med. Primatol. 7, 129-135. Arita, I., Gispan, R., Kalter, S. S., Wah, L. T., Marrenikova, S. S., Netter, R., and

778

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Pagaya, I. (1972). Outbreaks of monkeypox and serological surveys in nonhuman primates. Bull. W. H. O. 46, 625-631. Arita, I., Jezek, Z., Khodakevich, L., and Kalisa-Ruti, J. (1985). Human monkeypox: A newly emerged orthopox zoonosis in the tropical rainforests of Africa. Am. J. Trop. Med. Hyg. 34, 781-789. Asher, D. M., Gibbs, C. J., Jr., Lang, D. J., and Gajdusek, D. C. (1974). Persistent shedding of cytomegalovirus in the urine of healthy rhesus monkeys. Proc. Soc. Exp. Biol. Med. 145, 794-801. Assis, M. E L., and Barro, R. (1987). Karyotype pattern of Saimiri usutus. Int. J. Primatol. 8, 552. Ausman, L. M., Gallina, D. L., Samonds, K. W., and Hegsted, D. M. (1979). Assessment of the efficiency of protein utilization in young squirrel and macaque monkeys. Am. J. Clin. Nutr. 32, 1813-1823. Ausman, L. M., Gallina, D. L., and Nicolosi, R. J. (1985). Nutrition and metabolism of the squirrel monkey. In "Handbook of Squirrel Monkey Research" (L. A. Rosenblum and C. L. Coe, eds.), pp. 349-378. Plenum Press, New York. Baer, J. E (1994). Husbandry and medical management of the owl monkey. In "Aotus: The owl monkey" (J. E Baer, R. E. Weller, and I. Kakoma, eds.), pp. 134-164. Academic Press, San Diego. Baggs, R. B., Hunt, R. D., Garcia, E G., Hajema, E. M., Blake, B. J., and Fraser, C. E. O. (1976). Pseudotuberculosis (Yersinia enterocolitica) in the owl monkey (Aotus trivirgatus). Lab. Anim. Sci. 26, 1079-1083. Bagnall, B. G., and Grunberg, W. (1972). Generalized Trichophyton mentagrophytes ringworm in capuchin monkeys (Cebus nigrivitatus). Br. J. Dermatol. 87, 655-670. Bahnson, R. R., Ballou, B. T., Ernstoff, M. S., Schwentke, E N., and Hakala, T. R. (1988). A technique for catheterization and cystoscopic evaluation of cynomolgus monkey urinary bladders. Lab. Anim. Sci. 38, 731-733. Baker, H. J., Bradford, L. G., and Montes, L. E (1971). Dermatophytosis due to Microsporum canis in a rhesus monkey. J. Am. Vet. Med. Assoc. 159, 16071611. Baldwin, J. D. (1968). The social behavior of adult male squirrel monkeys (Saimiri sciureus) in a seminatural environment. Folia Primatol. 9, 281314. Baldwin, J. D. (1985). The behavior of squirrel monkeys (Saimiri) in natural environments. In "Handbook of Squirrel Monkey Research" (L. A. Rosenblum and C. L. Coe, eds.), pp. 35-53. Plenum Press, New York. Baldwin, J. D., and Baldwin, J. I. (1981). The squirrel monkeys, genus Saimiri. In "Ecology and Behavior of Neotropical Primates" (A. E Coimbra-Filho and R. A. Mittermeier, eds.), Vol. 1, pp. 277-330. Academia Brasileira de Ciencias, Rio de Janiero. Banish, L. D., Sims, R., Bush, M., Sack, D., and Montali, R. J. (1993a). Clearance of Shigellaflexneri carriers in a zoologic collection of primates. J. Am. Vet. Med. Assoc. 203, 133-136. Banish, L. D., Sims, R., Sack, D., Montali, R. J., Phillips, L., Jr., and Bush, M. (1993b). Prevalence of shigellosis and other enteric pathogens in a zoologic collection of primates. J. Am. Vet. Med. Assoc. 203, 126-132. Barhona, H., Melendez, L. V., Hunt, R. D., and Daniel, M. D. (1976). The owl monkey (Aotus trivirgatus) as an animal model for viral diseases and oncologic studies. Lab. Anim. Sci. 26, 1104-1112. Barnard, D., Knapka, J., and Renquist, D. (1988). The apparent reversal of a wasting syndrome by nutritional intervention in Saguinus mystax. Lab. Anim. Sci. 38, 282-288. Baron, E. J., Peterson, L. R., and Finegold, S. M. (1994). In "Diagnostic Microbiology" (J. P. Shanahan, ed.), pp. 596-597. Mosby, St. Louis. Baskerville, A., and Newell, D. G. (1988). Naturally occurring chronic gastritis and C. pylori infection in the rhesus monkey: A potential model for gastritis in man. Gut 29, 465-472. Baskerville, M., Wood, M., and Baskerville, A. (1983). An outbreak of Bordetella bronchiseptica pneumonia in a colony of common marmosets (Callithrixjacchus). Lab. Anim. 17, 350-355. Baskin, G. B. (1987). Disseminated cytomegalovirus infection in immunodeficient rhesus monkeys. Am. J. Pathol. 129, 345-352.

Baskin, G. B. (1991). Disseminated histoplasmosis in an SIV-infected rhesus monkey. J. Med. Primatol. 20, 251-253. Baskin, G. B. (1993). Encephalomyocarditis virus infection, nonhuman primates. In "Monographs on Pathology of Laboratory Animals: Nonhuman Primates" (T. C. Jones, U. Mohr, and R. D. Hunt, eds.), Vol. 2. pp. 104107. Springer-Verlag, Berlin and New York. Baskin, G. B., Wolf, R. H., Worth, C. L., Soike, K., Gibson, S. V., and Bieri, J. G. (1983). Anemia, steatitis, and muscle necrosis in marmosets (Saguinus labiatus). Lab. Anim. Sci. 33, 74-80. Baskin, G. B., Roberts, E. D., Kuebler, D., Martin, L. N., Blauw, B., Heeney, J., and Zurcher, C. (1995). Squamous epithelial proliferative lesions associated with rhesus Epstein-Barr virus in simian immunodeficiency virusinfected rhesus macaques. J. Infect. Dis. 172, 535-538. Bass, E G., and Stark, D. M. (1980). A simplified technique for shortening canine teeth of rhesus monkeys. Lab. Anim. 9, 49-50. Battles, A. H., Greiner, E. C., and Collins, B. R. (1988). Efficacy of ivermectin against natural infection of Strongyloides in squirrel monkeys (Saimiri sciureus). Lab. Anim. Sci. 38, 474-476. Bayne, K., Haines, M., Dexter, S., Woodman, D., and Evans, C. (1995). A retrospective analysis of the wounding incidence of nonhuman primates housed in different social conditions. Lab. Anim. Sci. 24, 40-44. Bellinger, D. A., and Bullock, B. C. (1988). Cutaneous Mycobacterium avium infection in a cynomolgus monkey. Lab. Anim. Sci. 38, 85-86. Bellini, S., Hubbard, G. B., and Kaufman, L. (1991). Spontaneous fatal coccidioidomycosis in a native-born hybrid baboon (Papio cynocephalus anubis/Papio cynocephalus cynocephalus). Lab. Anim. Sci. 41, 509-511. Benjamin, S. A., and Lang, C. M. (1971). Acute pasteurellosis in owl monkeys (Aotus trivirgatus). Lab. Anim. Sci. 21, 258-262. Bennett, B. T., Cuasay, L., Welsh, T. J., Belhun, E Z., and Schofield, L. (1980). Acute gastric dilatation in monkeys: A microbiologic study of gastric contents, blood, and feed. Lab. Anim. Sci. 30, 241-244. Bennett, B. T., Abee, C. R., and Henrickson, R. V. (1998). "Nonhuman Primates in Biomedical Research: Diseases." Academic Press, San Diego. Bercovitch, E B. (1985). Reproductive studies in adult female and adult male olive baboons. Ph.D. dissertation, Univ. of California, Los Angeles. Bergeland, M. E., Barnes, D. M., and Kaplan, W. (1970). Spontaneous histoplasmosis in a squirrel monkey. Primate Surveillance Zoonosis Report 1, January-February 1970, pp. 10-11. Center for Disease Control, Atlanta. Bingham, G. A., and Rabstein, M. M. (1964). A study of the effectiveness of thiabendazole in the rhesus monkey. Lab. Anim. Care 14, 357-365. Birrell, A. M., Hennessy, A., Gillin, A., Horvath, J., and Tiller, D. (1996). Reproductive and neonatal outcomes in captive bred baboons (Papio hamadryas). J. Med. Primatol. 25, 287-293. Bistner, S. I., and Ford, R. B. (1995). "Kirk and Bistner's Handbook of Veterinary Procedures and Emergency Treatment." Saunders, Philadelphia. Blanchard, J. L., Soile, K., and Baskin, G. B. (1987). Encephalomyocarditis virus infection in African green and squirrel monkeys: Comparison of pathologic effects. Lab. Anim. Sci. 37, 635-639. Blampied, N. LeQ., Allchurch, A. E, and Tagg, J. (1983). Diagnosis, treatment, and control of Pterygodermatites (Mesopectines) alphi (Nematodes: Spirurida) in the collection of Callitrichidae and Callimiconidae at the Jersey Wildlife Preservation Trust. J. Jersey Wildl. Trust 20, 90-91. Blevins, W. E., Widmer, W. R., Jakovljevic, S., and Peter, A. T. (1998). A course for those new to diagnostic ultrasound. In "Veterinary Diagnostic Ultrasound." School of Veterinary Medicine, Purdue Univ., West Lafayette, Indiana. Bloomsmith, M. A., Keeling, M. E., and Lambeth, S. P. (1990). Environmental enrichment for singly housed chimpanzees. Lab. Anim. 19, 42-46. Bloomsmith, M. A., Brent, L. Y., and Schapiro, S. J. (1991). Guidelines for developing and managing an environmental enrichment program for nonhuman primates. Lab. Anim. Sci. 41, 372-377. Boinski, S. (1987). Habitat use by squirrel monkeys (Saimiri oerstedii) in Costa Rica. Folia Primatol. 49, 151-167. Boncyk, L. H., McCullough, B., Grotts, D. D., and Kalter, S. S. (1975). Local-

16. NONHUMAN PRIMATES ized nocardiosis due to Nocardia caviae in a baboon (Papio cynocephalus). Lab. Anim. Sci. 25, 88-91. Bonney, R. C., Dixson, A. F., and Fleming, D. (1979). Cyclic changes in the circulating and urinary levels of ovarian steroids in the adult female owl monkey (Aotus trivirgatus). J. Reprod. Fertil. 56, 271-280. Bonney, R. C., Dixson, A. F., and Fleming, D. (1980). Plasma concentrations of oestradiol-17B, oestrone, progesterone, and testosterone during the ovarian cycle of the owl monkey (Aotus trivirgatus). J. Reprod. Fertil. 60, 101-107. Boothe, H. W. (1990). Exploratory laparotomy in small animals. Compend. Contin. Educ. Pract. Vet. 12, 1057-1066. Bourne, G. H. (1969). "The Chimpanzee." S. Karger, New York. Brack, M. (1987). Hepatitis viruses. In "Agents Transmissible from Simians to Man" (M. Brack, ed.), pp. 83-89. Springer-Verlag, New York. Brack, M., and Rietschel, W. (1986). Ivermectin for the control of Strongyloides fulleborni in rhesus monkeys. Kleinter-Prax (Short communication) 31, 29 (in German), cited by Battles et al., 1988. Brady, A. G. (2000). Research techniques for the squirrel monkey (Saimiri sp.). ILAR J. 41, 10-18. Brady, A. G., Pindak, F. F., Abee, C. R., and Gardner, W. A., Jr. (1988). Enteric trichomonads of squirrel monkeys (Saimiri sp.): Natural infestation and treatment. Am. J. Primatol. 14, 65-71. Brammer, D. W., O'Rourke, C. M., Heath, L. A., Chrisp, C. E., Peter, G. K., and Hofing, G. L. (1995). Mycobacterium kansasii infection in squirrel monkeys (Saimiri sciureus sciureus). J. Med. PrimatoL 24, 231-235. Brand, H. M. (1981). Husbandry and breeding of a newly-established colony of cotton-topped tamarins (Saguinus oedipus oedipus). Lab. Anim. 15, 7-11. Brendt, R. E, McDonough, W. E., and Walker, J. S. (1974). Persistence of Diplococcus pneumoniae after influenza virus infection in Macaca mulatta. Infect. Immun. 10, 369-374. Breznock, A. W., Henrickson, R. V., Silverman, S., and Schwartz, L. W. (1975). Coccidioidomycosis in a rhesus monkey. J. Am. Vet. Med. Assoc. 167, 657661. Broderson, J. R., Chapman, W. L., Jr., and Hanson, W. L. (1986). Experimental visceral leishmaniasis in the owl monkey. Vet. Pathol. 23, 293-302. Bronsdon, M. A., and DiGiacomo, R. E (1993). Pasteurella multocida infections in baboons (Papio cynocephalus). Primates 34, 205-209. Bronson, R. T., May, B. D., and Ruebner, B. H. (1972). An outbreak of infection by Yersinia pseudotuberculosis in nonhuman primates. Am. J. Path. 69, 289-303. Brown, B. G., and Swenson R. B. (1995). Surgical management. In "Nonhuman Primates in Biomedical Research: Diseases" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 297-304. Academic Press, San Diego. Brown, P., Gibbs, C. J., Jr., Rogers-Johnson, P., Asher, D. M., Sulima, M. P., Bacote, A., Goldfarb, L. G., and Gajdusek, D. C. (1994). Human spongiform encephalopathy: The National Institutes of Health series of 300 cases of experimentally transmitted disease. Ann. NeuroL 35, 513-529. Bruestle, M. E., Golden, J. G., Hall. A., and Bankneider, A. R. (1981). Naturally occurring Yaba tumor in a baboon (Papio papio). Lab. Anim. Sci. 31, 292294. Buchl, S. J., and Howard, B. (1997). Hematologic and serum biochemical and electrolyte values in clinically normal domestically bred rhesus monkeys (Macaca mulatta) according to age, sex, and gravidity. Lab. Anim. Sci. 35, 528-533. Buchl, S. J., Keeling, M. E., and Voss, W. R. (1997). Establishing specific pathogen free (SPF) nonhuman primate colonies. Inst. Lab. Anim. Res. J. 38, 22-27. Bulte, J. W., Miller, G. E, Vymazal, J., Brooks, R. A., and Frank, J. A. (1997). Hepatic hemosiderosis in non-human primates: Quantifications of liver iron using different field strengths. Magn. Reson. Med. 37, 530536. Butler, T. M., and Hubbard, G. B. (1991). An epizootic of histoplasmosis duboisii (African histoplasmosis) in an American baboon colony. Lab. Anim. Sci. 41, 407-410.

779 Butler, T. M., Gleiser, C. A., Bernal, J. C., and Ajello, L. (1988). Case of disseminated African histoplasmosis in a baboon. J. Med. Primatol. 17, 153161. Cameron, K. R., Stamminger, T., Craxton, M., Bodemer, W., Honess, R. W., and Fleckenstein, B. (1987). The 160,000-Mr protein encoded at the right end of the herpesvirus saimiri genome is homologous to the 140,000-Mr membrane encoded at the left end of the EBV genome. J. Virol. 61, 2063-2070. Campos, M. E, Alvares, J. N., Arruda, M. E, and do Vale, N. B. (1981). Surto epizootico por Klebsiella sp. num nucleo de reproducao de saguis (Callithrixjacchus), em cativiero. Rev. Bioterios. 1, 95-99. Castleman, W. L., Anderson, J., and Holmberg, C. A. (1980). Posterior paralysis and spinal osteomyelitis in a rhesus monkey with coccidioidomycosis. J. Am. Vet. Med. Assoc. 177, 933-934. Centers for Disease Control and Prevention (CDCP) (1987). B virus infection in humansmPensacola, Florida. Morb. Mortal. Wkly. Rep. 36, 289-290, 295-296. Centers for Disease Control and Prevention (CDCP) (1991). Current trends update: Nonhuman primate inportation. Morb. Mortal. Wkly. Rep. 40, 684691. Centers for Disease Control and Prevention (CDCP) (1998). Fatal cercopithecine herpesvirus 1 (B virus) infection following a mucocutaneous exposure and interim recommendations for worker protection. Morb. Mortal. Wkly. Rep. 47, 1073-1076, 1083. Centers for Disease Control and Prevention/National Institutes of Health (CDCP/NIH) (1995). Biosafety in microbiological and biomedical laboratories. In "Laboratory Safety" (D. O. Fleming, J. H. Richardson, J. J. Tulis, and D. Vesley, eds.), HHS Publ. (CDC) 93-8395, pp. 293-346. ASM Press, Washington, D.C. Centers for Disease Control and Prevention/National Institutes of Health (CDCP/NIH) (1999). "Biosafety in Microbiological and Biomedical Laboratories" (J. Y. Richmond and R. W. McKinney, eds.). U.S. Government Printing Office, Washington, D.C. Chalmers, D. T., Murgatroyd, L. B., and Wadsworth, P. E (1983). A survey of the pathology of marmosets (Callithrix jacchus) derived from a marmoset breeding unit. Lab. Anim. 17, 270-279. Champoux, M., Suomi, S. J., and Schneider, M. L. (1994). Temperament differences between captive Indian and Chinese-Indian hybrid rhesus macaque neonates. Lab. Anita. Sci. 44, 351-357. Champoux, M., Higley, J. D., and Suomi, S. J. (1997). Behavioral and physiological characteristics of Indian and Chinese-Indian hybrid rhesus macaque infants. Dev. Psychobiol. 31, 49-63. Chandler, E W., and McClure, H. M. (1982). Adenoviral pancreatitis in rhesus monkeys: Current knowledge. Vet. Pathol. 19, (Suppl. 7), 171-180. Chandler, E W., McClure, H. M., Campbell, W. G., Jr., and Watts, J. C. (1976). Pulmonary pneumocystosis in nonhuman primates. Arch. PathoL Lab. Med. 100, 163-167. Chang, J., Wagner, J. L., and Kornegay, R. W. (1980). Fatal Yersinia pseudotuberculosis infection in captive bushbabies. J. Am. Vet. Med. Assoc. 177, 820-821. Chapman, W. L., Hanson W. L., Jr., and Hendricks, L. D. (1981). Leishmania donovani in the owl monkey (Aotus trivirgatus). Trans. R. Soc. Trop. Med. Hyg. 75, 124-125. Churchill, A. E. (1963). The isolation of parainfluenza 3 virus from fatal cases of pneumonia in Erythrocebus patas monkeys. Br. J. Exp. PathoL 44, 529537. Cicmanec, J. C. (1977). Medical problems encountered in a callitrichid colony. In "The Biology and Conservation of the Callitrichidae" (D. G. Kleinman, ed.), pp. 331-336. Smithsonian Institution Press, Washington, D.C. Cicmanec, J. C., and Campbell, A. K. (1977). Breeding the owl monkey (Aotus trivirgatus) in a laboratory environment. Lab. Anita. Sci. 27, 512-517. Clarke, M. R., and O'Neil, J. A. (1999). Morphometric comparison of Chineseorigin and Indian-derived rhesus monkeys (Macaca mulatta). Am. J. Primatol. 47, 335-346. Clarke, C. J., Watt, N. J., Meredith, A., McIntyre, N., and Burns, S. M. (1994).

780

BRUCE J. BERNACKu SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Respiratory syncytial virus-associated bronchopneumonia in a young chimpanzee. J. Comp. PathoL 110, 207-212. Clarkson, M. J., Thorpe, E., and McCarthy, K. (1967). A virus disease of captive vervet monkeys (Cercopithecus aethiops) caused by a new herpesvirus. Arch. Gersamte Virusforsch. 22, 219-234. Clayton, O., and Kuehl, T. J. (1984). The first successful in vitro fertilization and embryo transfer in a nonhuman primate. Theriogenology 21, 228. Coates, K. W., Galan, H. L., Shull, B. L., and Kuehl, T. J. (1995a). The squirrel monkey: An animal model of pelvic relaxation. Am. J. Obstet. Gynecol. 172, 588-593. Coates, K. W., Gibson, S., Williams, L. E., Brady, A., Abee, C. R., Shull, B. L., and Kuehl, T. J. (1995b). The squirrel monkey as an animal model of pelvic relaxation: An evaluation of a large breeding colony. Am. J. Obstet. Gynecol. 173, 1664-1670. Code of Federal Regulations (CFR) (1999). Title 9, Animals and Animal Products; Chapter 1, Animal and Plant Health Inspection Service, USDA; Subchapter A, Animal Welfare; Part 3, Standards, Subpart D. Office of the Federal Register, Washington, D.C. Coe, C. L., and Rosenblum, L. A. (1978). Annual reproductive strategy of the squirrel monkey (Saimiri sciureus). Folia Primatol. 29, 19-42. Coe, C. L., Smith, E. R., and Levine, S. (1985). The endocrine system of the squirrel monkey. In "Handbook of Squirrel Monkey Research" (L. A. Rosenblum and C. L Coe, eds.), pp. 191-218. Plenum Press, New York. Coelho, A. M., and Carey, K. D. (1990). A social tethering system for nonhuman primates used in laboratory research. Lab. Anim. Sci. 40, 388-394. Coimbra-Filho, A. E, and Mittermeier, R. A. (1977). Tree-gouging, exudateeating, and the short-tusked condition in Callithrix and Cebuella. In "The Biology and Conservation of the Callitrichidae" (D. G. Kleiman, ed.), pp. 105-115. Smithsonian Institution Press, Washington, D.C. Coman, J. L., Fortman, J. D., Alves, M. E. A. E, Bunte, R. M., and Bennett, B. T. (1998). Assessement of a canine crown reduction technique in nonhuman primates. Contemp. Top. Lab. Anim. Sci. 37, 67-72. Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) (2000). Appendices I and II. CITES Secretariat, Geneva, Switzerland. (). Cooper, R. W. (1968). Squirrel monkey taxonomy and supply. In "The Squirrel Monkey" (L. A. Rosenblum and R. W. Cooper, eds.), pp. 1-29. Academic Press, New York. Cooper, J. E., and Needham, J. R. (1976). An outbreak of shigellosis in laboratory marmosets and tamarins. J. Hyg. Camb. 76, 415-424. Corcoran, K. D., and Thoen, C. O. (1991). Application of an enzyme immunoassay for detecting antibodies in sera of Macaca fascicularis naturally exposed to Mycobacterium tuberculosis. J. Med. Primatol. 20, 404408. Crockett, C. M., and Heffernan, K. S. (1998). Grooming-contact cages promote affiliative social interaction in individually housed adult baboons. Am. J. Primatol. 45, 176. Crompton, A. W., Taylor, C. R., and Jagger, J. A. (1978). Evolution of homeothermy in mammals. Nature 272, 333-336. Crow, S. E., and Walshaw, S. O. (1987). "Manual of Clinical Procedures in the Dog and Cat." J. B. Lippincott, Philadelphia. Dalgard, D. W., Hardy, R. J., Pearson, S. L., Pucak, G. J., Quander, R. V., Zack, P. M., Peters, C. J., and Jahrling, P. B. (1992). Combined simian hemorrhagic fever and Ebola virus infection in cynomolgus monkeys. Lab. Anim. Sci. 42, 152-157. Daniel, M. D. (1978). Prevention of fatal herpesvirus infections in owl and marmoset monkeys by vaccination. In "Recent Advances in Primatology" D. J. Chivers and E. H. R. Ford, eds.), Vol. 4, pp. 67-69. Academic Press, London. Da Silva, B. T. E, Sampaio, M. I. C., Schneider, M. E C., and Schneider, H. (1987). Preliminary analysis of genetic distance between squirrel monkeys. Int. J. Primatol. 8, 828. Dawson, G. A. (1977). Composition and stability of social groups of the

tamarin, Saguinus oedipus geoffroyi, in Panama: Ecology and behavioral implications. In "The Biology and Conservation of the Callitrichidae" (D. G. Kleiman, ed.), pp. 23-37. Smithsonian Institution Press, Washington, D.C. Deinhardt, E, Holmes, A. W., Devine, J., and Deinhardt, J. (1967a). Marmosets as laboratory animals. IV. The microbiology of laboratory-kept marmosets. Lab. Anim. Care 17, 48-70. Deinhardt, J. B., Devine, J., Passavoy, M., Pohlman, R., and Deinhardt, E (1967b). Marmosets as laboratory animals. I. Care of marmosets in the laboratory, pathology, and outline of the statistical evaluation of data. Lab. Anim. Care 17, 11-29. Deinhardt, E, Falk, L. G., Wolf, A., Schudel, A., Nonyama, M., Lai, P., Lapin, B., and Yakovleva, L. (1978). Susceptibility of marmosets to Epstein-Barr virus-like baboon herpesviruses. Primate Med. 10, 163-170. DeMarcus, T. A., Tipple, M. A., and Ostrowski, S. R. (1999). U.S. policy for disease control among imported nonhuman primates. J. Infect. Dis. 179 [Suppl], $281-$282. Demontoy, M. C., Berthier, J. L., and Letellier, E (1979). Vitamin A et spondylose ankylosante chez des ouistitis. Erkrank, Zoot. Verhandlungsher Int. Symp. 21st (Berlin), 13, 33-35. DeVore, I., and Hall, K. R. L. (1965). Baboon ecology. In "Primate behavior: Field studies of monkeys and apes" (I. DeVore, ed.). Holt, Rinehart and Winston, New York. Diamond, E. J., Aksel, S., Hazelton, J. M., Jennings, R. A., and Abee, C. R. (1984). Seasonal changes in serum concentrations of estradiol and progesterone in Bolivian squirrel monkeys (Saimiri sciureus) during the breeding season. Am. J. Primatol. 6, 103-113. Diamond, E. J., Aksel, S., Hazelton, J. M., Barnett, S. B., Williams, L. E., and Abee, C. R. (1985). Serum hormone patterns during abortion in the Bolivian squirrel monkey. Lab. Anim. Sci. 35, 619-623. Diamond, E. J., Aksel, S., Hazelton, J. M., Wiebe, R. H., and Abee, C. R. (1987). Serum oestradiol, progesterone, chorionic gonadotrophin, and prolactin concentrations during pregnancy in the Bolivian squirrel monkey (Saimiri boliviensis). J. Reprod. Fertil. 80, 373-381. DiGiacomo, R. E, and Missakian, E. A. (1972). Tetanus in a free-ranging colony of Macaca mulatta: A clinical and epizootiologic study. Lab. Anim. Sci. 22, 378-383. Dillehay, D. L., and Huerkemp, M. J. (1990). Tuberculosis in a tuberculinnegative rhesus monkey (Macaca mulatta) on chemoprophylaxis. J. Zoo Wildl. Med. 21, 480-484. Dixson, A. E (1983). The owl monkey. In "Reproduction in New World Primates" (J. P. Hearn, ed.), pp. 69-113. MTP Press, Hingham, MA. Dixson, A. E, and Fleming, D. (1981). Parental behavior and infant development in owl monkeys (Aotus trivirgatus griseimembra). J. Zool. (London) 194, 25-39. Dixson, A. E, and Gardner, J. S. (1981). Diurnal variations in plasma testosterone in a male nocturnal primate, the owl monkey (Aotus trivirgatus). J. Reprod. Fertil. 62, 83-86. Dixson, A. E, and Lunn, S. E (1987). Post-partum changes in hormones and sexual behavior in captive groups of marmosets (Callithrix jacchus). Physiol. Behav. 41, 577-583. Dixson, A. E, Gardner, J. S., and Bonney, R. C. (1980). Puberty in the male owl monkey (Aotus trivirgatus griseimembra): A study of physical and hormonal development. Int. J. Primatol. 1, 129-139. Doenges, J. L. (1939). Spirochetes in the gastric glands of Macaca rhesus and of man without related disease. Arch. Pathol. 27, 469-477. Douglas, J. D., Tanner, K. N., Prine, J. R., Van Riper, D. C., and Derwelis, S. K. (1967). Molluscum contagiosum in chimpanzees. J. Am. Vet. Med. Assoc. 151, 901 - 904. Downie, A. W. (1974). Serologic evidence of infection with Tana and Yaba pox viruses among several species of monkeys. J. Hyg. 72, 245-250. Downie, A. W., and Espana, C. (1972). Comparison of Tanapox and Yaba-like viruses causing epidemic disease in monkeys. J. Hyg. 70, 23-32.

16. NONHUMAN PRIMATES Doyle, L., Young, C. L., Jang, S. S., and Hillier, S. L. (1991). Normal vaginal aerobic and anaerobic bacterial flora of the rhesus macaque (Macaca mulatta). J. Med. Primatol. 20, 409-413. Dubois, A., Tarnawski, A., Newell, D. G., Fiala, N., Dabros, W., Stachura, J., Krivan, H., and Heman-Ackah, L. M. (1991). Gastric injury and invasion of parietal cells by spiral bacteria in rhesus monkeys: Are gastritis and hyperchlorhydria infectious diseases? Gastroenterology. 100, 884-891. Dubois, A., Fiala, N., Heman-Ackah, L. M., Drazek, E. S., Tarnawski, A., Fishbein, W. N., Perez-Perez, G. I., and Blaser, M. J. (1994). Natural gastric infection with Helicobacter pylori in monkeys: A model for spiral bacteria infection in humans. Gastroenterology 106, 1405-1417. DuMond, E V. (1968). The squirrel monkey in a seminatural environment. In "The Squirrel Monkey" (L. A. Rosenblum and R. W. Cooper, eds.), pp. 87145. Academic Press, New York. DuMond, E V., and Hutchinson, T. C. (1967). Squirrel monkey reproduction: The "fatted" male phenomenon and seasonal spermatogenesis. Science 158, 1067-1070. Duncan, M., Nichols, D. K., Montali, R. J., and Thomas, L. A. (1994). An epizootic of Salmonella enteritidis at the National Zoological Park. Proc. Am. Assoc. Zoo Vet., 256-248. Dyke, B. (1995). Animal identification and record keeping: Current practice and use. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 249-253. Academic Press, San Diego. Dysko, R. C., and Hoskins, D. E. (1995). Medical management: Collection of biological samples and therapy administration. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 270-283. Academic Press, San Diego. Eisele, P. H., Morgan, J. P., Line, A. S., and Anderson, J. H. (1992). Skeletal lesions and anemia associated with ascorbic acid deficiency in juvenile rhesus macaques. Lab. Anim. Sci. 42, 245-249. Elliot, M. W., Sehgal, P. K., and Chalifoux, L. V. (1976). Management and breeding of Aotus trivirgatus. Lab. Anim. Sci. 26, 1037-1040. Else, J. G., Tarara, R., Suleman, M. A., and Eley, R. M. (1986). Enclosure design and reproductive success in baboons used for reproductive research in Kenya. Lab. Anim. Sci. 36, 168-172. Epple, G. (1970a). Maintenance, breeding, and development of marmoset monkeys (Callitrichidae) in captivity. Folia Primatol. 12, 56-76. Epple, G. (t 970b). Quantitative studies on scent marking in the marmoset (Callithrix jacchus). Folia Primatol. 13, 48-62. Epple, G. (1973). The role of pheromones in the social communication of marmoset monkeys (Callitrichidae). J. Reprod. Fertil. 19 (Suppl.), 447-454. Epple, G. (1975). Parental behavior in Saguinusfuscicollis spp. Folia Primatol. 24, 221-238. Epple, G., and Katz, Y. (1980). Social influences on first reproductive success and related behaviors in the saddle-back tamarin (Saguinus fuscicolli Callitrichidae). Int. J. Primatol. 1, 171. Erkert, H. G. (1976). Beleuchtungsabhanglges aktivitalsoptimum bei nachtaffen (Aotus trivirgatus) (Light-induced activity optimum in night monkeys). Folia Primatol. 25, 186 - 192. Erwin, J. M., Blood, B. D., Southwick, C. H., and Wolfe, T. L. (1995). Primate conservation. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 113-127. Academic Press, San Diego. Espana, C. (1973). Herpes simiae infection in Macaca radiata. Am. J. Phys. Anthropol. 38, 447-454. Fear, E A., Pinkerton, M. E., Cline, J. A., Kriewaldt, E, and Kalter, S. S. (1968). A leptospirosis outbreak in a baboon (Papio sp.) colony. Lab. Anim. Care 18, 22-28. Feic.htinger, H., Li, S., Kaaya, E.~ Putkonen, P., Grunewald, K., Weyrer, K., Bottiger, D., Ernberg, I., Linde, A., Biberfeld, G., and Biberfeld, P. (1992). A monkey model of Epstein-Barr virus-associated lymphomagenesis

781 in human acquired immunodeficiency syndrome. J. Exp. Med. 176, 281286. Felsburg, E J., Heberling, R. L., Brack, M., and Kalter, S. S. (1973). Experimental genital herpes infection of the marmoset. J. Med. Primatol. 2, 5 0 60. Felsenfeld, A. D., and Schmidt, N. J. (1975). Immunological relationship between delta herpesvirus of patas monkeys and varicella-zoster virus of humans. Infect. lmmun. 12, 261-266. Fiennes, R. N. (1972). Rabies. In "Pathology of Simian Primates" (R. N. T.-W. Fiennes, ed.), pp. 646-662. Karger, Basel and New York. Fleagle, J. G. (1999). "Primate Adaptation and Evolution." 2nd ed., Academic Press, San Diego. Flecknell, E A., Parry, R., Needham, J. R., Ridley, R. M., Baker, H. E, and Bowes, E (1983). Respiratory disease associated with parainfluenza type I (Sendai) virus in a colony of marmosets (Callithrix jacchus). Lab. Anita. 17, 111-113. Flynn, R. J. (1973). "Parasites of Laboratory Animals." Iowa State Univ. Press, Ames. Ford, S. (1994). Taxonomy and distribution of the owl monkey. In "Aotus: The Owl Monkey" (J. E Baer, R. E. Weller, and I. Kakoma, eds.), pp. 1-60. Academic Press, San Diego. Fortman, J. D., Herring, J. M., Miller, J. B., Hess, D. L., Verhage, H. G., and Fazleabas, A. T. (1993). Chorionic gonadotropin, estradiol, and progesterone levels in baboons (Papio anubis) during early pregnancy and spontaneous abortion. Biol. Reprod. 49, 737-742. Fourie, E B., and Odendaal, M. W. (1983). Mycobacterium tuberculosis in a closed colony of baboons (Papio ursinus). Lab. Anita. 17, 125-128. Fox, J. G. (1975). Transmissible drug resistance in Shigella and Salmonella isolated from pet monkeys and their owners. J. Med. Primatol. 4, 165171. Fox, J. G., and Rohovsky, M. W. (1975). Meningitis caused by Klebsiella spp. in two rhesus monkeys. J. Am. Vet. Med. Assoc. 167, 634-636. Fox, J. G., and Wikse, S. E. (1971). Bacterial meningoencephalitis in rhesus monkeys: Clinical and pathological features. Lab. Anita. Sci. 21,558-563. Fox, J. G., Campbell, L. H., Snyder, S. B., Reed, C., and Soave, O. A. (1974). Tuberculous spondylitis and Pott's paraplegia in a rhesus monkey. Lab. Anita. Sci. 24, 335-339. Fox, J. G., Handt, L., Sheppard, B. J., Xu, S., Dewhirst, E E., Klein, H. (2001a). Helicobacter cinaedi isolated from the colon, liver, and mesenteric lymph node of a rhesus monkey with chronic colitis and hepatitis. J. Clin. Microbiol. 50, 421-429. Fox, J. G., Handt, L., Xu, S., Shen, Z., Dewhirst, E E., Dangler, C. A., Lodge, K., Motzel, S., Klein, H. (2001b). Novel Helicobacter spp. isolated from colonic tissue of rhesus monkeys with chronic idiopathic colitis. J. Med. Microbiol., in press. Fraser, C. E. O., Chalifoux, L., Sehgal, E, Hunt, R. D., and King, N. W. (1978). A paramyxovirus causing fatal gastroenteritis in marmoset monkeys. In "Primates in Medicine" (E. I. Goldsmith and J. Moor-Janowski, eds.), pp. 261-270. Karger, Basel. French, J. A. (1983). Lactation and fertility: An examination of nursing and interbirth intervals in cotton-top tamarins (Saguinus oedipus). Folia PrimatoL 40, 276-282. Fritz, E, and Fritz, J. (1979). Resocialization of chimpanzees: Ten years of experience at the Primate Foundation of Arizona. J. Med. Primatol. 8, 202221. Fulz, E N., Gordon, R. E, Anderson, D. C., and McClure, H. M. (1990). Prevalence of natural infection with SIVsmm and STLV-1 in a breeding colony of sooty mangabey monkeys. AIDS 4, 619-625. Gacad, M. A., Deseran, M. W., and Adams, J. S. (1992). Influence of ultraviolet radiation on vitamin D 3 metabolism in vitamin Da-resistant New World primates. Am. J. Primatol. 28, 263-270. Galland, G. G. (2000). Role of the squirrel monkey in parasitic disease research. ILAR J. 41, 37-43.

782

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Galton, M. M., Mitchell, R. B., Clark, G., and Riessen, A. H. (1948). Enteric infections in chimpanzees and spider monkeys with special reference to a sulfadiazine resistant Shigella. J. Infect. Dis. 83, 147-154. Garber, P. A. (1980). Locomotor behavior and feeding ecology of the Panamanian tamarin (Saguinus oedipus geoffroyi) (Callitrichidae: Primates). Int. J. Primatol. 1, 185-201. Garber, P. A. (1993). Feeding, ecology, and behaviour of the genus Saguinus. In "Marmosets and Tamarins: Systematics, Behaviour, and Ecology" (A. B. Rylands, ed.), pp. 273-295. Oxford Univ. Press, Oxford. Gauthier, C. A. (1999). Reproductive parameters and paracallosal skin color changes in captive female guinea baboons, Papio papio. Am. J. Primatol. 47, 67-74. Genain, C. P., and Hauser, S. L. (1996). Allergic encephalomyelitis in common marmosets: Pathogenesis of a multiple sclerosis-like lesion. Methods 10, 420-434. Genain, C. P., and Hauser, S. L. (1997). Creation of a model of multiple sclerosis in Callithrixjacchus marmosets. J. Mol. Med. 75, 187-197. Gengozian, N., and Batson, J. S. (1975). Single-born marmosets without hemopoietic chimerism: Naturally occurring and induced. J. Med. Primatol. 4, 252-261. Gengozian, N., Batson, J. S., and Smith, T. A. (1977). Breeding of tamarins (Saguinus spp.) in the laboratory. In "The Biology and Conservation of the Callitrichidae" (D. G. Kleiman, ed.), pp. 207-213. Smithsonian Institution Press, Washington, D.C. George, J. W., and Lerche, N. W. (1990). Electrolyte abnormalities associated with diarrhea in rhesus monkeys: 100 cases (1986-1987). J. Am. Vet. Med. Assoc. 196, 1654-1658. George, R., and Lam, S. K. (1997). Dengue virus infection in the Malayasian experience. Ann. Acad. Med. Singapore 26, 825-819. Georgi, J. R. (1980). "Parasitology for Veterinarians." Saunders, Philadelphia. Geretschlager, E., Russ, H., Mihatsch, W., and Przuntek, H. (1987). Suboccipital puncture for cerebrospinal fluid in the common marmoset (Callithrix jacchus). Lab. Anita. 24, 91-94. Gibb, W. R., Lees, A. J., Wells, E R., Barnard, R. O., Jenner, P., and Marsden, C. D. (1987). Pathology of MPTP in the marmoset. Adv. Neurol. 45, 187190. Gilbert, S. G., Reuhl, K. R., Wong, J. H., and Rice, D. C. (1987). Fatal pneumococcal meningitis in a colony-born monkey (Macaca fasicularis). J. Med. Primatol. 16, 333-338. Giles, R. C., Jr., Hildebrandt, P. K., and Tate, C. (1974). Klebsiella air sacculitis in the owl monkey (Aotus trivirgatus). Lab. Anim. Sci. 24, 610-616. Glad, W. R., and Nesland, J. M. (1995). Focal epithelial hyperplasia of the oral mucosa in two chimpanzees (Pan troglodytes). Am. J. Primatol. 10, 83- 89. Goldizen, A. W. (1987) Tamarins and marmosets: Communal care of offspring. In "Primate Societies" (B. B. Smuts, D. L. Cheney, R. M. Seyfarth, R. W. Wrangham, and T. T. Struhsaker, eds.), pp. 34-43. Univ. of Chicago Press, Chicago. Gonzalez, C. A., Hennessy, M. B., and Levine, S. (1981). Subspecies differences in hormonal and behavioral responses after group formation in squirrel monkeys. Am. J. Primatol. 1, 439-452. Good, R. C., and May, B. D. (1971). Respiratory pathogens in monkeys. Infect. Immun. 3, 87-99. Good, R. C., May, B. D., and Kawatomari, T. (1969). Enteric pathogens in monkeys. J. Bacteriol. 97, 1048-1055. Goodall, Jane (1986). "The Chimpanzees of Gombe." Belknap Press of Harvard Univ. Press, Cambridge, Massachusetts. Goodman, R. L., Hotchkiss, J., Karsch, E J., and Knobil, E. (1974). Diurnal variations in serum testosterone concentrations in the adult male rhesus monkey. Biol. Repro. 11, 624-630. Goodwin, W. J., and Coelho, A. M., Jr. (1982). Development of a large scale baboon breeding program. Lab. Anim. Sci. 32, 672-676. Goodwin, W. J., Haines, R. J., and Bernal, J. C. (1987). Tetanus in baboons of a corral breeding colony. Lab. Anim. Sci. 37, 231-232.

Goodwin, B. T., Jerome, C. E, and Bullock, B. C. (1988). Unusual lesion morphology and skin test reaction for Mycobacterium avium complex in macaques. Lab. Anim. Sci. 38, 20-24. Gough, A. W., Barsoum, N. J., Gracon, S. I., Mitchell, L., and Sturgess, J. M. (1982). Poxvirus infection in a colony of common marmosets (Callithrix jacchus). Lab. Anim. Sci. 32, 87-90. Gould, K. G., and Martin, D. E. (1986). Artificial insemination of nonhuman primates. In "Primates: The Road to Self-Sustaining Populations" (K. Benirschke, ed.), pp. 425-443. Springer-Verlag, New York. Gozalo, A., and Montoya, E. (1991). Klebsiella pneumoniae infection in a New World nonhuman primate center. Lab. Primate News. 30, 13-15. Gozalo, A., and Montoya, E. (1992). Mortality causes in the moustached tamarin (Saguinus mystax) in captivity. J. Med. Primatol. 21, 35-38. Gozalo, A., Block, K., Montoya, E., Moro, J., and Escamilla, J. (1991). A survey for Campylobacter in feral and captive tamarins. Primatol. Today 13, 675-676. Graczyk, T. K., Cranfield, M. R., Kempske, S. E., and Eckhaus, M. A. (1995). Fulminant Streptococcus pneumoniae meningitis in a lion-tailed macaque (Macaca silenus) without detected signs. J. Wildl. Dis. 31, 75-77. Gravell, M., Palmer, A. E., Rodriguez, M., London, W. T., and Hamilton, R. S. (1980). Method to detect asymptomatic carriers of simian hemorrhagic fever virus. Lab. Anim. Sci. 30, 988- 991. Green, S. W., Malkovska, I., O'Sullivan, M. G., and Brown, K. E. (2000). Rhesus and pig-tailed macaque parvoviruses: Identification of two new members of the erythrovirus genus in monkeys. Virology 269, 105-112. Greenstein, E. T., Doty, R. W., and Lowy, K. (1965). An outbreak of a fulminating infectious disease in the squirrel monkey (Saimiri sciureus). Lab. Anim. Care 15, 74-80. Hackerman, N. (1988). "Use of Laboratory Animals in Biomedical and Behavioral Research." Committee on the Use of Laboratory Animals in Biomedical and Behavioral Research, Institute of Medicine, National Academy of Sciences, Washington, D.C. Hainsey, B. M., Hubbard, G. B., Leland, M. M., and Brasky, K. M. (1993). Clinical parameters of the normal baboons (Papio species) and chimpanzees (Pan troglodytes). Lab. Anita. Sci. 43, 236-243. Hall, R. D., and Hodgen, G. D. (1979). Pregnancy diagnosis in owl monkeys (Aotus trivirgatus): Evaluation of the haemagglutination inhibition test for urinary human chorionic gonadotropin. Lab. Anim. Sci. 29, 345-348. Hall, W. C., Kovatch, R. M., Hermann, P. H., and Fox, J. G. (1971). Pathology of measles in rhesus monkey. Vet. Pathol. 8, 307-319. Hampton, J. K., Jr. (1964). Laboratory requirements and observations of Oedipomidas oedipus. Am. J. Physical Anthropol. 22, 239-243. Harding, L. and Liberman, D. E (1995). Epidemiology of laboratory-associated infections. In "Laboratory Safety: Principles and Practices" (D. O. Fleming, J. H. Richardson, J. J. Tulis, and D. Vesley, eds.), pp. 7-15. ASM Press, Washington, D.C. Harding, R. D., Hulme, M. J., Lunn, S. E, Henderson, C., and Aitken, R. J. (1982). Plasma progesterone levels throughout the ovarian cycle of the common marmoset (Callithrix jacchus). J. Med. Primatol. 11, 43-51. Hartwell, G., and Dalgard, D. (1979). Clinical Anatrichosoma cutaneum dermatitis in nonhuman primates. Annu. Proc. Am. Assoc. Zoo Vet. 83a86a. Harvey, P. H., Martin, R. D., and Clutton-brock, T. H. (1979). Life histories in comparative perspective. In "Primate Societies" (B. B. Smuts, D. L. Cheney, R. M. Seyfarth, R. W. Wrangham, and T. T. Struhsaker, eds.), pp. 181-196. Univ. of Chicago Press, Chicago. Hauck, H., and Klehr, N. (1977). Meerkatzenfavus als ursache fur die pilzinfektion eines menschen. Z. Allerg. Med. 53, 331-332. Hawkey, C. M., Hart, M. G., and Jones, D. M. (1982). Clinical hematology of the common marmoset Callithrix jacchus. Am. J. Primatol. 3, 179199. Hearn, J. P. (1977). The endocrinology of reproduction in the common marmoset Callithrix jacchus. In "The Biology and Conservation of the Cal-

16. NONHUMAN PRIMATES litrichidae" (D. G. Kleiman, ed.), pp. 163-171. Smithsonian Institution Press, Washington, D. C. Hearn, J. P. (1993). The common marmoset (Callithrix jacchus). In "Reproduction in New World Primates" (J. P. Hearn, ed.), pp.183-215. MTP Press, Hingham, MA. Hearn, J. P., and Burden, E J. (1979). "Collaborative" rearing of marmoset triplets. Lab. Anim. 13, 131-133. Hearn, J. P., and Lunn, S. E (1975). The reproductive biology of the marmoset monkey, Callithrixjacchus. In "Breeding Simians for Developmental Biology" (E T. Perkins and P. N. O'Donoghue, eds.), Lab. Anim. Handbook No. 6, pp. 191-202. Laboratory Animals Ltd., London. Hendrickx, A. G. (1971). "Embryology of the Baboon." Univ. of Chicago Press, London. Hendrickx, A. G., and Dukelow, W. R. (1995). Reproductive biology. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 147-191. Academic Press, San Diego. Hendrickx, A. G., and Kraemer, D. C. (1969). Observation on the menstrual cycles, optimal mating time, and pre-implantation of embryos of the baboon, Papio anubis and Papio cynocephalus. J. Reprod. Fertil. 6, 119128. Herman, P. H., and Fox, J. G. (1971). Panophthalmitis associated with diplococcic septicemia in a rhesus monkey. J. Am. Vet. Med. Assoc. 159, 560562. Hermindez-Camacho, J., and Cooper, R. W. (1976). The nonhuman primates of Colombia. In "Neotropical Primates: Field Studies and Conservation" (R. W. Thorington Jr. and P. G. Heltne, eds.), pp. 35-69. National Academy of Sciences, Washington D.C. Herring, J. M., Fortman, J. D., Anderson, R. J., and Bennett, B. T. (1991). Ultrasonic determination of fetal parameters in baboons. Lab. Anim. Sci. 41, 602-605. Hershkovitz, P. (1983). Two new species of night monkeys, genus Aotus (Cebidae, Plattyrrhini): A preliminary report on Aotus taxonomy. Am. J. Primatol. 4, 209-243. Hershkovitz, P. (1984). Taxonomy of squirrel monkeys genus Saimiri (Cebidae, Platyrrhini): A preliminary report with description of a hitherto unnamed form. Am. J. Primatol. 7, 155-210. Hetherington, C. M. (1978). Circadian oscillations of body temperature in the marmoset, Callithrix jacchus. Lab. Anim. 12, 107-108. Hill, S., Motzel, S., Klein, H., and Kornegay, R. (2000). Histopathologic assessment of experimental tuberculosis in three species of nonhuman primates. Contemp. Top. Lab. Anim. Sci. 39, 55. Hodgen, G. D., and Neimann, W. H. (1975). Application of the subhuman primate pregnancy test kit to pregnancy diagnosis in baboons. Lab. Anim. Sci. 25, 757-759. Hodgen, G. D., Wolfe, L. G., Ogden, J. D., Adams, M. R., Descalzi, C. C., and Hildebrand, D. E (1976). Diagnosis of pregnancy in marmosets: Hemagglutinin inhibition test and radioimmunoassay for urinary chorionic gonadotropin. Lab. Anim. Sci. 26, 224-229. Hodges, J. K., Czekala, N. M., and Lasley, B. L. (1979). Estrogen and luteinizing hormone secretion in diverse primate species from simplified urinary analysis. J. Med. Primatol. 8, 349-364. Holmberg, C. A., Henrickson, R., Lenninger, R., Anderson, J., Hayashi, L., and Ellingsworth, L. (1985). Immunologic abnormality in a group of Macaca arctoides with high mortality due to atypical mycobacterial and other disease processes. Am. J. Vet. Res. 46, 1192-1196. Holmes, A. W., Passovoy, M., and Capps, R. B. (1967). Marmosets as laboratory animals. III. Blood chemistry of laboratory-kept marmosets with particular attention to liver function and structure. Lab. Anim. Care 17, 41-47. Holmes, G. P., Hilliard, J. K., Klontz, K. C., Rupert, A. H., Schindler, C. M., Parrish, E., Griffin, G., Ward, G. S., Bernstein, N. D., Bean, T. W., Ball, M. R., Brady, J. A., and Wilder, M. H. (1990). B virus (Herpesvirus

783 simiae) infection in humans: Epidemiologic investigation of a cluster. Ann. Intern. Med. 112, 833-839. Holmes, G. P., Chapman, L. W., Stewart, H. A., Straus, J. A., Straus, S. E., Hilliard, J. K., and Davenport, D. S. (1995). Guidelines for the prevention and treatment of B virus infections in exposed persons. Clin. Infect. Dis. 20, 421-439. Horvath, C. J., Simon, M. A., Bergsagel, D. J., Pauley, D. R., King, N. W., Garcea, R. L., and Ringler, D. J. (1992). Simian virus 40-induced disease in rhesus monkeys with simian acquired immunodeficiency syndrome. Am. J. Pathol. 140, 1431-1440. Hubbard, G. B., Lee, D. R., Eichberg, J. W., Gormus, B. J., Xu, K., and Meyers, W. M. (1991). Spontaneous leprosy in a chimpanzee (Pan troglodytes). Vet. Pathol. 28, 546-548. Hubbard, G. B., Soike, K. E, Butler, T. M., Carey, K. D., Davis, H., Butcher, W. I., and Gauntt, C. J. (1992). An encephalomyocarditis virus epizootic in a baboon colony. Lab. Anim. Sci. 42, 233-239. Hugh-Jones, M. E., Hubbert, W. T., and Hagstad, H. V. (1995). "Zoonoses: Recognition, Control, and Prevention." Iowa State Univ. Press, Ames. Hunt, R. D., and Melendez, L. V. (1966). Spontaneous herpes-T infection in the owl monkey (Aotus trivirgatus). Vet. Pathol. 3, 1-26. Hunt, R. D., Chalifoux, L. V., King, N. M., and Trum, B. E (1975). Arrested spermatogenesis in owl monkeys (Aotus trivirgatus). J. Med. Primatol. 4, 399-400. Hunter, A. J., Martin, R. D., Dixon, A. E, and Rudder, B. C. (1979). Gestation and inter-birth intervals in the owl monkey (Aotus trivirgatus griseimembra). Folia PrimatoL 31, 165-175. Ihrig, M., Tassinary, L. G., Bernacky, B., and Keeling, M. E. (2001). Hematologic and serum reference intervals for the chimpanzee (Pan troglodytes) characterized by age and sex. Comp. Med. 51, 30-37. Ingram, J. (1977). Interactions between parents and infants and the development of independence in the common marmoset (Callithrix jacchus). Anim. Behav. 25, 811-827. Institute of Laboratory Animal Resources (ILAR) (1996). "Guide for the Care and Use of Laboratory Animals." National Academy Press, Washington, D.C. Institute for Laboratory Animal Research (ILAR) (1997). "Chimpanzees in Research, Strategies for Their Ethical Care, Management, and Use." A Report of the Institute for Laboratory Animal Research Committee on Long-Term Care of Chimpanzees. National Academy Press, Washington, D.C. Institute for Laboratory Animal Research (ILAR) (1998). "The Psychological Well-Being of Nonhuman Primates." A Report of the Institute for Laboratory Animal Research Committee on Well-Being of Nonhuman Primates, Publ. 98-40103. National Academy Press, Washington, D.C. Izawa, K. (1978). A field study of the ecology and behavior of the black-mantle tamarin (Saguinus nigricollis). Primates 19, 241-274. Jacobs, G. H., Tootell, R. B., and Blakeslee, B. (1979). Visual capacities of the owl monkey Aotus trivirgatus: Temporal contrast sensitivity. Folia Primatol. 32, 193-199. Jahrling, P. B., Geisbert, T. W., Jaax, N. K., Hanes, M. A., Ksiazek, T. G., and Peters, C. J. (1996). Experimental infection of cynomolgus macaques with Ebola-Reston filoviruses from the 1989-1900 U.S. epizootic. Arch. Virol. Suppl. 11, 115-134. Jaquish, C. E., Tardif, S. D., Toal, R. L., and Carson, R. L. (1996) Patterns of prenatal survival in the common marmoset (Callithrix jacchus). J. Med. Primatol. 25, 57-63. James, E. E., Alford, P. L., Reingold, A. L., Russell, H., Keeling, M. E., and Broome, C. V. (1984). Predisposition to invasive pneumococcal illness following parainfluenza 3 virus infection in chimpanzees. J. Am. Vet. Med. Assoc. 185, 1351-1353. Jezek, Z., Arita, I., Szczeniowski, M., Paluku, K. M., Ruti, K., and Nakanao, J. H. (1985). Human tanapox in Zaire: Clinical and epidemiological observations on cases confirmed by laboratory studies. Bull. W. H. O. 63, 10271035.

784

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Johnsen, D. O. (1995). History. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 1-14. Academic Press, San Diego. Johnson-Delaney, C. A. (1994). Primates. Vet. Clin. North Am. Small Anim. Pract. 24, 121-156. Jollie, W. P., Haar, J. L., and Craig, S. S. (1975). Fine structural observations on hemopoiesis in the chorioallantoic placenta of the marmoset. Am. J. Anat. 144, 9-38. Jones, A. M., and Wyand, D. S. (1966). Pulmonary nocardiosis in the rhesus monkey: Importance of differentiation from tuberculosis. Pathol. Vet. 3, 588-600. Jones, T. C., Thorington, R. W., Hu, M. M., Adams, E., and Cooper, R. W. (1973). Karyotypes of squirrel monkeys (Saimiri sciureus). Am. J. Phys. AnthropoL 38, 269-277. Jones, E. E., Alford, P. L., Reingold, A. L., Russell, H., Keeling, M. E., and Broome, C. V. (1984). Predisposition to invasive pneumococcal illness following parainfluenza type 3 virus infection in chimpanzees. J. Am. Vet. Med. Assoc. 185, 1351-1353. Kalter, S. S., and Heberling, R. L. (1990). Viral battery testing in nonhuman primate colony management. Lab. Anim. Sci. 40, 21-23. Kaplan, W., George, L. K., Hendricks, S. L., and Leeper, R. A. (1957). Isolation of Microsporum distortum from animals in the United States. J. Invest. Dermatol. 28, 449-453. Kark, J. A., Victor, M., Hines, J. D., and Harris, J. W. (1974). Nutritional vitamin B~2 deficiency in rhesus monkeys. Am. J. Clin. Nutr. 27, 470-478. Kaufmann, A. E, and Quist, K. D. (1969a). Pneumococcal meningitis and peritonitis in rhesus monkeys. J. Am. Vet. Med. Assoc. 155, 1158-1162. Kaufmann, A. E, and Quist, K. D. (1969b). Thrush in a rhesus monkey: Report of a case. Lab. Anim. Care 19, 526-527. Keeling, M. E., and McClure, H. M. (1974). Pneumococcal meningitis and fatal enterobiasis in a chimpanzee. Lab. Anim. Sci. 24, 92-95. Keeling, M. E., and Roberts, J. R. (1972). Breeding and reproduction of chimpanzees. In "The Chimpanzee" (G. H. Bourne, ed.), Vol. 5, pp. 127-152. Karger, Basel and Univ. Park Press, Baltimore. Keeling, M. E., and Wolf, R. H. (1975). Medical management of the rhesus monkey. In "The Rhesus Monkey" (G. H. Bourne, ed.), Vol. 2, pp. 11-96. Academic Press, New York. Kennedy, E M., Astbury, J., Needham, J. R., and Cheasty, T. (1993). Shigellosis due to occupational contact with nonhuman primates. Epidemiol. Infect. 110, 247-251. Kennett, M. (1996). Training shorts. ASLAP News. 29, 20-21. Kerber, W. T., Reese, W. H., and Van Natta, J. (1968). Balanitis, paronychia, and onychia in a rhesus monkey. Lab. Anim. Care 18, 506-507. Kessler, M. J., and Brown, R. J. (1979). Clinical description of tetanus in squirrel monkeys (Saimiri sciureus). Lab. Anim. Sci. 29, 240-242. Kessler, M. J., and Brown, R. J. (1981). Mycetomas in a squirrel monkey (Saimiri sciureus). J. Zoo Anim. Med. 12, 91-93. Kessler, M. J., Berard, J. D., and Rawlins, R. G. (1988). Effect of tetanus toxoid inoculation on mortality in the Cayo Santiago macaque population. Am. J. Primatol. 15, 93-101. Kestler, H., Kodama, T., Ringler, D., Marthas, M., Pederson, N., Lackner, A., Regier, D., Sehgal, E, Daniel, M., King, N., and Destrosiers, R. (1990). Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus. Science 248, 1109-1112. Kholkute, S. D. (1984). Diurnal and annual variations in plasma and androgen levels in the adult male marmoset (Callithrix jacchus). Int. J. Androl. 7, 431-438. King, G. (1975). Feeding and nutrition of the callitrichidae at the Jersey Zoological Park. 12th Annual Report, Jersey Wildlife Preservation Trust, pp. 81-90. Jersey, U.K. King, N. (1975). Morphological observations on the owl monkey testicles. Primate Rec. 6, 13. King, N. W., Hunt, R. D., Daniel, M. D., and Melendez, L. V. (1967). Overt her-

pes-T infection in squirrel monkeys (Saimiri sciureus). Lab. Anim. Care 17, 413-423. Kinzey, W. G. (1997). Saguinus. In "New World Primates: Ecology, Evolution, and Behavior" (W. G. Kinzey, ed.), pp. 289-296. Aldine de Gruyter, New York. Klein, H. J., and Murray, K. A. (1995). Medical management: Restraint. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 286-295. Academic Press, San Diego. Klein, E. C., Gebhart, C. J., and Duhamel, G. E. (1999). Fatal outbreaks of proliferative enteritis caused by Lawsonia intracellularis in young colonyraised rhesus macaques. J. Med. PrimatoL 28, 11-18. Klokke, A. H., and deVries, G. A. (1963). Tinea cap#is in a chimpanzee caused by Microsporum canis Bodin 1902 resembling M. obesum Conant 1937. Sabaraudia 2, 268-270. Klosterman, L. L., Murai, J. T., and Siiteri, P. K. (1986). Cortisol levels, binding, and properties of corticosteroid-binding globulin in the serum of primates. Endocrinology 118, 424-434. Klumpp, S. A., Weaver, D. S., Jerome, C. P., and Jokinen, M. P. (1986). Salmonella osteomyelitis in a rhesus monkey. Vet. Pathol. 23, 190-197. Knapp, L. A., Lehmann, E., Piekarczyk, M. S., Urvater, J. A., and Watkins, D. I. (1997). A high frequency of Manu-A *01 in the rhesus macaque detected by polymerase chain reaction with sequence-specific primers and direct sequencing. Tissue Antigens 50, 657-661. Konstant, W. R., and Mittermeier, R. A. (1982). Introduction, reintroduction, and translocation of neotropical primates: Past experiences and future possibilities. Int. Zoo Yearbook 22, 69-77. Kreeger, T. J. (1999). Chemical restraint and immobilization of wild canids. In "Zoo and Wild Animal Medicine: Current Therapy 4" (M. E. Fowler and R. E. Miller, eds.), pp. 429-435. Saunders, Philadelphia. Krook, L., and Barrett, R. B. (1962). Simian bone disease--a secondary hyperparathyroidism. Cornell Vet. 52, 459-492. Kuehne, R. W., Chatigny, M. A., Stainbrook, B. W., Runkle, R. S., and Stuart, D. G. (1995). Primary barriers and personal protective equipment in biomedical laboratories. In "Laboratory Safety: Principles and Practices" (D. O. Fleming, J. H. Richardson, J. J. Tulis, and D. Vesley, eds.), pp. 145170. ASM Press, Washington, D.C. Kumar, V., Ceulemans, F., and De Meurichy, W. (1978). Chemotherapy of helminthiasis among wild animals. IV. Efficacy of flubendazole 5% (R 17889) against Trichuris trichiura infections of baboons, Papio hamadryas L. Acta Zool. Pathol. Antverp. 73, 3-9. Kummer, H., Banaja, A. A., Abo-Khatwa, A. N., and Ghandor, A. M. (1981). A survey of hamadryas baboons in Saudi Arabia. Fauna Saudi Arabia 3, 441-471. LabDiet Product Reference Manual (1998). PMI Nutritional International, St. Louis. Lackner, A. A. (1988). Tissue distribution of simian AIDS retrovirus serotype 1 and its relationship to the pathogenesis of simian AIDS. Univ. of California at Davis. Langner, P. H., Brightman, A. H., and Tranquilli, W. J. (1986). Maxillofacial abscesses in captive squirrel monkeys. J. Am. Vet. Med. Assoc. 189, 1218. Latt, R. H. (1975). Runyon group III atypical mycobacteria as a cause of tuberculosis in a rhesus monkey. Lab. Anim. Sci. 25, 206-209. Lauerman, W. C., Platenberg, R. C., Cain, J. E., and Vincent, F. X. (1992). Agerelated disk degeneration: Preliminary report of a naturally occuring baboon model. J. Spinal Disorders 5, 170-174. Laule, G. (1993). Using training to enhance animal care and welfare. AWIC News. 4, 1-9. Lausen, N. C., Richter, A. G., and Lage, A. L. (1986). Pseudomonas aeruginosa infection in squirrel monkeys. J. Am. Vet. Med. Assoc. 189, 1216-1218. LeDuc, J. W., Lemon, S. M., Keenan, C. M., Graham, R. R., Marchwicki, R. H., and Binn, L. N. (1983). Experimental infection of the New World owl monkey (Aotus trivirgatus) with hepatitis ,k virus. Infect. Immun. 40, 766-772.

16. NONHUMAN PRIMATES Lee, D. R. (1993). Nonhuman primate models in biomedical research. Lecture at Baylor College of Medicine, Houston, Texas, November 10. Lee, D. R., Kuehl, T. J., and Eichberg, J. W. (1991). Real-time ultrasonography as a clinical and management tool to monitor pregnancy in a chimpanzee breeding colony. Am. J. Primatol. 24, 289-294. Lee, R. V., Prowten, W. M., Satchidanand, S., and Srivastava, B. I. S. (1985). Non-Hodgkin's lymphoma and HTLV-1 antibodies in a gorilla. N. Eng. J. Med. 312, 118-119. Lehner, N. D. M. (1984). Biology and diseases of the Cebidae. In "Laboratory Animal Medicine" (J. G. Fox, B. G. Cohen, and E M. Loew, eds.), pp. 321353. Academic Press, Orlando, Florida. Lehner, N. D. M., Bullock, B. C., Clarkson, T. B., and Lofland, H. B. (1967). Biological activities of vitamin D2 and vitamin D 3 for growing squirrel monkeys. Lab. Anim. Care 17, 483-493. Lehner, N. D. M., Bullock, B. C., and Clarkson, T. B. (1968). Ascorbic acid deficiency in the squirrel monkey. Proc. Soc. Exp. Biol. Med. 128, 512514. Leininger, J. R., Donham, K. J., and Rubino, M. J. (1978). Leprosy in a chimpanzee: Morphology of the skin lesions and characterization of the organism. Vet. Pathol. 15, 339-346. LeMaho, Y., Goffart, M., Rochas, A., Felbabel, H., and Chatonnet, J. (1981). Thermoregulation in the only nocturnal simian: The night monkey Aotus trivirgatus. Am. J. Physiol. 2411, R156-R165. Lerche, N. W., Osborn, K. G., Marx, P. A., Prahalda, S., Maul, D. H., Lowenstine, L. J., Munn, R. J., Bryant, M. L., Henrickson, R. V., Arthur, L. O., Gilden, R. V., Barker, C. S., Hunter, E., and Gardner, M. B. (1986). Inapparent carriers of simian acquired immunodeficiency syndrome type D retrovirus and disease transmission with saliva. J. Nat. Cancer Inst. 77, 489-496. Lerche, N. W., Yee, J. L., and Jennings, M. B. (1994). Establishing specific retrovirus-free breeding colonies of macaques: An approach to primary screening and surveillance. Lab. Anim. Sci. 44, 217-221. Letvin, N. L., Eaton, K. A., Aldrich, W. R., Sehgal, P. K., Blake, B. J., Schlossman, S. E, King, N. W., and Hunt, R. D. (1983). Acquired immunodeficiency syndrome in a colony of macaque monkeys. Proc. NatL Acad. Sci. 811,2718-2722. Levin, J. (1995). Medical management: Special techniques. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 304-316. Academic Press, San Diego. Levin, J. L., Hilliard, J. K., Lipper, S. L., Butler, T. M., and Goodwin, W. J. (1988). A naturally occurring epizootic of simian agent 8 in the baboon. Lab. Anim. Sci. 38, 394-397. Levy, B. M., and Artecona, J. (1964). The marmoset as an experimental animal in biological research: Care and maintenance. Lab. Anim. Care 14, 20-27. Liebenberg, S. P., and Giddens, W. E., Jr. (1985). Disseminated nocardiosis in three macaque monkeys. Lab. Anim. Sci. 35, 162-166. Lindburg, D. G. (1980). "The Macaques: Studies in Ecology, Behavior, and Evolution." Van Nostrand Reinhold, New York. Lindsey, J. R., and Melby, E. C., Jr. (1966). Naturally occurring primary cutaneous tuberculosis in the rhesus monkey. Lab. Anim. Care 16, 369-385. Lindsey, J. R., Hardy, P. H., Baker, H. J., and Melby, E. C., Jr. (1971). Observations on shigellosis and development of multiply resistant Shigellas in Macaca mulatta. Lab. Anim. Sci. 21, 832-844. Line, S., Dorr, T., Roberts, J., and Ihrke, P. (1984). Necrotizing cellulitis in a squirrel monkey. J. Am. Vet. Med. Assoc. 185, 1378-1379. Line, A. S., Paul-Murphy, J., Aucoin, D. P., and Hirsh, D. C. (1992). Enrofloxacin treatment of long-tailed macaques with acute bacillary dysentery due to multiresistant Shigellaflexneri IV. Lab. Anim. Sci. 42, 240-244. Lipman, B., Palmer, D., Noble, J., Haughton, V., and Collier, D. (1988). Effect of lumbar puncture on flow of cerebrospinal fluid. Invest. Radiol. 23, 359-360. Liu, S. K., Dolensek, E. P., Tappe, J. P., Stover, J., and Adams, C. R. (1984). Car-

785 diomyopathy associated with vitamin E deficiency in seven gelada baboons. J. Am. Vet. Med. Assoc. 185, 1347-1350. Lowenstine, L. J. (1993). Type D retrovirus infection, macaques. In "Monographs on Pathology of Laboratory Animals: Nonhuman Primates" (T. C. Jones, U. Mohr, and R. D. Hunt, eds.), Vol. 1, pp. 20-32. Springer-Verlag, Berlin and New York. Lowenstine, L. J., Lerche, N. W., Yee, J. L., Uyeda, A., Jennings, M. B., Munn, R. J., McClure, H. M., Anderson, D. C., Fulz, P. N., and Gardner, M. B. (1992). Evidence for a lentiviral etiology in an epizootic of immune deficiency and lymphoma in stump-tailed macaques (Macaca arctoides). J. Med. Primatol. 21, 1-14. Lyons, D. M., Mendoza, S. P., and Mason, W. A. (1992). Sexual segregation in squirrel monkeys (Saimiri sciureus): A transactional analysis of adult social dynamics. J. Comp. Psychol. 106, 323-330. Ma, N. S. E (1981). Chromosome evolution in the owl monkey, Aotus. Am. J. Primatol. 54, 293-303. Ma, N. S., and Harris, T. S. (1989). A putative homeolog of human chromosome 12 in the owl monkey. Cytogenet. Cell Genet. 50, 34-39. Ma, N. S. E, Jones, T. C., Miller, A. C., Morgan, L. M., and Adams, E. A. (1976). Chromosome polymorphism and banding patterns in the owl monkey (Aotus). Lab. Anim. Sci. 26, 1022-1036. Ma, N. S. F., Rossan, R. N., Kelley, S. T., Harper, J. S., B6dard, M. T., and Jones, T. C. (1978). Banding patterns of the chromosomes of two new karyotypes of the owl monkey, Aotus, captured in Panama. J. Med. Primatol. 7, 146155. Ma, N. S. E, Renquist, D. M., Hall, R., Sehgal, P. K., Simeone, T., and Jones, T. C. (1980). XX/XO sex determination system in a population of Peruvian owl monkeys, Aotus. J. Hered. 71, 336-340. Ma, N. S. F., Aquino, R., and Collins, W. E. (1985). Two new karyotypes in the Peruvian owl monkey (Aotus trivirgatus). Am. J. Anthropol. 9, 333-341. MacArthur, J. A., and Wood, M. (1983). Yersiniosis in a breeding unit of Macacafascicularis (cynomolgus monkeys). Lab. Anim. 17, 151-155. Machotka, S. V., Chapple, E E., and Stookey, J. L. (1975). Cerebral tuberculosis in a rhesus monkey. J. Am. Vet. Med. Assoc. 167, 648-650. Mahaney, M. C., Leland, M. M., Williams-Blangero, S., and Marinez, Y. N. (1993). Cross-sectional growth standards for captive baboons: II. Organ weight by body weight. J. Med. Primatol. 7-8, 415-427. Malaga, C. A., Weller, R. E., Buschbom, R. L., and Ragan, H. A. (1990). Hematology of the wild-caught karyotype I owl monkey (Aotus nancymaae). Lab. Anim. Sci. 411,204-205. Malaga, C. A., Weller, R. E., Buschbom, R. L., Baer, J. E, and Kimsey, B. B. (1997). Reproduction of the owl monkey (Aotus spp.) in captivity. J. Med. Primatol. 26, 147-152. Mansfield, K. G., and King, N. (1998). Viral diseases. In "Nonhuman Primates in Biomedical Research: Diseases" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 1-58. Academic Press, San Diego. Mansfield, K. G., Kuei-Chin, L., Newman, J., Schauer, D., MacKey, J., Lackner, A. A., Carville, A. (2001). Identification of enteropathogenic Escherichia coli in simian immunodeficiency virus-infected infant and adult rhesus macaques. J. Clin. Microbiol. 39, 971-976. Martin, D. P. (1986). Preventive medicine. In "Zoo and Wild Animal Medicine" (M. E. Fowler, ed.), pp. 667-669. Saunders, Philadelphia. Martin, D. P., and Kaye, H. S. (1983). Epizootic of parainfluenza-3 virus infection in gibbons. J. Am. Vet. Med. Assoc. 183, 1185-1187. Martin, L. N., and McNease, P. E. (1982). Genetically determined antigens of squirrel monkey (Saimiri sciureus) IgG. J. Med. Primatol. 11, 272-290. Martin, B. J., Dysko, R. C., and Chrisp, C. E. (1991). Pancreatitis associated with simian adenovirus 23 in a rhesus monkey. Lab. Anim. Sci. 41, 382384. McCarthy, T. J., Kennedy, J. L., Blakeslee, J. R., and Bennett, B. T. (1990). Spontaneous malignant lymphoma and leukemia in a simian T-lymphotrophic virus type 1 (STLV-1) antibody-positive olive baboon. Lab. Anim. Sci. 411,79-81.

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McChesney, M. B., Fujinami, R. S., Lerche, N. W., Marx, P. A., and Oldstone, M. B. (1989). Virus-induced immunosuppression: Infection of peripheral blood mononuclear cells and suppression of immunoglobulin synthesis during natural measles virus infection of rhesus monkeys. J. Infect. Dis. 159, 757-760. McClure, H. M. (1980). Bacterial diseases of nonhuman primates. In "The Comparative Pathology of Zoo Animals" (R. J. Montali and G. Migaki, eds.), pp. 197-217. Smithsonian Institution Press, Washington, D.C. McClure, H. M., and Keeling, M. E. (1971). Viral diseases noted at the Yerkes Primate Center colony. Lab. Anim. Sci. 21, 1002-1010. McClure, H. M., Weaver, R. E., and Kaufmann, A. E (1971). Pseudotuberculosis in nonhuman primates: Infection with organisms of the Yersinia enterocolitica group. Lab. Anim. Sci. 21, 376-382. McClure, H. M., Strozier, L. M., and Keeling, M. F. (1972). Enteropathogenic Escherichia coli infection in anthropoid apes. J. Am. Vet. Med. Assoc. 161, 687-689. McClure, H. M., Alford, P., and Swenson, B. (1976). Nonenteric Shigella infections in nonhuman primates. J. Am. Vet. Med. Assoc. 169, 938-939. McClure, H. M., Brodie, A. R., Anderson, D. C., and Swenson, R. B. (1986). Bacterial infections of nonhuman primates. In "Primates: The Road to Self-Sustaining Populations" (K. Benirschke, ed.), pp. 531-556. SpringerVerlag, New York. McConnell, S. J., Hickman, R. L., Wooding, W. L., and Huxsoll, D. L. (1968). Monkeypox: Experimental infection in chimpanzees and immunization with vaccinia virus. Am. J. Vet. Med. Res. 29, 1675 - 1680. McGuill, M. W., and Rowan, A. N. (1989). Biological effects of blood loss: Implications for sampling volumes and techniques. ILAR News 31, 5-18. McLaughlin, R. M. (1978). Mycobacterium bovis in nonhuman primates. In "Mycobacterial Infections in Zoo Animals" (R. J. Montali, ed.), pp. 151155. Smithsonian Institution Press, Washington, D.C. McLaughlin, R. M., and Marrs, G. E. (1978). Tuberculin testing in nonhuman primates: OT vs. PPD. In "Mycobacterial Infections in Zoo Animals" (R. J. Montali, ed.), pp. 123-127. Smithsonian Institution Press, Washington, D.C. McMahan, C. A., Wigodsky, H. S., and Moore, G. T. (1976). Weight of the infant baboon (Papio cynocephalus) from birth to fifteen weeks. Lab. Anim. Sci. 6, 928-931. McNamee, G. A., Wannemacher, R. W., Jr., Dinterman, R. E., Rozmiarek, H., and Montrey, R. D. (1984). A surgical procedure and tethering system for chronic blood sampling, infusion, and temperature monitoring in caged nonhuman primates. Lab. Anim. Sci. 34, 303-307. McNulty, W. P. (1972). Pox diseases in primates. In "Pathology of Simian Primates" (R. N. T.-W. Fiennes, ed.), pp. 612-645. Karger, Basel and New York. Melendez, L. V., Espana, C., Hunt, R. D., and Daniel, M. D. (1969). Natural herpes simplex infection in the owl monkey (Aotus trivirgatus). Lab. Anim. Care 19, 38-45. Melendez, L. V., Hunt, R. D., Daniel, M. D., and Trum, B. E (1970). New World monkeys, herpes viruses, and cancer. In "Infections and Immunosuppression in Subhuman Primates" (H. Balner and W. J. B. Beveridge, eds.), pp. 111-117. Munksgaard, Copenhagen. Melnick, D. J., and Pearl, M. C. (1987). Cercopithecines in multimale groups: Genetic diversity and population structure. In "Primate Societies" (B. B. Smuts, D. L. Cheney, R. M. Seyfarth, R. W. Wrangham, and T. T. Struhsaker, eds.), pp. 121-134. Univ. of Chicago Press, Chicago. Melnick, J. L., and Butel, J. S. (1988). Neurologic tumors in offspring after inoculation of mothers with killed-poliovirus vaccine. N. Eng. J. Med. 318, 1226. Mendoza, S. P., Lowe, E. L., Davidson, J. M., and Levine, S. (1978a). Annual cyclicity in the squirrel monkey (Saimiri sciureus): The relationship between testosterone, fatting, and sexual behavior. Horm. Behav. 11, 295303. Mendoza, S. P., Lowe, E. L., and Levine, S. (1978b). Social organization and

social behavior in two subspecies of squirrel monkeys (Saimiri sciureus). Folia Primatol. 30, 126 - 144. Mendoza, S. P., Lyons, D. M., and Saltzman, W. (1991). Sociophysiology of squirrel monkeys. Am. J. Primatol. 23, 37-54. The Merck Veterinary Manual (1991). Edited by C. M. Fraser, J. A. Bergeron, A. Mays, and S. E. Aiello, p. 133. Merck, Rahway, New Jersey. Meritt, D. A., Jr. (1976). The owl monkey, Aotus trivirgatus: Husbandry, behavior, and breeding. Proceedings of the National Conference of AAZPA, pp. 107-123. Meyers, W. M., Walsh, G. P., Brown, H. L., Binford, C. H., Imes, G. D., Jr., Hadfield, T. L., Schlagel, C. J., Fukunishi, Y., Gerone, P. J., Wolf, R. H., Gormus, B. J., Martin, L. T., Harboe, M., and Imaeda, T. (1985). Leprosy in a mangabey monkeymnaturally acquired infection. Int. J. Lepr. Other Mycobact. Dis. 53, 1-14. Migaki, G. (1986). Mycotic infections in nonhuman primates. In "Primates: The Road to Self-Sustaining Populations" (K. Benirschke, ed.), pp. 557570. Springer-Verlag, New York. Migaki, G., Asher, D. M., Casey, H. W., Locke, L. N., Gibbs, C. J., Jr., and Gajdusek, C. (1979). Fatal suppurative nephritis caused by Pseudomonas in a chimpanzee. J. Am. Vet. Med. Assoc. 175, 957-959. Migaki, G., Schmidt, R. E., Toft, J. D., and Kaufmann, A. E (1982). Mycotic infections of the alimentary tract of nonhuman primates: A review. Vet. Pathol. Suppl. 7, 93-103. Miller, R. E., and Boever, W. J. (1983). Cryptococcosis in a lion-tailed macaque (Macaca silenus). J. Zoo Anim. Med. 14, 110-114. Miller, G., Shope, T., Coope, D., Waters, L., Pagano, J., Bornkamm, G., and Henle, W. (1977). Lymphoma in cotton-topped marmosets after inoculation with Epstein-Barr virus: Tumor incidence, histologic spectrum, antibody responses, demonstration of viral DNA, and characterization of viruses. J. Exp. Med. 145, 948-967. Miller, G. E, Barnard, D. E., Woodward, R. A., Flynn, B. M., and Bulte, J. W. M. (1997). Hepatic hemosiderosis in common marmosets, Callithrix jacchus: Effect of diet on incidence and severity. Lab. Anim. Sci. 47, 138-142. Miranda, M. E., Ksiazek, T. G., Retuya, T. J., Khan, A. S., Sanchez, A., Fulhorst, C. E, Rollin, P. E., Calaor, A. B., Manalo, D. L., Roces, M. C., Dayrit, M. M., and Peters, C. J. (1999). Epidemiology of Ebola (subtype Reston) virus in the Philippines, 1996. J. Infect. Dis. 179(S1), S115-S119. Mittermeier, R. A., and van Roosmalen, G. M. (1981). Preliminary observations of habitat utilization and diet in eight Surinam monkeys. Folia Primatol. 36, 1-39. Mivart, St. George (1873). On Lepilemur and Cheirogaleus and on the zoological rank of the Lemuroidea. Proc. Zool. Soc. Lond., 484-510. Moisson, P., and Gysin, J. (1994). Reproductive performances and pathological findings in a breeding colony of squirrel monkeys (Saimiri sciureus). In "Current Primatology: Behavioral Neuroscience, Physiology, and Reproduction" (J. R. Anderson, J. J. Roeder, B. Thierry, and N. Herrenschmidt, eds.), Vol. 3, pp. 263-272. Univ. Louis Pasteur, Strasbourg. Montali, R. J., Scanga, C. A., Pernikoff, D., Wessner, D. R., Ward, R., and Holmes, K. V. (1993). A common source outbreak of callitrichid hepatitis in captive tamarins and marmosets. J. Infect. Dis. 167, 946-950. Montali, R. J., Connolly, B. M., Armstrong, D. L., Scanga, C. A., and Holmes, K. V. (1995). Pathology and immunohistochemistry of callitrichid hepatitis, an emerging disease of captive New World primates caused by lymphocytic choriomeningitis virus. Am. J. Pathol. 147, 1441-1449. Moore, G. T. (1975). The breeding and utilization of baboons for biomedical research. Lab. Anim. Sci. 25, 798-801. Morbeck, M. E., Zihlman, A. L., Summner, D. R., and Galloway, A. (1991). Poliomyelitis and skeletal asymmetry in Gombe chimpanzees. Primates 32, 77-91. Morgan, J. P., Silverman, S., and Zontine, W. J. (1975). "Techniques in Veterinary Radiology," pp. 339-342. Veterinary Radiology Assoc., Davis, California. Morton, W. R., Bronsdon, M., Mickelsen, G., Knitter, G., Rosenkranz, S., Kuller,

16. NONHUMAN PRIMATES L., and Sajuthi, D. (1983). Identification of Campylobacter jejuni in Macaca fascicularis imported from Indonesia. Lab. Anita. Sci. 33, 187-188. Motzel, S., Schachner, R., Kornegay, R., Fletcher, M., Kananaya, B., Gomez, J., Ngai, D., Handt, L., Wagner, J., and Klein, H. (1999). Assessment of methods for the diagnosis of tuberculosis in three species of nonhuman primates. Contemp. Top. Lab. Anita. Sci. 38, 28. Moynihan, M. A. (1964). Some behavior patterns of platyrrhine monkeys. 1. The night monkey (Aotus trivirgatus). Smithson. Misc. Collec. 146, 1-184. Mrema, J. E., Caldwell, C. W., Stogsdill, P. L., Kelley, S. T., and Green, T. J. (1987). Erythrocyte and erythrocyte morphologies of healthy and colonyborn owl monkeys (Aotus lemurinus griseimembra). J. Med. Primatol. 16, 13-25. Mulder, J. B. (1971). Shigellosis in nonhuman primates: A review. Lab. Anim. Sci. 21, 734-738. Mutombo, W. M., Arita, I., and Jezek, Z. (1983). Human monkeypox transmitted by a chimpanzee in a tropical rainforest area of Zaire. Lancet 1, 735737. Napier, J. R., and Napier, P. H. (1967). "A Handbook of Living Primates." Academic Press, New York. Napier, J. R., and Napier, P. H. (1985). "The Natural History of the Primates." MIT Press, Cambridge, Massachusetts. National Institutes of Health (NIH) (1994). In "NCRR Chimpanzee Breeding and Research Program Progress Report, July 1994." National Institutes of Health, Washington, D.C. National Research Council (NRC). (1997). "Occupational Health and Safety in the Care and Use of Research Animals" National Academy Press, Washington, D.C. Newton, W. M., Beamer, P. D., and Rhoades, H. E. (1971). Acute bloat syndrome in stumptailed macaques (Macaca arctoides): A report of four cases. Lab. Anita. Sci. 21, 193-196. Nicholson, N. (1982). Weaning and the development of independence in olive baboons. Ph.D. diss., Harvard University. Niedobitek, G., Agathanggelou, A., Finerty, S., Tierney, R., Watkins, P., Jones, E. L., Morgan, A., Young, L. S., and Rooney, N. (1994). Latent EpsteinBarr virus infection in cotton-top tamarins. Am. J. Pathol. 145, 969-978. 1999 Report of Progress, Southwest Foundation for Biomedical Research, San Antonio, TX. Novak, M. A., Kinsey, J. H., Jorgensen, M. J., and Hazen, T. J. (1998). Effects of puzzle feeders on pathological behavior in individually housed rhesus monkeys. Am. J. Primatol. 46, 213-227. Nyland, T. G., and Mattoon, J. S. (1995). "Veterinary Diagnostic Ultrasound." Saunders, Philadelphia. Obaldia, N., III ( 1991). Detection of Klebsiella pneumoniae antibodies in Aotus l. lemurinus (Panamanian owl monkey) using an enzyme linked immunosorbent assay (ELISA) test. Lab. Anim. 25, 133-141. Obeck, D. K. (1978). Galvanized caging as a potential factor in the development of the "fading infant" or "white monkey" syndrome. Lab. Anita. Sci. 28, 698 -704. Olson, L. C., Bergquist, D. Y., and Fitzgerald, D. L. (1986). Control of Shigella flexneri in Celebese black macaques (Macaca nigra). Lab. Anim. Sci. 36, 240-242. Osborn, K. G., Prahalda, S., Lowenstine, L. J., Gardner, M. B., Maul, D. H., and Henrickson, R. V. (1984). The pathology of acquired immunodeficiency syndrome in rhesus macaques. Am. J. Pathol. 114, 94-103. O'Sullivan, M. G., Anderson, D. C., Fikes, J. D., Bain, E T., Carlson, C. S., Green, S. W., Young, N. S., and Brown, K. E. (1994). Identification of a novel simian parvovirus in cynomolgus monkeys with severe anemia. A paradigm of human B19 parvovirus infection. J. Clin. Invest. 93, 1571-1576. O'Sullivan, M. G., Anderson, D. K., Lund, J. E., Brown, W. P., Green, S. W., Young, N. S., and Brown, K. E. (1996). Clinical and epidemiological features of simian parvovirus infection in cynomolgus macaques with severe anemia. Lab. Anim. Sci. 46, 291-297. O'Sullivan, M. G., Anderson, D. K., Goodrich, J. A., Tulli, H., Green, S. W.,

787 Young, N. S., and Brown, K. E. (1997). Experimental infection of cynomolgus monkeys with simian parvovirus. J. Virol. 71, 4517-4521. O'Toole, B. A., Fradkin, R., Warkany, J., Wilson, J. G., and Mann, G. V. (1974). Vitamin A deficiency and reproduction in rhesus monkeys. J. Nutr. 104, 1513-1524. Palmer, A. E., Allen, A. M., Tauraso, N. M., and Shelokov, A. (1968). Simian hemorrhagic fever. I. Clinical and epizootiologic aspects of an outbreak among quarantined monkeys. Am. J. Trop. Med. Hyg. 17, 404-492. Paul-Murphy, J. (1993). Bacterial enterocolitis in nonhuman primates. In "Zoo and Wild Animal Medicine: Current Therapy 3" (M. Fowler, ed.), pp. 334351. Saunders, Philadelphia. Peisert, W., Taborski, A., Pawlowski, Z., Karlewiczowa, R., and Zdun, M. (1983). Giardia infections in animals in Poznan Zoo. Vet. Parasitol. 13, 183-186. Perman, V., Osborne, C. A., and Stevens, J. B. (1974). Bone marrow biopsy. Vet. Clin. North Am. 4, 293-310. Perolat, P., Poingt, J., Vie, J., Jouaneau, C., Baranton, G., and Gysin, J. (1992). Occurrence of severe leptospirosis in a breeding colony of squirrel monkeys. Am. J. Trop. Med. Hyg. 46, 538-545. Peters, J. M., and Guerra, M. O. (1998). Growth of marmoset monkeys Callithrixjacchus in captivity. Folia Primatol. 69, 266-272. Phillips-Conroy, J. E., Jolly, C. J., Petros, B., Allan, J. S., and Desrosiers, R. C. (1994). Sexual transmission of SIVagm in wild grivet monkeys. J. Med. Primatol. 23, 1-7. Pierce, D. L., and Dukelow, W. R. (1988). Misleading positive tuberculin reactions in a squirrel monkey colony. Lab. Anim. Sci. 38, 729-730. Pinheiro, E. S., Simon, E, Cassaro, K., and Soares, M. E. G. (1993). Outbreak of diarrhea due to Campylobacter jejuni in lion-tamarins (Leontopithecus spp.) in captivity. Verh. Ber. Zootiere 35, 159-161. Pinkerton, M. (1972). Miscellaneous organisms. In "Pathology of Simian Primates Part II: Infectious and Parasitic Diseases" (R. N. T-W-Fiennes, ed.), pp. 283-313. Karger, Basel and New York. Plesker, R., and Carlos, M. (1992). A spontaneous Yersinia pseudotuberculosis infection in a monkey colony. Zentrabl. Veterinarmed. B. 39, 201-208. Plumb, D. C. (1995). In "Veterinary Drug Handbook," p. 604. Iowa State Univ. Press, Ames. Poelma, E G. (1975). Pneumocystis carinii infections in zoo animals. Z. Parasitenkd. 46, 61-68. Pook, A. G. (1976). Some notes on the development of hand-reared infants of four species of marmoset (Callitrichidae). 13th Annual Report, Jersey Wildlife Preservation Trust, pp. 38-46. Pope, C. E., Pope, V. Z., and Beck, L. R. (1986). Cryopreservation and transfer of baboon embryos. J. inVitro Fertil. Embryo Trans. 3, 33-39. Porter, J. A., Jr. (1969). Hematology of the night monkey, Aotus trivirgatus. Lab. Anita. Sci. 19, 470-472. Portman, O. W., Alexander M., Tanaka, N., and Osuga, T. (1980). Relationships between cholesterol, gallstones, biliary function, and plasma lipoproteins in squirrel monkeys. J. Lab. Clin. Med. 96, 90-101. Postal, J. M., Gysin, J., and Crenn, Y. (1988). Protection against fatal Klebsiella pneumoniae sepsis in the squirrel monkey Saimiri sciureus after immunization with a capsular polysaccharide vaccine. Ann. Inst. Pasteur Immunol. 139, 401-407. Potkay, S. (1992). Diseases of the Callitrichidae: A review. J. Med. Primatol. 21, 189-236. Rangan, S. R. S., Martin, L. N., Bozelka, B. E., Wang, N., and Gomus, B. J. (1995). Epstein-Barr virus-related herpesvirus from a rhesus monkey (Macaca mulatta) with malignant lymphoma. Int. J. Cancer 38, 425-432. Rasmussen, K. M., Thenen, S. W., and Hayes, K. C. (1980). Effect of folic acid supplementation on pregnancy in the squirrel monkey (Saimiri sciureus). J. Med. Primatol. 9, 169-184. Ratterree, M. S., Didier, P. J., Blanchard, J. L., Clarke, M. R., and Schaeffer, D. (1990). Vitamin C deficiency in captive nonhuman primates fed commercial primate diet. Lab. Anim. Sci. 40, 165-168. Rawlins, R. G., and Kessler, M. T. (1982). A five-year study of tetanus in the

788

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Cayo Santiago rhesus monkey colony: Behavioral description and epizootiology. Am. J. PrimatoL 3, 23-39. Reardon, M. J., Hall, R. D., and Davidson, C. E. (1979). Serum electrophoretic patterns of karyotypically defined owl monkeys (Aotus trivirgatus). Lab. Anim. Sci. 29, 617-620. Reid, H. A., Herron, A. J., Hines, M. E,, Jr., Orchard, E. A., and Altman, N. H. (1993). Leptospirosis in a white-lipped tamarin (Saguinus labiatus). Lab. Anim. Sci. 43(3), 258-259. Reindel, J. E, Fitzgerald, A. L., Breider, M. A., Gough, A. W., Yan, C., Mysore, J. V., Dubois, A. (1999). An epizootic of lymphoplasmacytic gastritis attributed to Helicobacterpylori infection in cynomolgus monkeys (Macaca fascicularis). Vet. PathoL 36, 1-13. Renne, R. A., McLaughlin, R., and Jenson, A. B. (1973). Measles virus-associated endometritus, cervicitis, and abortion in a rhesus monkey. J. Am. Vet. Med. Assoc. 163, 639-641. Renquist, D. (1990). Outbreak of hemorrhagic fever. J. Med. Primatol. 19, 7780. Renquist, D. M., and Whitney, R. A. (1978). Tuberculosis in nonhuman prim a t e s - a n overview. In "Mycobacterial Infections in Zoo Animals" (R. J. Montali, ed.), pp. 9-16. Smithsonian Institution Press, Washington, D.C. Renquist, D. M., and Whitney, R. A. (1987). Zoonoses acquired from pet primates. Vet. Clin. North Am. Small Anim. Pract. 17, 219-240. Richardson, J. H. (1995). Introduction. In "Laboratory Safety: Principles and Practices" (D. O. Fleming, J. H. Richardson, J. J. Tulis, and D. Vesley, eds.), pp. 1-6. ASM Press, Washington, D.C. Richardson, J. H., and Humphrey, G. L. (1971). Rabies in imported nonhuman primates. Lab. Anim. Sci. 21, 1082-1803. Richter, C. B., Humason, G. L., and Godbold, J. H., Jr. (1978). Endemic Pneumocystis carinii in a marmoset colony. J. Comp. PathoL 88, 221-223. Richter, C. B., Lehner, N. D. M., and Henrickson, R. V. (1984). Primates. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 298-393. Academic Press, San Diego. Rigau-Perez, J. G., Clark, G. G., Gabler, D. J., Reiter, P., Sanders, E. J., and Vorndam, A. V. (1998). Dengue and dengue hemorrhagic fever. Lancet 352, 971-977. Riddle, K. E., Keeling, M. E., Alford, P. L., and Beck, T. E (1982). Chimpanzee holding, rehabilitation, and breeding: Facilities design and colony management. Lab. Anim. Sci. 32, 525-533. Riordan, J. T. (1949). Rectal tuberculosis in monkeys from the use of contaminated thermometers. J. Infect. Dis. 73, 93-94. Roberts, J. A., Lerche, N. W., Markovits, J. E., and Maul, D. H. (1988). Epizootic measles at the CRPRC. Lab. Anim. Sci. 38, 492. Rogers, J., and Hixson, J. E. (1997). Baboons as an animal model for genetic studies of common human disease. Am. J. Hum. Genet. 61, 489-493. Rollin, P. E., Williams, R. J., Bressler, D. S., Pearson, S., Cottingham, M., Pucak, G., Sanchez, A., Trappier, S. G., Peters, R. L., Greer, P. W., Zaki, S., DeMarcus, T., Hendricks, K., Kelley, M., Simpson, D., Geisbert, T. W., Jahrling, P. B., Peters, C. J., and Ksiazek, T. G. (1999). Ebola (subtype Reston) virus among quarantined nonhuman primates recently imported from the Philippines to the United States. J. Infect. Dis. 179(S1), S108S144. Rosenberg, D. P. (1995). Critical care. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 316-334. Academic Press, San Diego. Rosenberg, D. P., and Blouin, P. (1979). Retrobulbar abscess in an infant rhesus monkey. J. Am. Vet. Med. Assoc. 175, 994-996. Rosenberg, D. P., Lerche, N. W., and Henrickson, R. V. (1980). Yersinia pseudotuberculosis infection in a group of Macaca fascicularis. J. Am. Vet. Med. Assoc. 177, 818-819. Rosenberg, D. P., Gleiser, C. A., and Carey, K. D. (1984). Spinal coccidioidomycosis in a baboon. J. Am. Vet. Med. Assoc. 185, 1379-1381. Rossan, R. N., Harper, J. S., Davidson, D. E., Jr., Escajadillo, A., and Christensen, H. A. (1985). Comparison of Plasmodium falciparum infections in

Panamanian and Colombian owl monkeys. Am. J. Trop. Med. Hyg. 35, 1037-1047. Roussilhon, C., Postal, J. M., and Ravisse, P. (1987). Spontaneous cryptococcosis of a squirrel monkey (Saimiri sciureus) in French Guyana. J. Med. Primatol. 16, 39-47. Rowe, N. (1996). "The Pictorial Guide to the Living Primates." Pogonias Press, New York. Russell, R. G., and DeTolla, L. J. (1993). Shigellosis. In "Nonhuman Primates" (T. C. Jones, U. Mohr, and R. D. Hunt, eds.), Vol. 1, pp. 46-53. SpringerVedag, Berlin. Russell, R. G., Rosenkranz, S. L., Lee, L. A., Howard, H., DiGiacomo, R. E, Bronsdon, M. A., Blakley, G. A., Tsii, C.-C., and Morton, W. R. (1987). Epidemiologyand etiology of diarrhea in colony-born Macaca nemestrina. Lab. Anim. Sci. 37, 309-316. Russell, R. G., Krugner, L., Tsai, C.-C., and Ekstrom, R. (1988). Prevalence of Campylobacter in infant, juvenile, and adult laboratory primates. Lab. Anim. Sci. 38, 711-714. Ryan, K. J., Benirschke, K., and Smith, O. W. (1981). Conversion of androstenedione-4-14C to estrone by the marmoset placenta. Endocrinology 69, 613-618. Rylands, A. B., Schneider, H., Langguth, A., Mittermeier, R. A., Groves, C. P., and Rodriguez-Luna, E. (2000). An assessment of the diversity of New World primates. Neotrop. Primates 8, 61-93. Sakakibara, I., Sugimoto, Y., Takasaka, M., and Honjo, S. (1984). Spontaneous nocardiosis with brain abscess caused by Nocardia asteroides in a cynomolgus monkey. J. Med. Primatol. 13, 89-95. Sauceda, R., and Schmidt, M. G. (2000). Refining macaque handling and restraint techniques. Lab. Anim. 29, 47-49. Saunders, K. E., Shen, Z., Dewhirst, E E., Paster, B. J., Dangler, C. A., and Fox, J. G. (1999). Novel intestinal Helicobacter species isolated from cotton-top tamarins (Saguinus oedipus) with chronic colitis. J. Clin. Microbiol. 37, 146-151. Savage, A., Shidelar, S. E., Soto, L. H., Causado, J., Giraldo, L. H., Lasley, B. L., and Snowdon, C. T. (1997). Reproductive events of wild cotton-top tamarins (Saguinus oedipus) in Colombia. Am. J. Primatol. 43, 329337. Scammell, J. G. (2000). Steroid resistance in the squirrel monkey: An old subject revisited. ILAR J. 41, 19-25. Schapiro, S. J. (2000). A few new developments in primate housing and husbandry. Scand. J. Lab. Anim. Sci. 27, 103-110. Schapiro, S. J., Bloomsmith, M. A., Suarez, S. A., and Porter, L. M. (1996). Effects of social and inanimate enrichment of the behavior of yearling rhesus monkeys. Am. J. PrimatoL 40, 247-260. Sch~itzl, H. M., Da Costa, M., Taylor, L., Cohen, F. E., and Prusiner, S. B. (1997). Prion protein gene variation among primates. J. Mol. BioL 265, 257. Schmidt, L. H. (1978). Plasmodium falciparum and Plasmodium vivax infections in the owl monkey (Aotus trivirgatus). I. The courses of untreated infections. II. Responses to chloroquine, quinine and pyrimethamine. III. Methods employed in the search for new schizonticidal drugs. Am. J. Trop. Med. Hyg. 26, 671-737. Schmidt, R. E., and Butler, T. M. (1971). Klebsiella-Enterobacter infections in chimpanzees. Lab. Anim. Sci. 21, 946-949. Schofield, J. C., Alves, M. E. A. E, Hughes, K. W., and Bennett, B. T. (1991). Disarming canine teeth of nonhuman primates using the submucosal vital root retention technique. Lab. Anim. Sci. 41, 128-133. Scimeca, J. M., and Brady, A. G. (1990). Neonatal mortality in the captive breeding squirrel monkey colony associated with an invasive Escherichia coli. Lab. Anim. Sci. 40, 546-547. Scott, L. M. (1984). Reproductive behavior of adolescent female baboons (Papio anubis) in Kenya. In "Female Primates: Studies by Female Primatologists" (M. E Small, ed.), Alan R. Liss, New York. Seal, U. S., and Flesness, N. R. (1986). Captive chimpanzee populations--past,

16. NONHUMAN PRIMATES present, and future. In "Primates: The Road to Self-Sustaining Populations" (K. Benerschke, ed.), pp. 47-55. Springer-Verlag, New York. Sedgwick, C., Parcher, J., and Durham, R. (1970). Atypical mycobacterial infection in the pig-tailed macaque (Macaca nemestrina). J. Am. Vet. Med. Assoc. 157, 724-725. Segal, E. E (1989). "Housing, Care, and Psychological Well-Being of Captive and Laboratory Primates." Noyes Publications, Park Ridge New Jersey. Sehgal, P. K., Bronson, R. T., Brady, P. S., Mclntyre, K. W., and Elliot, M. W. (1980). Therapeutic efficacy of vitamin E and selenium in treating hemolytic anemia of owl monkeys (Aotus trivirgatus). Lab. Anim. Sci. 30, 92-98. Seibold, H. R., Perrin, E. A., Jr., and Garner, A. C. (1970). Pneumonia associated with Bordetella bronchiseptica in Callicebus species primates. Lab. Anim. Care 20, 456-461. Sesline, D. H. (1978). Mycobacterium avium enteritis in nonhuman primates. In "Mycobacterial Infections in Zoo Animals" (R. J. Montali, ed.), pp. 157159. Smithsonian Institution Press, Washington, D.C. Shaikh, A. A., Allen-Rowlands, C., Dozier, T., and Kraemer, D. C. (1976). Diagnosis of early pregnancy in the baboon. Contraception 14, 391-402. Shaikh, A. A., Celaya, C. L., Gomez, I., and Shaikh, S. A. (1982). Temporal relationship of hormonal peaks to ovulation and skin deturgescence in the baboon. Primates 23, 444-452. Sherding, R. (1994). Anorectal diseases. In "Saunders Manual of Small Animal Practice" (S. J. Birchard and R. C. Sherding, eds.), pp. 777-786. Saunders, Philadelphia. Sigg, H., Stolba, A., Abegglen, J. J., and Dasser, V. (1982). Life history of hamadryas baboons: Physical development, infant mortality, reproductive parameters, and family relationships. Primates 23, 473-487. Simon, M. A., Daniel, M. D., Lee-Parritz, D., King, N. W., and Ringler, D. J. (1993). Disseminated B virus infection in a cynomolgus monkey. Lab. Anim. Sci. 43, 545-550. Sly, D. L., London, W. T., Palmer, A. E., and Rice, J. M. (1977). Disseminated cryptococcosis in a patas monkey (Erythrocebuspatas). Lab. Anim. Sci. 27, 694-699. Smith, M. O., and Lackner, A. A. (1993). Effects of sex, age, puncture site, and blood contamination on the clinical chemistry of cerebrospinal fluid in rhesus macaques (Macaca mulatta). Am. J. Vet. Res. 54, 1845-1850. Smith, P. C., Yuill, T. M., Buchanan, R. D., Stanton, J. S., and Chiacumpa, V. (1969). The gibbon (Hylobates lar); a new primate host for Herpesvirus hominia. I. A natural epizootic in a laboratory colony. J. Infec. Dis. 120, 292297. Snyder, S. B., Lund, J. E., Bone, J., Soave, O. A., and Hirsch, D. C. (1970). A study of Klebsiella infection in owl monkeys (Aotus trivirgatus). J. Am. Vet. Med. Assoc. 157, 1935-1939. Snyder, S. B., Omdahl, J. L., Law, D. H., and Froelich, J. W. (1980). Osteomalacia and nutritional secondary hyperparathyroidism in a semi-free-ranging troop of Japanese monkeys. In "The Comparative Pathology of Zoo Animals" (R. J. Montali and G. Migaki, eds.), pp. 51-57. Smithsonian Institution Press, Washington, D.C. Soave, O. (1978). Observations on acute gastric dilatation in nonhuman primates. Lab. Anim. Sci. 28, 331-334. Soave, O., Jackson, S., and Ghumman, J. S. (1981). Atypical mycobacteria as the probable cause of positive tuberculin reactions in squirrel monkeys (Saimiri sciureus). Lab. Anim. Sci. 31, 295-296. Soike, K. E, Rangan, S. R. S., and Gerone, P. J. (1984). Viral disease models in primates. Adv. Vet. Sci. Comp. Med. 28, 151-199. Soini, P. (1993). The ecology of the pygmy marmoset, Cebuella pygmaeus: Some comparisons with two sympatric tamarins. In "Marmosets and Tamarins: Systematics, Behavior, Ecology" (A. B. Rylands, ed.), pp. 257261. Oxford University Press, Oxford. Solleveld, H. A., van Zweiten, M. J., Heidt, P. J., and van Eerd, P. M. C. A. (1984). Clinicopathologic study of six cases of meningitis and meningoencephalitis in chimpanzees (Pan troglodytes). Lab. Anim. Sci. 34, 86-90.

789 Southers, J. L., and Ford, E. W. (1995). Environmental controls. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 262-263. Academic Press, San Diego. Southwick, C. H., and Lindburg, D. G. (1986). The primates of India: Status, trends, and conservation. In "Primates: The Road to Self-Sustaining Populations" (K. Benirschke, ed.), pp. 171-187. Springer-Verlag, New York. Staley, E. C., Southers, J. L., Thoen, C. O., and Easley, S. P. (1995). Evaluation of tuberculin testing and and measles prophylaxis procedures used in rhesus macaque quarantine/conditioning protocols. Lab. Anim. Sci. 45, 125130. Steele, M. D., Giddens, W. E., Jr., Valerio, M., Sumi, S. M., and Stetzer, E. R. (1982). Spontaneous paramyxoviral encephalitis in nonhuman primates (Macaca mulatta and M. nemestrina). Vet. Pathol. 19, 132-139. Stein, F. J., Lewis, D. H., Stott, G. G., and Sis, R. E (1981). Acute gastric dilatation in common marmosets (Callithrix jacchus). Lab. Anim. Sci. 31, 522-523. Stevenson, M. E (1977). The behavior and ecology of the common marmoset ( Callithrix jacchus jacchus) in its natural environment. Primate Eye 9, 5-6. Stoller M. K. (1995). The obstetric pelvis and mechanism of labor in nonhuman primates. Am. J. Phys. AnthropoL 101, 557-567. Stolz, L. P., and Saayman, G. S. (1970). Ecology and behavior of baboons in the northern Transvaal. Ann. Transvaal Mus. 26, 99-143. Stonerook, M. J., Weiss, H. S., Rodriguez, M. A., Rodriguez, J. V., Hernandez, J. I., Peck, O. C., and Wood, J. D. (1994). Temperature-metabolism relations in the cotton-top tamarin (Saguinus oedipus) model for ulcerative colitis. J. Med. Primatol. 23, 16-22. Strayer, E F., and Harris, P. J. (1979). Social cohesion among captive squirrel monkeys. Behav. Ecol. Sociobiol., 93-110. Strum, S. C. (1975). Life with the pumphouse gang: New insights into baboon behavior. Nat. Geog. 147, 672-691. Sutherland, S. D., Almeida, J. D., Gardner, P. S., Skarper, M., and Stabton, J. (1986). Rapid diagnosis and management of parainfluenza 1 virus infection in common marmosets (Callithrix jacchus) Lab. Anita. 20, 121-126. Swack, N. S., and Hsuing, G. D. (1982). Natural and experimental simian cytomegalovirus infections at a primate center. J. Med. Primatol. 11, 169177. Swenson, B., Strobert, E., and Orkin, J. (1979). "AALAS Workshop on Nonhuman Primate Parasitology." Emory Univ., Atlanta. Tardif, S. D., and Richter, C. B. (1981). Competition for a desired food in family groups of the common marmoset (Callithrixjacchus) and the cotton-top tamarin (Saguinus oedipus). Lab. Anita. Sci. 31, 52-55. Taylor, D. W., and Siddiqui, W. A. (1978). Comparative mitogen responses of lymphocytes from Colombian, Panamanian, and Peruvian owl monkeys. Lab. Anim. Sci. 28, 567-574. Taylor, N. S., Ellenberger, M. A., Wu, P. Y., and Fox, J. G. (1989). Diversity of serotypes of Campylobacterjejuni and Campylobacter coli isolated in laboratory animals. Lab. Anim. Sci. 39, 219-221. Taylor, W. M., and Grady, A. W. (1998). Catheter-tract infections in rhesus macaques (Macaca mulatta) with indwelling intravenous catheters. Lab. Anim. Sci. 48, 448-454. Terborgh, J. (1983). "Five New World Primates." Princeton Univ. Press, Princeton, New Jersey. Thomson, J. A., and Schemer, J. J. (1996). Hemorrhagic typhlocolitis associated with attaching and effacing Escherichia coli in common marmosets. Lab. Anim. Sci. 46, 275-279. Thurman, J. D., Morton, R. J., and Stair, E. L. (1983). Septic abortion caused by Salmonella heidelberg in a white-handed gibbon. J. Am. Vet. Med. Assoc. 183, 1325-1326. Toft, J. D., and Eberhard, M. L. (1998). Parasitic diseases. In "Nonhuman Primates in Biomedical Research: Diseases" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 111-205. Academic Press, Orlando, Florida. Traina-Dorge,V., Balanchard, J., Martin, L., and Murphey-Corb, M. (1992).

790

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Immunodeficiency and lymphoproliferative disease in an African green monkey dually infected with SIV and STLV-1. AIDS Res. Hum. Retroviruses 8, 97-100. Tribe, G. W., and Fleming, M. P. (1983). Biphasic enteritis in imported cynomolgus (Macaca fascicularis) monkeys infected with ShigelIa, Salmonella, and Campylobacter species. Lab. Anim. 17, 65-69. Tribe, G. W., MacKensie, P. S., and Fleming, M. P. (1979). Incidence of thermophilic Campylobacter species in newly imported simian primates with enteritis. Vet. Rec. 105, 333-337. Tsai, C.-C., Warner, T. F. C. S., Uno, H., Giddens, W. E., Jr., and Ochs, H. D. (1985). Subcutaneous fibromatosis associated with an acquired immune deficiency syndrome in pig-tailed macaques. Am. J. Pathol. 120, 30-37. Tsai, C.-C., Follis, K. E., Snyder, K., Windsor, S., Thouless, M. E., Kuller, L., and Morton, W. R. (1990). Maternal transmission of type D simian retrovirus (SRV-2) in pig-tailed macaques. J. Med. Primatol. 19, 203-216. Tucker, M. J. (1984). A survey of the pathology of marmosets (CaIlithrixjacchus). Lab. Anim. 18, 351-358. Turnquist, J. E., and Hong, N. (1995). Functional morphology. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 50-76. Academic Press, San Diego. U.S. Department of Agriculture (USDA) (1998). In "Animal Welfare Report," APHIS 41-35-059. Washington, D.C. Valerio, D. A., Dalgard, D. W., Voelker, R. W., McCarrol, N. E., and Good, R. C. (1979). Mycobacterium kansasii infection in rhesus monkeys. In "Mycobacterial Infections in Zoo Animals" (R. J. Montali, ed.), pp. 65-75. Smithsonian Institution Press, Washington, D.C. VandeBerg, J. L. (1995). Genetics of nonhuman primates. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 129-131. Academic Press, San Diego. VandeBerg, J. L., Moore, C. M., Williams-Blangero, S., Cheng, M. L., and Abee, C. R. (1990). Genetic relationships among three squirrel monkey types: Implications for taxonomy, biomedical research, and captive breeding. Am. J. Primatol. 22, 101-111. VandeWoude, S. J., and Luzarraga, M. B. (1991). The role of Branhamella catarrhalis in the "bloody-nose syndrome" of cynomolgus macaques. Lab. Anim. Sci. 41, 401-406. Van Roosmalen, M. G. M., van Roosmalen, T., Mittermeier, R. A., and Rylands, A. B. (2000). Two new species of marmoset, genus Callithrix erxleben (Callithrichidae, Primates), from the Tapaj6s/Madeira Interfluvium, South Central Amazonia, Brazil. Neotrop. Primates 8, 2-18. Voronin, L. G., Kanfor, I. S., Lakin, G. E, and Tikh, N. N. (1948). Spontaneous diseases of lower monkeys, their prophylaxis, diagnosis, and treatment. In "Experimentation on the Keeping and Raising of Monkeys at Sukhami." Academy Medical Science, Moscow. Wadsworth, P. E, Hiddleston, W. A., Jones, D. V., Fowler, J. S. L., and Ferguson, R. A. (1982). Haematological, coagulation, and blood chemistry data in red-bellied tamarins (Saguinus labiatus). Lab. Anim. 16, 327-330. Walker, J., and Spooner, E. T. C. (1960). Natural infection of the African baboon Papio papio with the large-cell form of Histoplasma. J. Pathol. Bacteriol. 80, 436-438. Wang, S., and Quan, G. (1986). Primate status and conservation in China. In "Primates: The Road to Self-Sustaining Populations" (K. Benirschke, ed.), pp. 213-220. Springer-Verlag, New York. Ward, J. A., and Hilliard, J. K. (1994). B virus-specific-pathogen-free (SPF) breeding colonies of macaques: Issues, surveillance, and results in 1992. Lab. Anim. Sci. 44, 222-228. Wasser, S. K. (1996). Reproductive control in wild baboons measured by fecal steroids. Biol. Reprod. 2, 393-399. Wasser, S. K., Norton, G. W., Rhine, R. J., Klein, N., and Kleindorfer, S. (1998). Ageing and social rank effects on the reproductive system of free-ranging yellow baboons (Papio cynocephalus) at Mikumi National Park, Tanzania. Hum. Reprod. Update 4, 430-438.

Weed, J., and Watson, E. A. (1998). Pair housing of adult owl monkeys (Aotus sp.) for environmental enrichment. Am. J. Primatol. 45, 212. Weigler, B. J. (1992). Biology of B virus in macaque and human hosts: A review. Clin. Infect. Dis. 14, 555-567. Weller, R. E. (1994). Infectious and noninfectious diseases of owl monkeys. In "Aotus: The Owl Monkey" (J. E Baer, R. E. Weller, and I. Kakoma, eds.), pp. 178-215. Academic Press, San Diego. Weller, R. E., Dagle, G. E., Malaga, C. A., and Baer, J. E (1990). Hypercalcemia and disseminated histoplasmosis in an owl monkey. J. Med. Primatol. 19, 675-680. Welshman, M. D. (1985). Management of newly imported primates. Ann. Technol. 36, 125-129. Wenner, H. A., Abel, D., Barrick, S., and Seshumurty, P. (1977). Clinical and pathogenetic studies of Medical Lake virus infections in cynomolgous monkeys (simian varicella). J. lnfec. Dis. 135, 611-622. Whary, M. T., Saunders, K. E., Esteves, M. I., Wood, J., Peck, O. C., and Fox, J. G. (1999). A novel Helicobacter sp. isolated from inflamed colonic tissue in cotton-top tamarins is associated with a specific humoral immune response. Gastroenterology 116, A845. Whiteley, H. E., Everitt, J. I., Kakoma, I., James, M. A., and Ristic, M. (1987). Pathologic changes associated with fatal Plasmodiumfalciparum infection in the Bolivian squirrel monkey (Saimiri sciureus boliviensis). Am. J. Trop. Med. Hyg. 37, 1-8. Whitney, R. A. (1995). Taxonomy. In "Nonhuman Primates in Biomedical Research: Biology and Management" (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 33-49. Academic Press, San Diego. Wiebe, R. H., Williams, L. E., Abee, C. R., Yeoman, R. R., and Diamond, E. J. (1988). Seasonal changes in serum dehydroepiandrosterone, androstenedione, and testosterone levels in the squirrel monkey (Saimiri boliviensis boliviensis). Am. J. Primatol. 14, 285-291. Wikse, S. E., Fox, J. G., and Kovatch, R. M. (1970). Candidiasis in simian primates. Lab. Anim. Care 20, 957-963. Wilbert, R., and Delorme, M. (1927). Note sur la spirochetose icteroh6morrhagique du chimpanzee. C.R. Soc. Bull. 98, 343-345. Wildt, D. E., Doyle, L. L., Stone, S. C., and Harrison, R. M. (1977). Correlation of perineal swelling with serum ovarian hormone levels, vaginal cytology, and ovarian follicular development during the baboon reproductive cycle. Primates 18, 261-270. Williams, L. E., and Bernstein, I. S. (1995). Study of primate social behavior. In "Nonhuman Primates in Biomedical Research: Biology and Management," (B. T. Bennett, C. R. Abee, and R. V. Henrickson, eds.), pp. 77-100. Academic Press, San Diego. Williams, L., Vitulli, W., McElhinney, T., Wiebe, R. H., and Abee, C. R. (1986). Male behavior through the breeding season in Saimiri boliviensis boliviensis. Am. J. Primatol. 11, 27-35. Williams, L. E., Abee, C. R., and Barnes, S. (1988). Allomaternal behavior in Saimiri boliviensis. Am. J. Primatol. 14, 445. Williams, L., Gibson, S., McDaniel, M., Bazzel, J., Barnes, S., and Abee, C. (1994). Allomaternal interactions in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis). Am. J. Primatol. 34, 145-156. Willy, M. E., Woodward, R. A., Thornton, V. B., Wolff, A. V., Flynn, B. M., Heath, J. L., Villamarzo, Y. S., Smith, S., Bellini, W. J., and Rota, P. A. (1999). Management of a measles outbreak among Old World nonhuman primates. Lab. Anim. Sci. 49, 42-48. Wilson, D. E., and Reeder, D. M. (1993). "Mammal Species of the World: A Taxonomic and Geographic Reference," 2nd ed. Smithsonian Institution Press, Washington, D.C. Wirth, H., and Buselmaier, W. (1982). Long-term experiments with a newly developed standardized diet for the New World primates Callithrix jacchus jacchus and Callithrix jacchus penicillata (marmosets). Lab. Anim. 16, 175-181. Wixson, S. K., and Griffith, J. W. (1986). Nutritional deficiency anemias of nonhuman primates. Lab. Anim. Sci. 36, 231-236. Wolfe, L. G., Ogden, J. D., Deinhardt, J. B., Fisher, L., and Deinhardt, F. (1972).

16. NONHUMAN PRIMATES

Breeding and hand-rearing marmosets for viral oncogenesis studies. In "Breeding Primates" (W. I. B. Beveridge, ed.), pp. 145-157. Karger, Basel. Wolfe, L. G., Deinhardt, E, Ogden, J. D., Adams, M. R., and Fisher, L. E. (1975). Reproduction of wild-caught and laboratory-born marmoset species used in biomedical research (Saguinus sp., Callithrix jacchus). Lab. Anim. Sci. 25, 802-813. Wolff, P. L. (1993). Parasites of New World primates. In "Zoo and Wild Animal Medicine Current Therapy 3" (M. E. Fowler, ed.), pp. 378-389. Saunders, Philadelphia. Wolfle, T. L. (1983). Nonhuman primates in research: Trends in conservation, importation, production, and use in the United States. Lab. Anim. 12, 19-27. Wolters, H. J. (1977). Some aspects of role taking behavior in captive family groups of the cotton-top tamarin Saguinus oedipus oedipus. In "Biology and Behavior of Marmosets" (H. Rothe, ed.), pp. 259-278. Mecke-Druck, Duderstadt, Germany. Wright, P. C. (1978). Home range, activity pattern, and agonistic encounters of a group of night monkeys (Aotus trivirgatus) in Peru. Folia Primatol. 29, 43-55.

791 Wright, E C. (1989). The nocturnal monkey niche in the New World. J. Human Evol. 18, 635-658. Wright, P. C. (1994). The behavior and ecology of the owl monkey. In "Aotus: The Owl Monkey" (J. E Baer, R. E. Weller, and I. Kakoma, eds.), pp. 97112. Academic Press, San Diego. Zack, P. M. (1993). Simian hemorrhagic fever. In "Monographs on Pathology of Laboratory Animals: Nonhuman Primates" (T. C. Jones, U. Mohr, and R. D. Hunt, eds.), Vol. 1. pp. 118-131. Springer-Vedag, Berlin and New York. Ziegler, T. E., Stein, E J., Sis, R. E, Coleman, M. S., and Green, J. H. (1981). Supplemental feeding of marmoset (Callithrixjacchus) triplets. Lab. Anim. Sci. 31, 194-195. Ziegler, T. E., Savage, A., Schemer, G., and Snowden, C. T. (1987). The endocrinology of puberty and reproductive functioning in female cotton-top tamarins (Saguinus oedipus) under varying socila conditions. Biol. Reprod. 37, 618-627. Zlotnik, I., Grant, D. P., Dayan, A. D., Earl, C. J. (1974). Transmission of Creutzfeldt-Jakob disease from man to squirrel monkey. Lancet 2, 435438.

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Chap ter 17 Biology and Diseases of Amphibians Dorcas P. O'Rourke and Terry Wayne Schultz

I.

II.

III.

Introduction .................................................

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Taxonomy

793

B.

U s e in R e s e a r c h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Availability and S o u r c e s

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L a b o r a t o r y M a n a g e m e n t and H u s b a n d r y

Biology

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795

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796

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796

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801

A.

A n a t o m y and Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

801

B.

Nutrition

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803

C.

Behavior ................................................

806

D.

Reproduction

807

E.

M a n a g e m e n t and R e p r o d u c t i o n o f X e n o p u s

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P h y s i c a l E x a m i n a t i o n and T e c h n i q u e s

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Diseases ....................................................

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Infectious Diseases

814

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Metabolic/Nutritional Diseases ..............................

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Traumatic Disorders

823

D.

Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

823

E.

Neoplasms

823

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References ..................................................

I.

INTRODUCTION

Amphibians are unique among vertebrate species in that they represent the transition between ancestral aquatic life-forms and more recently evolved terrestrial existence. The word amphibian is derived from the Greek "amphibios," which means "double life." This "double life" accurately describes the aquatic larval stage and postmetamorphic terrestrial lifestyle of many amphibians. This chapter presents an overview of amphibian biology and husbandry, followed by a specific section

LABORATORY ANIMAL MEDICINE, 2nd edition

823

on Xenopus management, and concludes with a discussion of amphibian diseases.

A.

Taxonomy

Class Amphibia is represented by approximately 4300 species contained in 3 orders: Gymnophiona, Caudata, and Anura (Table I). Caecilians comprise the order Gymnophiona. Caecilians are legless, burrowing amphibians that inhabit wet, tropical areas of Asia, Africa, and the Americas. Most are less than

Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ Table I

Scientific and CommonNames of Selected Amphibian Species Common names Hellbender Mudpuppy Axolotl Tiger salamander Red-backed salamander Red-spotted newt Fire-bellied toad African clawedfrog Bullfrog Leopard frog Green tree frog Green poison dart frog Giant toad

Scientific names Cryptobranchus alleganiensis Necturus maculosus Ambystoma mexicanum A. tigrinum Plethodon cinereus Notophthalmus viridescens Bombina orientalis Xenopus laevis Rana catesbeiana R. pipiens Hyla cinerea Dendrobates auratus Bufo marinus

50 cm in length and resemble earthworms, with blunt heads, degenerate eyes, and annular grooves along the body (Zug, 1993). Little is known about caecilian biology, and caecilians are rarely used in a research setting. Salamanders are in the order Caudata. There are approximately 400 species in Caudata, divided into three groups: sirens (eel-like amphibians), primitive salamanders (hellbenders and other related species), and advanced salamanders (mudpuppies, amphiumas, axolotls, newts, and many terrestrial species) (Zug, 1993). Sirens have external gills, no hindlimbs, and reduced forelimbs. They are totally aquatic and inhabit sluggish waterways of southern North America. There are 2 genera in the family Sirenidae, Siren and Pseudobranchus. Siren (true sirens) contains 2 species, and Pseudobranchus (dwarf sirens) is represented by a single species (Zug, 1993). There are 2 families of primitive salamanders. Cryptobranchidae includes hellbenders (Cryptobranchus sp.) of the United States and the giant salamanders (Andrias sp.) of Asia. Cryptobranchids are neotenic (condition where salamanders retain larval characteristics while becoming fully functional, reproducing adults). They are aquatic and have gill slits but no external gills. Respiration is almost exclusively cutaneous, and the skin lies in extensive, fleshy folds on the sides of the body. The head and body are flattened. Andrias can reach a length of 1.5 meters (5 feet); it is the largest salamander in the world. Hynobiidae, an exclusively Asian family comprising over 32 species, is the second family of primitive salamanders. Most members of this group are smaller than the cryptobranchids, have stout bodies, and undergo complete metamorphosis (Conant and Collins, 1991; Zug, 1993). Six families make up the advanced salamanders. Amphiumidae contains 3 species of Amphiuma, which superficially resemble sirens. Amphiuma, however, lack external gills and can reach an adult length of over 1 meter. Proteidae contains 2 genera, Proteus and Necturus. Proteus is a cave-dwelling salaman-

der found in Europe. Necturus maculosus, the mudpuppy, has a broad, flat head and well-developed external gills. Mudpuppies are aquatic and are found in east and central North America. There are 2 genera and over 34 species represented in Ambystomatidae. Several species demonstrate neoteny, including Ambystoma mexicanum (the axolotl) and A. tigrinum (the tiger salamander) (Fig. 1). Ambystomatids are predominantly terrestrial, with strong limbs and functional lungs. They are robust animals, and adults of many species can exceed 14 cm in length. Dicamptodontidae comprises 2 genera, Dicamptodon and Rhyacotriton. These salamanders resemble the ambystomatids but are found in moist forests of the Pacific coast. More than 300 species in North and South America and Italy make up the family Plethodontidae. Plethodontids occur in a wide variety of sizes and shapes; however, all are lungless, are quadrupedal, and possess a nasolabial groove. Included in this family are the genera Plethodon, Desmognathus, Eurycea, Gyrinophilus, Pseudotriton, Aneides, and Batrachoseps. Members of the family Salamandridae share some characteristics with Plethodontidae; however, salamandrids possess lungs and have numerous poison glands in their skin. Additionally, they may be brightly colored, an advertisement of their toxicity. Salamandra, Taricha, and Notophthalmus are representative genera of this family. Notophthalmus (newt) has an aquatic larval stage, terrestrial juvenile period (during which the animals are termed "efts"), and aquatic adult stage (Conant and Collins, 1991; Zug, 1993). There are over 3800 frog species in the order Anura, ranging from the Arctic Circle to extreme points in the southern hemisphere (Conant and Collins, 1991; Zug, 1993). All frogs share a common body plan designed for jumping, which allows movement an average of 2 to 10 times the body length. Classification of frog species remains controversial; however, frogs can be di-

Fig. 1. Ambystomatigrinum, the tiger salamander,is a native North American species used in research.

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17. BIOLOGY AND DISEASES OF AMPHIBIANS

vided basically into primitive frogs (3 families), transitional frogs (2 families), and two groups of advanced frogs (2 families in one and 16 in the other). In the primitive frogs, family Discoglossidae contains species used in research, including the fire-bellied toad, Bombina orientalis, and the midwife toad, Alytes obstetricans. The spadefoot toad, Scaphiopus, is in the family Pelobatidae, a member of the transitional frog group. The first group of advanced frogs contains the family Pipidae, in which the genera Pipa and Xenopus are found. Xenopus, although highly specialized, is the most primitive of the pipids, first occurring in the Cretaceous period. The second group of advanced frogs is represented by 16 families (Zug, 1993). Of these, Bufonidae, Dendrobatidae, Hylidae, Leptodactylidae, and Ranidae have species that can be encountered in a research environment. Bufonidae contains the true toads, including the genus Bufo. Toads have warty, thick skin with well-developed parotoid glands (a raised cluster of granular glands located on the head behind the eyes), and males have a Bidder's organ (ovarian tissue located on the cranial pole of the testis). Poison dart frogs (Dendrobates and Phyllobates) are members of the family Dendrobatidae. They are small, active frogs with bright color patterns, which alert would-be predators to the presence of highly toxic skin secretions (a characteristic termed aposomatism). The family Hylidae contains over 250 species of the genus Hyla. Hyla are tree frogs, recognizable by their slender bodies, their long limbs, and the expanded tips of their digits (Fig. 2). Ceratophrys, the horned frogs of South America, are large animals with fleshy protuberances over the eyes and phenomenally wide mouths. These members of the family Leptodactylidae are

Fig. 2. The barking tree frog, Hyla gratiosa, has expanded digit tips characteristic of the genus.

voracious terrestrial predators (Zug, 1993). Ranidae ("true frogs") includes members of the genus Rana. Species used in research are R. catesbeiana (bullfrog), R. grylio (pig frog), R. clamitans (bronze frog), and R. pipiens (northern leopard frog). Ranids are medium to large frogs with smooth skin. In some species (bullfrog, pig frog, and bronze frog), the tympanum of the male is larger than its eye, while the tympanum of the female is the same diameter as its eye (Conant and Collins, 1991).

B.

Use in Research

Amphibians have been used in research for many years. Xenopus laevis, the African clawed frog, is probably the most widely used and easily recognized amphibian research subject. Xenopus was originally used in pregnancy assays, when it was discovered that injection of a pregnant woman's urine into the dorsal lymph sac of a female Xenopus caused the frog to begin laying eggs. This method of pregnancy detection was soon replaced; however, the clawed frog remained popular with developmental biologists because of its ability to reproduce yearround when injected with commercially available hormones. Xenopus has also been used in cell and developmental biology research (Gurdon, 1996). Once normal clawed-frog development was documented (Nieuwkoop and Faber, 1994), Xenopus became the subject of developmental toxicology investigations and standardized as the FETAX (frog embryo teratogenesis assay: Xenopus) system (Dumont et al., 1983; Dawson and Bantie, 1987; Burkhart et. al., 1998). Currently, the most common use of Xenopus involves cell and molecular biology research. Oocytes of clawed frogs, when injected with foreign DNA or messenger RNA, will allow expression of this genetic material. Thus, Xenopus use has become an integral part of molecular biology laboratories (Sive et al., 2000), and clawed frogs are present in virtually all animal facilities supporting this type of research. Other amphibian species are used in research and teaching. Mudpuppies (Necturus) have traditionally been the subjects of comparative anatomy laboratories. Axolotls (Ambystoma mexicanum) are used in developmental research. Limb regeneration in axolotls and newts has been heavily investigated (Brockes, 1994). Frogs (usually bullfrogs and leopard frogs) have been extensively utilized in teaching physiology and in conducting physiology research (Karnes et al., 1992; Williams, 1997). Because of their ability to regurgitate easily, these species have also been used to study the effects of antiemetics (Kawai et al., 1994; Tai et al., 1995). A frog model has been developed and is being used to test antinociceptive (pain-relieving) effects of analgesics (Stevens, 1992). The pharmacologic and chemical properties of compounds secreted by amphibian skin are widely studied and characterized (Shen, 1995; Daly, 1995). Proteins isolated from frog eggs and embryos demonstrate anticancer activity (Rybak et al., 1996). Frogs have also been used aboard the

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space station M i r to explore the effects of microgravity posture, behavior, and motion sickness (Suzuki et al., 1993; IzumiKurotani et al., 1997). In addition to being used as research animal models, amphibians are studied extensively in the laboratory and the field. These studies focus on the ecology, behavior, and conservation of amphibians themselves, and are critically important as biologists strive to understand and correct the worldwide decline of amphibian species. It is essential that the laboratory animal veterinarian be familiar with the biology, husbandry, behavior, and medicine of amphibians in order to provide necessary support for these ongoing research projects.

C.

Availability and Sources

It is widely recognized that many amphibian populations throughout the world are drastically declining in numbers. Therefore, when choosing an amphibian as a model for teaching or research, special consideration should be given to acquiring animals that have been raised in captivity. Commercial vendors such as NASCO, Xenopus Express, Carolina Biological Supply, the University of Indiana Axolotl Colony, and Xenopus I sell colony-reared amphibians. A list of vendors can be accessed through the Institute of Laboratory Animal Resources (ILAR)

website. Using purpose-bred animals will protect wild populations and provide the researcher with a healthier, less stressed, and better-characterized animal model.

D.

Laboratory Management and Husbandry

Amphibians occupy a variety of niches in the wild, and laboratory housing requirements are equally varied. This section will describe general principles of amphibian husbandry, with emphasis on frogs and salamanders used commonly in research. Sources containing-in-depth information on biology and husbandry of a particular species should always be consulted prior to attempting to house that species.

1. PrimaryEnclosures Glass aquaria work very well as primary enclosures for amphibians, especially aquatic species. Plastic shoe boxes and sweater boxes also provide appropriate housing and have the advantage of being stackable. Small terrestrial salamander species such as P l e t h o d o n have been successfully maintained in medium and large plastic petri dishes (Jaeger, 1992). Larger aquatic frogs and salamanders are frequently housed in stainless steel, fiberglass, and Plexiglas tanks (Fig. 3). All cages should be constructed of impermeable, easily sanitized material, and

Fig. 3. EnclosedPlexiglashousing with a slanted floorworkswellfor Rana pipiens and R. catesbeiana.

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17. BIOLOGYAND DISEASES OF AMPHIBIANS should ideally be able to withstand multiple cage washings. Cages should be of adequate height to accommodate behavioral needs of climbing and jumping species, such as tree frogs and bullfrogs. Fitted, nonabrasive lids are required for most terrestrial and many aquatic species to prevent escape. Several commercial vendors, such as Pharmacal Research Laboratories, Inc., Marine Biotech, and Aquatic Habitat, Inc., offer customdesigned Xenopus housing with either flow-through or recirculating systems. Likewise, commercially available research-fish housing units can be adapted for Xenopus or other amphibian species. Amphibians require a moist habitat and can rapidly desiccate if left in a dry environment. Moistened sphagnum moss is a good substrate for many species. Some authors suggest using a layer of heat-treated soil covered with leaves or sphagnum moss. Leaves can be frozen for several days to eliminate arthropod parasites (Wright, 1996). Soil pH is important. In one study, the red-backed salamander (Plethodon cinereus) preferred the most basic pH range offered (pH 6-6.5). Juveniles avoided soils with a pH of less than 3.7. Very acidic pH ranges (pH 2.5-3) were acutely lethal, and a range of pH 3 - 4 caused death within 8 months (Wyman and Hawksley-Lescault, 1987). Jaeger (1992) recommends three layers of moistened filter paper for petri dish primary enclosures, and soft, moistened paper towels for larger containers (Fig. 4). Echternacht (personal communciation) uses precut, fitted pieces of foam rubber or sponge on the tank bottom, soaked with dechlorinated water (water level should be even with the top of the sponge) (Fig. 5). Because many terrestrial salamander and frog species are secretive, pieces of bark, polyvinyl chloride (PVC) pipe, or other types of hiding places should be provided. Many amphibians do not drink; water is absorbed through the skin (Pough, 1991). Water should be pro-

vided to terrestrial species in shallow dishes, through the moistened substrate, or by misting (Pough, 1991; Wright, 1996). Aquatic amphibians can be housed with or without substrate (depending on species). Aquatic species should also be given hiding places. Polyvinyl chloride pipe works well as a retreat. Semiaquatic frog and salamander species should be provided with a sloping floor or other means of facilitating emergence from the water (Culley, 1992; Wright, 1996). Bullfrogs and leopard frogs do very well in this type of environment. 2.

Water Quality

Fresh, dechlorinated water is preferred for amphibians. Although some species may tolerate low levels of chlorine, many are quite sensitive and will die from exposure to chlorinated water. Allowing open containers of water to age for 2 4 - 4 8 hr, aerating the water, adding sodium thiosulfate, and passing tap water through activated carbon filters are four methods of dechlorination (Fig. 6). Chloramines may be used in place of chlorine in some municipal water systems. Chloramines can be more toxic than chlorine and are best removed with an unused, activated charcoal filter or sodium thiosulfate. When sodium thiosulfate is added to chloramines, ammonia will be released. Zeolites can be used to remove excess ammonia (Gratzek, 1992). An analysis of local water quality should always be obtained prior to establishing an amphibian housing facility. Aquarium or swimming pool test kits will also provide approximate chlorine levels. Copper is toxic to amphibians, and care should be taken to avoid use of pipes made with this metal. Maintaining correct pH of water in the tank or cage is very important. If the preferred pH of a given species is not known, Wright (1996) recommends starting with a pH of 6.8-7.1 (neutral to slightly acidic), then adjusting to a more basic pH if the

Fig. 4. Smallspecies of salamanderscan be housed on moist filter paper in large petri dishes.

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Fig. 5. A saturatedpiece of foam serves as a substrate and keeps humidityelevated in amphibian cages.

animal appears irritated or is anorectic. Verhoeff-de Fremery et al. (1987) prefer housing amphibians at a pH of 7.5 - 8.5, and Horne and Dunson (1994) demonstrated that chronic exposure to low pH affected whole body water and sodium in a terrestrial salamander. Other parameters that can affect amphibian health are dissolved oxygen and ammonia. Test kits are commercially available to monitor water quality. Amphibians can be kept in static, recirculating, or flow-

through systems (Fleming, 1990; Sibold et al., 1993; Bartholomew et al., 1993; Stewart, 1994). Static systems work well for both small and large groups of animals, and many tanks can be plumbed to facilitate draining and refilling. A major disadvantage of this type of system is the need for frequent cleaning. Recirculating systems use filters to remove debris and nitrogenous waste from the water. Although less frequent cleaning is necessary with recirculating systems, filters can easily become overtaxed by high population densities and species that generate large amounts of waste. Flow-through systems run a constant stream of water into and out of the tank. Fresh water is always available with this method; however, a mechanism for dechlorination should be built in the line to assure removal of chlorine.

3.

Fig. 6. Waterfor aquatic amphibians can be dechlorinatedby allowingit to age in containers.

Temperature

Many amphibians spend their existence beneath leaf litter of forest floors or submerged in cool ponds and fast-moving streams. Consequently, most have preferred thermal zones lower than those of reptiles. Wright (1996) has found that tropical species can be maintained at 21~176 (700-85 ~F), while amphibians from temperate regions do well at 18~176 (65 ~ 72~ Pough (1989) recommends lower ranges: 20~176 (680-77 ~F) for tropical species, and 15~ ~C (590-68 ~F) for temperate species. Animals from temperate zones may require seasonal decreases of 5 ~ 1 7 6 (10~176 (Wright, 1996). Jaeger (1992) warns that temperatures in excess of 20 ~C (68 ~F) will prevent salamanders of the genus P l e t h o d o n from assimilating food rapidly enough to meet the needs of their increased metabolic rate. Mattison (1998) provides recommended tem-

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17. BIOLOGY AND DISEASES OF AMPHIBIANS

Table II Recommended Temperatures for Selected Amphibian Species a Species

Pipa pipa Bombina orientalis Rana catesbeiana R. pipiens Dendrobatids Bufo marinus Hyla versicolor Ceratophrys Ambystoma mexicanum A. tigrinum Salamandra salamandra a

Temperature (~ 28 25 15-25 15-25 25-30 23-30 21-27 23-30 10-25 15-25 20 maximum

From Mattison (1998).

peratures for several species (Table II). Other references contain specific information about preferred temperature ranges of less common amphibian species; these should always be consulted to ensure providing the most correct zone for a given species. 4.

Lighting

Most amphibians live in cool, dark environments in the wild; therefore, direct exposure to bright light should in general be avoided. For animal-room lighting, full-spectrum bulbs are suggested, especially if the particular needs of the species are not known (Wright, 1996). Shelter should be provided, so the animal may retreat from light if desired. Light cycles of 12 hr light12 hr dark are satisfactory in most cases. However, if breeding or mimicking the natural habitat of the animal is desired, light cycles will need to be manipulated accordingly. 5.

Airflow

Amphibians require moist habitats, and relative humidities of about 80% work well for most species (Pough, 1991; Wright, 1996). Normal animal-room airflows tend to cause evaporation and dry out wet environments. To prevent this desiccation of habitat and animals, room airflows should be reduced. Alternatively, amphibians can be housed in primary enclosures, which have minimal openings. These will retain moisture; however, care should be taken to ensure that temperatures inside the primary enclosures do not rise to unacceptable levels. 6.

Secondary Enclosures

Conventional animal rooms can be successfully adapted to house amphibians. Walls, floors, and ceilings should be impervious and easy to sanitize. Electrical outlets should have ground-fault interrupters, especially in rooms housing aquatic

species. Individual light timers and thermostats are recommended for each room, since species have different requirements. In cases where very cool and moist conditions are needed and cannot be achieved with normal room manipulations, environmental chambers can be utilized (Jaeger, 1992). 7.

Sanitation

Routine sanitation of amphibian primary enclosures requires special consideration. Many terrestrial species, particularly salamanders, are territorial and mark their environments with pheromones. Excessive cleaning will disrupt normal behavior and can be stressful to the animal (Jaeger, 1992). However, allowing excess buildup of excreta will result in accumulation of toxic metabolites as well as overgrowth of bacteria and fungi. Animals then are placed in a more compromised and stressful environment and can easily succumb to disease. Singly housed, terrestrial animals with ample floor space can be changed every 2 weeks (Jaeger, 1992) and spot-cleaned as necessary between. Group-housed animals require more frequent cleaning. Housing for aquatic species may require cleaning on a daily, weekly, or less frequent basis, depending on stocking density, frequency of feeding, and type of system (filtration, flow-through, etc.). In general, tanks should be cleaned at appropriate intervals to prevent water from fouling. Cleaning solutions should be carefully chosen, and extreme care must be taken to thoroughly rinse away chemical residues. Amphibians are exquisitely sensitive to many compounds, and their permeable skin makes them particularly susceptible to toxins. Phenolics are highly toxic and should not be used around amphibians. 8.

Handling

Amphibians should be handled carefully, and care must be taken to avoid disrupting their protective mucous layer or causing excess secretion of toxins. Gloves should be worn; they must be free of powder and moistened with dechlorinated water. Abrasive paper towels should not be used. Nets must be made of soft fine mesh, and should be an appropriate size to comfortably hold the animal. Aquatic species can be transferred or held in glass jars to prevent removal from water and to protect the sensitive gills. Small terrestrial amphibians can be manually restrained with one hand. Large salamanders should be firmly but gently grasped behind the head and around the pectoral girdle with one hand, and around the pelvic girdle with the other hand (Verhoeff-de Fremery et al., 1987; Crawshaw, 1993; Wright, 1996). Some species will release their tail as a predatoravoidance mechanism; therefore, the tail must not be used for restraint. Frogs, like salamanders, can be held around the pectoral girdle; however, the strong hindlegs must also be restrained to prevent kicking out and slipping through the handler's grasp (Fig. 7). Most amphibians can bite, and some can

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DORCAS P. O'ROURKE AND TERRY WAYNESCHULTZ to the animal using stainless steel or nonabsorbable suture. The suture must pass through a muscle mass in order to permanently anchor the beads; sewing to the skin only can result in sloughing within a few weeks. Passive integrated transponder (PIT) tags are being used with increasing frequency in the field and in the lab (Donnelly et al., 1994; Wright, 1996). Transponders can be placed intracoelomically using a large-diameter needle. Sterile microchip transponders can also be implanted in the dorsal lymph sac (Hoogstraten-Miller and Dunham, 1997). Although this method is more expensive than others, it allows unique identification of animals of virtually any size and body conformation. 10.

Fig. 7. Frogscan be held by supporting the pectoral girdle and restraining the powerful hindlimbs. inflict painful wounds. Additionally, many species secrete skin toxins, and in some species such as Bufo marinus, toxin can be ejected when the parotoid gland is pressed. Therefore, eye protection is recommended when handling these animals (Wright, 1996).

Quarantine

All newly arrived amphibians, especially those that are wildcaught, should undergo a quarantine period. The animals must be housed separately from existing colonies, and their room should be serviced last. Implements should not be shared between quarantine and other rooms. Animals intended for longterm use should be screened and treated, if necessary, for parasites. Diseases should be diagnosed and treated accordingly. The length of quarantine may vary, depending on intended use of the amphibians. Animals for acute studies may be quarantined for a shorter period, but long-term studies dictate a 2- to 4-week minimum quarantine period. Longer periods may be necessary for wild-caught amphibians, due to their questionable health status and unknown exposures.

9. Identification

Each primary enclosure housing amphibians should be labeled with appropriate information. Individual animals can be identified in a variety of ways. In animals with varying color patterns, these patterns can be drawn or photographed and used as unique identifiers (Donnelly et al., 1994). Toe clipping has historically been used in both field and laboratory situations (Donnelly et al., 1994; Wright, 1996). Many amphibians regenerate digits, so this method may be ineffective for long-term studies. Anesthesia (local or general) should be used for toe clipping, and instruments should be sterilized before use. Direct pressure will provide adequate hemostasis in small animals; larger species may require sutures or hemostatic clips. Careful attention must be paid to the animals postamputation for an extended period to ensure that inflammation, infection, and necrosis do not occur (Golay and Durrer, 1994; Wright, 1996). Tattooing and freeze or chemical branding have been used, but are nonpermanent. Skin transplantation (surgically transplanting various-shaped grafts of pale abdominal skin onto the darker dorsal skin) is labor-intensive and can result in graft rejection (Hoogstraten-Miller and Dunham, 1997). Glass or plastic beads have been used to identify individual frogs and salamanders (Verhoeff-de Fremery et al., 1987; Hoogstraten-Miller and Dunham, 1997). These beads are sewn

11.

Zoonoses/Allergies

Amphibians can potentially harbor zoonotic diseases. The most familiar of the amphibian zoonoses is atypical mycobacteriosis, caused by Mycobacterium fortuitum, M. marinum, or M. xenopi. Individuals handling aquatic amphibians that are shedding this disease can develop cutaneous lesions on the fingers and hands. In rare instances, the disease can spread to involve lymph nodes. Immunocompromised individuals are more at risk and can develop severe systemic disease. The best precaution against atypical mycobacteriosis is to wear gloves, especially if there are preexisting cuts or abrasions on the hands. Salmonellosis has been reported in amphibians, but with much less frequency than in reptiles (Woodward et al., 1997). Appropriate precautions include wearing gloves, washing hands thoroughly after handling amphibians or their surroundings, and screening animals periodically for Salmonella. Chlamydia psittaci has been isolated from clinically ill Xenopus laevis (Newcomer et al., 1982; Wilcke et al., 1983; Howerth, 1984). More recently, Chlamydia pneumoniae was identified in a giant barred frog (Berger et al., 1999). Although no reports have been identified that document amphibian-tohuman transmission, both agents should be recognized as potential human pathogens and appropriate precautions taken.

17. BIOLOGY AND DISEASES OF AMPHIBIANS

There have been occasional reports of allergies to frog skin and secretions (Armentia and Vega, 1997; Holtz et al., 1993). Individuals experiencing respiratory or cutaneous signs when working with frogs should seek medical advice concerning allergies.

II.

A.

BIOLOGY

Anatomy and Physiology

1. Integumentary System

The skin of most amphibians is smooth, moist, and glandular. Two primary types of skin glands are present in amphibians: mucous glands and granular glands. Mucous glands secrete a slimy protective layer, which prevents mechanical damage to the skin, facilitates retention of body fluids, and provides a barrier against pathogens. Granular glands synthesize and secrete a variety of compounds that protect against predators, as well as chemicals that have antibacterial and antifungal properties. Granular glands are usually found on the head and shoulders but can be scattered over the body (Zug, 1993; Clarke, 1997). The parotoid gland of toads (Bufo), located on the head behind the eyes, is a raised cluster of granular glands. Fire salamanders (Salamandra salamandra) also have parotoid glands. Defensive compounds found in parotoid and other granular glands can have neurotoxic, cardiotoxic, myotoxic, hallucinogenic, hypotensive, and vasoconstrictive activity (Clarke, 1997). Highly toxic alkaloids found in poison dart frogs (Dendrobatidae), Bufo marinus, and other species of frogs and salamanders can cause vomiting, respiratory paralysis, and death in some cases. In addition to antipredator activity, granular glands of some species secrete peptides, which exhibit antimicrobial activity. The magainins, peptides secreted by Xenopus laevis, inhibit growth of gram-positive and gram-negative bacteria, several fungi, and some protozoal species. Bombesin, caerulein, and bradykinin are among the peptides found in other amphibian species, including fire-bellied toads, midwife toads, and tree frogs. Skin secretions of Bufo have also demonstrated antibacterial and antifungal properties (Clarke, 1997). Other chemical compounds secreted by granular glands of various species include pheromones used in courtship and mating; dermorphin, a potent opioid that may function as an endogenous analgesic; and bioadhesives, which allow temporary entrapment of predators (Clarke, 1997), or permit certain species of small male frogs to adhere to females during breeding (Zug, 1993). Amphibians shed their skin in cycles, which may range from days to weeks. The skin commonly splits middorsally, and the

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animal uses its limbs to climb out of its skin. Shed skins are commonly eaten (Zug, 1993). Some amphibian species have a specialized area of permeable skin in the abdominal region, which is pressed against wet substrates and used to absorb water from the environment for rehydration (Jorgensen, 1997). 2.

Musculoskeletal System

The amphibian skeleton has undergone several modifications. The salamander skeleton is largely cartilaginous. Ribs are absent or greatly reduced in most frogs. Anuran adaptations for jumping include fusion of postsacral vertebrae into an elongate bone, the urostyle, which articulates with the sacral vertebra and the ilium; and fusion of the tibia and fibula into a single, strong bone, the tibiofibula. Many salamander species have tail autotomy, and are capable of regenerating a new tail if the original is lost. Moreover, certain species of newts also have the ability to regenerate limbs, jaws, and ocular tissues (Brockes, 1997). 3.

Respiratory System

Larval amphibians breathe primarily through gills. Adults can retain and use gills, lose gills and develop lungs, breathe with both gills and lungs, or have neither (Fig. 8). Adult plethodontids (lungless salamanders) lack both lungs and gills, and rely on cutaneous respiration. Skin, in fact, is the primary respiratory surface in most amphibians and must be kept moist. In species that use lungs for respiration, air is forced in and out of the lungs by movement of the buccopharyngeal floor (Zug, 1993). Lungs lack alveoli and are very fragile and easily ruptured (Wright, 1996). In many frog species, the trachea is short, and bifurcation occurs close to the glottis; this anatomic feature must be taken into account when performing endotracheal intubation. 4.

Cardiovascular System

Larval amphibians, like fish, have a two-chambered heart. Most adult amphibians have a three-chambered heart, consisting of paired atria and a single ventricle (plethodontid salamanders lack an atrial division, since they lack lungs). Hellbenders, mudpuppies, and sirens have a partial interventricular septum (Burggren and Warburton, 1994). Patterns of blood flow and mixing of oxygenated and deoxygenated blood vary among species, depending on degree of pulmonary respiration, physiological state, and anatomic structures (Zug, 1993). Hepatic portal veins drain blood from the rear half of the amphibian's body; this may impact the pharmacokinetics of drugs with hepatic excretion (Wright, 1996). Plasma osmolarity of amphibians is

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Fig. 8. Axolotlsare large, aquatic, neotenous salamandersthat respire through feathery external gills.

200 mOsm/kg (Walker and Whitaker, 2000). This difference from mammalian osmolarity should be considered when preparing media for in vitro work with amphibian tissues, as well as when administering replacement fluids to dehydrated amphibians. Amphibian Ringer's solution contains 6.6 gm NaC1, 0.15 gm CaC12, and 0.2 gm NaHCO3 per liter of water (Walker and Whitaker, 2000).

7. Excretory System

Salamanders and frogs have a mesonephric kidney and lack the ability to concentrate urine in excess of plasma levels. Aquatic amphibians excrete ammonia, and terrestrial amphibians excrete urea. Most amphibians have a bladder, which functions in water conservation. Many frogs, when frightened, will release urine to deter predators (Wright, 1996).

5. Lymphatic System

The lymphatic system of amphibians drains directly into the venous system. At venous junctions, lymph hearts contract and force lymph into the veins. Large sinuses, collection sites for lymph, are found throughout the amphibian's body. In frogs, a pair of these sinuses lies subcutaneously over the sacral area, lateral to midline. Substances injected into these dorsal lymph sacs will be transported to the venous circulation (Fig. 9). 6.

Gastrointestinal System

Adult amphibians are carnivorous and therefore have a relatively short gastrointestinal tract. The tongue is well developed in all species except pipids and is important for prehending food items. Xenopus and other pipids direct food items into the mouth with their front legs. Melanin is commonly found in the amphibian liver and other abdominal organs, and pronounced pigmentation is not unusual. Vomiting in amphibians is a common defensive mechanism, and it is not unusual for some frog species to evert part of the stomach during regurgitation (Bisazza et al., 1998).

8. Nervous System/Special Senses

Cerebral cortical structure in amphibians is dissimilar to that of higher vertebrates, and the function of the various areas is still controversial (Nieuwenhuys, 1994; Bruce and Neary, 1995). Amphibians have 10 cranial nerves. The hypoglossal nerve (cranial nerve XII), is formed by branches of the first 2 spinal nerves (Anderson and Nishikawa, 1997). A lateral line system (similar to that of fish) is well developed in larval amphibians and is retained by adults of many aquatic species (Fig. 10). The lateral line system is recognizable as a linear arrangement of neuromasts on the head and along the body of the animal. Neuromasts detect changes in water pressure and currents, and function in locating prey (Zug, 1993). Amphibians can detect higher-frequency sound transmitted through the air to the tympanic membrane, but low-frequency vibration is transmitted through the forelimbs and the cranium to the ear. The amphibian eye has two types of rods, red and green, which are responsible for color sensitivity (King et al., 1993). Cones detect only presence of light (Zug, 1993). A vomeronasal (Jacobson's) organ is responsible for odor detection.

803

17. BIOLOGY AND DISEASES OF AMPHIBIANS

summarizes hematologic values for several amphibian species, and Table IV lists serum chemistry values for Rana catesbeiana. In a recent study involving fall-collected bullfrog samples, Cathers et al. (1997) found significant differences between males and females for plasma proteins, sodium, and calcium. No differences were found in the remainder of the complete blood count (CBC) or serum chemistry values. Pfeiffer et al. (1990) investigated hematologic changes in Japanese newts following tail amputation. They observed a decrease in hematocrit during the first 10 days postamputation; hematocrit was restored by day 30. A transient lymphocytosis was also noted in the first few days following amputation. Basophil percentages were consistently high (49-64%) throughout this study, in contrast to that of Jerrett and Mays (1973), who found no basophils in two populations of hellbenders. Incomplete understanding of the function of the various cell types further complicates interpretation of amphibian hematologic and serum chemistry values. Amphibian species have nucleated red blood cells and thrombocytes. Amphibian lymphocytes, monocytes, and thrombocytes function in a fashion similar to that of their higher vertebrate counterparts. Neutrophils appear to respond to infection in a manner similar to that of mammalian neutrophils and reptilian heterophils. Eosinophil and basophil function is largely unknown, and interpretation of elevated percentages of these cell types cannot be extrapolated from mammalian literature (Campbell, 1991).

B. 1.

Fig. 9. The paired dorsal lymph sacs (arrows) of Xenopus and other frog species are a preferred site for injection. 9.

Normal Values

Longevity data are available for many amphibian species (Bowler, 1977; Kara, 1994; Smirina, 1994). Amphibians from northern climates tend to have longer life spans than those from southern latitudes, and larger aquatic salamanders live longer than their smaller, terrestrial counterparts. Xenopus laevis have been documented to live 15 years; Bombina, 11-13 years; bullfrogs, 16 years; and newts, 9 years (Kara, 1994; Smirina, 1994). Cryptobranchus can exceed 25 years; Desmognathus, 10 years; and Bufo americanus, 5 years (Zug, 1993). Age can be most accurately determined in amphibians by counting the layers in bone (Smirina, 1994). Hematologic and serum chemistry values for amphibians can be affected by a number of variables, including season, sex, environmental factors, and method of sample processing. Table III

Nutrition

Adult

Adult amphibians are carnivorous, and many are opportunistic feeders. In the wild, salamanders feed on a variety of vertebrate and invertebrate species. Ambystoma tigrinum has been documented to ingest worms, insects, snails, young field mice, and lizards. Plethodon cinereus eats ants, spiders, flies, beetles, and other small invertebrates. Notophthalmus viridescens feeds on aquatic insects and mollusks. Adult amphibians will frequently cannibalize larvae of their own and other species. Large salamanders such as Cryptobranchus and Amphiuma eat crawfish, fish, frogs, and mammals. Necturus feeds on both small and large prey items (Petranka, 1998). Frogs also feed on a variety of invertebrates, and larger species such as Rana catesbeiana have been noted to eat salamanders, snakes, turtles, and small birds and mammals. Bufo marinus has been reported to eat dog food (Bartlett and Bartlett, 1996). Most species will adapt to dietary modifications required for housing in a laboratory animal facility. Jaeger (1992) successfully kept several salamander species using a diet of Drosophila for smaller individuals, and crickets and earthworms for larger animals. Newts will eat chopped earthworms, fly larvae, and

804

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

Fig. 10. Manyaquatic amphibians,includingXenopuslaevis, havea lateral line system(arrow). Tubifex worms. Many terrestrial and semiaquatic frog species orient visually to prey and require moving food. Rana, Bufo, Bombina, and most other frog species will take crickets, earthworms, and waxworms. Dendrobates requires pinhead crickets (newly hatched). Mudpuppies and axolotls have been maintained on diets of beef muscle and organ meat; however, vitamins and minerals should be supplemented if these are fed (Verhoeff-de Fremery et al., 1987). Raw meat and organs should not be fed to young, growing amphibians, or calcium deficiency will result. Salmonella contamination is also a concern when feeding raw meat and organs. Axolotls will readily eat earthworms and adapt to commercially prepared diets. Most whole vertebrate prey items, if properly nourished, will constitute a balanced diet for amphibians. Bones provide calcium, phosphorus, and magnesium; liver and kidneys provide vitamins; pancreas provides zinc; and thyroids provide iodine. In contrast, the chitinous exoskeleton of many invertebrates is, for the most part, indigestible and contains little to no calcium or other nutrients (Donoghue, 1996). Because insects lack a calcium-rich skeletal structure, a vitamin-mineral mix should be dusted on the prey before feeding (Bartlett and Bartlett, 1996). Alternatively, insects can be fed a diet that is vitaminmineral rich ("gut loaded") immediately before being fed to amphibians. To avoid feeding prey items of poor nutritional quality, crickets, mealworms and similar species can be raised in-house on nutritious diets. Crickets can be kept in a large, deep container on a substrate of sawdust or vermiculite. A shallow dish with

moistened cotton balls or vermiculite serves as both a water source and a place to lay eggs. Crumpled newspaper, egg crates, or paper-towel tubes should be placed about the cage for hiding places. Crickets can be fed laying mash or crushed dog food (Fig. 11). Vegetables such as broccoli, carrots, and alfalfa sprouts can be added, and food can be sprinkled with calcium. Mealworms can be raised in a ventilated container with a fitted lid. Laying mash or chick starter and bran can be used for substrate, and should be covered with a paper or cloth towel. Vegetables and fruit can be added to provide moisture. Food should be replenished periodically (Bartlett and Bartlett, 1996; Mattison, 1998). Adult amphibians should be fed anywhere from daily to twice weekly, depending on species, age of animal, and ambient temperature. 2.

Larvae

Larval salamanders are carnivorous and eat a variety of prey items. Ambystoma tigrinum larvae in the wild consume aquatic mollusks, nematodes, insects, and eggs and larvae of their own and other amphibian species. Notophthalmus viridescens larvae eat small invertebrates, including copepods, snails, and water mites. Hatchling Cryptobranchus have large yolk sacs and apparently rely on yolk for nutrition for the first few months of life (Petranka, 1998). Tadpoles of several frog species tend to be more herbivorous initially after hatching, then convert to an omnivorous and later, a carnivorous diet.

Table Ill

CBC Values for Various Amphibian Species

Rana catesbeiana

Fall a Parameter Red Blood Cells (105/txl) White Blood Cells (103/1~1) Hemoglobin (g/dl) Packed Cell Volume (%) Plasma protein (g/dl) Segs (%) Bands (%) Lymphs (%) Monos (%) Eosinophils ( % ) Basophils (%) Differential/ WBC count method

Spring b

M

F

q

2.4-11.8

3.2-6.0

3.2-6.0

14-27

14-27

2.5-5.2

4.1-4.8

2-52 0-2 36-90 0-3 0 - 18 0-8 Natt & Herrick (direct)

2-52 0-2 36-90 0-3

9.5 40.4

Winter M

Spring F

4.57

4.06

5.98

5.71

10.08

9.65

41.5

31.7

Summer

Fall

Spring

M

F

M

F

M

F

3.17

2.64

6.04

4.61

6.57

5.91

6.4

7.6

12.72

12.30

47.4

44.4

16.1

17.9

6.59

4.80

29.0

24.1

11.6

10.2 8.44

11.04

44.9

33.7

Cryptobranchus alleganiensise

22.8

0.28-1.5 0.04 -0.3

6.2 22.4

m

6.63-12.16 40

30-55

28

2.7-44.8

m m

m

m

m m

m

m h

0 - 18

0-8 Natt & Herrick (direct)

From Cathers et al. (1997). ~ Carmena-Suero et al. (1980). CFrom Harris (1972). aFrom Pfeiffer et al. (1990). eFrom Jerrett and Mays (1973). a

M,F

m

2.4-11.8

Hyla Cynops septentrionalisb pyrrhogaster d

Rana pipiens c

m

m

m

m

m

m

m m

3 6 4 57 Wright/ Leishman (indirect)

46.4-83.7 3.0-19.7 1.0-9.7 0 Neutral red/ Formalin (indirect)

806

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ Table IV

Serum Chemistry Values for Rana catesbeianaa Parameter Sodium (mEq/liter) Potassium (mEq/liter) Chloride (mEq/liter) Total CO2 (mEq/liter) Albumin (gm/dl) Calcium (mg/dl) Creatinine (mg/dl) Aspartate Amino Transferase (U/l) Lactate Dehydrogenase (U/l) Phosphorus (mg/dl) Magnesium (mg/dl) Uric acid (mg/dl) Urea (mg/dl) Anion gap

Male

Female

100 -114 2.0-3.2 65 -86 15-32 1.0-2.1 6.5-8.5 0.7-3.0

107-115 2.0-3.2 65 - 86 15-32 1.0-2.1 8.2-9.6 0.7-3.0

22-91 10 -68 2.5-5.2 1.4-2.57 0-0.1 3 -6 1.3-24.2

22-91 10 - 68 2.5-5.2 1.4-2.57 0-0.1 3- 6 1.3-24.2

Instrument: Monarch Plus, Model 2000. From Cathers et al. (1997). aFall season.

In the laboratory animal facility, tadpoles of Rana pipiens, R. catesbeiana, Bombina, and other species will eat boiled romaine or other leafy dark green lettuce, ground rabbit chow mixed with gelatin, and in some cases, flaked fish food (Nace et al., 1974; Mattison, 1998). Spinach can cause oxalate toxicity and should be avoided. A standard diet has been developed

Fig. 1I.

for bullfrog tadpoles (Culley, 1992). Flores-Nava and GascaLeyva (1997) demonstrated that a Purina shrimp f e e d - g e l a t i n mixture provided best growth rates in bullfrog tadpoles when fed on a vertical platform. Dendrobates larvae are carnivorous and can be fed brine shrimp, flaked fish food, and egg yolk. Most small salamander larvae require young Daphnia, brine shrimp nauplii, and other very small prey items. As larvae grow, larger prey can be fed. Axolotl larvae will eat sectioned earthworms, Daphnia, and Tubifex (Nace et al., 1974; Mattison, 1998). Larval amphibians are voracious feeders and should be fed more frequently than adults. Some species require several small feedings throughout the day; others can be fed once or twice daily. Uneaten food must always be removed to prevent fouling of water.

C.

Behavior

Many flogs and salamanders, particularly terrestrial species, are territorial and should not be housed together. Both males and females of Plethodon cinereus are territorial and will vigorously defend their habitats. Fecal pellets and granular gland secretions are routinely used to mark home ranges. If another animal is encountered, agonistic posturing such as raising the tail and trunk can result. Aggression can escalate to biting. Plethodon cinereus bites the nasolabial groove of its competitor, thereby decreasing that animal's ability to locate food, and

Cricketscan be fed chicken laying mash or dog food to increase their nutritive value.

807

17. BIOLOGY AND DISEASES OF AMPHIBIANS ultimately affecting its survivability (Jaeger, 1981; Petranka, 1998).

D.

Reproduction

1. ReproductiveAnatomy and Physiology Sexual dimorphism exists in many amphibian species; this can be particularly evident during the breeding season. In general, female amphibians are larger than males. Male bullfrogs, pig frogs, and bronze frogs have a tympanum that is larger than the eye; the female counterpart is the same diameter as the eye. Differences in coloration between males and females exist in some frog and salamander species (e.g., Bufo and Triturus). The vocal sacs of male frogs become larger and more pigmented in breeding season. Hyla and related species often have yellowish vocal sacs, and the sacs of Bufo tend to be blackish. Cloacal glands in male salamanders become swollen, resulting in enlarged cloacal lips. Male plethodontid salamanders develop enlarged hedonic glands on the chin; secretions of this gland are rubbed on the female during courtship. Most male frogs develop keratin pads on their thumbs to assist in gripping females during amplexus (a characteristic prolonged breeding embrace). Enlarged teeth can be found in certain frog and salamander males in breeding readiness. In some plethodontid species, these teeth are used to abrade the skin of females and allow introduction of chin gland secretions into her bloodstream (Conant and Collins, 1991; Zug, 1993; Petranka, 1998).

Courtship and reproduction in amphibians range from simple to very elaborate. Internal fertilization occurs in many salamander species. Salamanders may engage in ritual behavioral displays such as the "hula" of Notophthalmus viridescens. In this dance, the male undulates his tail and body while swimming in front of a potential mate. If the female shows interest, the male deposits a spermatophore, and the female picks the packet up with her cloaca. Plethodon cinereus has a more complex courtship. Males use pheromone trails to locate receptive females. The male approaches the female, arches and undulates his tail, then rubs his hedonic gland secretions over her body (using his enlarged teeth to abrade her skin and introduce secretions into her system). The male next aligns himself along the female's body, keeping his tail arched and curled. The female places her chin on his dorsal surface above the vent, and the couple performs a "tail-straddle walk." Finally, the male deposits a spermatophore, the couple moves forward, and the female picks the packet up. The pair separates, the female deposits the fertilized eggs in clusters suspended by a pedicle, and she coils around the eggs until hatching (Petranka, 1998). Frog courtship is no less colorful than that of salamanders. In general, frogs have external fertilization. Males attract mates by vocalizing. When a receptive female is located, the male grasps her with his forelimbs in amplexus. The eggs are expelled, and the male releases sperm to fertilize them. Various adaptations of this basic plan include the courtship of the Surinam toad (Pipa pipa), a relative of Xenopus (Fig. 12). During amplexus, the pair swims in an upside-down circle, with the female releasing eggs

Fig. 12. The female Surinamtoad, Pipapipa, incubates her eggs in the skin of her back. (Photo courtesyof S. Echternacht.)

808

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

at the top of the circle. The male releases sperm, and the fertilized eggs drop and land on the back of the female, who is now at the bottom of the circle. The female's skin grows over the eggs, and the eggs and larvae are carried in this fashion until metamorphosis, when tiny froglets emerge. The midwife toad, Alytes obstetricans, exhibits another breeding strategy. After amplexus and fertilization, the male toad wraps the strands of eggs around his legs. He carries the eggs with him in this fashion, visiting ponds periodically to keep them moist until hatching (Mattison, 1998). The most unusual reported reproductive behaviors include the Darwin's frog and the gastric brooder, Rheobatrachus. The male Darwin's frog takes his newly hatched tadpoles into his mouth, where they migrate to the vocal sacs. The tadpoles remain in the vocal sacs until metamorphosis, then emerge from their father's mouth as froglets. The Rheobatrachus female swallows her fertilized eggs. The eggs and tadpoles are brooded in the female's stomach, where they feed on yolk, undergo metamorphosis, and emerge as froglets. During this brooding period, the female ceases all gastric activity (due to the release of prostaglandin E). Normal gastric activity resumes after the froglets have emerged (Duellman, 1992). 2.

Husbandry

Many amphibian species have been successfully bred in the laboratory. Arginine vasotocin regulates reproductive behavior in amphibians (Boyd and Moore, 1992). In some species, temperature plays a more important role than photoperiod in certain aspects of reproduction (Paniagua et al., 1990). When breeding axolotls, both males and females are kept separately at 22~ (72~ for a week, then placed together in 12~ (54~ water in a shaded container and left undisturbed. Breeding and egg deposition will occur within 2 days. A substrate should be provided for egg attachment. After oviposition, eggs should be removed and incubated in separate containers (Verhoeff-de Fremery et al., 1987; Mattison, 1998). Specific detailed information is also available for laboratory breeding of other amphibian species, including induction of ovulation and artificial insemination (Nace et al., 1974). 3.

Larval Amphibians and Metamorphosis

With few exceptions, larval amphibians are aquatic. Their skin is thin, fragile, and well vascularized to assist in respiration. Gills (internal or external) are present. All larvae lack eyelids. The skeleton is primarily to completely cartilaginous. Dorsal and ventral fins are present on the tail, and both the tail and body are heavily muscled for swimming. Lateral line systems are well developed in all amphibian larvae (Duellman and Trueb, 1986; Zug, 1993). Salamander larvae closely resemble adult animals, having four limbs and other common features. Premetamorphic tad-

poles (frog larvae), in contrast, appear very different from adult frogs. Most tadpoles have a fleshy oral disc surrounding their mouths. This disc can be located dorsally, ventrally, or anteriorly, depending on method of feeding. Teeth are not present; tadpoles have horny beaks and denticles that rasp algae and cut food into smaller pieces. Gills are initially external but are soon covered, along with the forelimbs, by an operculum. Hindlimbs appear late in the larval period (Zug, 1993). Larvae should be housed in well-aerated aquaria in appropriate stocking densities. Care should be taken to separate large from small larvae, especially in cannibalistic species. Pfennig and Collins (1993) discovered that cannibalism develops more slowly if sibling larvae are housed together exclusively. Metamorphosis requires the presence of thyroid hormone and iodine. Species such as the spadefoot toad, which lives in ponds prone to drying, can accelerate metamorphosis by environmentally induced release of corticotropin-releasing hormone (Denver, 1997). As larval development progresses (prometamorphosis) in tadpoles, external gills are resorbed and limbs develop. Immediately before emergence (metamorphic climax), the tail is resorbed, forelimbs break through the operculum, and the hindlimbs become functional (Duellman and Trueb, 1986; Zug, 1993). Water levels should be decreased as larvae undergo metamorphic climax and prepare to emerge. In many cases, a ramp or other object should be placed on the water to facilitate emergence to a terrestrial existence. Newly transformed amphibians can drown if this assistance is not provided. Metamorphosis is a time of immune stress in amphibians, and larvae that undergo metamorphosis at less-than-optimal size become immune-compromised (Rollins-Smith, 1998). Every effort should be made to prevent undue stressors and exposure to infectious agents during the metamorphic period.

E.

M a n a g e m e n t and Reproduction of Xenopus

As previously noted, the African clawed frog, Xenopus laevis, is used extensively in developmental, cellular, and molecular biology research. Current use of Xenopus has expanded into other areas as a result of several factors. First, Xenopus is easier to maintain than terrestrial amphibians. The species is hardy and thrives in captivity. The life cycle is relatively short, making it useful for studies involving multiple generations. The embryos are large and have a high yield of genetic material. Embryo development is well characterized and responses to environmental toxins make it ideal for teratogenesis studies. Finally, oocytes will express foreign nucleic acids when injected, making Xenopus invaluable in expression cloning research. These characteristics have resulted in Xenopus becoming a study animal in many facilities (Gurdon, 1996). Xenopus tropicalis, the only diploid Xenopus species, has re-

809

17. BIOLOGY AND DISEASES OF AMPHIBIANS

cently gained popularity in genetic and molecular research. Xenopus tropicalis is much smaller than X. laevis, has half the haploid genome size per nucleus, and has 20 chromosome pairs (X. laevis has 36 chromosome pairs). The generation time of X. tropicalis is 4 to 6 months. Females lay up to 3000 oocytes per ovulation, and embryos are less variable in development (Amaya et al., 1998). 1.

Natural History

The African clawed frog belongs to the family Pipidae. There are over 17 species of Xenopus and 6 subspecies of X. laevis, which occur throughout Africa. The subspecies are morphologically distinct, with X. laevis laevis being notably larger than the others. Color patterns and digit length also distinguish subspecific differences (Kobel et al., 1996). The clawed frog ranges from central to South Africa in a wide variety of habitats, including rivers, lakes, swamps, ditches, and wells. It appears to prefer still, cloudy water, although populations are found in clear streams. In lakes devoid of fish, Xenopus has evolved to occupy the fish niche. This species occurs at altitudes up to 3000 meters (Tinsley et al., 1996). Xenopus tolerates a fairly wide water-temperature range. Adults become stressed at prolonged temperatures less than 14 ~C (57 ~F) and greater than 26 ~C (81 oF). At these extremes, a decrease in oocyte quality is observed (Wu and Gerhart, 1991). Individuals have been known to survive in ice-covered ponds and desert ponds. When temperatures become too hot (30 ~C, 86 ~F), X. laevis will excavate pits in the cool mud on the bottom of ponds. In drought conditions, it will estivate (Tinsley et al., 1996). The preferred temperature range for this species is approximately 20~176 (68~176 (Etheridge and Richter, 1978). In the wild, Xenopus will breed in both acidic and alkaline water (however, tadpole survival rates decrease in water with a pH of 5). Xenopus laevis will also tolerate elevated salinity (40% seawater) for a short time (Tinsley et al., 1996). 2.

Anatomy and Physiology

Xenopus laevis laevis has a yellowish to darker, spotted to marbled dorsal coloring. The frog's ventral surface is solid yellowish white to spotted. This subspecies has a fifth toe that is much longer than the tibia. Female X. laevis laevis are larger than males and average 110 mm in length (Kobel et al., 1996). Females have large cloacal papillae, and males develop dark inner surfaces on their forearms (nuptial pads). The skin secretions of Xenopus include thyrotropin releasing hormone, caerulein, and xenopsin. Antimicrobial compounds (magainins) are also found in skin secretions (Kreil, 1996). Xenopus lacks a tongue; when feeding, it shreds prey with its hind claws and uses its front feet to shove food into its mouth (Tinsley et al., 1996). The eyes of the clawed frog are located

more dorsally on the head, are lidless, have a convex cornea, and are adapted for vision in air rather than water. Xenopus floats at the water's surface, and vision is directed upward; therefore, objects passing above will elicit a hiding response from the frog. There are two separate olfactory cavitiesmone for detecting scent in water, and one for airborne odors. The lateral line system is located dorsally and ventrally, and is retained in adult animals. The vocal apparatus of the clawed frog is designed for underwater sound production (Deuchar, 1975). The lungs, heart, and liver of Xenopus are large, and the urinary bladder is spherical. Xenopus must come to the water's surface and gulp air, because cutaneous respiration is not as well developed as in other species. Even tadpoles develop and utilize lungs as well as gills for breathing (Deuchar, 1975). During times of drought, Xenopus adapts physiologically by producing urea rather than ammonia. When ample water is available, the frog reverts to production of the more toxic ammonia, which is rapidly dissipated in the water (Tinsley et al., 1996). Life span of X. laevis in the wild is reported to be greater than 10 years. In captivity, the clawed frog can live 15 years or more (Deuchar, 1975; Tinsley et al., 1996). 3.

Housing and Husbandry

When acquiring Xenopus for the laboratory, it is important to verify the species of the animals, as different species may require different temperature and housing conditions. X. laevis are hardy frogs and can be kept successfully in a variety of housing situations. Tanks can be constructed of fiberglass, glass, plastic, or stainless steel (Fig. 13). Unless the sides of the tank are tall, lids should be provided. Screen, metal grills, and perforated plastic lids are commonly used. Xenopus will jump out of tanks if water levels are low (such as during cleaning) and when they are startled. Stackable plastic containers with lids that allow adequate air exchange are useful if space is limited in the facility. In the wild, X. laevis is commonly found in murky water. This dark water provides a visual barrier to predators (Tinsley et al., 1996). In the laboratory animal facility, clawed frogs prefer opaque containers to clear containers; one study demonstrated that darker tank colors actually enhanced frog growth (Hilken et al., 1995). Additionally, partially covered tanks and/or retreats such as PVC pipe, stainless steel rabbit feeders, styrofoam, sponge, round ceramic tiles, terra-cotta pots, and sandstone can provide adequate hiding places and decrease stress (Kaplan, 1993; Major and Wassersug, 1998). Both static and flow-through systems are used. To prevent fouling of water, static systems should be changed after animals have fed. Inexpensive modified, nonrecirculating systems have been described (Dawson et al., 1992; Rogers et al., 1997). Recirculating systems may incorporate charcoal or sand filters to clean and reuse water. Flow-through systems frequently contain a standpipe to drain accumulated water and feces, and a hose or

810

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

Fig. 13. Xenopuslaevis can be group-housedin stainless steel or fiberglasstanks.

other constant-drip water source. Both flow-through and recirculating systems are now commercially available through several companies. Regardless of the system, water should be dechlorinated before itis added to the tank. Water depth can vary from 5 to 20 cm. Although water that is too shallow will increase stress and escape response, no difference in growth was documented between water that was 5, 10, and 20 cm deep (Hilken et al., 1995). Population density within the Xenopus tank is a critical factor in growth and productivity. Suggested stocking densities of adult breeding frogs range from 1 frog per 3 liters to 4 frogs per 5-10 liters (McBride, 1978; Dawson et al., 1992; Hilken et al., 1995; Major and Wassersug, 1998). Increase in stocking densities will cause decrease in growth. Water temperature should be kept 20~176 (68~176 (Etheridge and Richter, 1978; Dawson et al., 1992; Hilken et al., 1994, 1995; Major and Wassersug, 1998). Ranges of 18 ~ 24~ (64~176 are considered acceptable for adequate growth. Some facilities keep Xenopus on a natural light cycle; however, most facilities use a constant 12 hr light-12 hr dark cycle, especially if breeding the frogs year-round (Major and Wassersug, 1998). Housing Xenopus directly under very bright light should be avoided (Hilken et al., 1994). A recent survey of Xenopus husbandry practices concluded that the most commonly used procedures include housing frogs in opaque plastic containers with static water systems, with approximately 1 frog per 4 liters water. Water temperature is maintained at 19~176 and light cycle is 12 hr light-12 hr dark.

Frogs are fed commercial dry chow 3 times a week, and tanks are changed after feeding. Cover is provided in about half of the facilities surveyed; however, many of the labs, which do not use cover, house the animals in opaque tanks (Major and Wassersug, 1998). Adult Xenopus should always be handled with soft nets or gloved hands to prevent skin abrasions and disruptions of the protective mucous layer. Xenopus are most commonly identified by sewing beads on a thread to the skin of the frog (Fig. 14). It has been found necessary to anchor the suture in a muscle mass of the forearm or hindleg to prevent sloughing of suture when skin is shed. Some laboratories have grafted pale skin from the ventral surface to the back of the frog. Individual animals are given unique graft patterns (Verhoeff-de Fremery and Griffin, 1987). This method is time-consuming and has more potential complications than the bead-and-thread method. Sterile microchip transponders can be implanted into the dorsal lymph sac (Hoogstraten-Miller and Dunham, 1997). 4.

Diet

In the wild, Xenopus eat a wide variety of prey items, including aquatic invertebrates, small crustaceans, and insects. Amphibians (including Xenopus tadpoles), small birds, and fish have also been documented as occasional prey items. Clawed frogs use olfaction rather then vision as the primary means of locating food; therefore, they will also scavenge carcasses (Tinsley et al., 1996).

811

17. BIOLOGY AND DISEASES OF AMPHIBIANS

5.

Fig. 14. Xenopus can be individually identified by sewing colored beads to the skin (arrow).

Laboratory-housed Xenopus have been maintained on a variety of diets. Historically, clawed frogs have been fed ground beef liver or heart. Adults can apparently be maintained for several years on these diets; however, tadpoles will develop calcium-phosphorus imbalance if fed only organ meat or muscle (calcium levels are very low and phosphorus levels high in these tissues). This type of food is best used as a supplement for adults fed a more balanced diet. Ground meat or liver can be presented to the frogs on an applicator stick or with a spoon to decrease fouling of the water (Sackin and Sackin, 1991; Dawson et al., 1992). Tanks should also be cleaned after animals have fed, because organ meat and muscle tend to plug drains (Dawson et al., 1992). Several animal facilities have converted from a raw meat diet to commercially prepared diets. Among those in use are salmon chow (Soft-Moist Salmon Diet, Rangen, Inc.), trout chow (Purina), Frog Brittle (Nasco), and Sinking Frog Food (Xenopus Express). Trout chow can be purchased in both floating and sinking forms, comes in various sizes, and does not break down in water. About 5-10 pellets per adult frog are sufficient for a single feeding. Clawed frogs may require a period of several days to 2 weeks to adapt to a new diet (Wu and Gerhart, 1991). Most facilities feed Xenopus from 2 to 5 times a week. Additional foods for clawed frogs include earthworms, mealworms, chick embryos, Tubifex worms, goldfish, and crickets. Feeding chitin-containing animals (crickets, mealworms) excessively can result in intestinal obstruction. Tanks should be cleaned after Xenopus are fed; however, care should be taken not to disturb the frogs for at least an hour, or they may regurgitate their food (Etheridge and Richter, 1978; McBride, 1978; Dawson et al., 1992).

Reproduction

Under ideal conditions, X. laevis will undergo metamorphosis at 2 months and will reach sexual maturity within 8 months of hatching. Cooler temperatures will slow development. In the wild, the breeding cycle corresponds to the onset of the rainy season and subsequent prey abundance (Tinsley et al., 1996). Females reach optimum egg production at 2 - 3 years and continue producing for several years. In captivity, breeding can be accomplished year-round using HCG injections. Females should be bred a maximum of 1 time per month (the ideal induced breeding interval is once every 1 to 4 months). To induce ovulation and breeding, HCG is injected into the dorsal lymph sac of both males and females. Two injections are given to each, spaced 1-5 hr apart. Males receive 400 units of HCG at each injection. Females receive 150250 units on the first injection, followed by 650-850 units on the subsequent injection. Injected frogs are placed into FETAX solution (Table V) and allowed to breed (Dawson and Bantle, 1987). In the laboratory, frogs should be bred in containers with false bottoms, to allow passage of the eggs and prevent ingestion by the parents. Each egg has an individual jelly capsule; therefore, eggs are less likely to clump as they do in other frog species. Larvae hatch 3 days after spawning. 6.

Embryos

Embryos can be collected by gently flushing them from the bottom of the breeding chamber, using a plastic meat baster. They should be placed in a separate container and covered with FETAX solution. Handling large numbers of embryos is easier if their jelly coat is first removed. This can be accomplished by gently swirling them in a 250 ml flask containing 100 ml of 2% (2 g/100 ml) cysteine solution at pH 8.1 (Dawson et al., 1992). A number of factors, including the thickness of the jelly coat and the number of embryos, affects the time it takes to dejelly the embryos (Dawson et al., 1992). Normally, 1-3 min are

Table V FETAX Solution a

Component

Concentration (mg/liter)

NaC1 NaHCO3 KC1 CaC12 CaSO4. H20 MgSO4

625 96 30 15 60 75

aUse deionized, distilled water. From Dumont et al. (1983).

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DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

sufficient, but the process should not go on longer than necessary. After swirling, the cysteine solution is decanted and the embryos rinsed 5 to 8 times with FETAX solution. Dejellied embryos should be placed into a container (e.g., glass petri dish) with a large surface area and covered with FETAX solution. The L-cysteine (the free-base form [Sigma Chemical, St. Louis, Missouri]) should be stored in a refrigerator and cysteine solution prepared fresh just prior to use. Best results are obtained using cysteine powder that is less than 6 months old. 7.

Tadpole Biology

Xenopus tadpoles have functional lungs as well as gills, and will periodically surface to breathe. They grow best when provided access to air, and deprivation of surface air results in increased buccopharyngeal respiration and consequent decreased feeding (Feder et al., 1984). Tadpoles orient themselves parallel to one another and hover in a characteristic head-down fashion, using their undulating tails to direct food particles to their mouths (Fig. 15). They are very efficient filter feeders and eat materials suspended in the water. From hatching through day 4, tadpoles absorb yolk sac; they begin feeding on day 5. In the wild, plankton makes up the bulk of the diet of the Xenopus tadpole. In captivity, strained baby-food green vegetables or algae powder have been used (Dawson et al., 1992; Hilken et al., 1995). Nettle powder, baker's yeast, and bonemeal in a 7:2:1 ratio have also been used successfully (Wu and Gerhart, 1991). A pelleted tadpole diet can be fed to larger larvae. Tadpoles are generally fed daily, and water should be changed after they feed. Xenopus tadpoles can be stocked at a density of 1-10 per liter, with more frequent water changes recommended for higher stocking densities (Wu and Gerhart, 1991). Jars are preferred to

nets when handling or transferring tadpoles, to avoid abrading the skin. When tadpoles are undergoing metamorphosis, they should be placed in shallow water to prevent drowning. 8.

Oocyte Harvest

For nonsurgical oocyte harvest, female frogs can be induced with HCG injections as described previously, then allowed to lay their eggs naturally. Eggs can also be carefully manually expressed from HCG-primed females. Tricaine methane sulfonate (MS222) can be used to anesthetize Xenopus for surgical oocyte harvest. The female frog is immersed in a buffered solution of MS222; a surgical plane of anesthesia is usually reached within 10-15 min. The animal can then be removed and anesthesia maintained if necessary by dripping the solution on the skin. Oocyte harvests can normally be completed in 30 min or less and should not require supplemental anesthesia. Recommended concentrations of MS222 are 0.5-2.0 gm per liter (Crawshaw, 1993), buffered with sodium bicarbonate at 0.42-1.05 gm per liter (Crawshaw, 1992; Schaeffer, 1997). It has been found that 1 gm per liter MS222 buffered with 0.7 gm per liter sodium bicarbonate provides adequate anesthesia for oocyte harvest. Caution should be exercised when handling crystalline MS222, to prevent inhalation and eye and skin exposure. Once the procedure is concluded, the frog can be recovered from anesthesia by rinsing in clean, dechlorinated water and placing into a recovery tank with a very shallow water level. In order to reduce stress, the water used for both induction and recovery can be taken from the Xenopus tank. Frogs should be closely observed during induction and recovery, to ensure that drowning does not occur.

Fig. 15. Xenopustransform from filter-feedinglarvae (A) to carnivorousfroglets (B) during metamorphosis.

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17. BIOLOGY AND DISEASES OF AMPHIBIANS

Fig. 16. Surgical harvest of Xenopus oocytes must be done aseptically.

Aseptic technique must be used whenever harvesting oocytes surgically (Fig. 16). A mask and sterile gloves should be used; gloves are changed between frogs. One procedure involves autoclaving instruments prior to use. They can be sterilized in a glass-bead sterilizer between animals to eliminate the possibility of inadvertently introducing toxic cold sterilants into the surgical site or onto the patient's permeable skin. Although Xenopus skin contains antimicrobial agents, performing a single skin prep with dilute povidone iodine or chlorhexidine solution is recommended. Soaps and scrubs must not be used, for they will destroy the protective mucous layer and be systemically absorbed. The animal is draped with a sterile drape, and an incision is made paramedian in the lower abdominal quadrant on either side (sides can be alternated to allow maximum healing time). Forceps are used to grasp the ovary and exteriorize the oocyte masses. The desired number of oocytes are excised, and the remainder carefully replaced in the coelomic cavity. Muscle and skin layers are closed separately with 4-0 nylon in a simple interrupted pattern. Absorbable suture can be used in the muscle layer. Analgesics can be given, but care should be exercised to ensure that the frog's swimming and other motor functions are not impaired to the point of risking drowning. Analgesics that have proven efficacy in leopard frogs and would likely work in Xenopus include morphine (10 mg/kg intracoelomic q12 hr), flunixin meglumine (25 mg/kg intracoelomic q4 hr), butorphanol (25 mg/kg intracoelomic q12 hr), and xylazine (10 mg/kg intracoelomic q12-24 hr). Flunixin causes an increase in activity, while butorphanol and xylazine have a calming effect (Terril-Robb et al., 1996). The frog is then rinsed with clean dechlorinated water and placed into the shallow recovery container. Response to stimuli should return within 15-30 min, and ability to swim and function fully, within a few hours. Once re-

covered, the frog can be returned to its home tank. Frogs are rested for at least a month between surgeries. Sutures should be removed in about 6 weeks if they have not sloughed out with shed skin. No published information was found on complications associated with multiple oocyte harvests in frogs. The Institutional Animal Care and Use Committee at the University of Tennessee currently allows a maximum of six survival surgeries per frog. Frogs have been necropsied after four surgical oocyte harvests, and only occasional minor adhesions have been identified. More controlled studies are warranted to clarify this issue and define specific end points.

F.

Physical Examination and Techniques

1. Physical Examination Physical examination of an amphibian should begin with observing the animal in its primary enclosure. Attitude, posture, equilibrium (especially if in water), locomotion, body color, respiration, and behavior should be noted. The enclosure should be checked for appropriate temperature, humidity, and cleanliness. Presence and nature of feces and vomitus should be recorded. Once this initial assessment has been made, the animal can be removed from the cage for closer examination. Restraint should be done as described previously (Section I,D,8). Care should always be taken to support the animal's entire body, and to avoid disrupting the protective mucous layer. When handling larger or more-aggressive amphibians, it is advisable to have one person restrain while the second performs the physical exam. The surface of the skin should be thoroughly examined for ulcerations, abrasions, redness, or other lesions. Heart rate can be

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DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

determined while examining the ventral body surface. Abdominal palpation can be attempted; however, many amphibians will inflate the abdomen as a defense mechanism, making palpation difficult. The corneas should be clear, and a blink reflex should be present (except for aquatic species such as Xenopus, which lack eyelids). Nares should be free of exudate. The mouth can be gently opened with a thin, flat plastic speculum, and the oral cavity examined for lesions, excessive salivation, or exudate. Care should be taken to avoid breaking the thin delicate maxillary bones. Withdrawal and righting reflexes can be tested (Raphael, 1993; Wright, 1996). The amphibian should be weighed and its body condition assessed before returning it to its cage. Body condition can be determined by observing the prominence of the skeletal system (particularly the pelvic bones in frogs), and by palpating muscles and abdominal contents (Crawshaw, 1993). 2.

Blood Collection

Blood samples should be collected in lithium heparinized syringes. The midventral abdominal vein can be used to collect blood in both frogs and salamanders. A small-gauge needle appropriate for the amphibian's size (usually a 26- to 27-gauge) is inserted at a point midway between the sternum and pelvis in a cranial direction, and the sample is collected. The ventral caudal vein in salamanders can be used as a phlebotomy site. Frogs have a prominent lingual venous plexus situated beneath the tongue. The tongue can be gently drawn forward with a cottontipped applicator, the plexus punctured with a needle, and the blood sample collected in a heparinized capillary tube (Wright, 1996). Cardiocentesis can be used in both frogs and salamanders. Because of the hazards associated with this technique, animals should be anesthetized. The amphibian must be in dorsal recumbency, and the heartbeat identified. A 25- to 27-gauge needle is inserted beneath the xiphoid at an angle of approximately 10~ ~ to the ventral body wall, and the sample withdrawn from the ventricle (Raphael, 1993; Wright, 1996). If a blood sample cannot be obtained in frogs, lymph should be aspirated from the dorsal lymph sac. Systemic infections can be identified by performing culture and sensitivity on lymph collected in this manner (Raphael, 1993). 3.

also been used in amphibians (Crawshaw, 1993; Raphael, 1993; Wright, 1996). 4.

Injections/Gavaging

Common routes of injection include subcutaneous, intramuscular, intracoelomic, and dorsal lymph sac. Subcutaneous injections can be given in the skin overlying the shoulder or pelvis. The muscles of the forelimbs in frogs and the epaxial muscles in salamanders are locations for intramuscular injections (Wright, 1996). Intracoelomic injections should be given off midline in the lower abdomen, and dorsal lymph sac injections are given subcutaneously in the caudodorsal part of the frog's body (over the pelvic area). Gavaging can be accomplished using standard rodent stainless steel gavage tubes, or with IV Teflon catheters in small species (Wright, 1996). 5.

Euthanasia

Amphibians are most commonly euthanatized by immersion in an overdose of MS222 or other immersion anesthetic. Concentrations exceeding 3gm/liter constitute overdoses for most amphibian species. As done for anesthesia, the acidity of the MS222 should be buffered to prevent irritation. Inhalant anesthetics may be used for euthanasia. Isoflurane, halothane, or methoxyflurane can be delivered into an induction chamber for air-breathing species; they can also be bubbled into aquatic systems. Euthanasia solutions such as sodium pentobarbital can be injected via the coelom or the dorsal lymph sacs. Amphibians are extremely resistant to hypoxia; therefore, certain physical methods that may be permissible in mammals are contraindicated. Decapitation alone is not considered sufficient; animals must be anesthetized prior to decapitation or double-pithed afterward (Andrews et al., 1993; Beaver et al., 2000). Cooper et al. (1989) recommend complete destruction of the brain by concussion if physical means are necessary. Because of the tolerance of many species to cold temperatures, hypothermia is not appropriate as either an anesthetic or euthanasia agent (Schaeffer, 1997; Cooper et al., 1989; Andrews et al., 1993; Beaver et al., 2000). Quick-freezing of anesthetized amphibians in liquid nitrogen may be acceptable (Cooper et al., 1989; Andrews et al., 1993; Beaver et al., 2000).

Other Diagnostic Tests

Fecal examination is performed as for other species. Specimens collected for culture and sensitivity should be incubated at both standard and room temperatures. Impression smears, skin scrapings, and abdominocentesis are performed as for other species. Biopsies can be taken under MS222 anesthesia, and the skin closed with nonabsorbable suture or tissue glue. Radiology is very useful in identifying foreign bodies, impactions, and pneumonia, and for assessing skeletal abnormalities. Dental film works well for small amphibians. Fluoroscopy, endoscopy, and transillumination (using an intense, cool light source) have

lIl.

A. 1.

DISEASES

Infectious Diseases

Bacterial

a.

Redleg

i. Etiology. The organism most commonly isolated from amphibians with redleg is Aeromonas hydrophila, a gram-

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17. BIOLOGY AND DISEASES OF AMPHIBIANS

negative bacterial rod. Aeromonas is frequently found in the aquatic environment and can be a normal inhabitant of the intestinal tract of healthy frogs (Hubbard, 1981; Hird et al., 1981). Stress and subsequent immunosuppression predispose amphibians to colonization by Aeromonas. The organism is transmitted to susceptible animals through the tank water. Aeromonas has been responsible for mass mortality in both wild and captive populations (Hubbard, 1981; Nyman, 1986; Rafidah et al., 1990). ii. Epizootiology and transmission.

iii. Pathogenesis. Waterborne Aeromonas will colonize the skin and visceral organs of frogs and salamanders. Course of the disease can be either acute or chronic (Rafidah et al., 1990). In acute disease, septicemia frequently results (hence the name "redleg").

Signs of acute infection include petechiation and ulceration of the skin, particularly evident on the legs and abdomen (Fig. 17). Lethargy, anorexia, and ascites are also seen. Ocular and periocular inflammation is often noted with this disease. Chronically infected animals exhibit ascites and neurologic signs (Hubbard, 1981; Nyman, 1986; Rafidah et al., 1990; Crawshaw, 1993; Williams and Whitaker, 1994; Wright, 1996). iv. Clinical signs.

vii. Treatment. Appropriate treatment is based on culture and sensitivity. Tetracycline (50 mg/kg PO BID) can be effective against Aeromonas. Valuable animals should be concomitantly treated with aminoglycosides (5 mg/kg IM q48 hr). Chloramphenicol at 50 mg/kg IM, IP q24 hr has also been effective against Aeromonas (Raphael, 1993; Crawshaw, 1993; Wright, 1996). Stoskopf et al. (1987) found that gentamicin at 2.5 mg/kg IM q72 hr provided therapeutic blood levels in Necturus housed at 3 ~C. Another study demonstrated that Rana pipiens required 3 mg/kg IM SID to achieve therapeutic levels when housed at 22~ (Teare et al., 1991). Riviere et al. (1979) showed that immersion in a solution of gentamicin sulfate (1 mg/ml) would provide therapeutic levels in R. pipiens; however, Teare et al. ( 1991) demonstrated that this concentration resuited in increasing serum levels and death after 120 hr. viii. Control. Affected animals should be isolated and husbandry practices reviewed to ensure that appropriate water quality, temperature, stocking density, and food are provided. The environment should be thoroughly cleaned and disinfected.

v. Necropsy findings. Hepatic necrosis, splenic congestion, and other lesions consistent with septic thrombi are commonly seen (Wright, 1996).

ix. Prevention. Amphibians should be housed in clean, dechlorinated water with proper stocking density and temperature. They should be provided nutritious food on a feeding schedule appropriate for the species. Newly arrived animals must be quarantined separately and thoroughly examined before introduction into the existing colony. Animals should be colony-reared rather than wild-caught if at all possible.

vi. Differential diagnoses. Organisms that cause similar lesions include Flavobacterium, Pseudomonas, and Proteus (Crawshaw, 1993; Wright, 1996).

x. Research complications. Redleg can severely decimate research populations of adult and larval amphibians. Data can be affected by using chronically infected animals.

Fig. 17. Bacterialsepticemiacausesthe subcutaneoushemorrhageseen in redleg(arrow).

816

b.

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

Pseudomonas

i. Etiology. Pseudomonas, a gram-negative rod, is commonly found in the aquatic and terrestrial environment. ii. Epizootiology and transmission. The organism is a waterborne opportunist, which typically causes secondary infections in immunosuppressed animals. iii. Pathogenesis. In Necturus, the organism colonizes the gills (Anver and Pond, 1984). Pseudomonas has also been implicated in ulcerative disease in axolotls (Crawshaw, 1993). iv. Clinical signs. In axolotls and other amphibian species, Pseudomonas is associated with skin sloughing and ulceration. Necturus become septicemic and die (Raphael, 1993; Anver and Pond, 1984). v. Necropsy findings. Lesions in the mudpuppy include necrotic gray foci on the gills and cutaneous hyperemia (Anver and Pond, 1984). vi. Differential diagnoses. Other bacterial agents that may cause ulceration, skin sloughing, and hyperemia include Aeromonas and Proteus. vii. Treatment. Gentamicin and chloramphenicol are the antibiotics of choice in treating Pseudomonas. Doses are the same as for Aeromonas. viii. Control. Control is the same as for Aeromonas and other waterborne opportunistic bacteria. ix. Prevention.

Prevention is the same as for Aeromonas.

x. Research complications. As with Aeromonas, Pseudomonas can cause significant morbidity and mortality in research amphibians, thus preventing accurate data collection.

c.

Mycobacteriosis

i. Etiology. The mycobacteria most frequently responsible for disease in amphibians are Mycobacterium xenopi, M. fortuitum, and M. marinum. More recently, M. chelonae has been reported as a pathogen in a colony ofXenopus laevis (Green et al., 2000). These acid-fast organisms are common saprophytes of soil and water (Crawshaw, 1993; Wright, 1996). ii. Epizootiology and transmission. Transmission of mycobacteriosis is most likely through traumatized skin of an immunocompromised animal. The disease does not appear to be highly infectious from animal to animal (Raphael, 1993).

iii. Pathogenesis. The organisms colonize amphibian skin and form nodules; they can also spread to the viscera and form granulomas in organs (Crawshaw, 1993). A recent study with Rana pipiens demonstrated chronic, nonlethal granulomatous disease in immunocompetent frogs; steroid-treated frogs developed an acute, lethal disease (Ramakrishnan et al., 1997). iv. Clinical signs. Typically, amphibians suffering from mycobacteriosis will demonstrate wasting in spite of a good appetite. Pneumonia may develop. Nodules or ulcers may be seen on the skin surface. Extensive skin lesions can interfere with cutaneous respiration. As the disease progresses, animals become more debilitated and eventually die (Crawshaw, 1993; Raphael, 1993). v. Necropsy findings. Gross lesions include yellowish white dermal and visceral granulomas and ulcerations. Visceral granulomas are most commonly found in the liver. Acid-fast organisms frequently are present in the granulomas (Anver and Pond, 1984). vi. Differential diagnoses. Fungal infections can cause cutaneous ulcers and granulomas. Lesions can be cultured and examined histologically with acid-fast stains to distinguish between mycobacterial and mycotic infections. Mycobacterium fortuitum will grow at both room and human body temperature when cultured. vii. Treatment. Valuable animals can be treated with amikacin (5 mg/kg SC, IM, IP q 2 4 - 4 8 hr) or enrofloxacin (5 mg/kg SC, IM q24 hr) (Raphael, 1993). Because of the zoonotic potential, most affected animals are culled. viii. Control. Disease can be controlled by isolating and treating or culling affected animals, and by cleaning and disinfecting the environment. ix. Prevention. Good husbandry (clean environment, appropriate food and temperature, lack of abrasive objects in tank, and low stocking density) will help prevent mycobacteriosis. x. Research complications. Debilitated animals are inappropriate as research subjects. Additionally, this disease is zoonotic, particularly to immunocompromised humans. d.

Flavobacterium

i. Etiology. Flavobacterium is a gram-negative, aerobic rod that is found in both soil and water. The organism is pigmented yellowish orange. Flavobacterium meningosepticum and F. indologenes have caused disease in leopard frogs, and F. meningosepticum and F. oderans disease outbreaks have been identified in Xenopus laevis colonies (Green et al., 1999).

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17. BIOLOGY AND DISEASES OF AMPHIBIANS

ii. Epizootiology and transmission. The organism is typically present in water and can enter through a wound or abrasion. Disease can occur in animals that are not stressed (Olson et al., 1992). iii. Pathogenesis. Flavobacterium indologenes can spread to multiple organs, ultimately causing septicemia. Flavobacterium meningosepticum affects the nervous system (Olson et al., 1992; Taylor et al., 1993). iv. Clinical signs. Signs of F. indologenes infection include weight loss, edema, ascites, petechia, dyspnea, uveitis, corneal edema, and incoordination. Flavobacterium meningosepticum causes head tilt, circling, ataxia, anorexia, and ocular opacity (Olson et al., 1992; Taylor et al., 1993). Flavobacterium meningosepticum can also cause ascites, petechial hemorrhage, lethargy, and other signs of septicemia (Green et al., 1999). v. Necropsyfindings. Histologic changes associated with F. indologenes are vascular congestion and hemorrhage, panophthalmitis with conjunctival and corneal edema, cardiac and skeletal myositis, and hepatocellular degeneration and necrosis (Olson et al., 1992). Flavobacterium meningosepticum causes panophthalmitis, meningitis, and otitis (Taylor et al., 1993); macrophage and neutrophilic infiltration of liver, spleen, and kidney has also been described (Green et al., 1999). vi. Differential diagnoses. Aeromonas will cause septicemia; however, panophthalmitis and corneal edema are not consistently seen with Aeromonas. Culture and sensitivity will distinguish between the two organisms. vii. Treatment. Flavobacterium can be treated with trimethoprim sulfa (3 mg/kg SC, IM, PO q24 hr) (Raphael, 1993). viii. Control. Sick animals should be isolated and the environment cleaned and disinfected, as for other bacterial diseases. ix. Prevention. The same husbandry practices used to prevent Aeromonas should help prevent outbreaks of Flavobacterium. x. Research complications. This organism has the same epizootic potential as Aeromonas and can seriously affect research colonies. e.

Salmonella

i. Etiology. The genus Salmonella includes a single species (S. choleraesuis) that comprises 2300 serovars and is subclassified into 7 subgroups based on DNA similarity and host range (Miller and Pegues, 2000). Salmonella can cause disease in many vertebrates, including amphibians.

ii. Epizootiology and transmission. The organism is most commonly shed by the fecal-oral route. An aquatic environment facilitates transmission. iii. Pathogenesis. The organism colonizes the intestinal tract; it can also spread via the blood. iv. Clinical signs. Affected amphibians exhibit anemia, lethargy, anorexia, and diarrhea (Raphael, 1993; Crawshaw, 1993). v. Necropsy findings. Gross and histopathologic lesions are consistent with enteritis and septicemia. vi. Differential diagnoses. Other bacteria can cause septicemia. Blood or lymph cultures will identify the causative agent. Cloacal and fecal cultures will help determine the cause of diarrhea (Raphael, 1993). vii. Treatment. Appropriate antibiotics should be selected based on culture and sensitivity results. viii. Control. Isolation and disinfection of the environment should be done as for other bacterial agents. Certain Salmonella species are zoonotic; care should be taken when handling infected animals. ix. Prevention. Amphibians should be obtained from reliable, colony-bred sources; quarantined; and maintained in appropriate conditions. x. Research complications. Anemia and diarrhea may affect results in physiologic and other types of studies. 2.

Viral/Chlamydial

a.

Luckg Tumor Herpesvirus

i. Etiology. The Luck6 tumor herpesvirus (LTHV) has icosahedral morphology and is 95-110 nm. It occurs spontaneously in the northern leopard frog, Rana pipiens. ii. Ep&ootiology and transmission. The virus replicates during cool (hibernation) winter temperatures and is shed during spawning. When warmer temperatures of summer occur, viral replication ceases and tumor growth begins. If summer flogs are cooled down again, the inactive tumors will begin to demonstrate herpesvirus replication (Mizell, 1985; Williams et al., 1996). iii. Pathogenesis. The virus causes renal adenocarcinomas in R. pipiens. Tumor growth is rapid during the warm months of summer but stops during winter virus production. With warmer temperatures, tumor growth resumes, and most frogs die after spawning (Wright, 1996).

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DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

iv. Clinical signs. Affected frogs may not show signs until disease is well advanced. Emaciation, lethargy, ascites, and death are most commonly seen. v. Necropsy findings. At necropsy, one or more whitish tumors can be found on either or both kidneys. Tumors can be quite large and metastasize to other organs. Histologically, the tumor is a papillary adenocarcinoma. Ascitic fluid may contain neoplastic cells. In winter flogs, eosinophilic intranuclear inclusions may be seen in renal cells (Anver and Pond, 1984; Wright, 1996). vi. Differential diagnoses. Lucke tumor herpesvirus can be distinguished from other tumors by history and necropsy. vii. Treatment.

There is no treatment for this disease.

viii. Control. Affected frogs should be culled and not allowed to reproduce.

v. Necropsy findings. Frog necropsies demonstrated petechial and ecchymotic hemorrhage of the skeletal muscle and viscera, dermal ulceration and necrosis, digit necrosis, and erythema of the skin (Cunningham et al., 1996a). Microscopic examination of salamander tissues showed hypertrophy of epidermal, gill, and liver cells, with evidence of viral infection (Jancovich et al., 1997). vi. Differential diagnoses. Lesions appear consistent with those of Aeromonas. This bacterium is present in most described cases of iridovirus infection; however, Jancovich et al. (1997) were unable to reproduce disease using bacteria alone, and Cunningham et al. (1996a) postulate that the iridovirus causes the primary lesions, with secondary invasion by the opportunistic Aeromonas. vii. Treatment. Successful treatment of iridovirus infections in amphibians has not been documented. Secondary Aeromonas infections may be treated as described previously.

ix. Prevention. Purchase of laboratory-reared, disease-free R. pipiens will prevent this disease.

viii. Control. Affected animals should be isolated if possible and given supportive care. Care should be taken not to mechanically transmit virus through handling.

x. Research complications. Asymptomatic animals with early-phase tumors can yield poor research data, particularly if renal physiology studies are being conducted.

ix. Prevention. In laboratory populations, good quarantine and husbandry practices should help in prevention of outbreaks.

b.

Iridovirus

i. Etiology. The organism is an enveloped, icosahedral virus ranging from 140 to 180 nm. Iridovirus or iridovirus-like particles have been implicated in disease outbreaks involving R. temporaria, R. esculenta, and Ambystoma tigrinum stebbinsi (Fijan et al., 1991; Drury et al., 1995; Cunningham et al., 1996a; Jancovich et al., 1997). ii. Epizootiology and transmission. This virus has been associated with epizootics in wild populations of frogs and salamanders. Transmission appears to be horizontal and likely waterborne. Previously described tadpole edema virus and viral hemorrhagic septicemia of frogs demonstrate lesions that closely resemble those of currently described iridoviruses. iii. Pathogenesis. Initial lesions are found on the skin, with progression to viscera. iv. Clinical signs. Affected wild flogs were found emaciated, with varying degrees of cutaneous erythema and ulcerations (Cunningham et al., 1996a). Tiger salamanders initially developed small white polyps, which spread to cover most of the epidermis, then progressed to epidermal hemorrhaging, excess mucus production, sloughed skin, lethargy, and anorexia (Jancovich et al., 1997).

x. Research complications. Epizootics can decimate populations of amphibians and seriously impair accurate data collection. c.

Frog Erythrocytic Virus

i. Etiology. Frog erythrocytic virus is a large (450 nm), enveloped icosahedral virus with double-stranded DNA (GruiaGray et al., 1989). ii. Epizootiology and transmission. The virus is found in the cytoplasm of erythrocytes of wild populations of Rana. iii. Pathogenesis. The organism appears to be transmitted mechanically by mosquitoes and midges. The virus infects the red blood cells, with juvenile frogs being most frequently affected. Adults apparently acquire immunity with repeated exposure to the virus. iv. Clinical signs. anemia.

Affected frogs may be weak due to

v. Necropsy findings. Structural changes in erythrocytes due to presence of virus particles can result in anemia. vi. Differential diagnoses. This virus can be distinguished from other organisms producing similar changes by observing

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17. BIOLOGY AND DISEASES OF AMPHIBIANS

trapezoidal inclusions in the cytoplasm of red blood cells, and by observing structural changes in the cells themselves (GruiaGray and Desser, 1992). vii. Treatment.

No treatment is described.

viii. Control. Exclusion of midges and mosquitoes from frog enclosures should control spread of disease. ix. Prevention. Acquisition of colony-reared frogs and prevention of vector contact should effectively stop disease entry. x. Research complications. Anemic frogs will be less hardy and more prone to developing secondary infections, thereby complicating research projects.

d.

Chlamydia

i. Etiology. The agent most commonly involved is Chlamydia psittaci. Chlamydia is the sole genus of the order Chlamydiales (Howerth, 1984). Chlamydia psittaci is best described as the causative agent of psittacosis in birds, and is a zoonotic disease. ii. Epizootiology and transmission. Chlamydia psittaci is most commonly transmitted through the fecal-oral route. Experimental inoculation into the dorsal lymph sacs of Xenopus laevis has caused disease. iii. Pathogenesis. The organism colonizes the lung, liver, spleen, kidney, and heart of Xenopus and causes a pyogranulomatous inflammatory response. iv. Clinical signs. Signs include lethargy, bloating, disequilibrium, and erythema and patchy depigmentation of skin. v. Necropsy findings. Gross lesions consist of hepatosplenomegaly, cutaneous petechiation and ulceration, coelomic effusion, and subcutaneous edema. Dense, basophilic intracytoplasmic inclusions can be found in liver and spleen cells. Interstitial pneumonia, glomerulonephritis, and endocarditis have also been described (Newcomer et al., 1982; Howerth, 1984). vi. Differential diagnoses. Aeromonas and iridovirus infections can produce cutaneous lesions similar to those of Chlamydia psittaci. Diagnosis can be made by observing typical chlamydial inclusions in liver and spleen cells, and by absence of bacteria in lesions (Crawshaw, 1993; Wright, 1996). vii. Treatment. Oxytetracycline (50 mg/kg PO q12-24 hr) or doxycycline may be effective in treating chlamydiosis in amphibians (Raphael, 1993; Wright, 1996).

viii. Control. Affected amphibians should be isolated, and valuable animals can be treated. Chlamydia psittaci is zoonotic; therefore, appropriate precautions should be taken. ix. Prevention. Amphibians should be purchased from reliable, disease-free sources. Appropriate husbandry should be provided, and sick animals should receive a thorough diagnostic workup. x. Research complications. This disease can cause significant animal loss, as well as interfere with physiologic and reproductive studies. Diseased animals pose a potential threat to researchers and animal-care staff. 3.

Parasitic

Amphibians normally host a variety of parasites, without exhibiting signs of disease (Poynton and Whitaker, 1994; Tinsley, 1995). Determination of a pathogenic state is made by identifying parasite burden, concomitant stressors, and the inherent pathogenicity of the parasite in question. Many species of parasites infest amphibians; the following are common examples from each of the major parasite groups. a.

Protozoal

i. Etiology. Many protozoans infest amphibians. Significant species include Entamoeba ranarum (ameba); Trichodina (ciliate); Oodinium and Trypanosoma (flagellates); and Plistophora (microsporidian). ii. Ep&ootiology and transmission. Fecal-oral transmission is common in enteric protozoal infestations. Species of pathogenic protozoa can be transmitted through water and from aquatic vegetation or feeder fish to amphibians. iii. Pathogenesis. Entamoeba cysts are swallowed and directly colonize the colon. Trophozoites can spread to the kidney and liver. Oodinium and Trichodina are external parasites that affect the skin and gills of aquatic amphibians. Ingestion of infected fly larvae is the likely source of Plistophora myotropica in toads. Trypanosoma infects the blood of amphibians and has an indirect life cycle. iv. Clinical signs. Signs of amebiasis include dehydration, anorexia, and emaciation. Feces are loose and bloody; vomiting may also occur. Ascites may be noted with hepatic and renal involvement. Oodinium causes the skin and gills to become grayish in color. Debilitation occurs in chronic cases. Reddened gills and skin cloudiness and ulceration can be observed with Trichodina and other ciliates. Animals affected by trypanosomiasis may be asymptomatic or may die acutely. Plistophora causes anorexia, muscle wasting, and death.

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DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

v. Necropsy findings. Entamoeba causes lesions of the colonic mucosa, suppurative nephritis, and hepatic abscesses. Cysts may be found in the liver and kidney. Splenomegaly is seen in amphibians that die acutely from trypanosomiasis. Necropsy findings associated with Plistophora include muscle atrophy and pale streaks in myofibers. vi. Differential diagnoses. Enteric protozoa can be identified by fecal examination or colonic wash. Skin scrapings and gill biopsies will demonstrate external protozoa. Plistophora sporocysts can be seen in histologic sections of degenerated myofibers. Trypanosoma and other hemoparasites can be identified on Wright-Giemsa-stained blood smears. vii. Treatment. Amebiasis and other enteric protozoal infections can be treated with metronidazole (100-150 mg/kg PO q14 days or 50 mg/kg PO SID for 3 - 5 days). Aquatic species may be treated with 50 mg/liter bath for 24 hr. Enteric ciliated protozoa may be treated with a combination of tetracycline (50 mg/kg PO BID) and paramomycin (50-75 mg/kg PO SID). Trypanosomiasis may respond to a quinine sulfate bath (30 mg/liter for 1 hr). Oodinium, Trichodina, and other external protozoa can be treated with salt baths (10-25 mg/liter SID for 5 - 3 0 min) or acriflavin baths (constant 0.025% bath for 5 days). Copper sulfate has also been used, but this compound can be toxic in some amphibian species (Crawshaw, 1993; Raphael, 1993; Wright, 1996; Whitaker, 1999; Wright, 1999a). viii. Control. Affected animals should be separated from community groups; they should be handled last, and equipment should not be shared. Tanks should be cleaned and sanitized, and water should be changed more frequently. Vectors should be excluded from animal facilities. ix. Prevention. Incoming animals should be quarantined and evaluated for presence of disease and/or pathogenic organisms. Food items and aquarium plants should be treated before introduction (short salt bath followed by thorough rinsing and 1-2 hr acriflavin bath). Whenever possible, purchase colonyreared animals and food items from reliable sources. x. Research complications. Subclinical infections of hemoparasites can confound hematologic and physiologic data. Overt protozoal disease can decrease research populations and render data questionable. b.

Nematodes

i. Etiology. The three most commonly described nematodes of amphibians are Pseudocapillaroides xenopi, Rhabdias, and Foleyella. Pseudocapillaroides is a major parasite of Xenopus laevis; Rhabdias affects both frogs and salamanders; and Foleyella has been described in frogs (Crawshaw, 1993).

ii. Epizootiology and transmission. Pseudocapillaroides is contracted when the eggs are ingested along with sloughed skin from a host frog. The life cycle is direct, with the nematode living in the epidermis of Xenopus and shedding its eggs directly into the aquatic environment. Rhabdias larvae penetrate the host frog's skin and migrate to the lungs. Eggs are coughed up and swallowed; thus, eggs and larvae are found in the gastrointestinal tract. Larval Foleyella are found in the blood; adults live in the body cavity and lymph spaces. iii. Pathogenesis. Pseudocapillaroides xenopi burrows into the epidermis, causing desquamation, debilitation, and secondary infection. Larval nematodes can be found in the kidney. Rhabdias causes damage to pulmonary tissue, and Foleyella can cause debilitation. iv. Clinical signs. Xenopus affected with Pseudocapillaroides have a rough, thickened, pitted appearance to the skin on their dorsal surface, and large patches slough (Fig. 18). Burrows and parasites can sometimes be seen in the epidermis. Debilitation and invasion by opportunistic bacteria and fungi can follow. Pneumonia may be observed in frogs with heavy Rhabdias infection. Foleyella may be asymptomatic or cause weakness and general malaise (Stephens et al., 1987; Crawshaw, 1993; Wright, 1996). v. Necropsy findings. Pseudocapillaroides xenopi lesions are usually confined to the skin and consist of hyperkeratosis, vacuolation, and a mixed inflammatory cell infiltrate. Severe cases involve epithelial erosion and ulceration. Nematodes are present in the lesions. In severe cases, visceral evidence of septicemia may be present (Ruble et al., 1995). Larval nematodes are sometimes found in Bowman's spaces and wrapped around glomerular tufts in the kidneys (Brayton, 1992). vi. Differential diagnoses. Pseudocapillaroides can be diagnosed by wet-mount preparations of desquamated skin or skin scraping. Bipolar eggs and adults will be detectable with these methods. Rhabdias larvated eggs and larvae can be found by fecal examination. Eggs can also be found in tracheal washes. Foleyella can be demonstrated in fresh blood smears or in Wright-Giemsa-stained samples. vii. Treatment. Pseudocapillaroides was initially treated with thiabendazole, but efficacy problems coupled with reports of adverse reactions have resulted in reduction of its use (Ruble et al., 1995; Iglauer et al., 1997). Ivermectin (0.2 mg/kg into the dorsal lymph sac or IM; repeat in 14 days) has proven effective (Dawson et al., 1992; Wright, 1999a). Levamisole has also been suggested as a treatment for Pseudocapillaroides (Cunningham et al., 1996b; Iglauer et al., 1997). Iglauer et al., (1997) recommends levamisole (12 mg/liter water, with each frog having access to 4.17-6.25 liters of treated water for a minimum of

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17. BIOLOGY AND DISEASES OF AMPHIBIANS

Fig. 18. Pseudocapillaroidesxenopi causes epidermalthickening and sloughing.The Xenopus pictured here had concomitantbacterial septicemia. 4 days; treatment repeated in 10-14 days). Wright (1999a) recommends levamisole (8-10 mg/kg IM or intracoelomically q14-21 days or 100-300 mg/liter bath for 24 hr q7-14 days); however, he warns of toxicity problems (flaccid paralysis) at more prolonged exposures. Rhabdias and other nematodes can be treated with ivermectin (2.0 mg/kg topically q14 days, or 0.2-0.4 mg/kg IM or PO q14 days) (Letcher and Glade, 1992; Crawshaw, 1993; Wright, 1999a). Fenbendazole can also be used to treat nematodes (100 mg/kg PO q10-21 days or 50 mg/kg PO SID for 3 - 5 days; repeat in 14-21 days (Wright, 1999a).

viii. Control. Isolation and treatment of affected animals, sanitation of environment, and elimination of vectors will help control nematodes in amphibians. ix. Prevention. zoal diseases.

Prevention can be carried out as for proto-

x. Research complications. Nematode-infested animals can be unthrifty to clinically ill, and therefore poor research subjects. c.

Trematodes and Cestodes

i. Etiology. Amphibians can serve as hosts to both trematodes and cestodes. Polystoma and Gyrodactylus are common trematodes of amphibians, and Nematotaenia is a frequently encountered cestode. Recently, cercariae of the trematode Ribeiroia have been associated with limb abnormalities in Pacific tree frogs (Johnson et aL, 1999).

ii. Epizootiology and transmission. Polystoma is found in the bladder of frogs, and Gyrodactylus is found on the skin and gills of aquatic species of amphibians. Nematotaenia is found in the gastrointestinal tract of amphibians. iii. Pathogenesis. Trematode and cestode infestations may be subclinical. High numbers of cestodes may cause mechanical obstruction of the gastrointestinal tract or wasting and debilitation of the amphibian. iv. Clinical signs and necropsy findings. Polystoma is typically asymptomatic. Gyrodactylus can cause debilitation, dyspnea, anemia, and ulceration of the skin. Nematotaenia can cause unthriftiness and gastrointestinal obstruction. v. Differential diagnoses. Polystoma can be detected by urinalysis. Gyrodactylus requires skin scraping and gill biopsy, and Nematotaenia can be detected by fecal examination (Crawshaw, 1993; Whitaker, 1999; Wright, 1999a). vi. Treatment. Praziquantel (8-24 mg/kg PO, SC, or intracoelomically q14-21 days or 10 mg/liter bath for up to 3 hr; repeat in 14-21 days) has been used to treat trematodes and cestodes in amphibians (Wright, 1996; Wright, 1999a). vii. Control and prevention. Trematodes and cestodes can be controlled and prevented in the same manner as other parasites. viii. Research complications. Debilitated animals make inappropriate research subjects, and subclinical infestations may confound data.

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d.

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

Other

i. Etiology. Acanthocephalans, copepods, leeches, trombiculid mites, and toad flies are examples of other types of parasites that may infest amphibians. ii. Epizootiology and transmission. Acanthocephalans have an indirect life cycle and require an arthropod host. Copepods are seen in aquatic amphibians, and leeches may be found on wild-caught animals. Trombiculid mites are found in soil and leaf litter, and parasitize terrestrial species. Toad flies infest terrestrial anurans. iii. Pathogenesis. Acanthocephalans inhabit the gastrointestinal tract; the other parasites are external. Toad flies lay eggs in the nasal cavity of frogs; larvae eat the nasal passages and the frog's face until the frog dies. iv. Clinical signs and necropsy findings. Acanthocephalan infections may be subclinical; however, weight loss and enteritis can be seen. If the intestinal wall is perforated, peritonitis will result. Leeches and copepods are visible externally. Trombiculid mites can cause erythematous vesicles on the skin of affected amphibians. Toad fly larvae can be seen in the nasal passages of affected frogs. v. Differential diagnoses. Acanthocephalans are detected by fecal examination. Copepods can be detected on skin scrapings. Leeches, mites, and flies are readily visible. vi. Treatment. Salt baths (10-25 gm salt/liter for 15-30 min) can be used to remove copepods and facilitate removal of leeches. Topical ivermectin may be effective in treating trombiculid mites. Treatment for toad flies and acanthocephalans is generally unrewarding (Crawshaw, 1993; Raphael, 1993; Wright, 1996; Whitaker, 1999). vii. Control and prevention. Excluding vectors and intermediate hosts is effective in controlling toad flies and acanthocephalans. Avoiding wild-caught animals will reduce problems with leeches and copepods, and heat-treating or freezing leaf litter, soil, and other cage accouterments will eliminate trombiculid mites. viii. Research complications. As in all parasitic infestations, compromised research animals are poor subjects and yield questionable data. 4.

Fungal

i. Etiology. Most fungi that affect amphibians are soil and water saprophytes; infection commonly occurs secondary to stress or disease. The fungal infections most frequently iden-

tiffed in amphibians are saprolegniasis, chromomycosis, and phycomycosis.

ii. Epizootiology and transmission. Saprolegniasis is caused by several fungi, including Saprolegnia. Various pigmented fungi cause chromomycosis, and Basidiobolus is the agent most commonly isolated from cases of phycomycosis. iii. Pathogenesis and clinical signs. Saprolegnia colonizes preexisting skin lesions in aquatic amphibians. A cottony mat of fungal hyphae cover the lesion. Paler tufts are indicative of acute infections, while darker mats indicate chronicity (Crawshaw, 1993; Wright, 1996). Lesions of chromomycosis are usually raised dark nodules; however, they may be ulcerated (Ackermann and Miller, 1992; Wright, 1996). Debilitation and weight loss may also be observed. Phycomycosis produces lesions similar to those of chromomycosis (Wright, 1996). iv. Necropsy findings. Lesions tend to remain cutaneous in saprolegniasis, while visceral granulomas can be seen in chromomycosis. v. Differential diagnoses. mounts and fungal cultures.

Diagnosis can be made by wet

vi. Treatment. Saprolegniasis can be treated with saltwater baths (10-25 gm/liter for 5 - 3 0 min SID) or benzalkonium chloride (2 mg/liter bath for 10-60 min) (Wright, 1999a). Groff et al. (1991) successfully treated Basidiobolus ranarum with benzalkonium chloride (2 mg/liter bath for 30 min every other day for 3 treatments; repeat in 8 days). Several treatments for chromomycosis have been tried; results are unrewarding (Ackermann and Miller, 1992; Wright, 1996). vii. Control and prevention. Fungal infections can be minimized by keeping animals healthy and unstressed in a clean environment. viii. Research complications. Saprophytic fungi can colonize surgical wounds and other skin lesions, compromising the health of the research animals.

B.

Metabolic/Nutritional Diseases

Amphibians are susceptible to several metabolic and nutritional diseases, including metabolic bone disease, lipid keratopathy, spindly leg, gas bubble disease, and dehydration. Metabolic bone disease (MBD) is seen in both adult and larval amphibians that have been fed diets deficient in calcium or with an improper calcium-phosphorus ratio. Tadpoles require significant amounts of calcium in their diet. Animals deficient in calcium will mobilize the mineral from bones in order to keep

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17. BIOLOGY AND DISEASES OF AMPHIBIANS

serum calcium levels normal. Signs of MBD in tadpoles include folding fractures, scoliosis, and "rubber jaw" (inordinate mandibular flexibility due to calcium loss). Adult frogs exhibit abdominal bloating and tetany following exertional movement. Diagnosis of MBD is based on clinical signs and radiographs (mammography film works well when radiographing small amphibians). Radiographic changes include decreased bone density and pathologic fractures. Treatment of MBD consists of appropriate calcium supplementation (daily 5% calcium gluconate baths, injectable vitamin D/calcium, oral supplementation with tropical fish food slurry), and change to an appropriate diet. The disease can be prevented by feeding larval and adult amphibians appropriate diets (Crawshaw, 1993; Wright, 1996). Lipid keratopathy is seen in female frogs. Affected animals have corneal thickening and opacity, with vascularization, superficial pigmentation, and cholesterol clefts. In some cases, xanthomatosis is associated with corneal changes. Possible etiologies include lipid and cholesterol mobilization associated with egg production, and high levels of dietary fat (from feeding with milk-fed newborn mice) (Williams and Whitaker, 1994). Spindly leg is seen in young frogs, particularly poison dart frogs. Limbs develop abnormally, do not emerge properly at metamorphosis, are thin and poorly muscled, and have angular deformities. Etiology is unknown; theories include genetics, temperature, water quality, oversupplementation of vitamins, and malnutrition. The condition is untreatable, and euthanasia is recommended (Crawshaw, 1993; Wright, 1996). Gas bubble disease is produced by air supersaturation of water. Large amounts of gas accumulate in the vascular system, causing obstruction of blood flow and capillary hemorrhage. Air bubbles are evident in webbing of feet and skin, and permit entry of bacteria, resulting in septicemia. The disease can be prevented by ensuring that water is not supersaturated with air (Colt et al., 1984; Crawshaw, 1993; Raphael, 1993; Wright, 1996). Amphibians require moist environments and are predisposed to dehydration. Signs of dehydration include dull, dark skin, sunken eyes, lethargy, and dry, sticky mucus. Mild dehydration can be treated by immersion in clean, dechlorinated water. Animals in shock can be given dexamethasone (1-2 mg/kg intracoelomically) and hypotonic fluids. Two parts saline to 1 part 5% dextrose is given intracoelomically at 2 - 5 % of the animal's body weight. Subsequent fluid solutions should be 9 parts saline to 1 part sterile water. Antibiotic baths are recommended if epidermis is damaged (Wright, 1999b).

C.

Traumatic Disorders

Traumatic lesions in amphibians are primarily bite wounds caused by cagemates, and abrasions from rough surfaces and

cage tops. Appropriate wound closure (if warranted) and antibiotic therapy are indicated (Wright, 1994). Aggressive animals should be separated, and cages should be free of abrasive surfaces.

D.

Toxins

Amphibians are exquisitely sensitive to a number of toxins, including chlorine and chloramine, nitrite, ammonia, iodine, heavy metals (copper, lead, zinc), PVC adhesives, and pesticides (Whitaker, 1993; Stansley and Roscoe, 1996). Signs associated with toxicities include excess mucus production, irritability, dyspnea, convulsions, paralysis, petechiation, and regurgitation (Wright, 1996). Animals displaying acute signs of toxicity should be removed immediately from their environment and placed in a clean, toxin-free enclosure.

E.

Neoplasms

With the exception of Lucke's renal adenocarcinoma, spontaneous neoplasms are relatively sporadic in amphibians. Ovarian and hepatic tumors are fairly common. Spontaneous tumors are reported more frequently in frogs, and frogs appear more sensitive than salamanders to carcinogen-induced tumors (Anver, 1992). Examples of spontaneous amphibian tumors include skin adenomas, papillomas, fibromas, pulmonary carcinomas, and testicular tumors (Balls and Clothier, 1974).

REFERENCES

Ackermann, J., and Miller, E. (1992). Chromomycosisin an African bullfrog,

Pyxicephalus adspersus. Bull. Assoc. Reptil. Amphib. Vet. 2, 8-9. Amaya, E., Offield, M. E, and Grainger, R. M. (1998). Frog genetics: Xenopus tropicalis jumps into the future. Trends Genet. 14, 253-255. Anderson, C. W., and Nishikawa, K. C. (1997). The functional anatomy and evolution of hypoglossalafferents in the leopard frog, Rana pipiens. Brain Res. 771, 285-291. Andrews, E. J., Bennett, B. T., Clark, J. D., Houpt, K. A., Pascoe, P. J., Robinson, G. W., and Boyce, J. R. (1993). Report of the AVMApanel on euthanasia. J. Am. Vet. Med. Assoc. 202, 229-249. Anver, M. R. (1992). Amphibian tumors: A comparison of anurans and urodeles. In Vivo 6, 435-438. Anver, M. R., and Pond, C. L. (1984) Biology and diseases of amphibians. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 427-447. Academic Press, Orlando, Florida. Armentia, A., and Vega, J. M. (1997). Allergyto frogs. Allergy 52, 674. Balls, M., and Clothier, R. H. (1974). Spontaneoustumors in amphibia. Oncology 29, 501-519. Bartholomew, J. L., Driessen, J. L., Walker-Harrison, P., and Riggs, R. R. (1993). Continuousflow aquatic systemfor long-termhousing of Xenopus laevis. Contemp. Top. Lab. Anim. Sci. 32, 37.

824 Bartlett, R. D., and Bartlett, R R (1996). "Frogs, Toads, and Treefrogs." Barron's Educational Series, Hauppauge, New York. Beaver, B. V., et al. (2000). 2000 report on the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc. 218, 669-696. Berger, L., Volp, K., Matthews, S., Speare, R., and Timms, E (1999). Chlamydia pneumoniae in a free-ranging giant barred frog (Mixophyes iteratus) from Australia. J. Clin. Microbiol. 37, 2378-2380. Bisazza, A., Rogers, L. J., and Vallortigara, G. (1998). The origins of cerebral asymmetry: A review of evidence of behavioural and brain lateralization in fishes, reptiles, and amphibians. Neurosci. Biobehav. Rev. 22, 411-426. Bowler, J. K. (1977). "Longevity of Reptiles and Amphibians in North American Collections." Society for the Study of Amphibians and Reptiles, Oxford, Ohio. Boyd, S. K., and Moore, E L. (1992). Sexually dimorphic concentrations of arginine vasotocin in sensory regions of the amphibian brain. Brain Res. 588, 304-306. Brayton, C. (1992). Wasting disease associated with cutaneous and renal nematodes, in commercially obtained Xenopus laevis. Ann. N.Y. Acad. Sci. 653, 197-201. Brockes, J. R (1994). New approaches to amphibian limb regeneration. Trends Genet. 10, 169-173. Brockes, J. R (1997). Amphibian limb regeneration: Rebuilding a complex structure. Science 276, 81- 87. Bruce, L. L., and Neary, T. J. (1995). The limbic system of tetrapods: A comparative analysis of cortical and amygdalar populations. Brain Behav. Evol. 46, 224-234. Burggren, W. W., and Warburton, S. J. (1994). Patterns of form and function in developing hearts: Contributions from non-mammalian vertebrates. Cardioscience 5, 183-191. Burkhart, J. G., Helgen, J. C., Fort, D. J., Gallagher, K., Bowers, D., Propst, T. L., Gernes, M., Manger, J., Shelby, M. D., and Lucier, G. (1998). Induction of mortality and malformation in Xenopus laevis embryos by water sources associated with field frog deformities. Environ. Health Perspect. 106, 841848. Campbell, T. W. (1991). Hematology of exotic animals. Compendium 13, 950957. Carmena-Suero, A., Siret, J. R., Callejas, J., and Arpones-Carmena, D. (1980). Blood volume in male Hyla septentrionalis (tree frog) and Rana catesbeiana (bullfrog). Comp. Biochem. Physiol. 67A, 187-189. Cathers, T., Lewbart, G. A., Correa, M., and Stevens, J. B. (1997). Serum chemistry and hematology values for anesthetized American bullfrogs (Rana catesbeiana). J. Zoo Wildl. Med. 28, 171-174. Clarke, B. T. (1997). The natural history of amphibian skin secretions, their normal functioning, and potential medical applications. Biol. Rev. 72, 365379. Colt, J., Orwicz, K., and Brooks, D. (1984). Gas bubble disease in the African clawed frog, Xenopus laevis. J. Herpetol. 18, 131-137. Conant, R., and Collins, J. T. (1991). "A Field Guide to the Reptiles and Amphibians: Eastern and Central North America," 3rd ed. Houghton Mifflin, Boston. Cooper, J. E., Ewbank, R., Platt, C., and Warwick, C. (1989). "Euthanasia of Amphibians and Reptiles." UFAW, London. Crawshaw, G. J. (1992). Medicine and diseases of amphibians. In "The Care and Use of Amphibians, Reptiles, and Fish in Research" (D. O. Schaeffer, K. M. Kleinow, and L. Krulisch, eds.), pp. 41-48. Scientists Center for Animal Welfare, Bethesda, Maryland. Crawshaw, G. J. (1993). Amphibian medicine. In "Zoo and Wild Animal Medicine: Current Therapy 3" (M. E. Fowler, ed.), pp. 131-139. Saunders, Philadelphia. Culley, D. D. (1992). Managing a bullfrog research colony. In "The Care and Use of Amphibians, Reptiles, and Fish in Research" (D. O. Schaeffer, K. M. Kleinow, and L. Krulisch, eds.), pp. 30-40. Scientists Center for Animal Welfare, Bethesda, Maryland.

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

Cunningham, A. A., Langton, T. E. S., Bennett, E M., Lewin, J. E, Drury, S. E. N., Gough, R. E., and MacGregor, S. K. (1996a). Pathological and microbiological findings from incidents of unusual mortality of the common frog (Rana temporaria). Philos. Trans. R. Soc. Lond. B Biol. Sci. 351, 15391557. Cunningham, A. A., Sainsbury, A. W., Cooper, J. E. (1996b). Diagnosis and treatment of a parasitic dermatitis in a laboratory colony of African clawed frogs (Xenopus laevis). Vet. Rec. 138, 640-642. Daly, J. W. (1995) The chemistry of poisons in amphibian skin. Proc. Natl. Acad. Sci. 92, 9-13. Dawson, D. A., and Bantle, J. A. (1987). Development of a reconstituted water medium and preliminary validation of the frog embryo teratogenesis assaymXenopus (FETAX). J. Appl. Toxicol. 7, 237-244. Dawson, D. A., Schultz, T. W., and Schroeder, E. C. (1992). Laboratory care and breeding of the African clawed frog. Lab. Anim. 21, 31-36. Denver, R. J. (1997). Environmental stress as a developmental cue: Corticotropin-releasing hormone is a proximate mediator of adaptive phenotypic plasicity in amphibian metamorphosis. Horm. Behav. 31, 169-179. Deuchar, E. M. (1975). "Xenopus: The South African Clawed Frog." Wiley and Sons, London. Donnelly, M. A., Guyer, C., Juterbock, J. E., and Alford, R. A. (1994). Techniques for marking amphibians. In "Measuring and Monitoring Biological Diversity: Standard Methods for Amphibians" (R. W. Heyer, M. A. Donnelly, R. W. McDiarmid, L.-A. C. Hayek, and M. S. Foster, eds.), pp. 277284. Smithsonian Institution Press, Washington, D. C. Donoghue, S. (1996). Veterinary nutritional management of amphibians and reptiles. J. Am. Vet. Med. Assoc. 208, 1816 - 1820. Drury, S. E. N., Gough, R. E., and Cunningham, A. A. (1995). Isolation of an iridovirus-like agent from common frogs (Rana temporaria). Vet. Rec. 137, 72-73. Duellman, W. E. (1992). Reproductive strategies of frogs. Sci. Am. 261, 80-87. Duellman, W. E., and Trueb, L. (1986). "Biology of Amphibians." McGrawHill, New York. Dumont, J. N., Schultz, T. W., Buchanan, M., and Kao, G. (1983). Frog embryo teratogenesis assay: Xenopus (FETAX)--a short-term assay applicable to complex environmental mixtures. In "Symposium on the Application of Short-Term Bioassays in the Analysis of Complex Environmental Mixtures III." Plenum Press, New York. Etheridge, A. L., and Richter, M. A. (1978). "Xenopus laevis: Rearing and Breeding the African Clawed Frog." NASCO, Fort Atkinson, Wisconsin. Feder, M. E., Seale, D. B., Boraas, M. E., Wassersug, R. J., and Gibbs, A. G. (1984). Functional conflicts between feeding and gas exchange in suspension-feeding tadpoles, Xenopus laevis. J. Exp. Biol. 110, 91-98. Fijan, N., Matasin, Z., Petrinec, Z., Valpotic, I., and Zwillenberg, L. O. (1991). Isolation of an iridovirus-like agent from the green frog (Rana esculenta L.). Veterinarski Arhiv. 61, 151-158. Fleming, L. R., (1990). A standardized method for housing temperate region freshwater aquatic species. Lab. Anim. Sci. 40, 564. Flores-Nava, A., and Gasca-Leyva, E. (1997). Use of artificial grazing substrates in bullfrog tadpole culture. Aquaculture 152, 91-101. Golay, N., and Durrer, H. (1994). Inflammation due to toe-clipping in natterjack toads (Bufo calamita). Amphib. Reptil. 15, 81-83. Gratzek, J. B. (1992). Getting started with aquaria. In "Aquariology: The Science of Fish Health Management" (J. B. Gratzek and J. R. Matthews, eds.), pp. 3-19. Tetra Press, Morris Plains, New Jersey. Green, S. L., Bouley, D. M., Tolwani, R. J., Waggie, K. S., Lifland, B. D., Otto, G. M., and Ferrell, J. E. (1999). Identification and management of an outbreak of Flavobacterium meningosepticum infection in a colony of South African clawed frogs (Xenopus laevis). J. Am. Vet. Med. Assoc. 214, 18331838. Green, S. L., Lifland, B. D., Bouley, D. M., Brown, B. A., Wallace, R. J., and Ferrell, J. E. (2000). Disease attributed to Mycobacterium chelonae in South African clawed frogs (Xenopus laevis). Comp. Med. 50, 675-679.

17. BIOLOGY AND DISEASES OF AMPHIBIANS

Groff, J. M., Mughannam, A., McDowell, T. S., Wong, A., Dykstra, M. J., Frye, E L., and Hedrick, R. P. (1991). An epizootic of cutaneous zygomycosis in cultured dwarf African clawed frogs (Hymenochirus curtipes) due to Basidiobolus ranarum. J. Med. Vet. Mycol. 29, 215-223. Gruia-Gray, J., and Desser, S. S. (1992). Cytopathological observations and epizootiology of frog erythrocytic virus in bullfrogs (Rana catesbeiana). J. Wildl. Dis. 28, 34-41. Gruia-Gray, J., Petric, M., and Desser, S. (1989). Ultrastructural, biochemical, and biophysical properties of an erythrocytic virus of frogs from Ontario, Canada. J. Wildl. Dis. 25, 497-506. Gurdon, J. B. (1996). Introductory comments: Xenopus as a laboratory animal. In "The Biology of Xenopus" (R. C. Tinsley and H. R. Kobel, eds.), pp. 3 8. Clarendon Press, Oxford. Harris, J. A. (1972). Seasonal variation in some hematological characteristics of Rana pipiens. Comp. Biochem. Physiol. A 43A, 975-989. Hilken, G., Willmann, G. H. E, Dimigen, J., and Iglauer, E (1994). Preference of Xenopus laevis for different housing conditions. Scand. J. Lab. Anim. Sci. 21, 71-80. Hilken, G., Dimigen, J., and Iglauer, E (1995). Growth of Xenopus laevis under different laboratory rearing conditions. Lab. Anim. 29, 152-162. Hird, D. W., Diesch, S. L., McKinnell, R. G., Gorham, E., Martin, E B., Kurtz, S. W., and Dubrovolny, C. (1981). Aeromonas hydrophila in wild-caught frogs and tadpoles (Rana pipiens) in Minnesota. Lab. Anim. Med. 31, 166169. Holtz, J., Frechelin, E., Noel, B., and Savolainen, H. (1993). A case of frog allergy: Antigenic skin protein. Int. Arch. Allergy Immunol. 101, 299300. Hoogstraten-Miller, S., and Dunham, D. (1997). Practical identification methods for African clawed frogs (Xenopus laevis). Lab. Anim. 26, 36-38. Horne, M. T., and Dunson, W. A. (1994). Behavioral and physiological responses of the terrestrial life stages of the Jefferson salamander, Ambystoma jeffersonianum, to low soil pH. Arch. Environ. Contam. Toxicol. 27, 232-238. Howerth, E. W. (1984). Pathology of naturally occurring chlamydiosis in African clawed frogs (Xenopus laevis). Vet. Pathol. 21, 28-32. Hubbard, G. B. (1981). Aeromonas hydrophila infection in Xenopus laevis. Lab. Anim. Sci. 31, 297-300. Iglauer, E, Willmann, E, Hilken, G., Huisinga, E., and Dimigen, J. (1997). Anthelmintic treatment to eradicate cutaneous capillariasis in a colony of South African clawed frogs (Xenopus laevis). Lab. Anim. Sci. 47, 477-482. Izumi-Kurotani, A., Mogami, Y., Okuno, M., and Yamashita, M. (1997). Frog experiment onboard space station Mir. In "Advances in Space Biology and Medicine," Vol. 6, pp. 193-211. JAI Press, Greenwich, Connecticut. Jaeger, R. G. (1981). Dear enemy recognition and the costs of aggression between salamanders. Am. Nat. 117, 962-974. Jaeger, R. G. (1992). Housing, handling, and nutrition of salamanders. In "The Care and Use of Amphibians, Reptiles, and Fish in Research" (D. O. Schaeffer, K. M. Kleinow, and L. Krulisch, eds.), pp. 25-29. Scientists Center for Animal Welfare, Bethesda, Maryland. Jancovich, J. K., Davidson, E. W., Morado, J. E, Jacobs, B. L., and Collins, J. P. (1997). Isolation of a lethal virus from the endangered tiger salamander Ambystoma tigrinum stebbinsi. Dis. Aquat. Org. 31, 161-167. Jerrett, D. P., and Mays, C. E. (1973). Comparative hematology of the hellbender, Cryptobranchus alleganiensis in Missouri. Copeia 2, 331-337. Johnson, P. T. J., Lunde, K. B., Ritchie, E. G., and Launer, A. E. (1999). The effect of trematode infection on amphibian limb development and survivorship. Science 284, 802-804. Jorgensen, C. B. (1997). 200 years of amphibian water economy: From Robert Townson to the present. Biol. Rev. 72, 153-237. Kaplan, M. L. (1993). An enriched environment for the African clawed frog (Xenopus laevis). Lab. Anim. 22, 25-27. Kara, T. C. (1994). Ageing in amphibians. Gerontology 40, 161-173. Karnes, J. L., Mendel, E C., and Fish, D. R. (1992). Effects of low voltage

825 pulsed current on edema formation in frog hindlimbs following impact injury. Phys. Ther. 72, 273-278. Kawai, T., Kinoshita, K., Koyama, K., and Takahashi, K. (1994). Anti-emetic principles of Magnolia obovata bark and Zingiber officinale rhizome. Planta Med. 60, 17-20. King, R. B., Douglass, J. K., Phillips, J. B., and Baube, C. L. (1993). Scotopic spectral sensitivity of the optomotor response in the green treefrog Hyla cinerea. J. Exp. Zool. 267, 40-46. Kobel, H. R., Loumont, C., and Tinsley, R. C. (1996). The extant species. In "The Biology of Xenopus" (R. C. Tinsley and H. R. Kobel, eds.), pp. 9-33. Clarendon Press, Oxford. Kreil, G. (1996). Skin secretions of Xenopus laevis. In "The Biology of Xenopus" (R. C. Tinsley and H. R. Kobel, eds.), pp. 263-277. Clarendon Press, Oxford. Letcher, J., and Glade, M. (1992). Efficacy of ivermectin as an anthelmintic in leopard frogs. J. Am. Vet. Med. Assoc. 200, 537-538. Major, N., and Wassersug, R. J. (1998). Survey of current techniques in the care and maintenance of the African clawed frog (Xenopus laevis). Contemp. Top. Lab. Anim. Sci. 37, 57-60. Mattison, C. (1998). "The Care of Reptiles and Amphibians in Captivity," rev. 3rd ed. Blanford Press, London. McBride, G. (1978). "South African clawed frog Xenopus laevis." Ann Arbor Biological Center, Ann Arbor, Michigan. Miller, S. I., and Pegues, D. A., (2000). Salmonella species, including Salmonella typhi. In "Principles and Practice of Infectious Diseases," (G. L. Mandell, J. E. Bennett, and R. Dolin eds.), 5th ed. Churchill Livingstone, Philadelphia. Mizell, M. (1985). Luck6 frog carcinoma herpesvirus: Transmission and expression during early development. Adv. Viral Oncol. 5, 129-146. Nace, G. W., Culley, D. D., Emmons, M. B., Gibbs, E. L., Hutchison, V. H., and McKinnell, R. G. (1974). "Amphibians: Guidelines for the Breeding, Care, and Management of Laboratory Animals." Institute of Laboratory Animal Resources, Washington, D.C. Newcomer, C. E., Anver, M. R., Simmons, J. L., and Wilcke B. W., Jr. (1982). Spontaneous and experimental infections of Xenopus laevis with Chlamydia psittaci. Lab. Anim. Sci. 32, 680-686. Nieuwenhuys, R. (1994). The neocortex. An overview of its evolutionary development, structural organization, and synaptology. Anat. Embryol. 190, 307-337. Nieuwkoop, P. D., and Faber, J. (1994). "Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis." Garland Publ., New York. Nyman, S. (1986). Mass mortality in larval Rana sylvatica attributable to the bacterium, Aeromonas hydrophila. J. Herpetol. 20, 196-201. Olson, M. E., Gard, S., Brown, M., Hampton, R., and Morck, D. W. (1992). Flavobacterium indologenes infection in leopard frogs. J. Am. Vet. Med. Assoc. 201, 1766-1770. Paniagua, R., Fraile, B., and S~iez, E J. (1990). Effects of photoperiod and temperature on testicular function in amphibians. Histol. Histopathol. 5, 365378. Petranka, J. W. (1998). "Salamanders of the United States and Canada." Smithsonian Institution Press, Washington, D.C. Pfeiffer, C. J., Pyle, H., and Asashima, M. (1990). Blood cell morphology and counts in the Japanese newt (Cynops pyrrhogaster). J. Zoo Wildl. Med. 21, 56-64. Pfennig, D. W., and Collins, J. P. (1993). Kinship affects morphogenesis in cannibalistic salamanders. Nature 362, 836-838. Pough, E H. (1989). Amphibians: A rich source of biological diversity. In "Nonmammalian Animal Models for Biomedical Research" (A. D. Woodhead, ed.), pp. 245-278. CRC Press, Boca Raton, Florida. Pough, E H. (1991). Recommendations for the care of amphibians and reptiles in academic institutions. ILAR News 33, S 1-$21.

826 Poynton, S. L., and Whitaker, B. R. (1994). Protozoa in poison dart frogs (Dendrobatidae): Clinical assessment and identification. J. Zoo Wildl. Med. 25, 29-39. Rafidah, J., Ong, B. L., and Saroja, S. (1990). Outbreak of "red leg"man Aeromonas hydrophila infection in frogs. J. Vet. Malaysia 2, 139-142. Ramakrishnan, L., Valdivia, R. H., McKerrow, J. H., and Falkow, S. (1997). Mycobacterium marinum causes both long-term subclinical infection and acute disease in the leopard frog (Rana pipiens). Infect. Immun. 65, 767773. Raphael, B. L. (1993). Amphibians. In "The Veterinary Clinics of North America: Small Animal Practice" (K. E. Quesenberry and E. V. Hillyer, eds.), Vol. 23, pp. 1271-1286. Saunders, Philadelphia. Riviere, J. E., Shapiro, D. P., and Coppoc, G. L. (1979). Percutaneous absorption of gentamicin by the leopard frog, Rana pipiens. J. Vet. PharmacoL Ther. 2, 235-239. Rogers, W. P., Simpson, T. W., Jones, L. M., and Renquist, D. M. (1997). An innovative aquatic non-recirculating system for use in housing African clawed frogs (Xenopus laevis). Contemp. Top. Lab. Anim. Sci. 36, 72-74. Rollins-Smith, L. A. (1998). Metamorphosis and the amphibian immune system. Immunol. Rev. 166, 221-230. Ruble, G., Berzins, I. K., and Huso, D. L. (1995). Diagnostic exercise: Anorexia, wasting, and death in South African clawed frogs. Lab. Anim. Sci. 45, 592-594. Rybak, S. M., Pearson, J. W., Fogler, W. E., Volker, K., Spence, S. E., Newton, D. L., Mikulski, S. M., Ardelt, W., Riggs, C. W., Kung, H.-E, and Longo, D. L. (1996). Enhancement of vincristine cytotoxicity in drug-resistant cells by simultaneous treatment with onconase, an antitumor ribonuclease. J. Nat. Cancer Inst. 88, 747-753. Sackin, N., and Sackin, H. (1991). A new method for feeding captive frogs (Xenopus laevis). Lab. Anim. 20, 44-46. Schaeffer, D. O. (1997). Anesthesia and analgesia in nontraditional laboratory animal species. In "Anesthesia and Analgesia in Laboratory Animals" (D. E Kohn, S. K. Wixson, W. J. White, and G. J. Benson, eds.), pp. 337378. Academic Press, San Diego. Shen, T. Y. (1995). Medicinal chemical studies of anti-inflammatory and analgesic natural products. J. Chin. Chem. Soc. 42, 617-621. Sibold, A., Eickhoff, A., and Bonner, R. A. (1993). Successful care and husbandry of the red-spotted newt, Notophthalmus viridescens. Contemp. Top. Lab. Anim. Sci. 32, 36. Sive, H. L., Grainger, R. M., and Harland, R. M. (2000). "Early Development of Xenopus laevis: A Laboratory Manual." Coldspring Harbor Laboratory Press. Smirina, E. M. (1994). Age determination and longevity in amphibians. Gerontology 40, 133-146. Stansley, W., and Roscoe, D. E. (1996). The uptake and effects of lead in small mammals and frogs at a trap and skeet range. Arch. Environ. Contam. Toxicol. 30, 220-226. Stephens, L. C., Cromeens, D. M., Robbins, V. W., Stromberg, P. C., and Jardine, J. H. (1987). Epidermal capillariasis in South African clawed frogs (Xenopus laevis). Lab. Anim. Sci. 37, 341-344. Stevens, C. W. (1992). Alternatives to the use of mammals for pain research. Life Sci. 50, 901-912. Stewart, P. L., (1994). An efficient, economical multicompartment tank for housing Xenopus laevis. Lab. Anim. Sci. 33, A30. Stoskopf, M. K., Arnold, J., and Mason, M. (1987). Aminoglycoside antibiotic levels in the aquatic salamander Necturus necturus. J. Zoo Anim. Med. 18, 81-85. Suzuki, M., Harada, Y., and Sekitani, T. (1993). Vestibular endorgan of the frog after the space flight and postural alteration of the neurectomized frog--its morphological and functional resilience. J. Vestib. Res. 3, 253-258. Tai, T., Akita, Y., Kinoshita, K., Koyama, K., Takahashi, K., and Watanabe, K. (1995). Anti-emetic principles of Poria cocos. Planta Med. 61, 527-530.

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Taylor, E R., Simmonds, R. C., and Loeffler, D. G. (1993). Isolation of Flavobacterium meningosepticum in a colony of leopard frogs (Rana pipiens). Lab. Anim. Sci. 43, 105. Teare, J. A., Wallace, R. S., and Bush, M. B. (1991). Pharmacology of gentamicin in the leopard frog (Rana pipiens). Proc. Am. Assoc. Zoo Vet. 128129. Terril-Robb, L. A., Suckow, M. A., and Grigdesby, C. F. (1996). Evaluation of the analgesic effects of butorphanol tartrate, xylazine hydrochloride, and flunixin meglumine in leopard frogs (Rana pipiens). Contemp. Top. Lab. Anim. Sci. 35, 54-56. Tinsley, R. C. (1995). Parasitic disease in amphibians: Control by the regulation of worm burdens. Parasitology 111, S153-S178. Tinsley, R. C., Loumont, C., and Kobel, H. R. (1996). Geographical distribution and ecology. In "The Biology of Xenopus" (R. C. Tinsley and H. R. Kobel, eds.), pp. 35-59. Clarendon Press, Oxford. Verhoeff-de Fremery, R., and Griffin, J. (1987). Anurans (frogs and toads). In "The UFAW Handbook on the Care and Management of Laboratory Animals" (T. B. Poole, ed.), 6th ed., pp. 773-783. Churchill Livingstone, New York. Verhoeff-de Fremery, R., Griffin, J., and Macgregor, H. C. (1987). Urodeles (newts and salamanders). In "The UFAW Handbook on the Care and Management of Laboratory Animals" (T. B. Poole, ed.), 6th ed., pp. 759-772. Churchill Livingstone, New York. Walker, I. D. E, and Whitaker, B. R. (2000). Amphibian therapeutics. In "The Veterinary Clinics of North America: Exotic Animal Practice" (S. P. A. Fronefield, ed.), Vol. 3, pp. 239-255. Saunders, Philadelphia. Whitaker, B. R. (1993). The use of polyvinyl chloride glues and their potential toxicity to amphibians. Proc. Am. Assoc. Zoo Vet. 16-18. Whitaker, B. R. (1999). Parasitic problems of amphibians. Proc. North Am. Vet. Conf., 801-803. Wilcke, B. W., Jr., Newcomer, C. E., Anver, M. R., Simmons, J. L., and Nace, G. W. (1983). Isolation of Chlamydia psittaci from naturally infected African clawed frogs (Xenopus laevis). Infect. Immun. 41, 789-794. Williams, J. H. (1997). Contractile apparatus and sarcoplasmic reticulum function: Effects of fatigue, recovery, and elevated calcium. J. Appl. Physiol. 83, 444-450. Williams, D. L., and Whitaker, B. R. (1994). The amphibian eye--a clinical review. J. Zoo Wild. Med. 25, 18-28. Williams, J. W. III, Tweedell, K. S., Sterling, D., Marshall, N., Christ, C. G., Carlson, D. L., and McKinnell, R. G. (1996). Oncogenic herpesvirus DNA absence in kidney cell lines established from the northern leopard frog Rana pipiens. Dis. Aquat. Org. 27, 1-4. Woodward, D. L., Khakhria, R., and Johnson, W. M. (1997). Human salmonellosis associated with exotic pets. J. Clin. Microbiol. 35, 2786-2790. Wright, K. (1994). Amputation of the tail of a two-toed amphiuma, Amphiuma means. Bull Assoc. Reptil. Amphib. Vet. 4, 5. Wright, K. (1999a). Common bacterial and fungal diseases of captive amphibians. Proc. North Am. Vet. Conf., 810-813. Wright, K. (1999b). Fluid therapy for amphibians. Proc. North Am. Vet. Conf., 814-816. Wright, K. M. (1996). Amphibian husbandry and medicine. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 436-459. Saunders, Philadelphia. Wu, M., and Gerhart, J. (1991). Raising Xenopus in the laboratory. In "Methods in Cell Biology" (B. K. Kay and H. B. Peng, eds.), Vol. 36, pp. 3-18. Academic Press, San Diego. Wyman, R. L., and Hawksley-Lescault, D. S. (1987). Soil acidity affects distribution, behavior, and physiology of the salamander Plethodon cinereus. Ecology 68, 1819-1827. Zug, G. R. (1993). "Herpetology: An Introductory Biology of Amphibians and Reptiles." Academic Press, San Diego.

Chapter 18 Biology and Diseases of Reptiles Dorcas P. O'Rourke and Juergen Schumacher

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Use in Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Availabilityand Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Laboratory Management and Husbandry . . . . . . . . . . . . . . . . . . . . . . II. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Anatomy and Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Physical Examination and Diagnostic Techniques . . . . . . . . . . . . . . . III. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Metabolic/Nutritional Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Traumatic Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Neoplastic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

INTRODUCTION

Reptiles are the first class of vertebrates to evolve an amniotic, shelled egg; therefore, they no longer require an aquatic e n v i r o n m e n t for reproduction. Furthermore, in contrast to superficial external appearance, reptiles are more closely related to endothermic birds than to ectothermic amphibians.

A.

Taxonomy

M e m b e r s of class Reptilia are derived from two lineages, Anapsida and Diapsida. Turtles are anapsids, unmistakable due LABORATORY ANIMAL MEDICINE, 2nd edition

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to the presence of a b o n y shell covering the b o d y Diapsids include the saurians (crocodilians and, according to m a n y taxonomists, birds) and the lepidosaurians (tuataras, lizards, and snakes) (Zug, 1993). Chelonians (turtles) are represented by over 240 species occurring worldwide. They are divided into two broad taxonomic groups based on m e t h o d of head retraction. Pleurodira or sideneck turtles withdraw their head and neck and fold it onto the shoulder. T w o families of pleurodires are found in freshwater in the Southern Hemisphere: Pelomedusidae from South America, Africa, Madagascar, and the Seychelles Islands; and the more advanced Chelidae from South America, Australia, and New Guinea. The most familiar of the chelids is Chelus fimbriatus, Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

the matamata. This unusual turtle with fleshy head protuberances and pronounced keels on its shell inhabits the freshwater streams of South America (Ernst and Barbour, 1989; Zug, 1993). Cryptodira contains several families, all of which withdraw the neck into the shell in a vertical, S-shaped fashion. Cheloniidae and Dermochelyidae are the sea turtles; representatives of this group can weigh more than a ton. A second group of cryptodires includes the softshell turtles (Trionychidae) and the mud and musk turtles (Kinosternidae). Softshell turtles have a leatherlike shell that lacks typical horny scales. Mud and musk turtles are smaller, have a domed shell, and exude an unpleasant odor when disturbed. A third group of cryptodires, Chelydridae, is represented by the snapping turtle (Chelydra) and alligator snapping turtle (Macroclemys). Snapping turtles have large heads, long tails, and powerful jaws (Fig. 1). The final group of cryptodires contains the "pond" turtles (Emydidae) and the tortoises (Testudinidae). Representatives of this group include the North American sliders (Trachemys), painted turtles (Chrysemys), box turtles (Terrapene), and the European Hermann's tortoise (Testudo hermanni) (Ernst and Barbour, 1989; Zug, 1993). Crocodilians are medium to large, quadrupedal reptiles adapted to an aquatic habitat. The order Crocodylia comprises three families: Alligatoridae, Crocodylidae, and Gavialidae. Two species of alligators and 6 species of caimans make up Alligatoridae. Caiman crocodilus (common caiman) and Alligator mississippiensis (American alligator) have been most commonly used in research. The narrow-snouted gharial is the sole representative of Gavialidae. Crocodylidae is the largest family, containing 8 genera and 22 species that occur throughout the world in tropical regions (Ross and Magnusson, 1989; Zug, 1993). The remainder of reptiles are classified as lepidosaurs. Included in this group are the orders Sphenodontida and Squa-

mata. The tuatara, a unique, lizard-like reptile, is the sole representative of Sphenodontida; the only 2 species of tuatara are both found in New Zealand. On the other hand, squamates (lizards and snakes) are the largest group of reptiles, with over 5000 species occurring worldwide. Lizard families composing Squamata include Gekkonidae (geckos), Iguanidae (iguanas, anoles), Chamaeleonidae (chameleons), Helodermatidae (Gila monsters), Varanidae (monitors), Scincidae (skinks), and Teiidae (tegus) (Zug, 1993). Snake families commonly seen in research settings are Boidae (boa constrictors, anacondas), Pythonidae (reticulated pythons, Burmese pythons, ball pythons), Elapidae (coral snakes, sea snakes, cobras), Viperidae (rattlesnakes, copperheads, puff adders, Gaboon vipers), and Colubridae (king snakes, rat snakes, garter snakes, water snakes). Boas and pythons are large, primitive snakes; some species can exceed lengths of 20 feet. Vipers are a group of venomous snakes with large erectile fangs. Elapids have smaller, fixed fangs and venom that is primarily neurotoxic. Colubridae is the largest family, with 290 genera and approximately 1700 species. Most colubrids are nonvenomous; however, some venomous species, including the rearfanged brown tree snake (Boiga irregularis) are members of this family (Zug, 1993; Greene, 1997). A list of common and scientific names is presented in Table I.

B.

Use in R e s e a r c h

For many years, herpetologists have studied reptiles in the field and maintained captive populations in the research laboratory. Most of these investigations have focused on the natural history, behavior, and reproduction of animals in their native habitats. Specific questions that cannot be answered in a field

Fig. 1. The snappingturtle has powerfuljaws and can inflict a seriousbite.

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18. BIOLOGY AND DISEASES OF REPTILES

Table I Commonand Scientific Namesof Selected Reptile Species Common names

Scientific names

Green iguana Anole Broad-head skink Leopard gecko Red-eared slider Painted turtle Box turtle Hermann's tortoise Gopher tortoise Garter snake Banded water snake Rat snake Corn snake King snake Boa constrictor Ball python American alligator Common caiman

Iguana iguana Anolis carolinensis Eumeces laticeps Eublepharis macularius Trachemys scripta elegans Chrysemys picta Terrapene carolina Testudo hermanni Gopherus polyphemus Thamnophis sirtalis Nerodia fasciata Elaphe obsoleta Elaphe guttata Lampropeltis getula Boa constrictor Python regius Alligator mississippiensis Caiman crocodilus

situation are addressed by bringing specimens into the laboratory for more intensive study. Thus, a substantial amount of research involving reptiles is dedicated to understanding and conserving the species themselves. A number of reptiles have been used as animal models and for teaching purposes. Red-eared sliders (Trachemys scripta elegans) and painted turtles (Chrysemys picta) are commonly used to teach physiology and anatomy (Fig. 2). Turtles have also been used to investigate effects of microgravity on orientation (Mori, 1995). Lizards have been the subjects of numerous investigations into stress and behavior, and snakes are commonly

used for chemoreception and behavior studies (Greenberg et al., 1989). Snake venoms have been intensively studied and their various components identified and used in production of antivenoms, as therapeutic agents, and for development of models of disease, such as myoglobinuria (Ponraj and Gopalakrishnakone, 1996). Neuroanatomy and neurophysiology research has frequently used crocodilians as animal models. Use of alligators has resulted in development of artificial blood and perfection of transmyocardial perfusion techniques (Dyer, 1995; Kohmoto et al., 1997). Alligator mississippiensis has also been the subject of environmental studies, ranging from examination of effects of mutagens (Winston et al., 1991) to assessment of endocrine disruptors on reproduction (Crain and Guillette, 1998) (Fig. 3). C.

Availability and Sources

Acquisition of reptiles for research and teaching has traditionally involved capturing animals from the wild. With chronic overharvesting of species such as the red-eared slider (Trachemys scripta elegans), many wild populations are suffering severe declines. When choosing a reptile species for research or teaching, reputable breeders should be given preference whenever possible. Animals purchased from these individuals are generally healthier and better adapted to captivity. A list of vendors is available through the Institute for Laboratory Animal Research website; reptile hobbyist magazines also contain names of suppliers and products. Many reptiles are captured in the wild by investigators and brought back to the laboratory for study. Alternatively, a species may be manipulated (sometimes rather extensively) in its natural habitat. Federal, state, and local permits may be required for collection and field studies, depending on location and the species involved. Investigators should be aware of and abide by all regulations governing the reptile they are studying (Greene, 1995). D.

Laboratory Management and Husbandry

Reptiles represent a diverse group of animals with speciesspecific husbandry requirements. Several excellent texts (referred to throughout this chapter) are available that address anatomy, physiology, behavior, reproduction, and captive maintenance of various species. These texts should be consulted prior to acquiring a given species of reptile in the laboratory animal facility. 1. Primary Enclosures

Fig. 2. The painted turtle, Chrysemys picta, is frequently used in research.

Reptiles can be maintained in a variety of primary enclosures. Glass aquaria are the most commonly used type of housing. Aquaria are readily available, come in a variety of sizes, and are easily sanitized. The two major drawbacks to glass aquaria are

830

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

Fig. 3.

Alligatorsare used for transmyocardialperfusion and environmentaltoxicologystudies.

that they are breakable and bulky. A workable alternative to glass aquaria is plastic shoe boxes and sweater boxes. Flexible plastic brands such as Rubbermaid withstand repeated cage washing and do not warp if lids are replaced immediately after removal from the cage washer. These cages are unbreakable, and with perforations for ventilation made in the sides, are stackable and occupy less space than aquaria. Several types of reptile housing are commercially available. Many of the hard

Fig. 4.

plastic cages do not withstand cage-washer temperatures. Fiberglass and Plexiglas housing units are also available commercially. These will withstand repeated cage washing, but the triangular-shaped fiberglass cages with sliding Plexiglas front entry take up more space than aquaria (Fig. 4). Additionally, approaching many reptiles directly from the front evokes more defensive responses than reaching in quietly from above. Stainless steel cages can be adapted for larger species of reptiles, and

Snakescan be maintainedin shoe-boxcages and fiberglasshousingunits.

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18. BIOLOGY AND DISEASES OF REPTILES

turtles and crocodilians can be housed in galvanized stainless steel or fiberglass tanks. With few exceptions, reptiles are escape artists and must be housed in cages with secure lids. Lids can be made of screen or a solid impervious material that is partially screened or otherwise ventilated. Lids should fit snugly, and for many species, should be secondarily secured with latches or weights. Many substrates can be used for reptiles. Newspaper or brown paper work very well and are fairly absorbent. Hardwood shavings and paper bedding are very absorbent, easy to spot-clean, and most important, allow the animals to burrow. Indooroutdoor carpet can be precut to fit cages, is easy to remove and sanitize, and works especially well for species such as garter snakes, which are very active and generate relatively large amounts of waste. Additionally, carpet offers traction for normal locomotion (Rossi, 1992). Hardwood chips are sometimes used but may inadvertently be ingested during feeding. Use of corncob bedding is strongly discouraged by many authors, because of accidental ingestion and development of gastrointestinal impaction. Corncob is also hygroscopic and will desiccate young animals. Likewise, kitty litter should never be used as a substrate (Page and Mautino, 1990; Anderson, 1991; Rossi, 1992; Boyer, 1991). Occasionally, a fastidious species may require a specific substrate such as sand, soil, sphagnum moss, or cypress mulch (Rossi, 1992) (Fig. 5). Sphagnum and cypress will inhibit growth of pathogens; sand and soil should be heattreated or washed prior to use. Most aquatic species do not require a substrate in the aquarium or tank; in fact, substrates can make cleaning more difficult (Boyer and Boyer, 1992). Terrestrial reptiles must be provided with a water bowl. Species that lap drops of water from leaves in the wild should be misted in addition to offered water in a container (Rossi, 1992).

Water bowls should be shallow enough to allow easy access (especially for terrestrial turtles), heavy enough to prevent tipping, and wide enough to accommodate all occupants of the cage simultaneously (Fig. 6). Reptiles spend time soaking, particularly prior to shedding, and should have access to fresh water at all times. In order to decrease stress and allow normal behavioral activity, reptile cages should contain certain accessories. Reptiles must have a hide box in which to retreat and feel secure (Page and Mautino, 1990; Anderson, 1991; Boyer, 1991; Rossi, 1992). Sanitizable, plastic hide boxes are available commercially in a variety of sizes. Polyvinyl chloride (PVC) pipe cut lengthwise in half works well, especially for larger species. Ceramic pots, heat-treated pieces of bark, and cardboard containers also make excellent refuges. Items that cannot be sanitized should be discarded when soiled (Fig. 7). Arboreal (treedwelling) reptiles should be provided with branches or dowels on which to climb. Species that bask should be provided with basking platforms. This is particularly important for many aquatic reptiles. A haul-out area or platform, easily accessible from the water and large enough to comfortably hold all tank occupants, must be provided to aquatic species in order to allow normal drying and behavioral thermoregulation (Boyer and Boyer, 1992). 2.

Water Quality

Aquatic reptiles are more tolerant than amphibians of chlorinated water; therefore, dechlorination of aquarium water is not necessary. In fact, chlorine may help retard growth of pathogens in the aquatic environment. When aquatic turtles are being housed, water should be at least as deep as the width of the

Fig. 5. Speciesof crawfish snake, Regina, require speciallyprepared tannin water and a cypress mulch basking spot.

832

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

shell; this will allow an overturned animal to right itself and not drown (Boyer and Boyer, 1992). Turtles and crocodilians generate large amounts of waste; therefore, static systems with frequent complete water changes or flow-through systems are preferred over recirculating systems. If recirculating systems are used, water quality should be monitored on a routine basis. 3.

Temperature

Reptiles tend to prefer warmer temperatures than do amphibians. Warm temperatures are necessary for normal physiologic processes, such as digestion, growth, reproduction, and immune function (Kanui et al., 1991; Dickinson and Fa, 1997). Each reptile species has a preferred optimal temperature range at which it should be maintained (Mattison, 1998). Examples of these ranges are provided in Table II. A thermal gradient should be provided within the cage. Low-wattage incandescent bulbs focused over a basking area can provide a site for behavioral thermoregulation (Fig. 8). Heat pads or strips placed under part of the cage will result in a temperature gradient. Aquarium heaters will increase water temperature for aquatic species, and a haul-out basking site will serve as the warm spot. "Hot rocks" (electrically heated basking surfaces that directly contact the animal) should be avoided because thermal burns have been reported with use of these devices (Boyer, 1991; Barten, 1996a). Reptiles should never be allowed to come into direct contact with any heat source; life-threatening thermal burns can result. 4.

Fig. 6. Waterbowls should be large enough for reptiles to soak in.

Lighting

Many reptile species need exposure to ultraviolet light in the appropriate UVB spectrum (290-320 nm) in order to endogenously manufacture vitamin D 3 (Gehrmann, 1996). Several species of lizards and turtles will develop metabolic bone disease if

Table II

Preferred Temperatureand Humidity for Selected Reptilian Speciesa Species Iguana iguana Anolis carolinensis Eublepharis macularius Boa constrictor Elaphe sp. Lampropeltis sp. Thamnophis sirtalis Testudo sp. Terrapene carolina Trachemys scripta Fig. 7. All reptiles should be provided with a hide box or other retreat,

a

From Divers (1996).

Temperature (~ 29-35 23-29 25-30 28-30 25-30 25-30 21-28 20-28 24 - 30 20-24

Humidity(%) 60-85 70-80 20-30 50-80 30-70 30-70 50-80 30-50 50 - 80 60- 90

18.

BIOLOGY AND DISEASES OF REPTILES

Fig. 8.

833

Baskinglights shouldbe providedfor manyreptile species.

deprived of ultraviolet light (Harcourt-Brown, 1996), particularly if an inappropriate diet (low in calcium or improper calcium-phosphorus ratio) is being fed. The ultraviolet light source should be placed 18-24 inches above the reptile and should not pass through plastic or glass (this will absorb the UV radiation) (Fig. 9). Examples of UV lights include Vitalites (Duro-Test), Chroma-50 (General Electric), and Colortone 50 (Philips). These may also be used in combination with a

Fig. 9.

black light (Boyer, 1991; Boyer and Boyer, 1992; Boyer, 1992); however, prolonged exposure to black light can result in ocular problems (Anderson, 1991; Fletcher, 1994; Divers, 1996). Artificial ultraviolet light sources should be replaced approximately every 6 months (Divers, 1996). Some species do not appear to require UV light if a proper diet is fed (Henkel and Schmidt, 1995). The biology, natural history, and nutritional requirements of a given species should be carefully

Manyreptiles require ultravioletlights to manufactureendogenousvitaminD 3.

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

834

researched when deciding on whether or not to supplement with UV light. Most reptiles do well with a 12 hr light-12 hr dark cycle. Breeding animals should have their light cycles adjusted accordingly.

5.

Airflow and Humidity

Many reptiles are terrestrial (ground-dwelling) or fossorial (burrowing); consequently, they require lower ventilation rates than mammals or birds. In fact, airflows appropriate for mammals can easily desiccate small reptiles. Most reptiles (with the exception of some desert-dwelling species) do well in a relative humidity range of 30-70%. Humidity requirements for selected species are listed in Table II. Low relative humidity can result in dysecdysis (difficult shedding), and humidity that is too high can result in "blister disease" (Rossi, 1996).

6.

Secondary Enclosures

Conventional animal rooms can be adapted for housing reptiles. Temperature and light cycles should be controlled for each room. Aquatic species can be kept in rooms with floor drains; tanks can be plumbed using PVC pipes to drain directly into floor drains. Sinks in animal rooms facilitate flushing and refilling aquatic housing units.

7.

Sanitation

Whenever possible, reptile cages should be sanitized in a cage washer. If this is not feasible, cages and tanks can be cleaned with quaternary ammonium compounds or a dilute sodium hypochlorite solution. A 3% sodium hypochlorite solution (1 part commercial bleach and 29 parts water) works well for sanitizing cages, hide boxes, water bowls, indoor-outdoor carpet, and equipment. Objects should be thoroughly rinsed and dried before returning to cages. Phenolic and cresolic compounds are very toxic to reptiles and must never be used around these animals, even to clean secondary enclosures (Page and Mautino, 1990; Anderson, 1991; Divers, 1996). Sanitation frequency of terrestrial reptile primary enclosures remains somewhat controversial. Although reptiles produce less waste than mammals, infrequent cleaning can result in buildup of pathogens such as Salmonella. Some individuals advocate prolonged intervals between cleaning, based on research demonstrating increased exploratory behavior (and presumably stress) following removal of feces and odor during cage cleaning (Conant, 1971; Pough, 1991). Other authors, however, have documented that the increased activity associated with being

subjected to a clean environment or other novel stimulus can result in positive behaviors (Huff, 1980; Radcliffe and Murphy, 1983). Chiszar et al. (1980) clearly demonstrated that the stimulus for increased exploratory behavior was the presence of a clean cage rather than the handling associated with the cleaning event, However, no information is currently available correlating this activity with acute or chronic stress in snakes (Chiszar et al., 1995). Until further studies clarify this issue, the most logical approach is to sanitize at intervals frequent enough to prevent pathogen buildup, while spot-cleaning as necessary between sanitizing. In general, changing cages at 1- to 2-week intervals works well for most species. Cages for garter snakes and other reptiles that generate large amounts of waste may need to be cleaned weekly. Many reptile cages and tanks are washed and sanitized by hand. Cleaning effectiveness should be monitored on a routine basis; cultures are one method of monitoring (Fig. 10). 8.

Handling

When handling any species of reptile, it is important to support the animal's body as much as possible. Reptiles should not be picked up or restrained by the tail. Many species of lizards have tail autotomy, and attempted tail restraint invariably ends in the appendage breaking off and the animal escaping. Lizards should have both the pectoral and pelvic girdles supported, with the tail gently held to prevent slapping. Very aggressive individuals may have to be restrained behind the head to prevent defensive biting. Lizards typically bite and hold on, and even relatively small animals can inflict a painful wound. Many lizards also have long, sharp claws and can scratch the handler. Small crocodilians can be held much the same as lizards, with more attention focused on head and tail restraint (crocodilians can administer a very powerful slap with the tail) (Fig. 11). Crocodilians may also roll when being held, in an effort to escape, and rough scales with dermal bones on the dorsal surface of the crocodilian can abrade hands. Large crocodilians should never be handled by one person alone. Tape can be wrapped around the animal's snout (taking care to avoid the nares) to prevent biting. Crocodilians have powerful jaws for crushing prey, yet relatively weak muscles for opening the mouth; therefore, muzzles work well. Many turtles can be restrained by holding the sides of the shell. However, species such as snapping turtles, softshell turtles, and mud and musk turtles have exceptionally long necks and can reach around to bite. These animals should be held by the back of the shell, taking care to avoid being scratched by claws on the hindfeet. When holding snakes, as much of the animal's body as possible should be supported. Many snakes are more comfortable if allowed to move about in the restrainer's hands. Snakes should never be held behind the head unless absolutely necessary. Grabbing a snake too tightly behind the head can damage tissues, restrict breathing, and elicit a much more

18. BIOLOGY AND DISEASES OF REPTILES

835

Fig. 10. Effectivenessof cage cleaning shouldbe monitored, particularly in cages that are washed by hand.

panicked escape response. Many snakes are quite docile if approached quietly and restrained gently (Fig. 12). 9.

Identification

Reptiles can be identified in a variety of ways. Shell notching, tail notching, and scale clipping have been used to identify turtles, crocodilians, and snakes, respectively. If the process of notching or clipping includes cutting dermal bone or extends into dermal tissues, anesthesia, analgesia, and appropriate asep-

tic technique should be used. Toe clipping is another means of marking that is controversial. Removal of too many toes will result in impaired locomotion; this can severely impact feeding, climbing, and other necessary survival functions. Anesthesia should be used for this painful procedure unless it clearly threatens the postrelease survival of the animals. Appropriate aseptic technique should be used, to prevent postamputation infections. Species with large, well-vascularized digits should not be subjected to toe amputation. Reptiles with skin pigmentation, such as some box turtles,

Fig. 11. Limbedreptiles shouldbe held by supportingboth the pectoral and pelvic girdles.

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

836

Fig. 12. Snakesshouldbe held with as much of the body supportedas possible and not restrained behind the head.

snakes, and lizards, can be identified based on skin color and pattern description. Implantable transponders offer permanent identification with minimal impact on the animal. Temporary identification methods include toe web tags in young crocodilians (these normally last for a few months) and nontoxic dyes. One of the reptile facilities in the University of Tennessee program records the animal's number in permanent ink on small pieces of yellow electrical tape and then affixes the tape to the head of the snake (Layne, personal communication). The tape is shed with the animal's skin, and a new piece is replaced immediately after shedding. 10.

Quarantine

All new reptiles entering a facility should be quarantined. Many animals purchased from vendors are in fact wild-caught, and depending on the practices of the holding facility, could be more severely stressed and diseased than animals collected directly from the wild. Animals that are colony-bred are normally healthier; however, few reptiles arrive with complete health reports. Therefore, it is best to quarantine all animals prior to introduction into an existing colony. Jacobson (1993b) recommends a 90-day quarantine period for snakes. On arrival, all animals should be weighed and receive physical examinations and appropriate diagnostic tests. External and internal parasites should be tested for and treated. Infectious disease should be diagnosed and treated. Feeding records should be kept, and any relevant observations should be recorded. A reptile with clinical signs of disease should never be introduced into an existing collection. 11.

Zoonoses

Salmonellosis is the zoonotic disease most frequently associated with reptiles (Mermin et al., 1997; Austin and Wilkins, 1998). Turtles, snakes, and lizards can carry the organism in their gastrointestinal tracts, and shed only when stressed (Chio-

dini and Sundberg, 1981; Austin and Wilkins, 1998). Transmission to humans is through the fecal-oral route, and indirect transmission through handling contaminated objects is common. Signs of salmonellosis in humans include fever, vomiting, cramps, and diarrhea. Individuals who are immunosuppressed, elderly, very young (less than 1 year old), or taking medication to increase gastric pH are at greater risk of infection. The disease can progress and cause dehydration, meningitis, osteomyelitis, and sepsis (Anonymous, 1992, 1995; Austin and Wilkins, 1998). Treatment of reptiles to eliminate the carrier state has not been successful and can result in antibiotic-resistant strains of the organism (D'Aoust et al., 1990). Immunocompromised individuals, pregnant women, and children under 5 years of age should avoid contact with reptiles (Anonymous, 1992, 1995). A set of dedicated cleaning equipment and supplies should be used for reptiles, and hands must be washed thoroughly after contacting reptiles or the equipment (Austin and Wilkins, 1998). Aquatic species of reptiles have been implicated in cases of atypical mycobacteriosis (Mycobacterium chelonae) and Edwardsiella tarda infections. Although these infections are rare, the organisms may pose a health hazard, particularly for immunocompromised individuals or people with underlying disease (Miller et al., 1990; Darrow et al., 1993). Appropriate precautions, such as avoiding direct contact with contaminated water (particularly if fingers and hands have preexisting injuries), wearing gloves, and washing hands thoroughly after handling aquatic reptiles or their caging and equipment, should help prevent disease transmission. 12.

Venomous Species

Numerous snake species and two species of lizards are venomous. Venoms have been broadly classified as hemotoxic and neurotoxic; however, most are complex mixtures of digestive enzymes and have varying effects on tissues and organs. For

18. BIOLOGYAND DISEASES OF REPTILES this reason, it is essential to be thoroughly familiar with the biology, behavior, and toxicity of any venomous species housed in the laboratory animal facility. Essential considerations for management of venomous reptiles include housing in unbreakable, locked cages; provision of equipment such as snake hooks, long forceps, large plastic garbage cans, and acrylic tubes for handling, feeding, and restraint; protocols for routine cage changing, animal handling, and accidental envenomation; and thorough training of all personnel involved with these species. Special caging can be designed for highly aggressive or specialized species (Mason et al., 1991; Greene, 1997). One of the reptile facilities at the University of Tennessee houses and studies venomous snakes. The animals are housed in specially constructed, locked cages. Two trained individuals must be present when any venomous snake is handled. A detailed protocol for accidental envenomation is prominently posted on the animal room door (which is kept locked). Antivenom is kept on hand for transport to the hospital with the victim in the event of a bite. Procedures will vary among species, depending on relative toxicity and behavior of the venomous animal being housed.

II.

A.

BIOLOGY

Anatomy and Physiology

1. Integumentary System Reptiles are covered primarily by scales, which are made of [3-keratin. The elastic form found in most vertebrates, a-keratin,

837

covers the skin between scales that do not overlap. Softshell and leatherback sea turtles have cx-keratin covering their shells, and snakes and lizards have an epidermal structure with a-keratin on the inside and [3-keratin on the outside (Zug, 1993). In crocodilians and turtles, epidermal growth is continuous, as is the shedding of pieces of skin. In contrast, lizards and snakes have a synchronized pattern of ecdysis (shedding). Germinal cells undergo a resting phase prior to synchronously beginning to divide. A second epithelial layer is formed beneath the original; this renewal phase takes 1-2 weeks. During this time, the skin is dull and the eyes appear cloudy (opaque) (Fig. 13). Lymph then diffuses between the layers and enzymatically cleaves them. The skin and eye opacities resolve, and shedding usually occurs 3 - 4 days following this clearing (Zug, 1993; Rossi, 1996). Crocodilians, some lizards, and turtles have osteoderms (bony plates in the dermis); these are usually found on the dorsal and lateral surfaces of the animals. Skin glands are present and have a variety of functions. Many turtles have musk glands in the inguinal and axillary regions. Male tortoises and both sexes of crocodilians have glands in the mandibular area; crocodilians also have cloacal glands. A linear array of secretory pores is evident on the inside of the thighs in male lizards of certain species. Snakes and some lizards have paired scent glands that empty through the cloaca and seem to function in defense and sexual recognition. Salt glands are found in a variety of locations (tongue, orbit, nasal passage) in several species of marine or desert reptiles (sea snakes, crocodiles, lizards, turtles). These glands excrete excess salt, because reptiles cannot concentrate urine above blood osmolality (Jacobson, 1984; Zug, 1993).

Fig. 13. The ocular scales of the snake become cloudy (opaque)prior to shedding(arrow).

838 2.

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER Musculoskeletal System

Reptiles have undergone a variety of musculoskeletal adaptations during evolution. Among the most notable is the bony and muscular arrangement that allows crocodilians to close their mouths with incredible crushing force. Another is the refinement of cranial bones in snakes that enables them to ingest large prey items. Flexible ligaments, rather than a mandibular symphysis, connect the independently moving mandibles. Each mandible also has a joint in the middle, which permits outward bowing and expansion. Additionally, the mandibles are independently attached to outward-slanted, free-swinging quadrate bones (Greene, 1997). These adaptations allow snakes to swallow prey items much larger than their own heads. Primitive snakes such as boas and pythons have remnants of pelvic girdles, which are visible radiographically. Boas and pythons also have spurs, clawlike hindlimb remnants, which are used during courtship. The vertebrae and ribs of turtles are fused to a bony shell. The upper part is referred to as the carapace, and the lower part of the shell is called the plastron. Box turtles have a hinge in the front part of the plastron, which closes to seal off access to the body when the legs and head are retracted.

valve, a fold of tissue at the caudal aspect of the tongue, which meets the palate to form a watertight seal and permits these animals to hold prey in their mouths while submerged (Schaeffer, 1997). The primary respiratory organ in reptiles is the lung. Most reptiles have paired lungs; many snakes have an elongate right lung and either no or a small vestigal left lung. The lungs of snakes and many lizards are saclike structures that may end in rather extensive air sacs (Fig. 15). Turtles and crocodilians have compartmentalized lungs. In turtles, movement of the head and limbs causes air to be forced in and out of the lungs. Crocodilians have a muscular septum that functions like a mammalian diaphragm (Stoakes, 1992; Zug, 1993). Many reptiles, particularly aquatic species, are tolerant of anoxia and can go for prolonged periods without breathing (Zug, 1993; Wasser et al., 1997; Hicks and Wang, 1998). This physiologic adaptation can significantly impact methods of anesthesia and euthanasia for these species. Most reptiles do not routinely vocalize. Obvious exceptions are crocodilians, some species of turtles and lizards, and the snake Pituophis. Pituophis is the first snake described to have a functional vocal cord (Young et al., 1995). 4.

3.

Respiratory System

Compared to that of mammals, the glottis of reptiles is easy to visualize and access (Fig. 14). It lies in the cranial part of the mouth in snakes and many lizards, which enables them to hold large prey items and still respire, and is behind the base of the tongue in turtles and crocodilians. Crocodilians have a basihyal

Cardiovascular System

With the exception of crocodilians, all reptiles have a threechambered heart, consisting of paired atria and a single ventricle. Reptiles also have paired aortas (Fig. 16). Crocodilians have essentially a four-chambered heart, with the foramen of Panizza being the sole intraventricular connection. The functioning of this septum, along with paired aortas arising from

Fig. 14. The glottis of snakes and otherreptiles is easyto visualize.

18. BIOLOGY AND DISEASES OF REPTILES

839

Fig. 15. The snake lung is elongate and ends in an air sac.

opposite separate ventricles, results in shunting of blood to cephalic and coronary circulation during anoxic events such as diving (Axelsson et al., 1991; Zug, 1993; Axelsson et al., 1996). Lizards have a midventral abdominal vein that lies just inside the abdominal wall. This vein should be avoided when making surgical incisions (Barten, 1996b). 5.

Gastrointestinal System

Snakes and a few lizard species demonstrate remarkable adaptation of the salivary glands. In these species, digestive enzymes have been modified into venoms. Vipers, elapids, and some other snakes have true venom glands, which secrete

venom through a duct to a single fang (generally fixed in elapids and erectile in viperids). Duvernoy's gland, composed of branched tubules, is present under the skin of the maxillary region, near the angle of the jaw. A duct connects Duvernoy's gland to a sometimes enlarged, grooved maxillary tooth. Duvernoy's gland is present in many species of colubrids (Greene, 1997). Venom glands are found in the lower jaw of Gila monsters and beaded lizards; these species chew to ensure envenomation. Crocodilians, snakes, and lizards have teeth; turtles have a horny beak that is used to bite off chunks of food. The esophagus of snakes is thin-walled and distensible, to accommodate large prey. Crocodilian stomachs are round,

Fig. 16. All reptiles except crocodilians have a three-chamberedheart, and all have paired aortas (arrows).

840

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

muscular, and thick-walled, and often contain gastroliths to aid in digestion (Lane, 1996). Crocodiles have a number of connective tissue septa that separate the body cavity into several separate components (Van Der Merwe and Kotze, 1993; Mushonga and Horowitz, 1996). Unlike that in mammals, the pancreas in snakes is discrete and compact (Moscona, 1990). The sequence of glucagon and insulin appearance and development of the alligator pancreas is identical to that of mammals and birds (Jackintell and Lance, 1994). The liver of snakes is elongate, in accordance with other anatomic features of this group (Fig. 17). Alligators have fibrous trabeculae that course through the liver (Beresford, 1993). Some turtle species have pigmented cell aggregations in the spleen and liver; these are macrophages, which increase in number as animals age (Christiansen et al., 1996). The small intestine and colon tend to have larger volumes in herbivorous species of reptiles (Jacobson, 1984; Barboza, 1995), as for other classes of vertebrates. In some snake species that eat infrequently, energy is conserved through atrophy of the small intestine and related organs between meals (Secor and Diamond, 1997). In all reptiles, products of the digestive, excretory, and reproductive tracts empty into the cloaca.

6.

Excretory System

Reptiles have paired, metanephric kidneys. Aquatic species tend to excrete ammonia, semiaquatic species excrete urea, and terrestrial species excrete primarily uric acid (Coulson and Hernandez, 1983; Jacobson, 1984). A renal portal system that drains venous blood from the caudal half of the reptile's body directly through the kidneys has been described. Recent evidence, however, suggests that at least in some species, venous blood may be diverted past the kidney directly to the liver in cer-

tain circumstances (Holz et al., 1997). Both situations should be considered when administering drugs that undergo renal or hepatic metabolism and excretion (Fig. 18). Many species of turtles and lizards have a urinary bladder; snakes and crocodilians do not.

7.

Nervous System/Special Senses

The reptilian brain has cerebral hemispheres and 12 cranial nerves. The spinal cord extends to the tip of the tail and contains locomotor control centers, thereby allowing animals to respond at the spinal level (Davies, 1981; Lawton, 1992). Most reptiles have lidded eyes and nictitating membranes. Snakes and some lizards (e.g., geckos) are the exception; the lidless eyes are protected by a transparent scale or spectacle. Snakes also lack nictitating membranes and scleral ossicles present in other reptilian species (Millichamp and Jacobson, 1983). Harderian glands are present in the orbits of many reptilian species and may function in vomerolfaction (Rehorek, 1997). Many reptiles have a vomeronasal organ, which has an accessory olfactory function (Zug, 1993). In snakes, particles of odor are picked up by the forked tongue and carried to the vomeronasal organ, located in the palate. Some species of snakes have pit organs that house infrared heat receptors. In pythons, a row of pits occurs in the labial (lip) scales; and in pit vipers, a single organ is located in the loreal scale between the eye and nostril. Many lizard species have a parietal or "third" eye, which contains photoreceptors that may permit enhanced detection of dawn and dusk (Solessio and Engbretson, 1993). Other lizards have dermal photoreceptors that may function in regulation of basking behavior (Tosini and Avery, 1996). Snakes lack external ears. Vibration is transmitted through

Fig. 17. The snake liver is large and elongate.

841

18. BIOLOGY AND DISEASES OF REPTILES

Fig. 18. Healthysnake kidney (A) and kidney affected with renal gout (B) from inappropriate antibiotic administration.

the body to the quadrate bone and then to the columella and inner ear (Greene, 1997). 8.

Normal Values

Reptilian red blood cells are nucleated and are lower in number than in mammals and birds. Lizards normally have a higher number of red blood cells than do snakes, who have a larger number than do turtles. Total erythrocyte counts can vary with sex, season, and nutritional status (Duguy, 1970; Sypek and Borysenko, 1988; Campbell, 1996). The white blood cells of reptiles include heterophils, eosinophils, basophils, thrombocytes, lymphocytes, monocytes, and in some species, azurophils. In most species, heterophils have fusiform, eosinophilic cytoplasmic granules; the cytoplasmic granules of squamates can be pleomorphic. Heterophils respond to inflammation or infection and function as phagocytic cells, similar to the neutrophils of mammals. Eosinophils resemble heterophils but have spherical eosinophilic cytoplasmic granules; the function of reptilian eosinophils is unclear. Basophils are small cells filled with basophilic granules, which frequently obscure the nucleus. Some species of reptiles have normally high basophil counts. These cells appear to function in reptiles as they do in mammals. Thrombocytes are the reptilian equivalent of platelets. Lymphocytes, the most numerous leukocytes in many species of reptiles, resemble and function like those of mammals. Monocytes tend to be large cells with vacuolated cytoplasm and are involved in granuloma formation and immune response. Squamates have a unique leukocyte, the azurophil, which appears to be a type of monocyte (Hawkey and Dennett, 1989). Like erythrocytes, many leukocytes in reptiles are af-

fected by temperature, season, sex, and nutritional status of the animal (Montali, 1988; Raphael et al., 1994; Campbell, 1996; Anderson et al., 1997). Additionally, factors such as site of venipuncture, type of anticoagulant, and method of analysis can affect blood cell counts in reptiles (Hawkey and Dennett, 1989; Bolten et al., 1992; Gottdenker and Jacobson, 1995; Muro et al., 1998). Representative CBC and plasma chemistry values are presented in Tables III and IV. B.

Nutrition

Reptiles fill a variety of ecological niches and range from carnivores to herbivores. Crocodilians begin life preying on insects and other invertebrates, small frogs, and fish. As crocodilians grow, prey size increases and may include other reptiles, birds, and mammals. The gharial is a specialized feeder, with fish being its mainstay. Other crocodilians, such as the Nile crocodile, eat prey as large as wildebeest. Large saltwater and Nile crocodiles will also prey on humans; most attacks on people are attributed to these two species. In captivity, small to medium crocodilians can be fed whole fish or rodents. Crocodilians can be adapted to dry chows; however, these should contain over 40% protein (Staton et al., 1990; Donoghue and Langenberg, 1996). In some areas, commercial "alligator chow" may be available. Young crocodilians will eat daily; older animals can have longer intervals between meals. Snakes are carnivorous and insectivorous, and several species have very specialized diets. Colubrids (king snakes, rat snakes) and boas feed readily on rodents; king snakes will also eat other snakes. Fish, frogs, and earthworms are common food items for

842

D O R C A S P. O ' R O U R K E A N D J U E R G E N S C H U M A C H E R T a b l e III

CBC Values for Selected Reptilian Species

Parameter

Iguana

Co ruc ia

C ro c ody lus

Te rrap e ne

Trac h e m y s

Te stuclo

Python

iguana a

ze b rata b

p o rosus r

carolina a

scripta a

he r m a n n i e

re gius f

0.84-1.43 24-60 7.4 - 11.6

0.6-1.3 17-41 4.7-12.2

0.27-0.83 20-38 5.0 -8.5

0.37-0.78 12-26 5.9-8.9

0.49 24.44 6.14

10.5-28.0 3.41-8.79

152-600

240-311

309-587

211-296

514.8

17-56

26.1-31.9

~

42-111 3.93-22.4 2-40 16-58 0-18 4-26 0-6 8-42

72-92 6.4-25.7 70-84 13-29 0-2.7 0-1.6 0-4.7 ~

79-131 7.5 56 11 ~ 8 9.4

Red Blood Cells (106/gl) 0.8-2.0 Hematocrit (gm%) 24-37 Hemoglobin (gm/dl) ~ Mean Corpuscular Volume (fl) ~ Mean Corpuscular Hemoglobin Concentration (gm/dl) ~ Mean Corpuscular Hemoglobin (pg) m White Blood Cells (103/gl) 4.5-10 Lymphocytes (%) 40-65 Heterophils (%) 30-45 Eosinophils (%) 0-2 Basophils (%) 1-4 Monocytes (%) 1-4 Azurophils (%) 15-25

m

26.32 96-118 9.7 39.5 34 ~ 1.5 1 ~

122.64 7.24 22.1 50.3 21.0 2.5 3.7

4.6-38.2 1-49 40-82 0-4 0-2 7-34

From Frye (1995). bFrom Wright and Skeba (1992). c From Millan et al. (1997). d From Wallach and Boever (1983). eFrom Muro et al. (1998). fFrom Johnson and Benson (1996). a

Table IV

Chemistry Values for Selected Reptilian Species

Parameter Total protein (gm/dl) Albumin (gm/dl) Glucose (mg/dl) Cholesterol (mg/dl) Alkaline phosphatase (U/liter) Aspartate Aminotransferase (U/liter) Lactate Dehydrogenase (U/liter) Creatine Phosphokinase (U/liter) Calcium (mg/dl) Phosphorus (mg/dl) Sodium (mEq/liter) Potassium (mEq/liter) Chloride (mEq/liter) Uric Acid (mg/dl) Total CO2 (mEq/liter) From Frye (1995). bFrom Wright and Skeba (1992). c From Millan et al. (1997). d From Marks and Citino (1990). e From Johnson and Benson (1996). ;From Drew (1994). a

Iguana iguana a

4.5-8.0 2.0 -3.5 65 - 155

240 - 450 < 3800 < 1000 10.5-13.6 5.3-6.8 152-164 2.5-3.8 0.5-3.2

Corucia zeb rata b

70 - 122 11-252 < 4 -76 27-940 11.0-21.2 2.8-6.7 145-167 1.4 -5.0 123-129 <0.3-3.1 ~

Crocodylus p o rosus c

Testudo radiata d

Python re gius e

Drymarchon co rais f

4.1-7.0 1.4-2.3 1.1-218 42-277 31-180 23-157 ~ m 9.64 -13.8 3.7-9.0 143-161 3.8-7.2 88-127 ~

3.2-5.0 0.8-1.3 46.2 -92.8 60.2-153.5 72-120 42-134 213.4 -591.5 ~ 10.8-14.4 2 . 6 - 4.3 121-132 5.1-5.8 92-99 0-0.6 24 - 2 9

4.3-5.9 m 25 - 3 2

5.9 -12.3 1.7-4.6 28-89

63-145 15-77

80 -161 6-163 13-1055 68-1923 30 -337 8.3-68.6 143-170 4.3-14.3 100 -129 2.2-17.1 15-24

11.3-15.3 2.3-5.3

1.8-5.0

18. BIOLOGY AND DISEASES OF REPTILES

garter snakes, and water snakes do well on fish. Specialized feeders among snakes include Eastern hognose snakes, which subsist on toads and frogs; mud snakes, which eat amphiumas; and members of the genus Regina, which feed on crawfish. The egg-eating snake, Dasypeltis, has a unique feeding adaptation as well as a singular diet. The snake swallows bird eggs 3 to 4 times the size of its head, then uses elongated blunt spines on the ventral processes of its vertebrae to pierce the eggshell. The contents are swallowed, and powerful muscles contract to crush the shell, which is subsequently ejected (Greene, 1997). Whenever possible, snakes should be fed euthanized rodents, to avoid prey-induced trauma. Frozen prey items should always be thawed to at least room temperature, to avoid putrefaction of food in the stomach. Fish should be obtained from parasite-free sources or frozen for several days prior to feeding, in order to prevent transmission of parasites. Most snakes will eat every 1 to 2 weeks; younger animals may need to eat more frequently. Many turtles are omnivorous or herbivorous. Box turtles will eat earthworms, dog or cat food, vegetables, berries, and fruits. Herbivorous tortoises prefer thawed frozen mixed vegetables, fresh dark leafy greens, and some fruit (Mattison, 1998) (Fig. 19). Aquatic species such as the red-eared slider (Trachemys scripta elegans) and the painted turtle (Chrysemys picta) eat whole minnows, dog food, trout chow, and earthworms (Mattison, 1998). Dark leafy greens or aquatic plants should also be offered (Parmenter and Avery, 1990). In general, small turtles should be fed daily; larger animals can be fed 2 - 3 times a week. Lizards range from total herbivores to strict carnivores. Green iguanas (Iguana iguana) are herbivorous and should be fed a diet of predominantly dark leafy greens (such as collards and turnips) mixed with other vegetables, supplemented by occasional fruit (Boyer, 1991). Anoles are insectivorous and can be fed crickets, if the crickets are dusted with calcium powder or fed a calcium-rich diet ("gut-loaded") prior to being offered to the lizard (Allen, 1997). Some species, such as the day geckos (genus Phelsuma), are primarily insectivorous but also eat ripe fruit (McKeown, 1984). Baby-food fruit can be substituted for fresh fruit (Henkel and Schmidt, 1995). Monitor lizards are carnivorous and opportunistic feeders. Small lizards should be fed daily (Mattison, 1991); large carnivorous species can go longer between meals.

C. Reproduction Sex determination in reptiles can be accomplished by any one of several methods, depending on species examined. Male box turtles and tortoises of many species have a concave plastron that serves to stabilize the male when he mounts the female during breeding. The tail of male turtles is longer and the cloacal opening more caudally placed than in females. Male red-eared sliders and other species have elongate claws on their forelegs;

843

Fig. 19. Many species of turtles and lizards are herbivorous and require a variety of fresh vegetables.

these claws are used to stroke the sides of the female's head during courtship. Males of several lizard species, such as iguanas, have a large row of femoral pores on the inside of the hindlegs. The femoral pores of females are smaller or absent. Male snakes have a longer, thicker, more gradually tapering tail than do females (Rossi and Rossi, 1995; Mattison, 1998). A single, fleshy penis is present in male turtles and crocodilians. Snakes and lizards have paired, membranous hemipenes, which lie in the base of the tail and are everted during copulation (only one hemipenis is used per breeding) (Fig. 20). Many species with hemipenes can be sexed by gently inserting a smooth probe into the cloaca and directing the probe caudally. If the snake is a male, the probe will easily pass along the hemipenis for a distance of about 3 - 4 scale rows or more (Rossi and Rossi, 1995). Other sexing methods include manually everting the hemipenis of neonatal snakes, and injecting saline into the tail behind the hemipenes to hydrostatically evert

844

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

atures produce females, and higher temperatures produce males. In the wild, temperature differentials across the nest result in a mixture of both sexes. In the laboratory, most reptile eggs can be artificially incubated. The eggs should be removed and placed in an incubation chamber (usually a plastic shoe box with a loose-fitting lid), taking care to preserve their original orientation. A moist bed of sphagnum moss or vermiculite will serve as an incubation medium. Humidity should be about 90-100%, and temperatures should average about 30~ (Rossi, 1992). Alligators require slightly higher temperatures. Many snake eggs hatch 2 to 3 months after being laid (Fig. 21). Neonatal reptiles are essentially miniature reproductions of adults, fully capable of surviving on their own. Some species may require diet modification; however, most eat essentially the same food type as adults.

Fig. 20. Snakesand lizards have paired hemipenes.

them. Crocodilians can be sexed by digitally palpating the cloaca (Rossi and Rossi, 1995; DeNardo, 1996b). Reproductive strategies vary among reptile species. As breeding concludes, the male garter snake (Thamnophis sirtalis) leaves a solidified plug of ejaculate in the female's cloaca to prevent subsequent breeding by another male (Zug, 1993). The whiptail lizard, Cnemidophorus uniparens, is parthenogenetic. Postovulatory females act as surrogate males, courting preovulatory females and thereby stimulating ovulation and production of genetically identical offspring. All turtles and crocodilians lay eggs; some lizards and many snakes bear live young. Anoles lay 1-2 eggs at a time throughout the breeding season, while iguanas lay 2 0 - 4 0 eggs in a single clutch. Rat snakes lay 5 - 4 4 eggs per breeding, and garter snakes deliver up to 80 live young at a time (Mattison, 1991; Rossi, 1992). Some species of day gecko (Phelsuma spp.) lay 2 eggs at a time. The female lies on her back and uses her hindlegs to roll the pliable eggs into spheres, which she presses together until they harden. She then hides the eggs in a selected hiding place to incubate (Henkel and Schmidt, 1995). Females of some python species wrap around their eggs and shiver, thereby producing metabolic heat and facilitating egg incubation. They also protect the eggs from predators (Greene, 1997). Female crocodilians vigorously guard their nests throughout the incubation period. When the young are ready to emerge, they begin vocalizing. The female assists the young in digging out of the nest, and in some cases will pick them up and carry them to the water. Some females remain with their brood for up to 2 years and defend them from potential predators (Mcllhenny, 1987; Lang, 1987; Burghardt and Layne, 1995). Incubation temperature determines the sex of offspring in many reptile species. Alligator eggs incubated at lower temper-

D.

Behavior

Many species of turtles and snakes are not territorial and will tolerate being group-housed. Snakes that are group-housed must be fed separately. Snakes that are ophiophagic (snakeeating) should be housed individually. Young crocodilians are found in sibling groups in the wild and do well if kept together in the laboratory. Some lizard species are quite territorial, and males should never be housed together. In anoles, acute stress causes an epinephrine-induced dark eyespot to become visible (Greenberg and Wingfield, 1987). Chronic stress in alligators can cause chronic corticosteroid elevation and subsequent immunosuppression (Lance, 1990). Therefore, when maintaining reptiles in the laboratory, attention should be paid to providing the appropriate social as well as physical environment for these species.

Fig. 21. All turtles and crocodilians and many species of snakes and lizards

lay eggs.

18. BIOLOGY AND DISEASES OF REPTILES

E.

Physical Examination and Diagnostic Techniques

When handling reptiles, one should be familiar with the species to be examined (e.g., knowledge of the defense mechanism), and the size of the animal should be considered. Snakes should be restrained at two points of the body. Some snakes are relatively docile and can easily be examined and manipulated while moving through the examiner's hands. Aggressive snakes should be restrained gently behind the head while supporting the body. Snake hooks can be used to manipulate or transfer the snakes into a container. For larger specimens, it is advisable to use two hooks to provide better support for the body. Transparent Plexiglas tubes are very useful for restraining snakes during visual examination, administration of injections, or collection of diagnostic specimens. Tubes can be customized with slits and holes through which injections can be given. These tubes are especially useful for venomous species. Lizards, particularly large specimens, are capable of inflicting serious bite injuries, and heavy leather gloves may be necessary to protect the handler from bites and scratches. Some species will also use their tails as weapons. While most turtles and tortoises can be examined with minor physical restraint, the handler should be aware that they can inflict serious bites. Chelonians will retract into their protective shell, thereby making examination and treatment quite challenging. Chemical immobilization may be necessary to facilitate handling and to reduce stress to the turtle. All crocodilians should be considered dangerous, and extreme care should be taken when handling members of this group. The mouth should be taped to prevent bites. Several people are necessary to restrain larger specimens.

845

Some species of crocodilians require chemical restraint or anesthesia to undergo a physical examination or diagnostic procedures such as blood and biopsy collection, radiography, and ultrasound. Knowledge of the unique anatomy and physiology of reptiles is essential for successful anesthesia (Bennett, 1991, 1996; Schumacher, 1996a). The most commonly used injectable agents include ketamine HC1, either alone or in combination with drugs such as diazepam and butorphanol. Induction and recovery times may be prolonged with most injectable agents. Propofol, an ultra-shortacting nonbarbiturate agent, has rapid induction and recovery times; however, it must be given intravenously or intraosseously (Fig. 22). The inhalation agent of choice in reptiles is isoflurane, which can be administered for induction and maintenance of anesthesia (Fig. 23). In most anesthetized reptiles, endotracheal intubation is easy to perform (Fig. 24). Cardiopulmonary parameters, in particular, heart rate and respiratory rate, should be monitored throughout the anesthetic event, including the recovery period.

1. PhysicalExamination A thorough physical examination is important in the evaluation of the reptilian patient. The examiner should be familiar with the normal anatomy and physiology of the species being examined. A systematic approach is most helpful in identifying any abnormalities. The integument, including the shell of cheIonians, should be inspected for the presence of lesions or abnormal growths, which may be indicative of infectious and/or metabolic disorders. This should be followed by inspection of the eyes (e.g., retained spectacles, periorbital abscesses), nares,

Fig. 22. Administrationof propofol in the jugular vein in a desert tortoise (Gopherus agassizii) for induction of anesthesia.

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

846

Muscle mass in snakes and lizards and presence or absence of shell abnormalities in chelonians should also be recorded. In addition, the attitude of the reptile should be evaluated with consideration of the normal behavior of the species. An alert animal should be actively exploring its environment and not demonstrating signs of lethargy or weakness. 3.

Fig. 23. Galapagostortoise (Geochelone elephantopus) under isoflurane

Blood Collection

Blood collection techniques, including sample handling and processing, have been reviewed by Jacobson (1993a). A venous blood sample can be collected from most reptile species using physical restraint alone. The volume of blood that can safely be withdrawn is determined by the size of the reptile. The total blood volume of reptiles ranges between 5 and 8% of total body weight, and 10% of the total blood volume can safely be collected from a reptile (Jacobson, 1993a). Snakes can be bled from the ventral tail vein or via cardiocentesis. In the latter technique, the snake is positioned in dorsal recumbency, and the location of the heart is confirmed by either palpation or visualization of its beating and movement of the scales. The heart is stabilized between two fingers, and a small-gauge needle is inserted midline between the ventral scales. The needle is advanced at a 45 ~ angle and aimed at the apex of the heart. When the needle is placed correctly, blood should fill the hub of the syringe with each heartbeat. Repeated attempts should be avoided, to prevent excessive myocardial damage and hemorrhage. Turtles and tortoises can be bled from the ventral tail vein, jugular vein, brachial plexus, and occipital sinus. The brachial plexus consists of a network of blood and lymph vessels. If lymph is aspirated into the needle, the sample should be discarded. Lizards can be bled from the ventral tail vein or from the ventral abdominal vein. Crocodilians are commonly bled from the ventral tail vein or from the occipital sinus.

anesthesia.

4.

and oral cavity. Lizards and snakes can be easily palpated, and knowledge of normal anatomy is essential in order to appreciate any abnormal changes. The cloaca of all reptiles should be inspected for evidence of prolapsed tissue (e.g., reproductive or intestinal) or diarrhea. 2.

Body Condition Assessment

Assessment of body condition by visual examination alone is sometimes difficult in reptiles. Nutritional and hydration status should be noted since poor environmental conditions, such as excessively high or low humidity and temperatures outside the preferred temperature range, may contribute to poor feeding responses and inadequate hydration status. Incomplete shedding, including retained spectacles, are commonly seen in sick reptiles and animals kept in suboptimal environmental conditions.

Injection Techniques

Because of the presence of a renal portal system, injections, especially those of potentially nephrotoxic substances, should be given into the epaxial muscles of the cranial half of the body in snakes or the muscles of the front legs in chelonians, lizards, and crocodilians. If large volumes are to be administered, it may become necessary to divide them into multiple injection sites. Fluids and medications can also be given intraosseously. In lizards and chelonians, an intraosseous catheter can be placed into the tibia. Intravenous injection can be given into the ventral tail vein, ventral abdominal vein, or the jugular vein. In snakes, the jugular vein can be catheterized after a cut-down procedure. 5.

Fecal Examination

Fecal samples for flotation, wet mounts, bacterial culture, and cytology can be collected from the cage. In order to obtain a

18. BIOLOGY AND DISEASES OF REPTILES

847

Fig. 24. Greentree python (Chondropython viridis) after placementof an endotracheal tube.

fresh sample, a colonic wash should be performed. The cloaca should be dilated with a speculum and the colonic opening identified. A sterile, lubricated catheter of appropriate size is carefully inserted into the colon, and sterile saline is injected by syringe. Fecal material can be collected by repeated aspirations and submitted for diagnostic evaluations. 6.

Tracheal Culture/Wash

A tracheal wash is a useful tool for the diagnosis and treatment of respiratory infections. The sample should be submitted for cytology, culture, and sensitivity testing. Based on these resuits, appropriate antimicrobial therapy can be initiated. Aseptic techniques must be followed in order to obtain a useful sample. In snakes, the mouth can be held open with an appropriate speculum, and a catheter of appropriate size can be inserted into the trachea while the animal is awake. Most other species of reptiles need to be anesthetized to visualize and gain access to the glottis. Anesthetized animals should be intubated and the catheter inserted through a sterile endotracheal tube. Sterile saline (approximately 5 ml/kg total body weight) is injected through the catheter and repeatedly aspirated into a sterile syringe. The collected material can then be submitted for microbiologic and cytologic evaluation. 7.

Collection of Biopsy Specimens

Biopsy specimens should be obtained for histologic and microbiologic evaluation of skin, visceral organs, and masses. Biopsies can be collected by direct visualization (e.g., skin), by ultrasound guidance for visceral organs (e.g., kidneys), or by endoscopy (e.g., stomach) (Fig. 25). The specimen should be placed in 10% neutral-buffered Formalin for histopathologic

evaluation. Impression smears can be made for cytologic evaluation, and samples should be submitted for culture and sensitivity if an infectious process is suspected. Additionally, samples may be saved in a suitable fixative for electron microscopic examination (ultrastructural morphology of masses and organs, or viral screening). 8.

Endoscopy

Endoscopy is a valuable tool for visualization of internal or- t gans and collection of diagnostic specimens such as biopsies and cultures (Jenkins, 1996). In most cases, the size of the reptile is the limiting factor, although small endoscopes (those used in avian medicine and surgery) are suitable for most small reptiles. Tracheoscopy, gastroscopy, and colonoscopy can be performed on the anesthetized reptilian patient. An endoscope with an appropriate diameter can be used for visualization of the coelomic cavity and visceral organs by a flank incision in lizards or through the inguinal region in chelonians. 9.

Radiography/Ultrasound

Radiography and ultrasonography are important diagnostic tools in the field of reptile medicine (Silverman and Janssen, 1996). In order to apply radiography and ultrasonography effectively, it is essential to know the normal anatomy of the reptile species being evaluated. Most reptiles do not require anesthesia for radiography. Snakes may be placed inside a Plexiglas tube to obtain dorsoventral and lateral views. Radiography is primarily useful to evaluate the skeletal system in snakes and to identify disease processes involving the respiratory tract. With radiography alone, visceral organs are often difficult to discern. Contrast studies are of great diagnostic value in cases of gastrointestinal disease and for identification of foreign bodies and

848

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

Fig. 25. Collection of an ultrasound-guided kidney biopsy in a green iguana (Iguana iguana).

masses. In chelonians, as in snakes, radiographs are most helpful in evaluating the respiratory and skeletal systems. Craniocaudal views are obtained in addition to the standard views in order to fully appreciate the respiratory tract. In lizards and crocodilians, radiography is a useful tool to evaluate skeletal changes (e.g., metabolic bone disease) and visceral organs and to diagnose foreign bodies and masses (Fig. 26). Soft tissue changes often can be better demonstrated with the use of ultrasonography. Most reptiles can be examined with manual restraint alone. As is the case with radiography, knowledge of the normal anatomy and pathophysiology of disease is essential to diagnose abnormalities. Also, biopsies of visceral organs or masses can be more safely obtained with ultrasound guidance. 10.

Nutritional Support

Reptiles, especially those kept under suboptimal environmental conditions, often develop nutritional imbalances and/or diseases that may require nutritional support (Donoghue and Langenberg, 1996). Many reptiles diagnosed with noninfectious and infectious diseases are anorectic and require supportive care, including fluid therapy and nutritional support. Following identification and correction of environmental deficiencies and underlying disease processes, anorectic reptiles should be force-fed. Care should be taken to ensure that the animal has a functional gastrointestinal tract. Reptiles with systemic disease or those kept in poor environmental conditions are commonly seen with gastrointestinal stasis. Drugs that promote gastric emptying (e.g., cisapride) have been used in reptiles with variable success. Most reptiles can be fed via a flexible orogastric feeding tube, and medications can be added to the diet. If the animal requires long-term force-feeding, placement

of a pharyngostomy tube is indicated in order to reduce stress for the animal. Placement of a pharyngostomy tube requires short-term sedation and facilitates feeding for longer periods of time without major side effects.

11. Euthanasia

Sodium pentobarbital overdose (intravenously or intracoelomically) is the most effective means of euthanatizing reptiles. Many species are tolerant to hypoxia and therefore are resistant to physical methods such as CO2 inhalation. General anesthesia (Schumacher, 1996a) followed by a secondary, physical method (exsanguination, decapitation, pithing) will ensure death and remove concerns associated with hypoxia tolerance. As discussed for amphibians, hypothermia is not appropriate for euthanasia of reptiles (Cooper et al., 1988; Andrews et al., 1993; Schaeffer, 1997; Beaver, et al., 2000).

III.

A.

DISEASES

Infectious Diseases

1. Bacterial/Mycoplasmosis

Reptiles are commonly diagnosed with bacterial and Mycoplasma spp. infections. While most disease processes are associated with gram-negative organisms, gram-positive isolates, anaerobes, and Mycoplasma spp. play an important role in reptile diseases (Rosenthal and Mader, 1996). The presence of

18. BIOLOGY AND DISEASES OF REPTILES

Fig. 26. Femaleprehensile-tailed skink (Corucia zebrata) with coelomic effusion (A) causedby a rupturedfollicle, and postoperativeview (B).

some organisms (gram-negative isolates) alone is not indicative of disease, since often they are part of the normal flora in reptiles. However, presence of clinical signs, changes in hematological parameters, and detection of potential pathogens may indicate an infectious disease process, and antimicrobial treatment should be initiated. Most commonly, immunosuppression caused by improper environmental conditions has been associated with development of bacterial diseases in reptiles. Culture and sensitivity testing should always precede antimicrobial treatment of bacterial diseases. In some cases, it may be necessary to administer broad-spectrum antimicrobials known to be effective against common reptilian pathogens while culture and sensitivity results are pending (e.g., enrofloxacin 5 mg/kg IM every 48 hr for 10 treatments; ceftazidime 20 mg/kg IM every 72 hr for 10 treatments; amikacin 5 mg/kg IM first dose, then 2.5 mg/kg IM thereafter for 10 treatments). Selection of an appropriate antibiotic depends on the size of the reptile, the route of administration, and the health status of the patient. a.

849

spp., Aeromonas spp., Morganella spp., Klebsiella spp., Salmonella spp., Proteus spp., and Escherichia coli. Stomatitis, pneumonia, upper respiratory tract disease, abscesses (Fig. 27), and gastrointestinal disease have been associated with the above organisms (Keymer, 1978; Hilf et al., 1990). Pseudomonas spp., Aeromonas spp., Serratia spp., and Providencia spp. are common isolates from the oral cavities and intestinal tracts of reptiles. Pseudomonas spp., Aeromonas spp., and Klebsiella spp. are associated with respiratory tract disease in reptiles, while Serratia spp. is most commonly isolated from subcutaneous abscesses. Salmonellosis is a potentially zoonotic disease, and many serotypes of Salmonella have been isolated from different orders of reptiles. Salmonella spp. appears to be part of the normal flora of many reptiles, and a positive culture for these organisms does not necessarily indicate clinical disease. Signs of salmonellosis are relatively rare in reptiles, and no effective treatment to eliminate Salmonella from an infected reptile has been described. Mycobacterium spp., although common in the environment, are not commonly isolated from reptiles (Brownstein, 1978). Most often M. chelonae, M. marinum, and M. thamnopheos have been associated with disease in reptiles. The integumentary, gastrointestinal, and respiratory systems may be affected. A diagnosis of mycobacteriosis can be made by demonstration of mycobacterial organisms by acid-fast staining of biopsy specimens, aspirates, or scrapings. There is no effective treatment for reptile mycobacteriosis, and because of the zoonotic potential, euthanasia of infected animals should be considered. Prevention of mycobacteriosis includes a strict quarantine protocol, proper environmental conditions, and strict hygiene procedures.

Gram-Negative Bacteria

Many gram-negative organisms are commonly isolated from clinically healthy reptiles. Examples include Pseudomonas

Fig. 27. Easternbox turtle (Terrapenecarolina) with an auricular abscess.

850 b.

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

Gram-Positive Bacteria

Gram-positive organisms are commonly isolated from reptiles, particularly from the integument. However, they are not considered major pathogens in most cases. Isolation of coagulase-positive Staphylococcus spp. accompanied by signs of disease indicates pathogenicity, and treatment should be initiated. If ~-hemolytic streptococci, which should be considered pathogens, are isolated from a sick reptile, appropriate antimicrobial therapy is warranted. c.

Anaerobic Bacteria

Anaerobic bacteria are important components of reptile disease, but their significance has not been fully elucidated (Stewart, 1990). Bacteroides spp. are most commonly isolated, while Clostridium spp. are often associated with gastrointestinal disease and endotoxemia (Schumacher, 1996c). d.

Mycoplasmosis

Mycoplasma spp. have been shown to cause upper respiratory tract disease in chelonians and are associated with high morbidity and mortality (Jacobson et al., 1991 a). Affected tortoises will have clear to purulent nasal discharge, swollen eyelids, and in advanced cases, occlusion of the upper airways. Diagnosis can be made by presence of clinical signs and serologic testing. An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of M. agassizii-specific antibodies (Schumacher et al., 1993). Treatment includes administration of antimicrobials (e.g., enrofloxacin 5 mg/kg IM every 48 hr for 10 treatments), supportive care, and isolation of affected animals. 2.

Viral

A variety of DNA and RNA viruses have been described in reptiles. While most studies report a viral agent to be associated with a clinical or pathological condition in a single animal or a defined group of animals, few agents have actually been identified as disease-causing. Koch's postulates have been fulfilled in only a few cases. However, considering the relatively high number of reports of viral agents in reptiles and the limited number of studies being performed, viral infections play an important role in reptile disease processes. Reviews of viral agents detected in reptiles have been published elsewhere (Jacobson, 1986, 1993c; Schumacher, 1996b). Only those agents associated with clinical disease and with important implications in captive collections of reptiles are reported here. a.

Herpesvirus

Herpesviruses have been reported in a variety of chelonians, including tortoises as well as freshwater and marine turtles. In

freshwater turtles, no specific clinical signs were associated with herpesvirus infection, and a diagnosis was made on postmortem histologic examination of multiple organs (Cox et al., 1980; Frye et al., 1977; Jacobson et al., 1982). Intranuclear inclusions characteristic of herpesvirus were detected in cells of the liver, spleen, lungs, and kidneys. A definitive diagnosis of herpesvirus was made by electron microscopic demonstration of viral particles within hepatocytes. In tortoises, herpesvirus infection plays a significant role in captive collections, especially in Europe where large numbers of tortoises have become infected (Cooper, 1988). Clinical signs are often limited to the upper respiratory tract. Nasal and ocular discharge, as well as signs of respiratory distress (including open-mouth breathing), may be seen. In infected tortoises, necrotizing, caseous lesions within the oral cavity are present. A diagnosis can be made by histologic evaluation of biopsy specimens and detection of intranuclear inclusions. The route of transmission is unknown but is most likely through ingestion of viral material from the feces of infected tortoises. Treatment consists of strict quarantine procedures, debridement of lesions, prevention of secondary bacterial infections, and supportive care. In green sea turtles, herpesvirus is associated with gray-patch disease and lung, eye, and trachea disease (LET), as well as fibropapillomatosis. The first two appear to affect juvenile turtles kept in crowded conditions (Rebell et al., 1975; Jacobson, 1986), whereas fibropapillomatosis has been described in free-ranging marine turtles (Jacobson et al., 1991b). b.

Adenovirus

Adenoviruses have been reported in crocodilians, snakes, and lizards (Jacobson et al., 1984; Jacobson and Gaskin, 1985; Jacobson and Gardiner, 1990; Schumacher et al., 1994a). Nile crocodiles died without clinical signs of illness and at necropsy, signs of gastrointestinal disease were noted. Histologically, intranuclear inclusions suggestive of adenovirus were seen within hepatocytes and intestinal epithelial cells. Adenovirus-like particles were demonstrated by electron microscopy. Rosy boas from a zoologic collection of snakes also died without prior signs of illness. These findings were similar to those seen in lizard species. Although the route of transmission has not been determined in reptiles, it is likely that adenovirus may cause disease in immunocompromised animals. Treatment in all cases should consist of supportive care, administration of antimicrobials, and correction of environmental conditions. c.

Poxvirus

Poxvirus infection has been seen in captive spectacled caiman with gray-white skin lesions (Jacobson et al., 1979). Lesions were located predominantly around the head, including the mandible, maxilla, and palpebrae. Diagnosis can be made by histologic evaluation of affected skin samples and demon-

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18. BIOLOGY AND DISEASES OF REPTILES

stration of typical inclusions. A definitive diagnosis can be made only by electron microscopy and demonstration of viral particles. d.

Paramyxovirus

Paramyxovirus infections have been reported in a variety of snake species (Jacobson et al., 1992). Most commonly, viperid snakes are infected, and major losses in collections of snakes have been associated with the agent. The virus is transmitted through respiratory secretions. Clinically, affected snakes show signs of respiratory tract disease, including open-mouth breathing, caseous material in the oral cavity, and respiratory sounds. At necropsy, the main findings are often limited to the respiratory tract and include edematous lungs and fluid accumulation in all major air passageways. Histologically, interstitial pneumonia with proliferation of lining epithelial cells can be seen. Diagnosis can be made by serology or viral isolation. There is no specific treatment except isolation of suspected snakes and a strict quarantine protocol. e.

Retrovirus

Retroviruses have been described in snakes and have been associated with a myxofibroma in a Russel's viper, a rhabdomyosarcoma in a corn snake, and a lymphosarcoma in a California king snake (Zeigel and Clark, 1969; Lunger et al., 1974). The route of transmission in these cases is unknown, and no specific treatment is available. The most significant retroviral infection is seen in the family Boidae. Snakes of this family have been diagnosed with a disease called inclusion body disease of boid snakes (Schumacher et al., 1994b). This disease has been reported from the United States, Africa, Europe, and Australia. Significant losses have been associated with inclusion body disease in captive collections of boids. The virus associated with this disease morphologically resembles members of the family Retroviridae. The route of transmission has not been determined, but mites may play a role as vectors. Clinical signs in boas include chronic regurgitation, disorientation, failure to right themselves, and flaccid paralysis of the musculature. Affected pythons show more severe and more rapidly progressing signs of central nervous system disease than do boas, usually within 2 - 4 weeks after exposure to the agent. The presence of large eosinophilic, intracytoplasmic inclusions in epithelial cells of all major organs, including the brain and spinal cord, is characteristic of this disease. A nonsuppurative meningoencephalitis is commonly seen in infected boas and pythons. Antemortem diagnosis can be made by collection of biopsy specimens from the esophagus, stomach, liver, and kidney and by demonstration of typical intracytoplasmic inclusions. Detection of inclusions in biopsy specimens is more reliable as a diagnostic tool in boas than in pythons. In infected pythons, inclusions are often limited to the brain. Postmortem diagnosis is

made by histologic evaluation of multiple organs and detection of typical inclusions. A definitive diagnosis can be made only by demonstration of viral particles by electron microscopy or by viral isolation from infected snakes. This disease is highly contagious among boas and pythons, and there is no treatment known. Infected snakes should be separated, and euthanasia of affected snakes is recommended to prevent further spread of disease. A strict quarantine protocol should be followed (minimally 90 days) before introducing boid snakes into an existing collection.

3.

Parasitic

Evaluation of reptiles for parasites is an important component of assessing the health status of the animal, especially wildcaught specimens. Proper quarantine procedures, including screening of fecal samples for parasites, should be followed, especially before introducing a new reptile into an existing colony. Parasite infestations often go undiagnosed due to unspecific signs, including chronic weight loss, anorexia, regurgitation, and diarrhea. It is essential to collect proper specimens (fresh fecal samples and biopsies, if necessary) to make an accurate diagnosis (Greiner and Schumacher, 1997; Jacobson, 1983).

a.

Protozoan

Although many species of amoebas can be found in reptiles, Entamoeba invadens is the most clinically important parasite. Entamoeba invadens is associated with high mortality in af-

fected snakes and lizards. The infective cysts are passed in the feces and are very stable in the environment. Transmission occurs by ingestion of infective cysts. Due to the direct life cycle of this organism, a colony may rapidly sustain high mortality. Clinical signs include anorexia, weight loss, and dehydration accompanied by mucoid, bloody feces. Strict hygiene and disinfection of all contaminated cages and equipment, as well as isolation of infected and exposed animals, should be followed in order to prevent spread of disease. Diagnosis can be made by demonstration of trophozoites in a direct smear of a fecal sample. The coccidian organism Cryptosporidium causes severe disease in reptiles. Although clinical signs may be absent during initial stages of the disease, in advanced stages, chronic regurgitation, weight loss, and a firm midbody swelling caused by gastric hypertrophy may be seen. Diagnosis can be made by demonstration of oocysts in fecal smears of infected animals, by gastric lavage, or by collection of gastric biopsies via gastroscopy for histopathology. There is no effective treatment, and the zoonotic potential of cryptosporidiosis should be considered. Metronidazole (100 mg/kg PO, repeat in 2 and 4 weeks) is recommended for the treatment of most protozoan infections in reptiles.

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DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

Nematodes

Nematode infections are commonly found in the gastrointestinal system of all orders of reptiles. Heavy infestation with ascarids may result in high morbidity and mortality. Extensive larval migration in the host may cause considerable damage to affected tissues. While mild infestations are subclinical, heavy infestations have been associated with severe inflammatory responses in the mucosa of the esophagus and stomach. Secondary bacterial infections may also be present. Clinically, regurgitation, chronic wasting, and poor body condition are often seen. Strongyles, especially the snake hookworm Kalicephalus spp., are important nematode parasites in reptiles. Kalicephalus spp. has a direct life cycle and is most often found in the gastrointestinal tract, where it has been associated with erosions and ulceration of intestinal epithelial cells. Clinical signs include weight loss, wasting, and anorexia. Lungworms, most commonly of the genus Rhabdias, are found in snakes and lizards. Clinical signs include labored breathing, exudate within the oral cavity, and presence of secondary bacterial infections resulting in severe bacterial pneumonia. Filarids are commonly diagnosed in reptiles, but signs of disease are usually absent. Macdonaldius oschei was found to cause severe dermatitis in pythons; however, in the natural hosts, Mexican viperid and colubrid snakes, this parasite was found in the posterior vena cava and renal veins. In pythons, however, it was found in mesenteric veins, eliciting a granulomatous response. Diagnosis can be made by demonstration of microfilariae in a blood smear of an infected snake. Fenbendazole (50-100 mg/kg PO, repeat in 2 weeks) is recommended for treatment of nematode infections.

c.

Cestodes

Cestodes have been reported in chelonians, lizards, and snakes but not in crocodilians. Reptiles can be either definite or intermediate hosts. Within the order Proteocephalidea, the genus Ophiotaenia is most commonly seen in snakes following ingestion of frogs, which serve as the intermediate host. Most infections are subclinical. The genera Bothridium and Botriocephalus within the pseudophyllidean order are known to infect pythons and can be associated with chronic enteritis. Tapeworms of the family Mesocestoidea can be found in all vertebrates. Mesocestoides infect snakes and lizards, causing subcutaneous nodules and presence of many encysted larvae within the coelomic cavity. Treatment for cestode infections consists of praziquantel administration (8 mg/kg IM, PO, repeat in 2 and 4 weeks).

d.

Trematodes

Although found in reptiles, trematodes are usually not considered major pathogens. Members of digenetic trematodes,

however, including the fluke refifer, are associated with ulcerative lesions within the lung and predisposition of the reptile to secondary bacterial infections. Spirorchidae are found as adults in the circulatory system of affected reptiles, especially turtles. Released eggs may become entrapped in arterioles where they may cause severe inflammatory responses. Almost any visceral organ may be affected. Diagnosis is made b~r demonstration of eggs on fecal examination or within tissue sections. Renal flukes (Styphylodora spp.) can be found within renal tubules and ureters of snakes. In severe cases, an interstitial nephritis with accumulation of cellular debris within tubules may be seen. Trematodes can be treated with praziquantel (8 mg/kg IM, PO, repeat in 2 and 4 weeks).

e.

Acanthocephala

The defnitive host of spiny-headed worms are amphibians. However, they can also be found in snakes and chelonians. While most infections are subclinical, heavy infestations may result in granuloma formation and ulceration of intestinal epithelial cells. If larvae are present in unsuitable hosts, they may encyst in viscera and subcutaneous tissue. The worm can be found within subcutaneous nodules. Diagnosis can also be made by demonstration of characteristic eggs in a direct fecal smear. While there appears to be no safe and effective treatment, administration of ivermectin (0.2 mg/kg IM, PO, repeat in 2 weeks; do not use in turtles and skinks!) may be attempted.

f

Pentastomes

Pentastomids are found in all reptiles. Larvae migrate from the intestinal tract to the lungs and/or air sacs or the subcutaneous tissue, where they mature. Minor infection may be asymptomatic, but heavy infestation may cause pronounced inflammatory responses, including damage to lungs, air sacs, and skin. Pentastomes can be removed from subcutaneous nodules by surgery or from the air sacs and lungs via endoscopy.

g.

Mites and Ticks

Both mites and ticks are found on captive as well as wild reptiles. Mites may cause a major problem in captive collections of reptiles, especially snakes. The snake mite, Ophionyssus natricis, is commonly seen in private and zoologic collections of snakes. Poor hygiene and/or introduction of infected reptiles into a collection are predisposing factors. Treatment of mite infestation is difficult, and all infected and exposed reptiles, as well as the environment, should be included in the treatment regimen. Ivermectin (0.2 mg/kg IM, SQ, repeat in 2 weeks; and topically, 0.5 ml of 10% solution/quart water for 10 days) has been recommended. Organophosphate products (e.g., Vapona strip) for treatment of the environment should be used with caution due to potential toxic side effects. Treatment should not ex-

18. BIOLOGYAND DISEASES OF REPTILES ceed 4 days! Ticks can be found on wild and captive reptiles and are best treated by manual removal. 4.

Fungal

Fungal infections are often associated with suboptimal husbandry, including overcrowding, high humidity and tempera, tures, poor hygiene, and other stressors. Prolonged antimicrobial therapy may also predispose the reptile to fungal infections. In most cases, the skin, the respiratory tract, or the gastrointestinal tract is affected. Mycotic dermatitis often manifests as skin lesions, characterized by hyperkeratosis, necrosis, or loss of pigmentation of the scales. Mucor spp., a mold, is ubiquitous in the environment and a common contaminant. However, Mucor spp. must be considered pathogenic if demonstrated histologically in sections of affected skin. Mucormycosis has been associated with high mortality. Candida albicans most commonly causes chronic fungal granuloma in the upper alimentary tract. Fusarium spp. are ubiquitous in soil and have been shown as common secondary invaders into damaged skin, especially in aquatic reptiles. Aspergillus sp. has been reported to cause dermatitis and pneumonia. Fungal infections can be treated with ketoconazole (15-30 mg/kg PO SID for 2 - 4 weeks), itraconazole (10 mg/kg PO SID for 4 weeks), nystatin for enteral fungal infections in tortoises (100,000 U/kg PO SID for 14 days), and malachite green (0.15 mg/liter water, 1 hr treatment for 14 days).

B. 1.

Metabolic/Nutritional Diseases

853

including splinting and cage rest. The prognosis for MBD is good, especially in juvenile animals, if the disease is diagnosed and treated early. In general, the more advanced the disease, the poorer the prognosis for recovery. 2.

Hypovitaminosis A

This condition is most commonly seen in chelonians, especially box turtles (Boyer, 1996b). Clinically, hyperkeratosis and squamous metaplasia of the respiratory, ocular, and gastrointestinal epithelia are most evident. Secondary bacterial infections may also be present. The most prominent clinical signs are those involving the ocular system and upper respiratory tract, including cellular debris beneath the eyelids, and nasal and ocular discharge. Additional signs include anorexia and lethargy. Diagnosis is made by evaluation of the diet, cytology of ocular discharge, or assay for serum vitamin A concentrations. Treatment includes vitamin A injections (2000 IU/kg SC once a week for 2 - 6 weeks), debridement of cellular debris from the eyes, treatment of secondary bacterial infections, and correction of dietary vitamin A deficiency. 3.

Gout

Both articular gout and visceral gout are seen in reptiles (Mader, 1996b) (Fig. 28). In most reptiles, uric acid is the end product of purine degradation. Uric acid and urate salts are insoluble in water and are cleared from the blood through renal tubules. In cases of hyperurecemia, uric acid crystallizes and forms insoluble crystals that are deposited in various organs.

Metabolic Bone Disease

Metabolic bone disease (MBD), often seen in herbivorous lizards and chelonians, is associated with a dietary deficiency of Ca, a negative calcium-phosphorus ratio in the diet, or a lack of exposure to ultraviolet UVB radiation (Boyer, 1996a; Barten, 1993). Metabolic bone disease is commonly seen in juvenile reptiles. A diagnosis of MBD is made by careful evaluation of the diet and the presence of characteristic clinical signs. In lizards, clinical signs include pliable mandibles, rounded skull, pathologic fractures (especially humerus and femur), reluctance to move, and fibrous osteodystrophy of the long bones. In advanced cases, paresis, muscle tremors, and seizures may be present. Shell abnormalities are the most prominent clinical feature in chelonians. Radiographs are useful to determine the grade of MBD. Treatment consists of dietary improvements, including administration of calcium-rich diets with a balanced calcium-phosphorus ratio, access to UVB radiation (natural sunlight is best), calcium supplementation (e.g., calcium glubionate 1 ml/kg PO BID for approximately 3 months, or calcium gluconate 100 mg/kg IM BID, TID), and supportive care. Fractures of the long bones should be managed conservatively,

Fig. 28. Jackson'schameleon (Chamaeleojacksonii) with gout deposits in

the tongue.

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DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

Gout may be caused by an overproduction of uric acid or by conditions that affect normal balance between production and excretion of uric acid (e.g., chronic renal disease, starvation, and diuretics). Treatment of gout in reptiles follows the same principles as treatment of gout in humans. Drugs should be administered that lower serum uric acid levels (e.g., allopurino120 mg/kg PO SID) and promote urate excretion (e.g., probenecid 250 mg/kg PO BID). Anti-inflammatory drugs, such as corticosteroids, are indicated for management of arthritis. Dosages used in reptiles are derived from those used in human medicine; however, efficiency has not been evaluated. Treatment should also include dietary changes, improvement of environmental conditions, administration of antimicrobials, and adequate hydration. The prognosis for reptiles with severe gout is very poor, and treatment should include proper analgesic management of the reptile. Mild cases of gout have to be managed long-term. 4.

Cloacal Prolapse

Prolapse of the cloaca is commonly seen in captive reptiles and is often associated with improper husbandry or infectious processes. There is always an underlying cause (e.g., bacterial enteritis, dystocia) for the prolapse, and successful treatment has to include correction of these conditions. If a reptile presents with a cloacal prolapse, it is most important to determine which organ has prolapsed: reproductive tract, colon, or urinary bladder. Once the tissue has been identified, it is necessary to evaluate the extent of tissue damage. Exposed tissue should be cleaned with antiseptic solution and lubricated to prevent drying. Necrotic tissue should be removed surgically. While some prolapses can be reduced manually, often a purse-string or transverse suture may be placed to prevent recurrence. 5. Dystocia

Dystocias are commonly diagnosed in captive reptiles and are in most cases associated with improper husbandry (DeNardo, 1996a). Infectious agents have also been found to cause dystocia. One has to differentiate between obstructive and nonobstructive dystocias. Obstructive dystocias result from oversized eggs or fetuses, or the inability of the female to pass the eggs because of anatomical abnormalities, infectious causes, or metabolic compromise. Nonobstructive dystocias are commonly seen in chelonians and lizards, especially green iguanas, and are characterized by the inability to pass normal-appearing eggs through a normal-appearing reproductive tract. Malnutrition, dehydration, and lack of suitable nest or digging sites have been associated with these conditions. A diagnosis of dystocia is sometimes difficult to make. Recent oviposition and presence of additional eggs within the reproductive tract are suggestive of dystocia in snakes. In lizards, behavioral changes help determine if the animal is truly egg-

bound. Gravid lizards (especially green iguanas) will normally not eat for up to 1 month. During this time, the animals remain alert and active. The presence of digging behavior followed by lethargy and weakness is an indicator of dystocia. Care should be taken to identify this crucial period since some lizards will start eating again, only to become ill within a short period of time. In chelonians, lack of a suitable nesting site and proper substrate may induce dystocia. Radiographs and ultrasonography .are helpful to identify any egg abnormalities as well as to determine the stage of follicular development. Once a diagnosis of dystocia is made, supportive care (including administration of fluids and calcium injections) is beneficial. Hormonal stimulation may be successful in some species and a complete failure in others. Oxytocin (5-20 IU/kg IM) or arginine vasotocin may be given. If hormonal stimulation fails to produce oviposition, ovocentesis may be attempted. A sterile large-gauge needle is inserted into the egg, the content aspirated, and the egg subsequently passed. In cases where the egg is visible through the cloaca, the collapsed egg may be removed with forceps. This is easier to perform in soft-shelled eggs, while hard-shelled eggs must be handled carefully to avoid cuts and rupture of the oviduct by pieces of the shell. In some cases, general anesthesia and consequently better muscle relaxation of the reptile may help in manipulation and removal of the eggs. If the above procedures fail, surgery is indicated. In snakes, multiple incisions may be necessary to facilitate removal of eggs. In lizards, a single paramedian incision is made to facilitate either salpingectomy or in cases where future breeding is not attempted, ovariosalpingectomy. In chelonians, an approach through the plastron or the inguinal region will facilitate removal of retained eggs. The prognosis for recovery and future breeding depends on the condition of the reproductive tract at the time surgery is performed. Correction of improper environmental conditions will help future breeding attempts, but in general, reptiles diagnosed with dystocia are likely to develop similar problems in the future. 6. Dehydration

Dehydration in reptiles is often the result of improper husbandry (low humidity, lack of water, and/or anorexia). Clinically, the skin may be wrinkled, the eyes may be sunken into the orbit, and mucous membranes may be dry. A definitive diagnosis of the degree of dehydration can be made only by collection of a venous blood sample and laboratory determinations, including packed cell volume, total protein, and electrolyte values. Fluid therapy should be initiated only with proof of volume depletion. In mild cases, soaking the animal in warm water may promote fluid intake. Oral administration of fluids is indicated in cases of mild dehydration. In cases of severe dehydration, the

18. BIOLOGYAND DISEASES OF REPTILES most effective way to administer fluids rapidly is through the intravenous and intraosseous routes. Intracoelomic fluid administration is an alternative route of fluid administration in cases of moderate dehydration.

7.

855

tion. Scrapings of the skin or full-thickness skin biopsies can be submitted for microbiologic and histologic evaluation. Topical administration of antimicrobial ointments should accompany systemic antimicrobial treatment.

C.

Dysecdysis

Snakes shed their skins in one piece, while most lizards and chelonians shed in several pieces. Dysecdysis may be a result of improper husbandry, such as inadequate temperatures and humidity or improper nutrition (Mader, 1996a). Shedding problems may also be caused by heavy infestation with ectoparasites and bacterial and/or fungal dermatitis. The latter may also develop as a result of untreated shedding problems. Treatment consists of improving environmental conditions and removal of retained pieces of skin, especially retained spectacles. The animal should be soaked in warm water and loose skin carefully removed. Retained spectacles should also be removed carefully. The eye should be moistened with eyewash solutions and eye ointment. The spectacle should come off easily when gently pulled with forceps. If in doubt, and the eye looks uninfected, one can also wait for the next shedding cycle, at which time the retained spectacle will often come off. In cases of bacterial or fungal dermatitis, systemic administration of antimicrobials or antifungal agents should be initiated. Cultures and sensitivity testing should be performed to assure appropriate drug selec-

1.

Traumatic Disorders

Bite Wounds

Bite wounds are most commonly inflicted by incompatible cagemates or prey animals. Care should be taken to become familiar with the social structure of the species of reptile kept in captivity. While immature reptiles can often be kept in a large community enclosure, adult, sexually mature animals will often display aggression to animals of the same sex. For example, juvenile iguanas can be kept together, whereas it is impossible to keep adult male iguanas together in one cage. Serious fights and injury may result. Females will often tolerate each other as long as there is enough space for each animal to establish its own territory. The same is true for most chelonians. Snakes may inflict serious bites to each other when not separated prior to feeding. Care should be taken not to house carnivorous reptiles of different ages together, as cannibalism may occur. Prey animals should not be fed alive since they may inflict serious bite injuries to a snake or lizard (Fig. 29). All bite wounds should be considered contaminated and appropriate treatment initiated.

Fig. 29. Boa constrictor (Boa constrictor) with severerat bite injuries.

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DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

This will include sterile saline flushes and debridement of necrotic tissue. Bite wounds are best managed as an open wound and allowed to heal by granulation. Initially, wet to dry bandages are applied and changed daily until there is healthy granulation tissue present. Wound dressings and topical antibiotic ointments (e.g., Silvadene cream) should be applied to promote healing and prevent infection. In most cases, systemic administration of a broad-spectrum antibiotic (e.g., enrofloxacin) is recommended. During the healing process, which may take several months, particular attention should be paid to a clean environment for the animal.

including wet to dry bandages, followed by topical application of antimicrobial ointments. Epoxy and fiberglass materials should be used only if one is absolutely certain that there is no infection or contamination present and healthy granulation tissue is present. If not treated appropriately with broad-spectrum antimicrobials (e.g., enrofloxacin, amikacin, ceftazidime) prior to shell repair, abscess formation, septicemia, and death of the animal may result. A compromise is the use of epoxy and fiberglass materials in aquatic turtles, where it is necessary to place the animal back into its aquatic environment as soon as possible to prevent electrolyte imbalances.

2.

3.

Shell Fractures

Shell fractures in chelonians may result from improper handling. Radiographs should be taken in order to evaluate the extent of the fracture. Loose fragments of shell may be removed or may be incorporated into the shell repair if there is vascular supply. In general, the standard principles of wound management apply to shell fractures. Wounds should be cleaned with sterile saline and evaluated carefully to see if they penetrate into the coelomic cavity. While soft tissue injuries and the shell fracture should be managed as an open wound, it will greatly facilitate the healing process if the shell fragments can be stabilized with the use of wires. Holes can be drilled into the shell with a dremel tool, and wires can be placed to reduce the fracture fragments. If at all possible, the wound should be treated as an open wound,

Burns

Burns are often caused by improper functioning of heating devices such as hot rocks or inappropriately placed heating lamps (Fig. 30). In all cases, care should be taken to prevent the animal from direct contact with the heating device. Heating tapes and lamps installed outside the enclosure or heating the entire room to the animals' preferred temperature zone are the safest solutions. In many cases, the burned area will become edematous, tissue will become necrotic, and secondary infections may develop. Proper wound care, including debridement of necrotic tissue, wet to dry bandages, and dressing with topical wound dressings is essential for wound healing. Administration of systemic antimicrobials is also indicated in most cases to prevent secondary bacterial infections. In addition, careful

Fig. 30. Greeniguana (Iguana iguana) with severeburn woundsresulting from a faultyheating device.

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18. BIOLOGY AND DISEASES OF REPTILES

evaluation of the reptile is warranted, especially with severe burn wounds, which will lead to significant loss of fluids and electrolytes. Supportive care, including fluid therapy and nutritional support, should be initiated.

4. Tail Injuries Fractures of the tail are commonly seen with improper handling of some lizard species, such as green iguanas (Barten, 1993). A tail may break off if it hits a sharp object or if the animal is inappropriately restrained by holding on to the tail. While some fractures may be splinted, loss of a segment of the tail should be treated conservatively. In species with tail autotomy, the wound should be treated as an open wound and never be sutured, so that the tail may regenerate. The treatment indicated in most cases is cleaning the wound with sterile saline, applying a topical antiseptic ointment, and providing a clean cage environment. In cases where the tail has to be amputated due to an infectious etiology (e.g., bacterial or fungal dermatitis, abscess formation) or neoplasia, systemic antimicrobial treatment is warranted.

D.

Toxins

Toxins are often associated with improper use of pesticides (such as organophosphates) in the treatment of external parasites or with toxins on plant material fed to herbivorous reptiles. Some plants are toxic to reptiles, and one should consult with a knowledgeable source before feeding unknown plants. Improper use of drugs (e.g., metronidazole, ivermectin) has also been associated with toxicities. Disinfectants such as iodine may also be toxic. Clinical signs of affected reptiles may be unspecific, such as lethargy and weakness, or may include tremors, convulsions, and seizures. Treatment consists of identification of the toxin; appropriate administration of an antidote, if available; supportive care (administration of fluids, antiseizure drugs such as diazepam, and if indicated, systemic antimicrobials to prevent secondary bacterial infections); and nutritional support.

E.

unknown, but viruses and environmental factors have been associated with development of tumors in reptiles. A diagnosis of neoplasia is always based on histopathologic or cytologic evaluation of suspected tissue. When performing a physical examination, care should be taken not to falsely misdiagnose a mass as a tumor. Granulomas, foreign bodies, or cysts may be falsely identified as tumors unless the examiner performs more diagnostic examinations, such as radiography, ultrasonography, aspirates, and/or biopsies, which are submitted for cytologic and histopathologic evaluation. In chelonians, tumors of the integumentary system include fibropapillomas and fibromas. Carcinomas of the gastrointestinal system and adenomas of the endocrine system have also been reported. In lizards, most commonly squamous cell carcinomas, fibrosarcomas, and lymphosarcomas have been reported. In snakes, lymphosarcomas and adenocarcinomas of the kidneys and gastrointestinal tract, as well as fibrosarcomas of the integumentary system, have been described. Although some reports describing chemotherapy, radiation therapy, and photodynamic surgery in the treatment of reptile neoplasms have been published, none of these have proven to be successful. At present, standard surgical removal of the neoplasm, possibly combined with chemotherapy or radiation therapy, as well as supportive care, is the recommended treatment.

Neoplastic Diseases

Neoplastic diseases are commonly seen in all orders of reptiles, especially chelonians, snakes, and lizards (Jacobson, 1981; Done, 1996). Most neoplastic diseases of domestic animals have also been reported in reptiles. Excellent reviews of neoplastic diseases in reptiles have been published, and the reader is referred to these for more detailed descriptions on specific neoplasia. The etiology for most reptilian neoplasia is

REFERENCES

Allen, M. E. (1997). From blackbirds and thrushes.., to the gut-loaded cricket: A new approach to zoo animal nutrition. Br. J. Nutr. 78, S135-S143. Anderson, N. L. (1991). Husbandry and clinical evaluation of Iguana iguana. Compend. Contin. Educ. Pract. Vet. 13, 1265-1272. Anderson, N. L., Wack, R. E, and Hatcher, R. (1997). Hematology and clinical chemistry reference ranges for clinically normal, captive New Guinea snapping turtles (Elseya novaeguineae) and the effects of temperature, sex, and sample type. J. Zoo WiIdl. Med. 28, 394-403. Andrews, E. J., Bennett, B. T., Clark, J. D., Houpt, K. A., Pascoe, P. J., Robinson, G. W., and Boyce, J. R. (1993). Report of the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc. 202, 229-249. Anonymous (1992). Lizard-associated salmonellosismUtah. Morb. Mortal. Wkly. Rep. 41, 610-611. Anonymous (1995). Reptile-associated salmonellosis-selected states, 19941995. Morb. Mortal. Wkly. Rep. 44, 347-350. Austin, C. C., and Wilkins, M. J. (1998). Reptile-associated salmonellosis. J. Am. Vet. Med. Assoc. 212, 866-867. Axelsson, M., Fritsche, R., Holmgren, S., Grove, D. J., and Nilsson, S. (1991). Gut blood flow in the estuarine crocodile, Crocodylus porosus. Acta Physiol. Scand. 142, 509-516. Axelsson, M., Franklin, C. E., Lofman, C. O., Nilsson, S., and Grigg, G. C. (1996). Dynamic anatomical study of cardiac shunting in crocodiles using high-resolution angioscopy. J. Exp. Biol. 199, 359-365. Barboza, P. S. (1995). Digesta passage and functional anatomy of the digestive tract in the desert tortoise (Xerobates agassizii). J. Comp. Physiol. B 165, 193-202.

858

Barten, S. L. (1993). The medical care of iguanas and other common pet lizards. In "The Veterinary Clinics of North America, Small Animal Practice, Exotic Pet Medicine I" (K. E. Quesenberry and E. V. Hillyer, eds.), pp. 12131249, Saunders, Philadelphia. Barten, S. L. (1996a) Thermal burns. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 419-421. Saunders, Philadelphia. Barten, S. L. (1996b) Lizards. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 47-61. Saunders, Philadelphia. Beaver, B. V., et al. (2000). 2000 report of the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc. 218, 669-696. Bennett, R. A. (1991). A review of anesthesia and chemical restraint in reptiles. J. Zoo Wildl. Med. 22(3), 282. Bennett, R. A. (1996). Anesthesia. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 241-247. Saunders, Philadelphia. Beresford, W. A. (1993). Fibrous trabeculae in the liver of the alligator (Alligator mississippiensis). Ann. Anat. 175, 357-359. Bolten, A. B., Jacobson, E. R., and Bjorndal, K. A. (1992). Effects of anticoagulant and autoanalyzer on blood biochemical values of loggerhead sea turtles (Caretta caretta). Am. J. Vet. Res. 53, 2224-2227. Boyer, T. H. (1991). Green iguana care. Am. Assoc. Reptile Vet. 1, 12-14. Boyer, T. H. (1992). Box turtle care. Bull. Assoc. Reptil. Amphib. Vet. 2, 14-17. Boyer, T. H. (1996a). Metabolic bone disease. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 385-392. Saunders, Philadelphia. Boyer, T. H. (1996b). Hypovitaminosis A and hypervitaminosis A. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 382-385. Saunders, Philadelphia. Boyer, T. H., and Boyer, D. M. (1992). Aquatic turtle care. Bull. Assoc. Reptil. Amphib. Vet. 2, 13-17. Brownstein, D. G. (1978). Reptilian mycobacteriosis. In "Mycobacterial Infections of Zoo Animals" (R. J. Montali, ed.), pp. 665-668. Smithsonian Institution Press, Washington, D.C. Burghardt, G. M., and Layne, D. G. (1995). Effects of ontogenetic processes and rearing conditions. In "Health and Welfare of Captive Reptiles" (C. Warwick, E L. Frye, and J. B., Murphy, eds.), pp. 165-185. Chapman and Hall, London. Campbell, T. W. (1996). Clinical pathology. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 248-257. Saunders, Philadelphia. Chiodini, R. J., and Sundberg, J. P. (1981). Salmonellosis in reptiles: A review. Am. J. Epidemiol. 113, 494-498. Chiszar, D., Wellborn, S., Wand, M. A., Scudder, K. M., and Smith, H. (1980). Investigatory behaviour in snakes II: Cage cleaning and the induction of defaecation in snakes. Anim. Learn. Behav. 8, 505-510. Chiszar, D., Tomlinson, W. T., Smith, H. M., Murphy, J. B., and Radcliffe, C. W. (1995). Behavioural consequences of husbandry manipulations: Indicators of arousal, quiescence, and environmental awareness. In "Health and Welfare of Captive Reptiles" (C. Warwick, E L. Frye, and J. B., Murphy, eds.), pp] 186-204. Chapman and Hall, London. Christiansen, J. L., Grzybowski, J. M., and Kodama, R. M. (1996). Melanomacrophage aggregations and their age relationships in the yellow mud turtle, Kinosternon flavescens (Kinosternidae). Pigment Cell Res. 9, 185190. Conant, R. (1971). Reptile and amphibian management practices at Philadelphia Zoo. Int. Zoo Yearbook 11, 224-230. Cooper, J. E., Geschmeissner, S., and Bone, R. D. (1988). Herpes-like virus particles in necrotic stomatitis of tortoises. Vet. Rec. 123, 554. Coulson, R. A., and Hernandez, T. (1983). "Alligator Metabolism Studies on Chemical Reactions in Vivo.'" Pergamon Press, Oxford. Cox, W. R., Rapley, W. A., and Barker, I. K. (1980). Herpesvirus infection in a painted turtle. J. Wildl. Dis. 16, 445. Crain, D. A., and Guillette L. J., Jr. (1998). Reptiles as models of contaminantinduced endocrine disruption. Anim. Reprod. Sci. 53, 77-86. D'Aoust, J.-Y., Daley, E., Crozier, M., and Sewell, A. M. (1990). Pet turtles: A continuing international threat to public health. Am. J. Epidemiol. 132, 233-238.

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER Darrow, M., Foulkes, G., and Ayoub, M. (1993). Zoonotic transmission of turtle-borne Edwardsiella tarda. Complications Surg. 12, 33-35. Davies, P. M. C. (1981). Anatomy and physiology. In "Diseases of the Reptilia" (J. E. Cooper and O. F. Jackson, eds.), Vol. 1. Academic Press, London. DeNardo, D. (1996a). Dystocias. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 370-374. Saunders, Philadelphia. DeNardo, D. (1996b). Reproductive biology. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 212-224. Saunders, Philadelphia. Dickinson, H. C., and Fa, J. E. (1997). Ultraviolet light and heat source selection in captive spiny-tailed iguanas (Oplurus cuvieri). Zoo Biol. 16, 391401. Divers, S. (1996). Basic reptile husbandry, history taking, and clinical examination. In Prac. 18, 51-65. Done, L. B. (1996). Neoplasia. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 125-141. Saunders, Philadelphia. Donoghue, S., and Langenberg, J. (1996). Nutrition. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 148-174. Saunders, Philadelphia. Drew, M. L. (1994). Hypercalcemia and hyperphosphatemia in indigo snakes (Drymarchon corais) and serum biochemical reference values. J. Zoo Wild. Med. 25, 48-52. Duguy, R. (1970). Numbers of blood cells and their variations. In "Biology of the Reptilia" (C. Gans and T. C. Parsons, eds.), Vol. 3, pp. 93-109. Academic Press, San Diego. Dyer, O. (1995). Crocodiles help to develop artificial blood. BMJ 310, 211. Ernst, C. H., and Barbour, R. W. (1989). "Turtles of the World." Smithsonian Institution Press, Washington, D.C. Fletcher, J. J. (1994). Fluorescent tubes. Vet. Rec. 134, 395. Frye, F. L. (1995). Iguana iguana: Guide for successful captive care. Krieger Pub. Co., Malabar, Florida. Frye, E L., Oshiro, L. S., Dutra, F. R., and Carney, J. D. (1977). Herpesviruslike infection in two Pacific pond turtles. J. Am. Vet. Med. Assoc. 171, 882. Gehrmann, E. H. (1996). Evaluation of artificial lighting. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 463-465. Saunders, Philadelphia. Gottdenker, N. L., and Jacobson, E. R. (1995). Effect of venipuncture sites on hematologic and clinical biochemical values in desert tortoises (Gopherus agassizii). Am. J. Vet. Res. 56, 19-21. Greenberg, N., and Wingfield, J. C. (1987). Stress and reproduction: Reciprocal relationships. In "Hormones and Reproduction in Fishes, Amphibians, and Reptiles" (D. O. Norris and R. E. Jones, eds.), pp. 461-503. Plenum Press, New York. Greenberg, N., Burghardt, G. M., Crews, D., Font, E., Jones, R. E., and Vaughn, G. (1989). Reptile models for biomedical research. In "Nonmammalian Animal Models for Biomedical Research" (A. D. Woodhead and K. Vivirito, eds.), pp. 289-308. CRC Press, Boca Raton, Florida. Greene, H. W. (1995). Nonavian reptiles as laboratory animals. ILAR J. 37, 182-186. Greene, H. W. (1997). "Snakes: The Evolution of Mystery in Nature." Univ. of California Press, Berkeley. Greiner, E., and Schumacher, J. (1997). Parasitology of reptiles. In "The Biology, Husbandry, and Health Care of Reptiles" (L. Ackerman, ed.), pp. 689-702. TFH Publications, Neptune City, New Jersey. Harcourt-Brown, E (1996). Ultraviolet lights for reptiles. Vet. Rec. 138, 528. Hawkey, C. M., and Dennett, T. B. (1989). "Color Atlas of Comparative Veterinary Hematology." Iowa State Univ. Press, Ames. Henkel, E-W., and Schmidt, W. (1995). "Geckoes." Krieger Pub. Co., Malabar. Hicks, J. W., and Wang, T. (1998). Cardiovascular regulation during anoxia in the turtle: An in vivo study. Physiol. Zoo. 71, 1-14. Hilf, M., Wagner, R., and Yu, V. (1990). A prospective study of upper airway flora in healthy boid snakes and snakes with pneumonia. J. Zoo Wildl. Med. 21(3), 318. Holz, P., Barker, I. K., Crawshaw, G. J., and Dobson, H. (1997). The anatomy and perfusion of the renal portal system in the red-eared slider (Trachemys scripta elegans). J. Zoo Wildl. Med. 28, 378-385. Huff, T. (1980). Captive propagation of the subfamily Boinae with emphasis on

18. BIOLOGY AND DISEASES OF REPTILES the genus Epicrates. In "Reproductive Biology and Diseases of Captive Reptiles" (J. B. Murphy and J. T. Collins, eds.), Vol. 1, pp. 125-134. Society for the Study of Amphibians and Reptiles, Oxford. Jackintell, L. A., and Lance, V. A. (1994). Ontogeny and regional distribution of hormone-producing cells in the embryonic pancreas of Alligator mississippiensis. Gen. Comp. Endocrinol. 94, 244-260. Jacobson, E. R. (1981). Neoplastic diseases. In "Diseases of the Reptilia," (J. E. Cooper and O. E Jackson, eds.), Vol. 2, pp. 429-468. Academic Press, San Diego. Jacobson, E. R. (1983). Parasitic diseases of reptiles. In "Current Veterinary Therapy 8: Small Animal Practice" (R. W. Kirk, ed.), p. 601. Saunders, Philadelphia. Jacobson, E. R. (1984). Biology and diseases of reptiles. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 449-476. Academic Press, San Diego. Jacobson, E. R. (1986). Viruses and viral associated diseases of reptiles. In "Acta Zoologica et Pathologica Antverpiensia. Maintenance and Reproduction of Reptiles in Captivity" (V. L. Bels and A. P. Vanden Sande, eds.), pp. 73-90. Antwerpen, Belgium. Jacobson, E. R. (1993a). Blood collection techniques in reptiles: Laboratory investigations. In "Zoo and Wild Animal Medicine" (M. E. Fowler, ed.), pp. 144-152. Saunders, Philadelphia. Jacobson, E. R. (1993b) Snakes. In "The Veterinary Clinics of North America: Small Animal Practice" (K. E. Quesenberry and E. V. Hillyer, eds.), Vol. 23, pp. 1179-1212. Saunders, Philadelphia. Jacobson, E. R. (1993c). Viral diseases of reptiles. In "Zoo and Wild Animal Medicine" (M. E. Fowler, ed.), pp. 153-159. Saunders, Philadelphia. Jacobson, E. R., and Gardiner, C. H. (1990). Adeno-like virus in esophageal and tracheal mucosa of a Jackson's chameleon (Chameleo jacksonii). Vet. Pathol. 27, 210. Jacobson, E. R., and Gaskin, J. M. (1985). Adenovirus-like infection in a boa constrictor. J. Am. Vet. Med. Assoc. 187, 1226. Jacobson, E. R., Popp, J., Shields, R. P., and Gaskin, J. M. (1979). Pox-like virus associated with skin lesions in captive caimans. J. Am. Vet. Med. Assoc. 175, 937. Jacobson, E. R., Gardiner, C. H., and Foggin, C. M. (1984). Adenovirus-like infection in two Nile crocodiles. J. Am. Vet. Med. Assoc. 185, 1421. Jacobson, E. R., Gaskin, J. M., and Roelke, M. (1986). Conjunctivitis, tracheitis, and pneumonia associated with herpesvirus infection in green sea turtles. J. Am. Vet. Med. Assoc. 189, 1020. Jacobson, E. R., Gaskin, J. M., and Wahlquist, H. (1982). Herpesvirus-like infection in map turtles. J. Am. Vet. Med. Assoc. 181, 1322. Jacobson, E. R., Gaskin, J. M., Brown, M. B., Harris, R. K., Gardines, C. H., LaPointe, J. L., Adams, H. P., and Reggiardo, C. (1991a). Chronic upper respiratory tract disease of free-ranging desert tortoises (Xerobates agassizii). J. Wildl. Dis. 27, 296-316. Jacobson, E. R., Buergelt, C., Williams, B., Bowler, K., and Schumacher, J. (1991b). Herpesvirus in cutaneous fibropapillomas of the green sea turtle (Chelonia mydas). Dis. Aquat. Org. 12, 1. Jacobson, E. R., Gaskin, J. M., Wells, S., Bowler, K., and Schumacher, J. (1992). Epizootic of ophidian paramyxovirus in a zoological collection: Pathological, microbiological, and serological findings. J. Zoo Wildl. Med. 23(3), 318. Jenkins, J. R. (1996). Diagnostic and clinical techniques. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 264-276. Saunders, Philadelphia. Johnson, J. H., and Benson, P. A. (1996). Laboratory reference values for a group of captive ball pythons (Python regius). Am. J. Vet. Res. 57, 13041307. Kanui, T., Mwendia, C., Aulie, A., and Wanyoike, M. (1991). Effects of temperature on growth, food uptake, and retention time of juvenile Nile crocodiles (Crocodylus niloticus). Comp. Biochem. Physiol. A Comp. Physiol. 99, 453-456. Keymer, I. (1978). Diseases of chelonians: Necropsy survey of terrapins and turtles. Vet. Rec. 103, 577.

859 Kohmoto, T., Argenziano, M., Yamamoto, N., Vliet, K. A., Gu, A., DeRosa, C. M., Fisher, P. E., Spotnitz, H. M., Burkhoff, D., and Smith, C. R. (1997). Assessment of transmyocardial perfusion in alligator hearts. Circulation 95, 1585-1591. Lance, V. A. (1990). Stress in reptiles. Prog. Clin. Biol. Res. 342, 461-466. Lane, T. J. (1996). Crocodilians. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 78-94. Saunders, Philadelphia. Lang, J. W. (1987). Crocodilian behaviour: Implications for management. In "Wildlife Management: Crocodiles and Alligators" (G. J. W. Webb, S. C. Manoles, and P. J. Whitehead, eds.), pp. 273-294. Surrey Beatty and Sons, Australia. Lawton, M. P. C. (1992). Neurological diseases. In "Manual of Reptiles" (P. H. Beynon, M. P. C. Lawton, and J. E. Cooper, eds.), pp. 128-137. British Small Animal Veterinary Assoc., Iowa State University Press, Ames. Lunger, P. D., Hardy, W. D., and Clark H. E (1974). C-type particles in a reptilian tumor. J. Natl. Cancer Inst. 52, 1231. Mader, D. R. (1996a). Dysecdysis: Abnormal shedding and retained eye caps. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 368-370. Saunders, Philadelphia. Mader, D. R. (1996b). Gout. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 374-379. Saunders, Philadelphia. Marks, S. K., and Citino, S. B. (1990). Hematology and serum chemistry of the radiated tortoise (Testudo radiata). J. Zoo Wild. Med. 21, 342-344. Mason, R. T., Hoyt, R. E, Jr., Pannell, L. K., Wellner, E. E, and Demeter, B. (1991). Cage design and configuration for arboreal reptiles. Lab. Anim. Sci. 41, 84-86. Mattison, C. (1991). "Keeping and Breeding Lizards." Blandford, London. Mattison, C. (1998). "The Care of Reptiles and Amphibians in Captivity." Blandford, London. Mcllhenny, E. A. (1987). "The Alligator's Life History." Ten Speed Press, Berkeley, California. McKeown, S. (1984). Management and propagation of the lizard genus Phelsuma. Acta Zool. Pathol. Antverp. 78, 149-162. Mermin, J., Hoar, B., and Angulo, F. J. (1997). Iguanas and Salmonella marina infection in children: A reflection of the increasing incidence of reptileassociated salmonellosis in the United States. Pediatrics 99, 399-402. Millan, J. M., Janmaat, A., Richardson, K. C., Chambers, L. K., and Fomiatti, K. R. (1997). Reference ranges for biochemical and haematological values in farmed saltwater crocodile (Crocodylus porosus) yearlings. Aust. Vet. J. 75, 814-817. Miller, A. C., Commens, C. A., Jaworski, R., and Packham, D. (1990). The turtle's revenge: A case of soft tissue Mycobacterium chelonae infection. Med. J. Aust. 153, 493-495. Millichamp, N. J., and Jacobson, E. R. (1983). Diseases of the eye and ocular adnexa in reptiles. J. Am. Vet. Med. Assoc. 18, 1205-1212. Montali, R. J. (1988). Comparative pathology of inflammation in the higher vertebrates (reptiles, birds, and mammals). J. Comp.. Pathol. 99, 1-26. Mori, S. (1995). Disorientation of animals in microgravity. Nagoya J. Med. Sci. 58, 71-81. Moscona, A. A. (1990). Anatomy of the pancreas and Langerhans islets in snakes and lizards. Anat. Rec. 227, 232-244. Muro, J., Cuenca, R., Pastor, J., Vinas, L., and Lavin, S. (1998). Effects of lithium herparin and tripotassium EDTA on hematologic values of Hermann's tortoises (Testudo hermanni). J. Zoo Wild. Med. 29, 40-44. Mushonga, B., and Horowitz, A. (1996). Serous cavities of the Nile crocodile (Crocodylus niloticus). J. Zoo Wild. Med. 27, 170-179. Page, C. D., and, Mautino, M. (1990). Clinical management of tortoises. Compend. Contin. Educ. Pract. Vet. 12, 221-228. Parmenter, R. R., and Avery, H. W. (1990). The feeding ecology of the slider turtle. In "Life History and Ecology of the Slider Turtle" (J. W. Gibbons, ed.), pp. 257- 266. Ponraj, D., and Gopalakrishnakone, P. (1996). Establishment of an animal model for myoglobinuria by use of a myotoxin from Pseudechis australis (king brown snake) venom in mice. Lab. Anim. Sci. 46, 393-398.

860

Pough, E H. (1991). Recommendations for the care of amphibians and reptiles in academic institutions. ILAR News 33, S1-$23. Radcliffe, C. W., and Murphy, J. B. (1983). Precopulatory and related behaviours in captive crotalids and other reptiles: Suggestions for future investigations. Int. Zoo Yearbook 23, 163-166. Raphael, B. L., Klemens, M. W., Moehlman, P., Dierenfeld, E., and Karesh, W. B. (1994). Blood values in free-ranging pancake tortoises (Malacochersus tornieri). J. Zoo Wildl. Med. 25, 63-67. Rebell, H., Rywlin, A., and Haines, H. (1975). A herpesvirus-type agent associated with skin lesions of green sea turtles in aquaculture. Am. J. Vet. Res. 36, 1221. Rehorek, S. J. (1997). Squamate Harderian gland: An overview. Anat. Rec. 248, 301-306. Rosenthal, K. L., and Mader, D. R. (1996). Microbiology. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 117-125. Saunders, Philadelphia. Ross, C. A., and Magnusson, W. E. (1989). Living crocodilians. In "Crocodiles and Alligators" (C. A. Ross, ed.), pp. 58-73. Facts on File, New York. Rossi, J. V. (1992). "Snakes of the United States and Canada: Keeping Them Healthy in Captivity. Volume I: Eastern Area." Krieger Publ. Co., Malabar, Florida. Rossi, J. V. (1996). Dermatology. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 104-I 17. Saunders, Philadelphia. Rossi, J. V., and Rossi, R. (1995). "Snakes of the United States and Canada: Keeping Them Healthy in Captivity. Volume II: Western Area." Krieger Publ. Co., Malabar, Florida. Schaeffer, D. O. (1997). Anesthesia and analgesia in nontraditional laboratory animal species. In "Anesthesia and Analgesia in Laboratory Animals" (D. F. Kohn, S. K. Wixson, W. J. White, and G. J. Benson, eds.), pp. 337-378. Academic Press, San Diego. Schumacher, J. (1996a). Reptile and amphibian anesthesia. In "Lumb and Jones' Veterinary Anesthesia" (J. C. Thurmon, ed.), pp. 670-685. Williams and Wilkins, Baltimore. Schumacher, J. (1996b). Viral diseases. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 224-234. Saunders, Philadelphia. Schumacher, I. M., Brown, M. B., Jacobson, E. R., Collins, B. R., and Klein, P. A. (1993). Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease. J. Clin. Microbiol. 31(6), 1454-1460. Schumacher, J., Jacobson, E. R., Burns, R., and Tramontin, R. R. (1994a). Adenovirus infection in two rosy boas (Lichanura trivirgata). J. Zoo Wildl. Med. 25(3), 461. Schumacher, J., Jacobson, E. R., Homer, B. L., and Gaskin, J. M. (1994b). Inclusion body disease of boid snakes. J. Zoo Wildl. Med. 25(4), 511. Schumacher, J., Papendick, R., Herbst, L., and Jacobson, E. R. (1996). Volvulus of the proximal colon in a hawksbill turtle (Eretmochelys imbricata). J. Zoo Wildl. Med. 27(3), 386-391.

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER Secor, S. M., and Diamond, J. (1997). Determinants of the postfeeding metabolic response of Burmese pythons, Python molurus. Physiol. Zool. 70, 202-212. Silverman, S., and Janssen, D. L. (1996). Diagnostic imaging. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 258-264. Saunders, Philadelphia. Solessio, E., and Engbretson, G. A. (1993). Antagonistic chromatic mechanisms in photoreceptors of the parietal eye of lizards. Nature 364, 442445. Staton, M. A., Edwards, H. M., Jr., Brisbin, I. L., Jr., Joanen, T., and McNease, L. (1990). Protein and energy relationships in the diet of the American alligator (Alligator mississippiensis). J. Nutr. 120, 775-785. Stewart, J. (1990). Anaerobic bacterial infections in reptiles. J. Zoo Wildl. Med. 21(2), 180. Stoakes, L. C. (1992). Respiratory system. In "Manual of Reptiles" (E H. Beynon, M. E C. Lawton, and J. E. Cooper, eds.), pp. 88-100. British Small Animal Veterinary Assoc., Iowa State University Press, Ames. Sypek, J., and Borysenko, M. (1988). Reptiles. In "Vertebrate Blood Cells" (A. E Rowley and N. A. Ratcliffe, eds.), pp. 211-256. Cambridge Univ. Press, Cambridge. Tosini, G., and Avery, R. A. (1996). Dermal photoreceptors regulate basking behavior in the lizard Podarcis muralis. Physiol. Behav. 59, 195-198. Van Der Merwe, N. J., and Kotze, S. H. (1993). The topography of the thoracic and abdominal organs of the Nile crocodile (Crocodylus niloticus). Onderstepoort J. Vet. Res. 60, 219-222. Wallach, J. D., and Boever, W. J. (1983). "Diseases of Exotic Animals: Medical and Surgical Management." Saunders, Philadelphia. Wasser, J. S., Guthrie, S. S., and Chari, M. (1997). In vitro tolerance to anoxia and ischemia in isolated hearts from hypoxia sensitive and hypoxia tolerant turtles. Comp. Biochem. Physiol. l18A, 1359-1370. Winston, G. W., Kirchin, M. A., Ronis, M. J. J. (1991). Microsomal activation of benzo[a]pyrene by Alligator mississippiensis: Mechanisms, mutagenicity, and induction. Biochem. Soc. Trans. 19, 746-750. Wright, K. M., and Skeba, S. (1992). Hematology and plasma chemistries of captive prehensile-tailed skinks (Corucia zebrata). J. Zoo Wild. Med. 23, 429-432. Young, B. A., Sheft, S., and Yost, W. (1995). Sound production in Pituophis melanoleucus (Serpentes: Colubridae) with the first description of a vocal cord in snakes. J. Exp. Zool. 273, 472-481. Zeigel, R. E, and Clark, H. E (1969). Electron microscopic observations on a C-type virus in cell cultures derived from a tumor-bearing viper. J. Natl. Cancer Inst. 42, 1097. Zug, G. R. (1993). "Herpetology: An Introductory Biology of Amphibians and Reptiles." Academic Press, San Diego.

Chapter 19 Biology and Management of the Zebrafish Keith M. Astrofsky, Robert A. Bullis, and Charles G. Sagerstrom

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Background .............................................

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E n v i r o n m e n t a l Factors I m p o r t a n t to H e a l t h . . . . . . . . . . . . . . . . . . . . . . . .

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Total W a t e r H a r d n e s s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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G. G e n e r a l Features of L a b o r a t o r y Facilities . . . . . . . . . . . . . . . . . . . . . . N u t r i t i o n and F e e d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acquisition

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Bacterial S e p t i c e m i a

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Aquatic Mycosis

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Trichodinosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Infectious Pancreatic Necrosis Virus and O t h e r Viruses . . . . . . . . . . .

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References ..................................................

LABORATORY ANIMAL MEDICINE, 2nd edition

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H e a l t h M a n a g e m e n t in the A q u a t i c A n i m a l Facility . . . . . . . . . . . . . . . . . .

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Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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I.

INTRODUCTION

A.

Background

The zebrafish, Brachydanio rerio (also referred to as Danio rerio and the zebra danio), is currently emerging as an increasingly popular model of vertebrate embryonic development, gene function analysis, and mutagenesis. Prior to the development of the zebrafish model in the 1970s, developmental geneticists relied on invertebrate models such as Drosophila melanogaster (fruit fly) and, more recently, Caenorhabditis elegans (nematode) for the investigation of early embryonic development. The prolific reproductive capacity of Drosophila and successful manipulation of its embryos combined to make embryo development and genetic analysis more practical in the laboratory. However, the application of this information to vertebrate embryonic development was limited. Since mouse embryos develop within a uterus and the African clawed frog (Xenopus laevis) has a slow reproductive capacity, neither of these more popular vertebrate models possesses the characteristics that made Drosophila such a practical model (Kahn, 1994). Because of its high fecundity and external fertilization, the zebrafish possessed the attributes of these existing models without their inherent drawbacks. Because the fundamental molecular mechanisms of embryonic development are similar for all vertebrates, the zebrafish has gradually become the lower vertebrate model of choice. Mutagenesis screens allow researchers to investigate uncharted areas of the genome without prior knowledge of the function of specific genes. Mice are utilized in more conventional laboratory techniques where knockout mutations of known genes of interest are created in order to study the effect of the gene in the resultant phenotype. Through the creation of mutant phenotypes via chemical mutagenesis, the functions of many genes associated with pigmentation, muscular, cardiovascular, and central nervous system (CNS) development have been investigated extensively (Driever et al., 1994; Postlethwait et al., 1997). In the early 1970s, George Streisinger (University of Oregon), a phage geneticist, identified the zebrafish as a vertebrate model to isolate mutations in genetic screens using systematic mutagenesis protocols. This line of work has been continued at the University of Oregon by several laboratories, including that of Charles Kimmel, who has also contributed much of the information involving the staging of early zebrafish embryo development. By the early 1990s, two laboratories (Christiane Nusslein-Volhard of the Max Planck Institute for Developmental Biology and Wolfgang Driever of Massachusetts General Hospital) began applying "saturation mutagenesis" screens to identify mutant phenotypes in zebrafish (Kahn, 1994). Using this methodology, researchers treated adult male zebrafish with a

chemical mutagen, and the F 3 generation was examined for developmental abnormalities. The function of various disrupted genes could be identified by observing mutant phenotypes in large numbers of developing zebrafish. By 1996, these screens had produced over 2000 mutations in several hundred genes that are necessary for normal embryonic development (Driever et al., 1996; Haffter et al., 1996). In 1994, John Postlethwait (University of Oregon) published the first genetic map that identified approximately 400 markers in 29 linkage groups. By 1996, the genetic map had already grown to incorporate approximately 1200 markers (Postlethwait et al., 1997).

B.

Natural History

The zebrafish is a freshwater species native to the Ganges River of India and extends into other surrounding mainland areas of southern Asia (Hamilton-Buchanan, 1822). The zebrafish is a member of the family Cyprinidae, which includes the danios and barbs. Adults are sexually dimorphic, with the females being slightly larger, more silvery, and slightly rounded. Adults usually do not exceed 3 - 4 cm in length. Males are more streamlined and usually more brightly colored than females. Healthy adults are usually a silver or gold (less common) tone with several bright blue/purple horizontal stripes extending from the operculum to the base of the caudal fin. Similarly colored stripes are frequently repeated on both the anal and dorsal fins. Males exhibiting dominant behavior over subordinate tankmates usually demonstrate brighter gold or yellow tones. Dorsally, a dark yellowish brown to olive tone is evident from the head to the caudal fin. The ventrum is usually a homogeneous, yellow-white tone. Two pairs of barbules are present on the lower jaw. Selective breeding by the aquarium trade has produced a number of varieties, including the veil-finned and the long-fin variants. Zebrafish are usually not aggressive and are very active swimmers occupying the upper strata of the water column. They are omnivorous and have proven to be quite hardy and prolific breeders in captivity. Through good nutrition and by manipulation of the light-dark cycle, females can be readily induced to spawn in the laboratory. After a period of total darkness, the initial appearance of light and persistent rubbing of the female by the male induces the female to spawn into an egg-collection chamber placed at the bottom of the containment unit. After inducing the female to spawn through tactile behavior, the males promptly fertilize the eggs. Egg-collection devices or rows of glass beads and marbles placed at the bottom of the unit are necessary to prevent the adults from consuming their own spawned eggs prior to collection.

19. BIOLOGY AND MANAGEMENTOF THE ZEBRAFISH II. EXPERIMENTAL MODEL IN BIOMEDICAL RESEARCH

A.

Model System

The zebrafish has been used as a biomedical model for several decades (Streisinger et al., 1981) but only recently has it received widespread attention, most notably from the completion of two large-scale mutagenic screens for genes that regulate early development and organogenesis (Driever et al., 1996; Haffter et al., 1996). As these genetic screens underscore, the zebrafish is used primarily as a model in developmental biology. However, biological processes relevant to the adult organism can also be addressed (e.g., hemostasis; Jagadeeswaran and Liu, 1997). Although much work has emphasized zebrafish as a model for basic animal development, its appeal goes beyond such uses and extends into its role as a model organism for human development and disease. 1.

Characteristics of Model Organism

The utility of the zebrafish as a model system depends not only on its shared features with other animals and humans, but also on several characteristics that permit a variety of experimental approaches. Adult zebrafish are small (approximately 1 inch long), which makes it possible to keep a larger number of fish than of other vertebrates, such as chicks or mice, in a given space. Further, zebrafish require little maintenance, except for water changes and feedings, and these can be automated. This makes zebrafish a very economical choice as a model organism. Zebrafish routinely lay a couple of hundred eggs, and females can be recrossed at weekly intervals. These large litter sizes are important for genetic experiments and also provide large numbers of embryos for molecular and biochemical assays. Since the embryos are laid in rapid succession, they also represent a fairly well synchronized population when utilizing segregated pair crosses. Zebrafish embryos develop outside the mother and contain yolk that nourishes them for several days. This means that all stages of development are accessible for analysis, without sacrificing the mother, as is often needed to obtain embryos that develop in utero. The embryos are optically transparent, which makes it possible to observe cells in the living embryo. Zebrafish embryos develop very rapidly, and 24 hr after fertilization the embryos have a fish-like appearance with most of the organ systems already functioning. Experiments that target development of a specific organ system (e.g., by transient overexpression of a particular gene) can therefore be scored within a day or two. The zebrafish genome is about half the size of mammalian genomes; thus the generation of physical maps and positional cloning projects in zebrafish require similar efforts to those in

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the mouse. Zebrafish reach sexual maturity in a relatively short time ( 2 - 4 months, depending on density of fish and rate of feeding). Taken together, these characteristics allow zebrafish to be probed by a variety of embryological, molecular, and genetic methods. 2.

Embryonic Development

After fertilization the zebrafish embryo undergoes a series of rapid cleavages. At 28.5~ (the standard temperature for zebrafish maintenance) the first cleavage occurs 45 min after fertilization, and the subsequent eight cleavages take place at regular 15-min intervals. After the ninth cleavage the cell cycle begins to lengthen, and cell divisions become asynchronous. This marks the onset of the midblastula transition (MBT) at about 3 hr postfertilization (hpf). One important aspect of the MBT is the activation of zygotic transcription; all processes taking place up to this point are regulated by maternal factors deposited in the egg. Gastrulation, the process whereby the three germ layers (endoderm, mesoderm, and ectoderm) take up their final positions in the embryo, begins after about 5.5 hpf and is complete at 10 hpf. Since the early cleavages do not bisect the yolk, the embryo proper sits as a ball of cells on top of the yolk until about 4.5 hpf, when the embryonic cells begin to spread down over the yolk. This process, termed "epiboly," is also completed at 10 hpf. During the period between 10 and 24 hpf, the internal organs begin to form, and morphological landmarks that preview adult structures become detectable. At 24 hpf the heart is beating, blood is starting to circulate, and the embryo becomes responsive to touches, signaling the presence of neuromuscular connections. Over the next several days the embryo continues to grow, and internal organs complete their development. Embryos hatch from their chorions on day 3 or 4 and begin to feed shortly thereafter on day 4 or 5 (Kimmel et al., 1995). 3.

Model for Human Disease and Development

The use of the zebrafish as a model for human biology stems from the fact that the zebrafish belongs to the teleostei, or bony fishes, and as such is a vertebrate. Since all vertebrates show extensive similarities in early development, organogenesis, and physiology, findings from zebrafish are applicable to human conditions. This has been demonstrated particularly for blood and the vascular system, where mutations resembling human disease conditions have been isolated. For instance, the "gridlock" mutation appears to mimic the human birth defect "coarctation of the aorta" (Weinstein et al., 1995), and mutations that lead to light sensitivity of red blood cells may be similar to human congenital erythropoietic porphyrias (Ransom et al., 1996). Although it is possible to extrapolate from fish to humans, there are clearly model organisms that display greater

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KEITH M. ASTROFSKY, ROBERT A. BULLIS, AND CHARLES G. SAGERSTROM

similarities to humans (e.g., lower mammals such as the mouse and various nonhuman primates). However, because of the unique characteristics of zebrafish, some experimental techniques can be applied more readily in this organism than in others. The choice of which system to employ as a model for human biology therefore depends on a balance between what techniques are required and how closely the chosen organism mimics the human situation.

B.

C o m m o n l y Used Experimental Methods

As already stated, most work on zebrafish to date has targeted early development and organogenesis, but with appropriate modifications, the techniques employed should be applicable to a wide variety of biological problems. The following section is intended to provide an overview of approaches currently taken with zebrafish. For experimental details the readers are referred to several zebrafish handbooks (Westerfield, 1995; Dietrich, 1999a, 1999b). 1.

Genetic Methods

Genetic screens represent a powerful tool to identify novel genes involved in a particular biological process. To date, the zebrafish is the only vertebrate model organism for which largescale genetic screens can be performed rapidly. To this end, founder fish are exposed to a mutagen and the progeny inbred to generate homozygous mutant fish (Driever et al., 1996; Haffter et al., 1996). The mutant offspring is then scored for defects in the biological process under study. Zebrafish are well suited for this type of analysis because large numbers can be maintained in a small space, the embryos are translucent, and they can be observed continuously during development. Several mutagenic screen variations have been used (Alexander et al., 1998). The basic approach is a two-generation breeding scheme where males are mutagenized and outcrossed to wild-type females. The offspring are inbred, and the F 3 generation is scored for mutations. This approach can be modified by instead raising the F2 generation as haploid fish, thereby eliminating the need for one intercross and saving a significant amount of time. Haploid fish are generated by fertilizing eggs in vitro with UVirradiated sperm. The resulting embryos develop fairly normally for several days before dying, however; some mutations may have a different phenotype in haploids than in diploids. Another variation is to cross the mutated males immediately after mutagenesis. This ensures that postmeiotic gametes, where the mutation is not yet fixed in the genome, will contribute to the zygote. Mutations will then become fixed in the genome of the developing embryo, which results in mosaic offspring. Since mosaic fish can carry a greater number of mutations than nonmosaic fish, it is possible to screen a large number of muta-

tions in a small number of fish. The trade-off is that the germline will also be mosaic, and not all offspring will carry a particular mutation. (N-Nitroso-N-ethylurea) (ENU), which primarily generates point mutations (Driever et al., 1996; Haffter et al., 1996), and radiation (Walker and Streisinger, 1983), which leads to larger defects (e.g., deletions and inversions), have been used as mutagens in zebrafish with great success. The drawback with these agents is that while they are efficient at inducing mutations, it is labor-intensive to identify the mutated genes. An alternative approach utilizes viral infection to integrate a piece of DNA with a known sequence into the genome (Gaiano et al., 1996). When the inserted DNA disrupts an essential gene, a phenotype is observed. The mutated gene can then be isolated by polymerase chain reaction (PCR)-based approaches in a short period of time. The mutagenesis step is more time-consuming in this type of screen, but the cloning is significantly simplified. Reverse genetics, whereby a known gene is disrupted in the germline by knockout genetics, has been of immense value to mouse biologists. Such technologies are not yet available in the zebrafish. Instead, PCR is used to screen for disruption of genes of interest in the offspring of fish mutated by radiation (Fritz et al., 1996). The ability to generate haploid embryos greatly simplifies this process. 2.

Embryological Methods

Various embryological methods have been applied for over a century to answer questions about how the development of vertebrate organisms is orchestrated. These methods have been applied mostly in amphibian species, primarily Xenopus laevis. Amphibians are particularly attractive for this purpose because they lay large eggs that develop externally. Since this is also true for zebrafish embryos, several of the methods originally applied to amphibians have been adapted for use in zebrafish embryos (Eisen, 1991; Sagerstr6m et al., 1996; Shih and Fraser, 1996). The application of these techniques to the zebrafish allows the unique combination of embryological and genetic tools (e.g., using cell transplantation to analyze cell-autonomous and noncell-autonomous defects in mutant fish lines) adapted for zebrafish. This can not be done in Xenopus, which is not a genetically accessible organism, and is complicated in the mouse by the intrauterine development of the embryos. In studying particular cells or embryonic regions, it is useful to know to what tissues they will contribute. This final differentiation state is referred to as the "fate" of the cell, and by determining the fate of each cell at a particular developmental stage, one can draw a fate map that shows what differentiation pathway each cell will adopt. Fate maps have been derived for the whole zebrafish embryo (Kimmel et al., 1990) and for individual tissues, e.g., the neurectoderm (Woo and Fraser, 1995) and the heart (Stainier et al., 1993). It is important to note that a fate map does not provide infor-

19. BIOLOGY AND MANAGEMENTOF THE ZEBRAFISH

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mation about the commitment of the labeled cell. To test the commitment state of a cell, it must be exposed to a new environment. This can be accomplished by transplantation (e.g., Ho and Kimmel, 1993; Eisen, 1991; Shih and Fraser, 1996) or in vitro culture of embryonic tissue explants (Sagerstr6m et al., 1996). If, following transplantation or explantation, the cells differentiate along the same pathway as dictated by the fate map, they are said to be "committed."

dominant negative constructs (e.g., for kinases) or suppressing constructs (e.g., fusion of the engrailed repressor domain to transcriptional regulators). In some instances, it has also been possible to inactivate proteins by microinjection of specific antibodies (Krauss et al., 1992), presumably by interfering with the normal function of the targeted protein. A different approach is to employ an antibody in conjunction with lasermediated inactivation (Jay, 1988).

3.

4.

Molecular Methods

In many instances, the isolation of new genes from zebrafish is done in a targeted fashion, attempting to isolate genes that function in a particular biological process. This can be done by a candidate approach (searching for genes with homologues of known function in other organisms) or by selectively enriching for genes of interest (e.g., by subtractive hybridization; Sagerstrom et al., 1997). Less directed approaches, aimed at isolating as many zebrafish genes as possible (e.g., by generating expressed sequence tags; Gong et al., 1997), have also been undertaken. The technologies (e.g., mRNA purification) and physical resources (e.g., cDNA libraries) required for these types of work are readily available. Analyses of gene function can be readily performed in zebrafish by transient or stable gene expression. This is accomplished by pressure-driven microinjection of DNA or RNA into immobilized embryos viewed under a steromicroscope. For stable expression, DNA encoding the gene of interest is microinjected, and embryos where the exogenous DNA has integrated in the germline are identified (e.g., Lin et al., 1994). For transient expression, DNA or in vitro transcribed RNA encoding the gene of interest is injected. DNA is more stable than RNA but is inherited less evenly, which leads to more widespread mosaicism. Coinjection of a tracer (usually RNA encoding green fluorescent protein (GFP)) is often used to determine the distribution of the experimental RNA or DNA. This is particularly attractive since zebrafish embryos are transparent through the first 24 hr and the GFP can be detected directly in the living embryo. Transient gene expression persists for an extended period of time in the embryo, and most developmental processes can be analyzed in this fashion. Another important consideration is that injected DNA constructs are not expressed until after the midblastula transition (MBT), while injected RNA is translated immediately upon injection. Various approaches have also been developed to temporally control activity of the injected gene (e.g., Kolm and Sive, 1995). Gain- and loss-of-function assays are often employed to study the function of particular genes, and these assays can be carried out in a variety of ways in the zebrafish. Gain-of-function analyses are often performed by ectopic overexpression of the wild-type gene, or, if the gene function is known, by mutations leading to constitutive activity (e.g., for kinases). Similarly, loss-of-function analyses can be performed by overexpressing

Toxicological Methods

The use of zebrafish in determining the effects of various toxins precedes its more recent use in studying early development. The recent accumulation of information about the development and biology of the zebrafish makes its use in toxicological analysis particularly attractive since the more that is known about normal biology and development, the better one can understand the effects of various compounds. The zebrafish is currently being used both as a sentinel species to screen for compounds with toxic effects (Mizell and Romig, 1997) and as a model organism for the in-depth analysis of the effects of particular compounds, such as dioxin (Henry et al., 1997). The zebrafish eggs and early embryos can be used to determine both mortality rates and frequencies of abnormalities in developing embryos. Since the protective chorion is impenetrable to some compounds, it may be necessary to dechorionate the embryo (a process that can be carried out by exposure to enzymes or by manual removal) or to microinject the compound under study into the intrachorionic space. The exposure of adult zebrafish to toxins is also used as a model for detection of tumorigenic compounds (Khudoley, 1984). Cell lines have been established from various zebrafish tissues and used in the analysis of cellular responses to toxins (Collodi et al., 1992). In the future, the combination of toxicology and genetics should permit the isolation of mutations that compensate for defects induced by toxins, which will allow for a better understanding of the toxic mechanisms of action. 5.

Calcium Imaging Methods

All healthy cells maintain stringent control of their intracellular calcium levels. This is especially true of the developing and differentiating cells of embryos. Calcium ions function as a "universal second messenger" carrying information across cells, tissues, organs, and organisms, as well as communicating between external and internal environments in order to orchestrate normal development and cell function. The calcium-sensitive bioluminescent protein, aequorin, is used to study the role played by calcium signaling. When this nontoxic protein comes into contact with calcium ions, it emits light. Ultrasensitive spatial and temporal information is then collected by photomultiplier tubes or photon imaging microscopy (Miller et al., 1994). In addition to its use in studying normal developmental

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KEITH M. ASTROFSKY, ROBERT A. BULLIS, AND CHARLES G. SAGERSTROM

signaling, calcium signaling can be used to study disruptions in embryogenesis. Zebrafish embryos come in direct contact with their surrounding aquatic environment, to which they are exquisitely sensitive. One of the first responses of a cell to toxicant-induced damage, which often results in cell death, (i.e., LD 50/LC50) is a loss of its ability to regulate intracellular calcium levels. Thus, the ability to reliably and routinely monitor calcium-signaling patterns in developing embryos represents an attractive, ultrasensitive methodology to access levels of a wide variety of toxicants in the environment. Under development are transgenic zebrafish that contain the jellyfish-derived protein (aequorin), which improves the efficiency of the bioassay by eliminating the unnecessary step of adding the aequorin experimentally (Miller et al., 1994). The emergence of such specialized strains will undoubtedly contribute to the advancement and the overall utility of the zebrafish model. 6.

Genomics

The haploid zebrafish genome contains approximately 1.7 x 10 9 bp divided among 25 chromosomes, and significant efforts aimed at developing genetic and physical maps, as well as the technologies to utilize these resources, have been undertaken. These maps are important aids for, among other things, the cloning of mutated genes. At present, the zebrafish genetic map is made up of several different types of markers spaced an average of 1.8 Mbp apart. Reagents such as bacterial artificial chromosome (BAC) libraries required for positional cloning of mutated genes are now available, as are zebrafish/mouse and zebrafish/hamster radiation hybrid panels. With the aid of these resources several mutations have been shown to reside in already-cloned genes (e.g., Talbot et al., 1995), and others have been isolated by positional cloning (e.g., Zhang et al., 1998).

III.

H E A L T H MANAGEMENT IN T H E AQUATIC ANIMAL FACILITY

A.

Health of the Aquatic Animal Model

Proper animal health will ensure vigorous animals and maximal reproductive yield, and minimize animal loss due to disease. Health is dependent not only on proper nutrition and animal selection, but also on proper water quality, water-system/ life-support maintenance, and water-system design. By operating closely monitored, well-maintained water systems and implementing a preventive medicine program, fish hygiene can be maintained at a high level. This management approach will minimize losses in valuable animals, research time, and money due to water-system failure or infectious disease (Detolla, 1995).

Unexpected loss of valuable aquatic research animals due to disease is one of the most common causes of research frustration when using aquatic animal models. Whether these losses occur due to slow, insidious mortality or by sudden, catastrophic loss because of an unexplained epizootic or failure of the life-support system, diseases of aquatic animal models have adversely influenced the validity of statistics, confounded resuits, and disrupted research schedules. Preventive medicine programs tailored to the aquatic animal have as much validity in the modern zebrafish facility as these programs do in a rodent facility. B.

H o s t - P a t h o g e n - E n v i r o n m e n t Interaction

Infection is always the result of a complex interaction of host, environment, and pathogen. All three factors may be out of balance in the highly artificial world of aquatic laboratory culture facilities. A primary goal of the aquatic animal manager is to maintain the aquatic system in a balanced state where host and potential pathogens coexist in the controlled laboratory environment (Fig. 1). Because aquatic systems are very complex ecosystems, pathogen-host relationships are difficult to access in the artificial laboratory environment. Consequently, controlling disease outbreaks can be a difficult endeavor. Since primary pathogens can induce disease when other environmental factors are balanced, they must be distinguished from secondary or opportunistic pathogens that cause disease when other metabolic or environmental factors are suboptimal. Poor health is often a reflection of one or more marginal environmental variables, such as improper temperature or pH, hardness/alkalinity or other water chemistry, nutrition, overstocking, biofouling, or bacterial overgrowth. Disease within the aquatic environment usually results when stresses within a marginal environment tip the balance in favor of the pathogen. Disease diagnosis can be viewed as the recognition of imbalance within the system. Prevention acts as a buffer to extend the limits of the balanced system. The aim of treatment is to restore balance to a disrupted system. Proper health management and disease control depend on effective implementation of all the above-mentioned laboratory practices: preventive medicine, diagnosis, and treatment.

IV.

ENVIRONMENTAL FACTORS IMPORTANT TO HEALTH

A.

Temperature

Temperature is critically important in the survival, development, growth, and successful reproduction of zebrafish. When

19. BIOLOGY AND MANAGEMENT OF THE ZEBRAFISH

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Fig. 1. Host-pathogen-environmentinteraction diagram. The aquatic ecosystemmaintains a delicatebalance between fish, potential pathogens, and environmental parameters. While relativelyfew diseases are the direct result of environmental imbalances (e.g., temperature shock, gas bubble disease), the environment has a direct influence on potential pathogen populations and the host's resistance to disease.

the water temperature is abruptly raised or lowered, zebrafish, as do most poikilotherms, show an internal shock reaction. The magnitude of this effect depends on the strain, its recent thermal history, and the magnitude of the temperature change. As a general rule, a change in temperature should be limited to ___1.5~ day, although many aquatic organisms can tolerate larger shifts in temperature quite well after an initial shock and a brief period of acclimation. The following generalizations may be useful in developing laboratory practices that promote good health. The optimal temperature for the zebrafish appears to be 75~176 (24-28~ The higher temperatures are recommended to stimulate egg laying/reproductive behavior and to facilitate development of fertilized embryos. A change in temperature affects tolerance to other factors. Increased temperature (within the tolerance range) speeds up metabolism and increases oxygen demand. As a general rule, metabolic rate doubles for each 10~ increase in temperature. Larvae are usually less temperature tolerant than their respective adult forms. Maintenance of the lifesupport system at slightly lower temperatures in the laboratory will increase the available oxygen, reduce the need for food, and minimize losses due to accidental increases in temperature. However, lower temperatures will decrease rate of growth and development. The limits of temperature tolerance are highly variable among populations and between seasons. Temperature change (usually temperature increases) is often a factor in the initiation of reproductive activity or other hormonal-induced activity.

The temperature of the water system should be monitored and recorded daily by the placement oi~a probe into each independent system. Small shifts in the temperature either above or below the optimal range are usually not detrimental. However, sudden, large shifts in environmental temperature can shock and quickly incapacitate zebrafish. The ambient room temperature should also be maintained at least 1~176 above that of the containment system water. Room air at a temperature of approximately 78~176 (24-27~ helps prevent condensation of water on the external surfaces of aquaria, walls, and floors. Condensation on these surfaces can serve as a medium for the growth of mold or fungi and negatively impact air quality. Room temperatures of greater than 80~ (27~ are generally not recommended due to the cost of maintaining such a high temperature, lower dissolved oxygen saturation associated with warmer water, higher metabolic rates of the fish, and laboratory worker discomfort.

B.

Dissolved Oxygen

In water, a fish will asphyxiate when the dissolved-oxygen content drops below a critical level. This level is speciesand strain-specific, subject to adaptation, and temperaturedependent. When a fish is taken out of water, the gill lamellae stick together because they lack internal skeletal support. Since this dramatically reduces the surface area available for oxygen

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KEITH M. ASTROFSKY, ROBERT A. BULLIS, AND CHARLES G. SAGERSTROM

diffusion, the fish will ultimately die of asphyxiation unless returned to the water. Therefore, the length of time out of water due to periodic sampling for experiments or when transferring animals should be as brief as possible. Fish avoid areas where oxygen depletion develops. This can be an aid in the identification of oxygen-deficient areas within water systems. Conversely, large numbers of fish gathered around aerators, air stones, or other points of air supply indicate serious oxygen depletion within the system. In the same manner, fish gathered at the water surface also indicate oxygen deprivation (Fig. 2). This behavior is elicited because the oxygen saturation of the water is slightly higher at the air-water interface. Although adequate oxygenation is essential, too much dissolved oxygen or other compressed gases, such as nitrogen, can be detrimental and even fatal if not corrected quickly. Massive aeration can produce clouds of very fine bubbles. Also, improperly maintained equipment, such as cavitating water pumps or air stones placed too close to the water pump inflow lines, can compress room air to the point where the water becomes supersaturated with dissolved gas (Stoskopf, 1993; unpublished observations). Under the high pressure of a mechanical air pump, compressed gases tend to remain in solution. Once expelled into the circulating water system, the compressed gas comes out of solution at lower pressure in the respiring fish. This results in a phenomenon similar to the "bends" in humans when air emboli develop in the smaller blood vessels such as in the gill lamellae. These emboli disrupt the flow of blood across the gill or in some instances can rupture the delicate vessels, resulting in hemorrhage, asphyxiation, and death. This condition is known as gas bubble disease (GBD) and frequently manifests as fish demonstrating difficulty in breathing (open mouth), hemorrhage around the gills, exophthalmos, small air bubbles associated with the scales or cornea, and sudden death. Definitive diagnosis of GBD can be difficult unless affected fish are necropsied soon after death. Overt lesions associated with GBD frequently become inapparent after 24 hr postmortem (Lasee, 1995). GBD must be quickly remedied by removal of all secondary

aeration devices (air stones, etc.) that contribute to adding more pressurized gas to the water. This will reduce the mixing of dissolved gas into the water system. Most cases of gas supersaturation can be directly attributed to air becoming entrapped in the plumbing system through faulty or aging equipment, such as piping, joint seals, or hose fittings usually located on the suction side of the pump. Gas supersaturation of the water system can also occur at any location that could permit air to enter and remain in the system under increased pressure. Cavitation of the water or air becoming entrapped in the pump is a frequent cause in precipitating gas supersaturation and can sometimes be diagnosed by listening to the pump. Air trapped within a running pump will usually result in a "gurgling" or "rattling" noise. Defective components resulting in air leakage need to be identified, repaired, or replaced. Frequently, operating systems need to be modified to avoid placement of air stones close to the pump inlet. Never allow a pump to run dry without water or to "gulp" air during water changes. A properly placed "bleeder valve" on the discharge side of the pump can permit entrapped air within the pump to be vented without shutting down the system. Once the excess air is purged from the system and the cause of the gas supersaturation identified and remedied, the increased levels of dissolved gas within the water will equilibrate with the room air. Aeration can be provided to shallow trays by simply maintaining circulation of the water. Oxygen diffusion is adequate at depths of less than 4 inches. Air should be pumped at the rate of 2 liters/min for each 100 liters of system water. A lower capacity can be utilized if fish are inactive or water temperature is at the lower threshold of thermal tolerance for that species or strain or by experimental design. Compressed air is hot and dry, which tends to accelerate the evaporation of water within the system. Air bubbling into aquaria will produce a fine mist that can carry fomites and aid in the transmission of disease. Mists can also result in the rusting of metal components. The resulting "scale" can build up and drop into tanks, resulting in increased levels of metallic ions that can be toxic. Therefore, all metallic, non-stainless steel components should be regularly evaluated for scale or "rust" buildup. These components should either be replaced or scraped routinely to prevent introduction into the water system

C.

Fig. 2. Zebrafishdisplaying abnormal surface-breathing behavior due to low dissolved-oxygenlevels within the water.Whenlow dissolved-oxygenlevels are present, fish will congregate in areas of highest saturation (e.g., airwater interface, waterinflowports).

pH

Zebrafish, like most freshwater species, prefer a pH range of pH 6.8-7.2 with pH 7.0 being optimal. As with temperature shifts outside the optimal range, sudden, drastic shifts in pH can be very detrimental to animal health. Higher pH ( > 8.0) also results in higher concentrations of un-ionized ammonia (NH3). Low pH (< 5.0) inhibits the activity of nitrifying bacteria, which tends to increase total ammonia levels due to accumulation.

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19. BIOLOGY AND MANAGEMENT OF T ~ ZEBRAFISH

In closed, recirculating water systems, the pH will gradually decrease due to the production of acids during the nitrification process as the bacteria within the biofilter convert ammonia to nitrate. The pH will also decrease in poorly aerated systems due to the production and accumulation of carbon dioxide (CO2) created by respiring fish. The pH of the water system has a direct influence on the susceptibility of fish to toxins. Common metallic contaminants such as zinc, copper, iron, and aluminum are more soluble in water under acidic conditions. Therefore, toxicity associated with exposure to these elements is more common in water systems maintained at a lower pH value.

D.

Conductivity

Conductivity is an indicator of the total amount of dissolved ions in a solution that includes sodium and other ionized minerals. It is a direct measure of the amount of electric current that a particular aqueous solution can conduct. Since direct measurement of salinity is difficult, conductivity is a convenient method to imprecisely measure the salinity of the water system and allows monitoring of changes in salinity due to water changes or evaporative loss. Most freshwater species have a salinity range for optimal growth and reproduction (Stoskopf, 1993). Conductivity is usually measured in terms of microseimens (ms) with zebrafish generally preferring conductivity of 3-500 ms for optimal growth and breeding.

E.

Total Water Hardness

The amount of calcium and magnesium salts in the water is referred to as the water hardness. Other cations also contribute to the total hardness of the water, but these are usually present only in very small quantities within normal freshwater. Commercially available test kits tend to measure hardness in terms of how much calcium carbonate (CaCO3) is present in the water. Water-quality reports usually express hardness levels in terms of parts per million (ppm) or milligrams per liter (mg/liter). Zebrafish are generally considered to be a "hard" water species with optimum calcium and magnesium levels between 80 and 200 ppm. Very soft water (0-10 ppm) can be detrimental to young developing fry since they rely on the water for essential mineral uptake during the early, growing phases of life. Low water hardness or calcium levels have also been found to be associated with low embryo survival rates and increased susceptibility to other environmentally induced disease as a result of poor water quality (Stoskopf, 1993 referencing Piper et al., 1982). When utilizing reverse osmosis (RO) or distilled water, the

minerals have been removed by the filtration process and need to be replaced. Calcium can be added to the water in the form of CaCO 3 or crushed coral preparations.

F.

A m m o n i a , Nitrite, and Nitrate

A thorough understanding of the major environmental inputs and ubiquitous microorganisms that metabolize proteins to form nitrogenous by-products by way of the nitrogen cycle is essential in evaluating or interpreting the levels of ammonia (NH3), nitrite (NO2), and nitrate (NO3) within a water system. In the nitrogen or nitrification cycle, a mixed population of bacteria develop within the biofilter of the system. A fully functioning biofilter should be capable of converting all toxic nitrogenous wastes produced within a closed system to nontoxic by-products. Uneaten, decaying food and excretion from the fish are the primary sources of nitrogen in the form of ammonia within the system. Ammonia is first converted to nitrite by Nitrosomonas spp. bacteria present in the biofilter. The nitrite is then converted to nitrate by the action of Nitrobacter spp. bacteria that are also normal flora of the established biofilter (Fig. 3). Although these bacteria are primarily responsible for the conversion of ammonia to nitrate, other bacterial species of the flora, including the potentially opportunistic heterotrophs, such as Aeromonas spp. and Pseudomonas spp., contribute to the conversion of nitrogenous wastes. These bacteria are also integral components of the normal flora of the fish and are constantly seeded into the biofilter through fish excretions. It is important to realize that the biofilter is a dynamic population of many species of bacteria that have a significant impact on water quality and subsequently, fish health. Any abrupt change in the aquatic environment can adversely affect the bacteria within the biofilter. The addition of large numbers of new fish too quickly or treatment of the water with unwarranted chemical agents (including antibiotics) can frequently result in disastrous shifts in the character of the bacterial flora within the biofilter. Hence, when populations of bacteria within the biofilter suddenly die off or "crash," significant and sudden changes in the pH of the water and levels of ammonia, nitrite, and nitrate frequently result. While high levels of either ammonia or nitrite are very toxic to fish, nitrate is relatively nontoxic. However, maintaining low levels of nitrate within the system is important to ensure proper health of the biofilter and to control the growth of algae. Therefore, periodic water changes are essential to prevent the accumulation of nitrate and other toxic metabolites within the environment. However, biofilter "crashes" can also be precipitated by changing the system water too frequently or changing too large a volume of water during a scheduled water exchange. Improper water changes can also mechanically disrupt a biofilter by too vigorous mixing, which dislodges the bacteria from the substrate.

KEITH M. ASTROFSKY, ROBERT A. BULLIS, AND CHARLES G. SAGERSTROM

870

Fig. 3. The nitrogen cycle. Decaying food and nitrogenous waste excretion are the primary sources of ammonia. Toxic ammonia is converted to nitrite by Nitrosomonas spp. The toxic nitrite is then converted to relatively nontoxic nitrate by Nitrobacter spp. Nitrate accumulates within the water unless removedby

regular water changes or by resident plant metabolism.

G.

General Features of Laboratory Facilities

1. Composition Wall coverings, floor treatments, door thresholds, and ceilings should be of materials impervious to water or rendered waterresistant as necessary. Epoxy-coated floors and epoxy-painted walls offer the advantages of easy sanitation and water resistance. Doors should have thresholds that are bermed to prevent water leakage.

2. Plumbing Only "food-grade" silicon sealer should be utilized, to avoid the introduction of potentially toxic chemical leachates. Copper piping and lead-based solders should be avoided. Rooms should be provided with adequate drainage. This may require floor drains in several locations. Steeply angled flooring to a central drain should be avoided because this creates unstable footing for heavy aquarium racks. Specialized drains are an important consideration. Discharges of water with infectious agents and or life stages of exotic (nonindigenous) species should not be discharged into surface waters. If outflow water is not plumbed to sanitary lines with proper chlorine disinfection or ozone treatment, specialized

systems to contain and treat contaminated wastewater should be constructed. Polishing filters (particulate and/or charcoal) as well as water softeners can be provided to ensure the availability of conditioned water as necessary.

3. Lighting Systems Photoperiod is of critical importance in maintaining normal physiological/environmental health in aquatic systems. Most appropriate are 12-16 hr of light per day supplied by either natural or balanced fluorescent lights. Rooms should be equipped with overhead lighting with a timer control. Depending on the protocol, photoperiod control is then accomplished either by control of room lighting or by timers on individual aquaria. In larger facilities with multiple modular rack systems extending from the floor to the ceiling, care must be given to the arrangement of lighting to ensure that adequate light penetrates to all tanks containing fish, especially those housed on the lower shelves near the floor. Conversely, fish housed on the top shelves too close to the light source may receive light that is too intense from overhead bulbs. Either too much or too little light can adversely influence the reproductive activity of the fish within the colony. In these facilities with rack-type shelving units, fluorescent lighting fixtures should be arranged parallel to

871

19. BIOLOGY AND MANAGEMENT OF THE ZEBRAFISH

the rows of shelving and directly over the aisles between two shelving units. This arrangement maximizes light penetration to fish housed on all levels of the shelving unit. 4.

Heating, Ventilation, Air Conditioning (HVAC) systems

Care should be taken to ensure that air exchange does not occur when spraying insecticides or herbicides in agricultural areas. Pest control or painting within or around a facility should also be of concern. Facility managers should be aware of any physical plant maintenance in or around the facility. Water systems need to be protected from the harmful effects of insecticides, herbicides, pipe treatment agents, etc. Air intal~.es for compressed air used in aeration of system water should be filtered and protected from airborne contaminants as well. Since many research facilities housing mammalian and avian species (i.e., rodents and poultry) can produce high levels of ammonia, close proximity to aquatic facilities should be avoided. All rooms should be provided with a source of clean, compressed air of low or medium pressure piped appropriately to provide for air delivery to all tank areas. Overhead polyvinyl chloride (PVC) piping with fittings for standard aquarium tubing provides for universal hook-up points for air stones that can be placed in aquaria when needed. Humidity control should be considered for all rooms containing aquaria. Large numbers of aquaria containing water at approximately 78 ~ F can produce a significantly high degree of humidity, especially during summer months. Poorly circulated room air and high humidity could result in significant mold proliferation within the facility. 5.

Electrical Service

All electrical service should ideally be supplied overhead and siaspended from the ceiling by knuckle fixtures or permanent fixtures high along the walls (approximately 4 ft) in waterproof conduits. All circuits should be ground-fault interrupted (GFI) and of appropriate amperage. In general, most aquatic facilities never have enough properly installed outlet space. Plan ahead and install greater than the standard numbers of outlets usually placed for a similar-sized room scheme. An emergency power source should be provided for critical life-support equipment supplying aeration, filtration, and lighting. Loss of power can result in catastrophic losses within hours, especially under the conditions of high water temperature and high stocking density that are encountered routinely in the laboratory setting. 6.

Water Tanks and Support

Tank support should be carefully considered in terms of weight distribution and construction materials. A standard rack of 40 ten-gallon aquaria filled to capacity weighs nearly 2 tons!

Support racks should be of heavy-duty plastic or metal such as aluminum or painted/treated steel angle iron. Wooden supports should be avoided since they tend to absorb moisture and harbor microorganisms, and they cannot be routinely disinfected effectively. Treated woods should also be avoided because of toxic leachates contaminating the water system. Even wellsealed or treated wood supports tend to degrade over time in the wet, high-temperature, and high-humidity environment of the aquatic facility. Nonleaching boric acid-treated wood or plastic may be a safe alternative if no other options exist. Various new products composed of plastics are emerging on the market, and each should be evaluated thoroughly for chemical leachates associated with the composition or manufacturing process of the product. 7.

Design of Water and Life-Support System

The design of the aquatic environment within the laboratory should be a major concern when establishing or renovating a zebrafish facility. Animal health, system maintenance, cost of the system, and animal numbers/uses should be factors considered when purchasing and designing a facility. With the rapid increase in the number of aquatic species being housed routinely in the laboratory research community, several manufacturers of aquatic life-support systems have emerged to offer an ever increasing array of products to support the needs of the laboratory community. The modular life-support-system approach to facility design appears to offer the best advantages. These systems allow for the implementation of comprehensive health-management protocols involving easy animal access and monitoring, disease surveillance, system maintenance, and cost-effective expansion of the facility as the need for more animals becomes necessary. Modular systems come in a variety of sizes and can be freestanding or wall-mounted. Custom modular systems can also be designed and constructed if fund limitations exist, but proper research into design specifications is a necessity. Most systems are completely self-contained, closed recirculating water systems and incorporate mechanical, biological, and chemical filtration options. Modular construction allows easy access to all pipes and mechanical components for routine replacement service, as well as in the case of system failure. With modular systems containing independent life-support equipment, failure of one life-support system will not result in catastrophic failure of the whole facility. As mentioned previously, these systems allow for expansion of a facility as necessary without the disruption of existing, established systems. The modules also allow for maximum use of available space if limitations do exist (Fig. 4). Within each modular rack system, the fish are housed in variable-sized containment units. These containers can house anywhere between 12 and 30 zebrafish and are usually composed of a high-impact, transparent plastic or acrylic that allows for easy

872

KEITH M. ASTROFSKY, ROBERT A. BULLIS, AND CHARLES G. SAGERSTROM

Fig. 4. Examples of modular-type housing units for laboratory zebrafish. Biological, mechanical, and chemical fltration units are all incorporated into these recirculating water systems.

cleaning and visualization of the animals. Polycarbonate construction appears to be the best construction, as acrylic frequently warps with age and repeated use. A limited number of manufacturers are now producing containment units that can be autoclaved. Some units also employ a self-cleaning design that sweeps uneaten food and feces out of the unit through the outflow tract. In this system design, a directional current is created by the inflow-outflow tracts and a baffle at the back of each individual container. While this option minimizes routine maintenance and cleaning, care must be taken to ensure that the smaller zebrafish are not inadvertently swept into the outflow tract. Modular life-support systems appear to be the most practical and economical choice in most circumstances. However, individual 1 0 - 3 0 gallon tanks still maintain utility in laboratory research colonies for a number of reasons. If a small colony is to be maintained, single aquaria offer an extremely inexpensive alternative and can be set up on the laboratory workbench if necessary (Fig. 5). In infection studies requiring independent life support, multiple individual aquaria can be maintained for disease challenge in studies not involving large numbers of animals. For those studies involving multiple environmental parameters, it may be easier and more economical to maintain side-by-side aquaria than larger, multiple modular units. Even in a facility with multiple modular systems, individual aquaria are extremely useful as quarantine or "hospital" units for treatment of fish in isolation from the rest of the facility. In terms of disaster planning and prevention, the availability of backup or redundant life-support equipment such as water

pumps or an alternative power source can mean the difference between inconvenience and catastrophic loss. Especially in larger facilities or those housing valuable transgenic or mutant strains, backup equipment allows greater freedom to conduct routine maintenance on otherwise continuously operated equipment and allows for quicker reaction in the event of life-support failure at an inopportune time. While maintaining redundant key components may not appear economical, it remains the best option available to thwart those unforeseen failures that have resuited in complete loss of valuable animals and research time.

Fig. 5. Basic 10-gallon aquarium setup for small-scale zebrafish housing. These 10-20 gallon aquaria are also very useful as "hospital" or quarantine tanks in large facilities.

873

19. BIOLOGY AND MANAGEMENT OF THE ZEBRAFISH Every day, approximately 5-10% of the total volume of water in a recirculating water system should be drained off and replaced. This input of a fresh volume of water daily helps to ensure the maintenance of adequate oxygen saturation, minimizes nitrogenous waste buildup, and replaces important trace mineral compounds. Prior to introducing fresh volumes of water from a reservoir tank to the system, the pH and temperature of the water should be equilibrated. Addition of large volumes of improperly buffered water or water of an inappropriate temperature can dramatically alter water chemistry and induce shock in the resident fish. Reservoir water should be located in the same temperature-controlled room as the water for the current system, to avoid temperature differentials. 8.

Sentinel Tank Incorporation

A key component to any comprehensive health-management program for the monitoring of animal health status is the maintenance of "sentinel" animals within each modular water system. These animals occupy one tank within each separate water system. For disease-status monitoring purposes, these animals may be routinely evaluated for various potential pathogens to which the transgenic animals within the system might have been exposed. Since these sentinel animals have been exposed to the same environmental conditions as the study fish, the use of valuable transgenic animals for routine disease surveillance and diagnostic workup in the event of a disease outbreak is eliminated. Sentinel animals should be placed within a water system for routine culling for diagnostic purposes performed according to an established schedule. Some sentinel animals should be subjected to the water-system environment for extended time periods to ensure the adequate exposure to potential pathogens with a chronic, subclinical, or insidious onset of disease (e.g., Mycobacterium spp.). 9.

Specialized Equipment

Rooms should be provided with an area where tanks can be cleaned, sanitized, and dried. In larger facilities, these cleaning areas can be centralized. Rooms should also be provided with a net disinfection system. Such a system should be implemented to provide for a "one-time use" of nets to prevent the spread of infectious agents within a room or among rooms of a larger facility. A supply of sanitized or autoclaved nets should be made available in a designated clean bucket. Following each use, nets should be placed in another bucket for cleaning in a disinfectant solution (Rocal, Clorox, etc.). Nets should never be mixed among tanks, rooms, or experiments or especially, with quarantined animals. To prevent the possibility of toxicity, properly disinfected nets should also be rinsed thoroughly with water prior to dipping into the water system or catching fish.

10.

Specialized Areas

An area should be set aside to provide bench space for equipment for water-quality analysis. In large facilities, a waterquality laboratory can be centralized. A dry area should be provided for report writing, other waterprotected activities, record keeping, and storage. Dry areas within an aquatic facility can become extremely difficult to allocate, so plan ahead. This area should display in a protected fashion the procedures, emergency procedures, phone trees, feeding schedules, water-quality reports, and other important data. Phones and computer stations should be located here as well. A quarantine area should be provided and clearly designated. This area can be a single designated-use tank in small facilities or a designated quarantine area. Large, permanent facilities should designate a separate room for quarantine of incoming animals or treatment of in-house animals. All equipment and materials should be designated for quarantine use only and never be introduced back into the main facility. This room should be isolated from all other life-support systems within the facility. Ideally, this room should be under negative pressure for containment purposes and be located in a noncentralized, lowtraffic area. These precautions help limit the number of people moving within the quarantine space and limit the possibility of inadvertent transfer of potentially contaminated equipment or animals. II.

Water Source

The water source for the facility must be carefully considered. Ideally, deep wells are the best source of freshwater because they contain few infectious agents and toxic chemicals such as sewage or agricultural compounds. In most cases, municipal tap water must be treated for the removal of agents such as chlorine, copper, and chloramines that are toxic to fish. Distilled and reverse osmosis (RO) water systems may be used within the aquatic facility. The advantage of the use of these water sources is purity through the removal of toxic chemicals and microorganism contaminants. However, these water sources have virtually all the trace minerals removed by design. Since these compounds are extremely important to animal health and maintenance of the buffering capacity of the water system, the water needs to be properly conditioned by the addition of mineral compounds. The addition of commercially available conditioning preparations (i.e., Instant Ocean, sea salts, etc.) to the water prior to use helps prevent nutritional-induced disease or rapid, problematic shifts in pH within the water system. It is important to remember that with evaporative loss of system water, the salinity of the system will continue to increase. Therefore, the addition of salts to the water needs to be properly monitored to prevent exposure of the fish to a salinity level beyond the range of normal tolerance.

874 12.

KEITH M. ASTROFSKY, ROBERT A. BULLIS, AND CHARLES G. SAGERSTROM Use of Freshwater Snails

While some research laboratories routinely use freshwater snails (Planorbella spp.) to help control the unsightly growth of algae on tanks and maintain good water quality by consuming uneaten food and other organic debris, there are potential disadvantages to their use in the laboratory facility. Once introduced into an established water system, freshwater snails can reproduce quite readily and frequently get out of control. As their numbers increase and the snails grow larger, they often migrate into the piping of the water system where the hard shells can disrupt and, in some instances, block the flow of water through the recirculating system. These conditions necessitate disruption of the established aquatic environment to remove snails from clogged piping. The control of algae and the accumulation of excess nitrogenous wastes can be better regulated by adjusting the amount of light and by decreasing the frequency and amount of feed introduced into the water system. Another potential hazard of utilizing snails in the laboratory setting is that snails act as the intermediate host or vector for the larval stages of a number of parasitic organisms, such as digenetic trematodes. Likewise, a limited number of reports indicate that invertebrates can carry bacterial pathogens such as Mycobacterium spp. (Michelson, 1961). Empirical evidence indicates that these freshwater snails, once established within the water system, could then act as a reservoir of infection. Although Mycobacterium spp. have been isolated from several species of freshwater snails (six species, including Helisoma spp., Australorbis sp., and Rtomphalaria sp.) under both experimental and natural infection studies, there has been as yet no report of invertebrate vectors transmitting mycobacterial disease to aquatic vertebrate organisms such as teleost fish. Lesions and acid-fast bacteria were associated with both natural and experimental infection of the snails (Michelson, 1961).

(L-ascorbic acid) degrades in the presence of oxygen, moisture, light, and elevated temperatures. Deficiencies of vitamin C in fish have been associated with reduced growth, reduced egg viability, scoliosis, lordosis, fin/tail erosion, and mortality (Tacon, 1992). Once a container is opened, commercial flake diets should be refrigerated and used within 1 month. Manufacturers of high-quality feed should provide lot and time-dated coding to prevent use of outdated feed. Newly hatched zebrafish present somewhat of a problem in trying to feed a dry diet formulation. Since these young fish are usually 0.5-1.0 cm in length, obtaining a diet that is small enough to be readily consumed can be difficult. For these reasons, young fish up to 10-14 days of age are fed an exclusive diet of Paramecium spp. (ciliated protozoan) grown in culture within the laboratory. A wide degree of variation can exist among various strains as to when young fish can be converted from paramecia and brine shrimp to dry feed. This usually occurs at 10-21 days of age. The best approach is to offer fry a combination of Paramecium and increasing amounts of dry diet until the young fish readily accept an exclusive diet of dry feed. The proper nutritional management of fish at 10-21 days of age is critical, and high mortality is frequently encountered during this stage of development. If offering fish a mixed diet of both live and artificial feed, offer live food and artificial diet at different feedings. Some laboratories maintain that reproductive performance can be enhanced by supplementation of the dry diet with fresh live foods. Brine shrimp nauplii, Drosophila larvae, or rotifers are the most common and readily obtainable live food sources. Other laboratories report that reproductively active and healthy adults are routinely maintained on an exclusive dry, flaked diet.

B.

V.

NUTRITION AND FEEDING

A.

Diet F o r m u l a t i o n

As with all other laboratory animal species, it is important to provide zebrafish with a complete and balanced diet. Adults and juveniles (> 10-14 days of age) can be maintained on an artificially formulated diet in the form of either dry flakes or pellets that are readily available from a variety of commercial aquarium product suppliers. In addition to providing the 40 essential dietary nutrients (vitamins, minerals, etc.), these formulated diets provide roughage to aid in digestion and pigment sources for natural coloration. The vitamin, lipid, and pigment content of commercial feeds can degrade if stored improperly or over an extended period of time. For example, vitamin C

Feeding Interval

Feeding frequency is an important area of concern for the laboratory manager. Fish should not be fed more food than they can consume in 3-5 min. Food that is not readily eaten quickly sinks and is taken up by the recirculating water system, where it fouls both mechanical and biological filters. Uneaten food quickly decays and is a major source of nitrogen waste in the water system. This decaying food can adversely influence water quality by contributing large amounts of ammonia that can disrupt the biofilter and cause sudden pH shifts. Frequent or inappropriate feeding is a significant cause of poor water quality in terms of excess nitrogenous products, high bacteria counts, and pH fluctuation. While young and breeding zebrafish may require multiple feedings daily, feeding adult zebrafish once or twice daily maximizes both fish health and healthy waterquality parameters. Feeding fish up to 3 or more times daily has been described routinely to maintain optimal fertility in reproductively active fish, but it is important to consider the adverse

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19. BIOLOGYAND MANAGEMENTOF THE ZEBRAFISH effects this practice may have in contributing high levels of nitrogenous waste to the water system. VI.

ACQUISITION

A concerted effort should be undertaken in every established facility to limit the introduction of fish from outside sources. The introduction of new animals always constitutes a source of pathogen introduction into an established specific pathogenfree (SPF) facility. However, wild-type or transgenic fish can be safely introduced into a facility by using stringent quarantine/ surveillance programs and by obtaining animals from quality zebrafish retailers or from the facilities of research colleagues that also implement a comprehensive fish health-management protocol. Many laboratories are now routinely quarantining incoming adult zebrafish. These fish are then bred in quarantine, and the fertilized eggs are harvested. The eggs are treated with a dilute bleach solution and hatched. These fry are then introduced into the main facility. While this approach certainly offers protection against the introduction of many potential pathogens, this treatment protocol should not be interpreted as a procedure to eliminate all risk of infectious disease. For example, vertical transmission of Mycobacterium spp. has been demonstrated in live-bearing (viviparous) fish such as guppies and platyfish. However, vertical transmission in oviparous species such as zebrafish has not been determined (Stoskopf, 1993; Noga, 1996).

A.

Wild-TypeSuppliers

It is important to consider that most of the retailers currently supplying wild-type zebrafish stocks to the laboratory research community are primarily suppliers of ornamental tropical fish to the aquarium pet trade. As a result, the significance placed on health management of fish within these farms varies greatly according to the individual companies. Therefore, never assume that animals arriving at your facility are healthy. While some suppliers will make an effort to identify and treat clinically observable disease in their fish stocks, fish with subclinical disease obtained from suppliers with limited health monitoring could enter an established research facility undetected. Due to the limited ability of these retailers to implement a comprehensive health-monitoring program for economic and practical issues, it is extremely important to evaluate all incoming fish for latent or subclinical infection before their introduction into an established system. The acquisition of zebrafish from local pet stores for outcrossing is strongly discouraged. Due to the nature of the pet trade, local aquarium shops usually have a high degree of turnover in both incoming and outgoing animals. This usually

results in little or no disease screening, overstocked aquaria, and fish stressed from shipping conditions. Pathogens that are either established in the system or that arrive with newly introduced fish can spread quickly among stressed or debilitated resident fish. Because of the high volume of fish within these shops, the fish frequently become infected with the more common aquatic pathogens, such as Mycobacterium spp., Capillaria spp., and Ichthyophthiirius spp. Fish obtained from a local aquarium shop should be considered as high-risk candidates for harboring pathogens and quarantined for a minimum of 3 0 - 4 5 days prior to introduction into the research facility.

B.

Transfer among Research Facilities

Due to the rapid expansion in the number and size of zebrafish facilities worldwide, the numbers of transgenic animals and their resultant phenotypes are dramatically increasing. Various research groups worldwide are constantly exchanging these transgenic fish among their respective facilities. Therefore, it is important to ascertain the current health status of the facility from which you are obtaining fish. Establishing a comprehensive health-monitoring system among various institutions will allow for the safest and most expedient transfer of animals among facilities. Likewise, it is essential to share accurate colony health information with a facility to which you are sending fish. False, outdated, or inaccurate information concerning health status, especially with regard to infectious pathogens, can result in significant loss of valuable animals, research time, and funds in an unsuspecting facility receiving diseased animals.

VII.

INFECTIOUS DISEASES

In assessing the health of a colony, it is important to evaluate the appearance and behavior of the animals on a daily basis and conduct diagnostic evaluations of unexplained mortalities as well as routine diagnostic workups on sentinel animals (Astrofsky, 2002). Several nonspecific signs in abnormal appearance or behavior can be useful in the initial diagnostic workup to help determine the causative agent or event. It is important to remember that these are nonspecific clinical signs of disease and are useful only in forming an initial list of differential diagnoses. More specific diagnostic tests must be implemented to accurately identify the causative agent (Fig. 6). The following section contains information pertaining to the most significant pathogens that are of concern to the laboratory zebrafish facility. It is an attempt to provide clear, concise information regarding the identification of clinical signs, diagnostic workup, and treatment options available to the facility

KEITH M. ASTROFSKY,ROBERTA. BULLIS, AND CHARLESG. SAGERSTROM

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Generalized Clinical Signs Opercular flaring Sloughed mucus Clamped f'ms Petechiation or hemorrhage Changes in body color Scale loss Improper buoyancy Lethargy Surface breathing Sudden death

Differential Diagnoses respiratory distress, parasites, bacterial infection chemical irritation, parasites parasites bacterial infection, parasites bacterial infection, hormonal influence parasites, mechanical trauma baroregulatory (swim bladder) failure, parasites disease, stress, starvation oxygen depletion chemical toxicity, abrupt change in water quality

Fig. 6. Generalizedclinical signs and abnormal behaviors frequently observed in diseased fish and potential differential diagnoses that should be considered. operator. The following list is only a survey of pathogens that have been previously described in zebrafish or are pathogens that can infect laboratory zebrafish. As the number of laboratory zebrafish facilities continues to increase, new, emerging diseases will continue to be characterized (Astrofsky, 2000; Matthews, 2001). Likewise, treatment options will continue to be explored for those infectious agents that have long been the scourge of aquatic facilities.

A.

Mycobacteriosis (Fish Tuberculosis)

Atypical (or environmental) mycobacterial infections of fish are most commonly associated with Mycobacterium marinum, M. fortuitum, or M. chelonae. Due to its long incubation period and chronic, subclinical form, this insidious disease can remain undetected within established facilities for extended periods of time. Mycobacterial infections of fish have been identified worldwide in over 150 species of saltwater and freshwater fish (Talaat et al., 1997). Once established, Mycobacterium spp. become a resident of the microbial flora within the water system. The clinical signs of mycobacteriosis in zebrafish can be highly variable. Chronically diseased animals usually have poor growth rate, chronic wasting, and emaciation. There is usually a decrease in reproductive rates and a slightly increased mortality rate within the affected colony (Talaat et al., 1998). Acutely diseased animals often demonstrate the generalized clinical condition known as "dropsy syndrome," which consists of abdominal distension and scale edema. This edema results in a lifting or "porcupine-like" effect to the scales. Petechiation or ulceration of scales and fin erosion is often evident (Fig. 7). Preliminary diagnosis of mycobacteriosis is based on the identification of clinical signs consistent with the disease. Histologic examination of affected kidney, liver, and splenic tissue often yields acid-fast positive staining, rod-shaped bacteria in af-

fected tissues. The atypical aquatic mycobacterium species may display staining characteristics similar to those of gram-positive bacteria (Fig. 8). The presence of numerous, well-developed granulomas in the liver, spleen, kidney, and reproductive organs is also a common histologic finding in chronically infected fish (Fig. 9). However, definitive confirmation of mycobacterium infection is currently made only by culture of the organisms on Lowenstein-Jensen (LJ) or other selective media and subsequent biochemical analysis. Atypical Mycobacterium spp. are extremely slow growing organisms in culture and may require 3 0 - 4 5 days for definitive culture results to be obtained. Frequently, acid-fast bacteria may not be readily identified histologically in affected tissues but yield positive culture results when tested. Currently, PCR-based assays with mycobacteria species-specific primers have been described for use with infected tissue, bacterial colonies from culture, or purified DNA (Talaat et al., 1997; Astrofsky et al., 2000). These molecularbased assays may hold future clinical utility in screening incoming animals for possible mycobacterial infection prior to introduction into an established research colony. Unfortunately, effective treatment of infected facilities can be accomplished only by eradication of infected stocks and subsequent disinfection of all substrates within the facility. Various attempts at treatment with a number of antibiotics have had lim-

Fig. 7. Gross photograph of a confirmed Mycobacterium fortuituminfected zebrafish demonstrating "dropsylike" clinical signs of abdominal distension, scale edema, and petechiae around operculum and pectoral fin.

19. BIOLOGY AND MANAGEMENT OF THE ZEBRAFISH

877

Fig. 8. Photomicrographsof acid-fast stained caudal (trunk) kidney (left) and heart (right) from confirmedMycobacteriumfortuitum-infected zebrafish. In the kidney, numerous extracellular, acid-fast positive bacilli are present in Bowman's space and within interstitial macrophages (arrows). The section of heart similarly demonstrates severe septicemia with bacilli present in the lumen (arrows) and filling the cytoplasm of endothelial cells (E) lining the myocardium (M). Magnification: X 330 (left), • 350 (right). ited success in controlling the infection but not eliminating it. Since various mycobacteria species have been isolated from the environmental biofilms that form within water systems (Schultze-R6bbecke et al., 1992), disinfection of the tank and filter system is necessary. Prior to sanitation of the system, all associated filter material and disposable equipment should be

Fig. 9. Photomicrographof a well-demarcated granuloma from the gonad of a confirmed Mycobacterium chelonae-infected zebrafish. These granulomas are composed of closely spaced collections of macrophages, including numerous epithelioid and foamy macrophages, and peripheral circumferential bands of fibrosis surrounding necrotic centers with coagulated anucleate or karyopyknotic cells, amorphous granular debris, and hypereosinophilic coagulum.

discarded. Disinfection of the water system and surfaces should be conducted with a bleach solution (1/4 cup Clorox per gallon of system water). Run the system with bleach for 3 days with aeration and then drain. Refill the system with water and run for another 3 days. Drain the system and refill. The outer surfaces of all tanks and related hardware should be treated with the same bleach solution. During the sanitation procedure, adequate ventilation or closure of workbenches within the treated areas is necessary due to the concentrated fumes of the bleach solution. Restock the system and culture fish after several months to monitor for reinfection of the system. It is important to realize that once an infection has occurred within a facility, it is very difficult to eliminate the infection. Cutaneous mycobacterial infections of humans due to atypical mycobacteria have been well documented. Known as "fish handler's granuloma" or "swimmer's granuloma," these infections are usually self-limiting and result only in a localized area of erythema and swelling on the affected extremity. However, more serious clinical diseases such as persistent cutaneous granulomas, osteomyelitis, and tenosynovitis have also been reported. Life-threatening disease has also been reported in immunocompromised individuals. Zoonotic transmission of M. marinum and M. fortuitum to fish handlers or persons in close contact with infected fish or aquaria has been documented. All laboratory personnel in contact with the fish or associated hardware should be aware of the potential health risk. Precautionary measures, such as the wearing of gloves, should be implemented when treating outbreaks.

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KEITH M. ASTROFSKY, ROBERT A. BULLIS, AND CHARLES G. SAGERSTROM B.

Bacterial Septicemia

Several genera of bacteria contain significant pathogens that result in generalized systemic infections in fish. Streptococcus iniae and Edwardsiella tarda are capable of infecting all species of fish, and sporadic outbreaks frequently result in high mortality. However, the vast majority of bacterial infections result from secondary or opportunistic bacteria such as Aeromonas hydrophila, Flexibacter columnaris, Flavobacterium spp., and Pseudomonas spp. These ubiquitous species exist within the water and life-support system and usually cause clinical disease only in stressed or immunocompromised fish as a result of improper environmental conditions, concurrent disease, or trauma (Pullium et al., 1999). Clinical signs of sepsis include exophthalmos, increased respiratory effort, abdominal distension ("dropsy syndrome"), and "pinpoint" hemorrhages over the body and around the eyes, mouth, anus, opercula, or base of the fins. At necropsy, affected fish demonstrate congested and hemorrhagic internal organs. The liver is usually pale, and the spleen is dark red. Blood-tinged abdominal fluid is frequently evident within the peritoneal cavity. Definitive diagnosis is based on primary isolation in triptic soy broth or blood agar incubated at 20~176 for 2 4 - 4 8 hr and subsequent biochemical analysis. Incubation of poikilotherm cultures at 37~ or use of commercial test strips not intended for use in these species may result in unreliable identifications. For the treatment of primary bacterial pathogens (S. iniae and E. tarda) or secondary infections in valuable fish, several antibiotic therapies have been implemented. Sulfadiazinetrimethoprin (prolonged immersion at 6 - 1 2 mg/liter daily for 3 - 7 days) and enrofloxacin (bath for 5 hrs at 2.5-5.0 mg/liter daily for 5 - 7 days) have been useful in treating infections. Oxytetracycline hydrochloride, prolonged immersion at 10100 mg/liter of system water (higher dose with harder water) for 3 - 7 days, is frequently used. If necessary, repeat treatment on third day after a 50% water change. Turn off all lights in the laboratory to prevent photoinactivation of drug (Noga, 1996; Mashima and Lewbart, 2000). In evaluating microbial culture results of the water system or fish suffering from secondary bacterial infections, it is important to assess the health of the fish in relation to the physical environment. It is far more efficacious to remedy the underlying environmental or physical stress that allows opportunistic microorganisms to colonize the host than it is to implement haphazard or unwarranted antimicrobial therapy. Additionally, indiscriminant antibiotic use frequently results in the selection of resistant microbial strains that colonize the established water system.

C.

fection usually occurs as a result of the introduction of infected fish into a facility. In most infections, there are no overt clinical signs of disease. However, heavy infections result in significant disease with nonspecific signs such as reduced growth potential, decreased reproductive rate, emaciation, and chronic wasting (Westerfield, 1995). However, acute clinical disease with mortality can occur in which affected fish lose body condition rapidly. Definitive diagnosis is confirmed by microscopic examination of fresh feces for eggs that have a characteristic elongated oval shape with polar plugs or opercula present (Fig. 10). Visualization of the adult worms which are motile, thin, transparent, and approximately 1 cm in length within the intestines at necropsy are also diagnostic. A combination of trichlorfonmebendazole (by Aquarium Products) is most effective in eradicating these parasites (Goven and Amend, 1982; Westerfield, 1995). Dissolve one Fluketabs tablet per 38 liters once daily for 3 days and then repeat in 10 days. Panacur (fenbendazole) used as a prolonged immersion at a concentration of 2 mg/liter once per week for 3 weeks has been used to treat infections. Ivermectin has also been described in the literature for use in aquatic animals, but no studies have been conducted in zebrafish. It is also important to note that ivermectin appears to have a lower therapeutic index in aquatic animals (Noga, 1996). Removal of the carbon filters prior to treatment and replacement after 72 hr is necessary to prevent premature uptake and binding of the drug. Repeat treatment in 10 days to prevent reinfection by newly hatched larvae.

D.

Velvet Disease (Gold Dust Disease)

Members of the Danio family are particularly susceptible to these parasitic dinoflagellates of the genus Piscinoodinium (for-

Intestinal Nematodes

Pseudocapillaria tormentosa is a nematode that commonly infects zebrafish (Kent, unpublished observations, 2002). In-

Fig. 10. Wet mount of nematode eggs of the genus Capillaria demonstrating characteristic operculatedappearance.

19. BIOLOGYAND MANAGEMENTOF THE ZEBRAFISH merly genus Oodinium [Westerfield, 1995]). Piscinoodinium limneticum and P. pillulare are the two freshwater species that have been recognized to cause disease in freshwater fish. The trophont (feeding stage) extends filamentous rootlike structures (rhizoids) into the skin, which embed in the host epithelium and result in sloughing of large segments of skin or gill. The parasite then drops off the host to become a sessile tomont form that undergoes numerous divisions producing motile dinospores. The infective dinospores must attach to a host within 2 4 - 4 8 hr. Fish infected with Piscinoodinium commonly flash (flip over and rub up against sides of tanks) due to epithelial irritation and demonstrate a shiny, "velvety or dustlike," yellow/red-browncolored appearance along the flanks, which is most apparent when viewed head on (Fig. 11). Decreased egg production, lethargy, and retracted fins (especially dorsal fin) may also be noted. If the gill epithelium is colonized, raised opercula and increased respiratory effort are frequently evident. Microscopic identification (• 100) of the trophont is the basis of diagnosis. The trophont is an oval (or pear-shaped), yellow/brown structure evident on gill and fin clippings or mucus scrapings in which the organisms may appear in "grapelike" clusters (Fig. 12). Gross visualization of the parasite can be enhanced by directing a flashlight onto the dorsal aspect of an affected fish in a darkened room. A fine, dusty, granular effect will be evident. Several treatment options are available for affected facilities. The parasite can sometimes be eradicated by immersion in a saltwater solution (3.8-19 gm of salt per gallon of water). Lower concentrations of salt should be used initially until the sensitivity of the fish population to the treatment is established (Noga, 1996). Raising the environmental temperature 1.5~ every 2 4 - 3 6 hr for 3 days speeds up the life cycle of the parasite and aids in eradication during treatment. Other treatment

Fig. 11. Grossphotograph of the dorsal fin of a tiger barb (Barbus tetrazona) of the family Cyprinidae demonstrating gold-colored, spherical Piscinoodinium trophonts.

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Fig. 12. Wet mountof a fin demonstratingtrophontsof the marinedinoflagellate Amyloodinium ocellatum anchored to the surface of the affected tissue (arrow). Piscinoodinium trophontshave a similarmorphology.

regimes include quinine hydrochloride using a prolonged immersion at 1 gm/100 liters for 3 days (Westerfield, 1995), and formalin using a prolonged immersion at 1 ml/10 gallons of water (treat every other day for 3 days and change 50% of system water on alternate days [Noga, 1996]). Due to its potential teratogenic and mutagenic effects, malachite green dye, commonly used alone or in conjunction with formalin in the aquarium pet trade, should not be used to treat animals being used for genetic research.

E. AquaticMycosis Saprolegnia spp. are classified as Oomycetes and are ubiquitous water molds. These organisms frequently become established in mature biofilters. Although Saprolegnia spp. are relatively easy to culture and identify, definitive species identification of clinical isolates is extremely difficult due to markedly different morphologic and physiologic characteristics. Infections of zebrafish are the result of opportunistic colonization of damaged skin due to trauma, poor husbandry, or concurrent bacterial/parasitic infection (Stoskopf, 1993). These molds do not routinely colonize living, healthy tissue, and it has been suggested that various isolates may be more pathogenic than others. Affected fish demonstrate thin white filaments that build up to puffy, white, "cottony" mattes on affected tissue (Fig. 13). Microscopic evaluation of skin wet mounts on low power demonstrates numerous broad aseptate hyphae, often with sporebearing sporangia at the tips (Fig. 14). Aseptate, branching hyphae are usually visible with H & E staining and are very pronounced with silver staining (Wardrip et al., 1999). Although uncommon, other ubiquitous aquatic fungi can cause opportunistic disease in zebrafish (Dykstra et al., 2001).

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KEITH M. ASTROFSKY, ROBERT A. BULLIS, AND CHARLES G. SAGERSTROM

Fig. 13. Gross photograph of goldfish (Carassius auratus) demonstrating the characteristic fluffy, white, cottonlike appearance of Saprolegnia water mold. These fish were subsequently cultured positive for goldfish ulcer disease (Aeromonas sp.).

solution (3.8-19 gm of salt per gallon of water) is the simplest and best prophylactic treatment. Lower concentrations of salt should be utilized initially until the sensitivity of the fish population to the treatment is established (Noga, 1996). Formalin, prolonged immersion at 1 ml/10 gallons of water (treat every other day for 3 days and change 50% of system water on alternate days) or in combination with salt immersion, has been used (Noga, 1996) but is not the method of choice. However, the best treatment for water mold infections remains a malachite green dye bath at 5 0 - 6 0 mg/liter for 10-30 sec. Again, the use of dyes as treatment agents should be carefully considered due to their potential teratogenic and mutagenic effects. These water molds are also a common problem in zebrafish egg rearing. Decaying organic matter such as infertile or dead eggs provides an excellent substrate for the growth and multiplication of these fungi. Maintaining stringent water quality in egg-rearing chambers with adequate oxygenation and low organic debris is essential in minimizing problems (Lasee, 1995).

F.

Saprolegnia spp. are ubiquitous microorganisms and spread very rapidly within the water system. Heavily infected fish with large areas of colonization are usually severely debilitated due to secondary bacterial sepsis and osmotic imbalance. These animals usually have a very poor prognosis for recovery with treatment and should be euthanatized. Identification of the underlying cause of fungal colonization should be investigated and remedied (i.e., trauma, environment, concurrent pathogen). Immersion in a saltwater (noniodized table salt, Instant Ocean)

Fig. 14. Wet mount of cottonlikemat demonstratingnonbranching, aseptate hyphae of the Saprolegnia water mold.

White Spot Disease (Ich)

Ichthyophthiirius multifiliis is a ciliated protozoan parasite in which the trophont colonizes the epithelium of the fins, skin, or gills of the host fish. This mature trophont ruptures the overlying epithelium of the host and within 6 hr attaches to a substrate, such as the gravel, filter, or tank. This tomont undergoes multiple divisions to produce numerous tomites. The tomites differentiate into free-swimming theronts that must colonize a suitable host within 48 hr. The infective theronts penetrate the epithelium of the host and feed on tissue and fluids (Cover and Guat-Lian, 1995). Trophonts appear as multifocal, raised, white, 1 mm diameter, mucoid nodules on the skin and gills. Flashing and increased respiratory effort may be evident if the gill epithelium is colonized (Fig. 15). Presumptive diagnosis is based on the appearance of the "white spots." Definitive diagnosis is based on identification of the trophont form, which is readily identified on microscopic evaluation of skin wet mounts by its characteristic horseshoe-shaped nucleus (Fig. 16). Treatment involves raising the environmental temperature 1.5~ every 2 4 - 3 6 hr for 3 days to hasten the life cycle of the parasite in conjunction with immersion in a salt solution of 7.6 gm of salt per gallon of water (Noga, 1996). However, some strains of ich can tolerate high-salinity water treatments. Formalin using a prolonged immersion at 1 ml/10 gallons of water (treat every other day for 3 days and change 50% of system water on alternate days), is also effective in eradicating this parasite (Noga, 1996). Due to its potential teratogenic and mutagenic effects, malachite green dye should not be used in laboratory zebrafish, as previously mentioned.

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19. BIOLOGY AND MANAGEMENT OF THE ZEBRAFISH G.

Trichodinosis

There are many species of this ciliated protozoan that are parasites of the gills or skin of various freshwater and marine fish. The trichodinids comprise six genera (Trichodina, Trichodinella, Tripartiella, Paratrichodina, Hemitrichodina, and Vachomia) that all have a similar morphology. Several nonpathogenic species have been identified that routinely colonize the gastrointestinal and reproductive tracts. While the larger, skinassociated trichodinids appear to have a broad host range, the smaller gill-associated species appear to be more host-specific. These organisms have a direct life cycle, reproduce by multiple divisions, and do not produce "resting spores." Trichodinids do not appear to persist in the environment off the host for greater than 2 4 - 4 8 hr. Other aquatic animals (amphibian larvae) and substrates have been implicated as reservoirs of disease in some fish species (Noga, 1996; Stoskopf, 1993). Although infection usually results in relatively minor clinical disease, heavy infection of stressed or diseased fish can result in poor body condition, low reproductive rates, and mortality. Clinical signs are highly variable and include respiratory disFig. 15. Grossphotograph of an Ichthyophthiirius multifiliis-infected ze- tress, skin erosion, excessive mucus production, sloughed brafish demonstratingnumerouswhite, mucoid, sphericalnodules alongthe en- scales, and frayed fins. Histologically, hyperplastic gill epithetire external surface. Note flared operculum and retracted dorsal fin denoting lium may be evident. irritation. Trichodinid infection is easily diagnosed through wet mounts of skin scrapings or gill clippings. Morphologically, these organisms appear circular when viewed dorsoventrally and domeshaped when viewed laterally. They range in size from approximately 30 to 90 ~tm in diameter and are ringed by several concentric rows of cilia along the periphery. Several large filamentous attachment structures called dentricles may also be evident. In wet-mount preparations, these organisms have a characteristic rotating motion. Infection is most commonly associated with poor water quality, high stocking densities, or concurrent disease. Trichodinid infection is most common in pond-raised fish where there is a high organic load in the water and does not usually pose a threat to established colonies in the laboratory setting. However, pond-raised fish that are not examined prior to introduction to the laboratory can pose a health risk. Higher infection and mortality rates seem to be associated with young fish and usually result from secondary bacterial colonization of damaged epithelium. Treatment usually involves maintaining or restoring optimal water-quality parameters. Heavily infected fish can be treated by a salt immersion (3.8-19 gm of salt per gallon of water). Lower concentrations of salt should be utilized initially until the sensitivity of the fish population to the treatment is established (Noga, 1996). Formalin with prolonged immersion at 1 ml/10 Fig. 16. Wet mount of a skin scraping having characteristic variable-sized gallons of water (treat every other day for 3 days and change trophonts of Ichthyophthiirius multifiliis with a typicalhorseshoe-shapedmacro- 50% of system water on alternate days [Noga, 1996]), is also nucleus. Note variable size of trophonts (arrows). effective.

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KEITH M. ASTROFSKY, ROBERT A. BULLIS, AND CHARLES G. SAGERSTROM

HQ Infectious Pancreatic Necrosis Virus

REFERENCES

and Other Viruses

There have been empirical reports of infectious pancreatic necrosis virus (IPNV) infection in zebrafish. These reports have indicated only incidental histopathologic findings. A disease of major importance in salmonid (trout and salmon) aquaculture, IPNV has never been associated with clinical disease or mortality in the zebrafish. The principle source of I P N V is infected salmonid brood stock, and overt disease as a result of I P N V is currently a health problem only in salmonid facilities (Wolf, 1988). The host range of fish that can b e c o m e infected with this bisegmented, double-stranded R N A birnavirus is quite large, and subclinically infected fish of any species can be a source of I P N V for other species (Wolf, 1988). Wild-type zebrafish suppliers that grow zebrafish outdoors, in conjunction with salmonid-related aquaculture, or in shared water supplies, are the primary source of these incidental findings at necropsy. The use of these wild-type stocks for the purpose of outcrossing should therefore be avoided. The significance of other k n o w n viruses in ornamental tropical fish is not well documented. M u c h of the knowledge concerning pathogenic viruses in teleost fish is the result of research in both marine and freshwater aquaculture species due to the economic impact of these agents on the commercial industry (Stoskopf, 1993). However, viral-induced disease and mortalities in ornamental fish have been described in association with iridoviruses (goldfish) and rhabdoviruses (carp and cichlids).

ACKNOWLEDGMENTS

The authors wish to offer their sincere gratitude to the following individuals for their input and evaluation of this chapter. Without the addition of their thoughts and personal experiences, many sections of this chapter would have been incomplete: A m y Chin, Phylonix, Cambridge, Massachusetts; Robbert Creton, Brown University, Providence, Rhode Island; William Mebane, Marine Biological Laboratory, Woods Hole, Massachusetts; Andrew Miller, Hong Kong University of Science & Technology, Kowloon, H o n g Kong; and Benjamin Pratt, Whitehead Institute, Cambridge, Massachusetts. A special thanks from the authors to the following individuals for the use of several photographs in this chapter: Edward Noga, North Carolina State University, Raleigh, North Carolina, and Todd Wenzel, Fish Pros, Raleigh, North Carolina.

Alexander, J., Stainier, D. Y., and Yelon, D. (1998). Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning. Dev. Genet. 22, 288-299. Astrofsky, K. M., Schrenzel, M. D., Bullis, R. A., Smolowitz, R. M., and Fox, J. G. (2000). Diagnosis and management of atypical Mycobacterium spp. infections in established laboratory zebrafish (Brachydanio rerio) facilities. Comp. Med. 50, 666-672. Astrofsky, K. M., Schech, J. M., Sheppard, B. A., Obenschain, C. A., Chin, A. M., Kacergis, M. C., Laver, E. R., Bartholomew, J. L., and Fox, J. G. (2001). High mortality secondary to Tetrahymena sp. infection in laboratory zebrafish (Brachydanio rerio). Contemp. Top. Lab. Anim. Sci. 40, 68. Astrofsky, K. M., Harper, C. M., Rogers, A. B., and Fox, J. G. (2002). Diagnostic techniques for clinical investigation of laboratory zebrafish (Brachydanio rerio). Lab Animal. In press. Collodi, P., Kamei, Y., Ernst, T., Miranda, C., Buhler, D., and Barnes, D. (1992). Culture of cells from zebrafish (Brachydanio rerio) embryo and adult tissues. Cell Biol. Toxicol. 8, 43-61. Cover, C., and Guat-Lian, K. (1995). Diagnostic exercise: Mortality in rainbow trout. Lab. Anim. Sci. 45, 98-100. DeTolla, L., Srinivas, S., Whitaker, B., Andrews, C., Hecker, B., Kane, A., and. Reimschuessel, R. (1995). Guidelines for the care and use of fish in research. ILAR J. 37, 159-173. Dietrich, H. W., Westerfield, M., and Zon, L. T. (1999a). The zebrafish: Biology. In "Methods in Cell Biology" (L. Wolson and P. Matsudaira, ed.), Vol. 59. Academic Press, San Diego. Dietrich, H. W., Westerfield, M., and Zon, L. T. (1999b). The zebrafish: Genetics and genomics. In "Methods in Cell Biology" (L. Wolson and P. Matsudaira, ed.), Vol. 59. Academic Press, San Diego. Driever, W., Stemple, D., Schier, A., and Solnica-Kriezel, L. (1994). Zebrafish: Genetic tools for studying vertebrate development. Trends Genet. 10, 152159. Driever, W., Solnica-Krezel, L., Schier, A. E, Neuhauss, S. C., Malicki, J., Stemple, D. L., Stainier, D. Y., Zwartkruis, E, Abdelilah, S., Rangini, Z., Belak, J., and Boggs, C. (1996). A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37-46. Dykstra, M. J., Astrofsky, K. M., Schrenzel, M. D., Bullis, R. A., Fox, J. G., Farrington, S., Sigler, L. S., Rinaldi, N. G., and McGinnis, M. R. (2001). High mortalities in a large-scale zebrafish colony (Brachydanio rerio) associated with Lecythophora mutabilis (Van Bayma). Comp. Med. 51, 361-368. Eisen, J. S. (1991). Determination of primary motoneuron identity in developing zebrafish embryos. Science 252, 569-572. Fritz, A., Rozowski, M., Walker, C., and Westerfield, M. (1996). Identification of selected gamma-ray induced deficiencies in zebrafish using multiplex polymerase chain reaction. Genetics 144, 1735-1745. Gaiano, N., Amsterdam, A., Kawakami, K., Allende, M., Becker, T., and Hopkins, N. (1996). Insertional mutagenesis and rapid cloning of essential genes in zebrafish. Nature 383, 829-832. Gong, Z., Yan, T., Liao, J., Lee, S. E., He, J., and Hew, C. L. (1997). Rapid identification and isolation of zebrafish cDNA clones. Gene 201, 87-98. Goven, B., and Amend, D. (1982). Mebendazole/trichlorofon combinations: A new anti-helminthic for removing monogeneic trematodes from fish. J. Fish Biol. 20, 373-378. Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, E J., Jiang, Y. J., Heisenberg, C. P., Kelsh, R. N., Furutani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C., and Nusslein-Volhard, C. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1-36.

19. BIOLOGY AND MANAGEMENT OF THE ZEBRAFISH

Henry, T. R., Spitsbergen, J. M., Hornung, M. W., Abnet, C. C., and Peterson, R. E. (1997). Early life stage toxicity of 2,3,7,8-tetrachlorodibenzop-dioxin in zebrafish (Danio rerio). Toxicol. Appl. Pharmacol. 142, 56-68. Ho, R. K., and Kimmel, C. B. (1993). Commitment of cell fate in the early zebrafish embryo. Science 261, 109-111. Jagadeeswaran, P., and Liu, Y. C. (1997). A hemophilia model in zebrafish: Analysis of hemostasis. Blood Cells Mol. Dis. 23, 52-57. Jay, D. G. (1988). Selective destruction of protein function by chromophoreassisted laser inactivation. Proc. Natl. Acad. Sci. US.A. 85, 5454-5458. Kahn, P. (1994). Zebrafish hit the big time. Science 264, 904-905. Khudoley, V. V. (1984). Use of aquarium fish, Danio rerio and Poecilia reticulata, as test species for evaluation of nitrosamine carcinogenicity. Natl. Cancer Inst. Monogr. 65, 65-70. Kimmel, C. B., Warga, R. M., and Schilling, T. E (1990). Origin and organization of the zebrafish fate map. Development 108, 581-594. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ulman, B., and Schilling, T. E (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253-310. Kolm, P. J., and Sive, H. L. (1995). Efficient hormone-inducible protein function in Xenopus laevis. Dev. Biol. 171, 267-272. Krauss, S., Maden, M., Holder, N., and Wilson, S. W. (1992). Zebrafish pax[b] is involved in the formation of the midbrain-hindbrain boundary. Nature 360, 87-89. Lasee, B. (1995). "Introduction to Fish Health Management." U.S. Fish and Wildlife Service, Omalaska. Lin, S., Yang, S., and Hopkins, N. (1994). lacZ expression in germline transgenic zebrafish can be detected in living embryos. Dev. Biol. 161, 77-83. Mashima, T. Y., and Lewbart, G. A. (2000). Pet fish formulary. In "Veterinary Clinics of North America: Exotic Animal Practice" (S. E. Fronefield, ed.), pp. 117-130. Saunders, Philadelphia. Matthews, J. L., Brown, A. M. V., Larison, K., Bishop-Stewart, J. K., Rogers, P., and Kent, M. L. (2001). Pseudoloma neurophilia n.g., n.sp., a new genus and species of microsporidia from the central nervous system of the zebrafish (Danio rerio). J. Euk. Microbiol. 48, 229-235. Michelson, E. (1961). An acid-fast pathogen of fresh-water snails. Am. J. Trop. Med. 10, 423-427. Miller, A., Karplus, E., and Jaffree, L. (1994). Use of aequorin for Ca ++ imaging. In "Methods in Cell Biology" (R. Nuccitelli, ed.), Vol. 40. Academic Press, San Diego. Mizell, M., and Romig, E. (1997). The aquatic vertebrate embryo as a sentinel for toxins: Zebrafish embryo dechorionation and perivitelline space microinjection. Int. J. Dev. BioL 41, 411-423. Noga, E. (1996). "Fish Disease: Diagnosis and Treatment." Mosby, St. Louis. Piper, R., McElwain, J., Orne, L., McCraren, J., Fowler, L., and Leonard, J. (1982). "Fish Hatchery Management." U.S. Fish and Wildlife Service, Washington, D.C. Postlethwait, J. H., and Talbot, J. (1997). Zebrafish genomics: From mutants to genes. Trends Genet. 13, 183-190. Pullium, J. K., Dillehay, D. L., and Webb, S. (1999). High mortality in zebrafish (Danio rerio). Contemp. Top. Lab. Anim. Sci. 38, 80-83.

883 Ransom, D. G., Haffter, E, Odenthal, J., Brownlie, A., Vogelsang, E., Kelsh, R. N., Brand, M., van Eeden, E J., Furutani-Seiki, M., Granato, M., Hammerschmidt, M., Heisenberg, C. P., Jiang, Y. J., Kane, D. A., Mullins, M. C., and Nusslein-Volhard, C. (1996). Characterization of zebrafish mutants with defects in embryonic hematopoiesis. Development 123, 311-319. Sagerstr6m, C. G., Grinblat, Y., and Sive, H. (1996). Anteroposterior patterning in the zebrafish, Danio rerio: An explant assay reveals inductive and suppressive cell interactions. Development 122, 1873-1883. Sagerstr6m, C. G., Sun, B. I., and Sive, H. L. (1997). Subtractive cloning: Past, present, and future. Annu. Rev. Biochem. 66, 751-783. Schultze-R6bbecke, R., Janning, B., and Fischeder, R. (1992). Occurrence of mycobacteria in biofilm samples. Tubercle Lung Dis. 73, 141-144. Shih, J., and Fraser, S. E. (1996). Characterizing the zebrafish organizer: Microsurgical analysis at the early shield stage. Development 122, 13131322. Stainier, D. Y., Lee, R. K., and Fishman, M. C. (1993). Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development 119, 31-40. Stoskopf, M. (1993). "Fish Medicine." Saunders, Philadelphia. Streisinger, G., Walker, C., Dower, N., Khauber, D., and Singer, E (1981). Production of clones of homozygous diploid zebrafish (Brachydanio rerio). Nature 291, 293-296. Talaat, A., Reimschuessel, R., and Trucksis, M. (1997). Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis. Vet. Microbiol. 58, 229-237. Talaat, A. M., Reimschuessel, R., Wasserman, S. S., and Trucksis, M. (1998). Goldfish, Carassius auratus, a novel animal model for the study of Mycobacterium marinum pathogenesis. Infect. Immun. 66, 2938-2942. Talbot, W. S., Trevarrow, B., Halpern, M. E., Melby, A. E., Farr, G., Postlethwait, J. H., Jowett, T., Kimmel, C. B., and Kimelman, D. (1995). A homeobox gene essential for zebrafish notochord development. Nature 378, 150157. Walker, C.," and Streisinger, G. (1983). Induction of mutations by gamma-rays in pregonial germ cells of zebrafish embryos. Genetics 103, 125-136. Wardrip, C., Seps, S., Skrocki, L., Nguyen, L., Waterstrat, P., and Li, X. (1999). Diagnostic exercise: Fluffy, white, cotton candy-like growth on the gills, fins, and skin of salamanders (Ambystoma tigrinum). Contemp. Top. Lab. Anim. Sci. 38, 81-83. Weinstein, B. M., Stempel, D. L., Driever, W., and Fishman, M. C. (1995). Gridlock, a localized, heritable vascular patterning defect in the zebrafish. Nat. Med. 1, 1143-1147. Westerfield, M. (1995). "The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish," 3rd ed. Univ. of Oregon Press, Eugene. Wolf, K. (1988). "Fish Viruses and Viral Diseases." Cornell Univ. Press, Ithaca, New York. Woo, K., and Fraser, S. E. (1995). Order and coherence in the fate map of the zebrafish nervous system. Development 121, 2595-2609. Zhang, J., Talbot, W. S., and Schier, A. E (1998). Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation. Cell 92, 241-251.

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Chapter 20 Biology and Health of Laboratory Fishes Michael K. Stoskopf

I.

II.

III.

IV.

V.

VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A.

Taxonomy

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B.

Use in Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A.

General E r g o n o m i c and Safety Considerations . . . . . . . . . . . . . . . . . .

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B.

Water Sources and H a n d l i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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C.

Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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M a n a g e m e n t and H u s b a n d r y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

893

A.

S y s t e m Startup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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B.

Water M a n a g e m e n t

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C.

Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D.

Social G r o u p i n g E n r i c h m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

M e d i c a l Protocols

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894 894 894

A.

Health Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B.

Quarantine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

895

C.

Pain

895

D.

Anesthesia

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895

Zoonotic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

898

A.

Bacterial Z o o n o s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

898

B.

Parasitic Z o o n o s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

899

C.

F u n g a l and Viral Z o o n o s e s

899

D.

Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...,

.............................

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.

Mycobacteriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

899 899 899

B. Ichthyophthirius multifiliis Infestation . . . . . . . . . . . . . . . . . . . . . . . . . C. lchthyobodo necatrix Infestation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

900

D.

Dactylogyridiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

902

E.

Gyrodactylodiasis

902

E

N e m a t o d e Infections

G.

Microsporidiosis

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H.

Myxosporidiosis

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........................................ ......................................

901

903 903 904

I.

Lymphocystis ............................................

904

J.

Infectious Pancreatic Necrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

904

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

LABORATORY ANIMALMEDICINE, 2nd edition

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Facility D e s i g n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Copyright 9 2002 Michael K. Stoskopf. All rights reserved. ISBN 0-12-263951-0

886

MICHAEL K. STOSKOPF I.

B.

INTRODUCTION

A.

Taxonomy

Fish is a broad, encompassing term for the most diverse and largest taxonomic grouping of the vertebrates. In common usage, the term includes all of the members of four taxonomic classes, the Myxini, Cephalaspidomorphi, Elasmobranchiomorphi, and Osteichthyes. More than 2400 species are known from the United States and Canada alone (Robins et al., 1991). The Osteichthyes alone, the class of the bony fishes, comprises more extant species than all of the mammals, birds, reptiles, and amphibians combined. This massive biodiversity is not a quirk of taxonomic whim. The anatomic and physiologic differences among species of fish are every bit as dramatic as the differences among mammalian species. This characteristic of fish offers tremendous opportunity for the researcher seeking a particular anatomic, physiologic, or disease model, but it also presents serious challenges for laboratory animal managers and veterinarians seeking to maintain the health and well-being of the animals selected. Although certain themes of medicine and health management transcend the species differences of the fishes, it is important to keep in mind the adage, "A fish is not a fish." What it means is that all fish species are not equivalent in their basic biologic and husbandry needs. It is important to know what fish species you are dealing with when making a diagnosis or designing a health protocol. Fish are susceptible to the entire range of infectious and noninfectious diseases known to affect terrestrial species, and the diversity of pathogens capable of causing disease in fish is at least as great as that of the terrestrial species familiar to most laboratory animal specialists. It is not possible to cover the vast array of diseases and their different manifestations among the broad diversity of all fishes within the confines of this chapter. There are major texts devoted to clinical medicine (Stoskopf 1993; Noga 1996), pathology (Roberts 1989; Ferguson 1989), and more specialized disciplines such as virology (Wolf, 1988) and bacteriology (Austin and Austin 1987; Roberts 1982) of fishes. It is more appropriate for the reader to use these texts for more detailed and complete coverage of the wide range of disease manifestations affecting fishes and the therapeutic approaches employed. The zebrafish, a popular laboratory fish species bred for research use, and many of the common diseases of freshwater fishes are covered in Chapter 19. The focus of this chapter will be on significant issues of health management of fishes in laboratory animal facilities, with particular attention to wild-caught or truly random-source fishes as laboratory animals. This unfortunately remains a common situation for most fish species used in research today.

Use in Research

The popularity of fish as laboratory animals continues to grow. They have become particularly useful in toxicology studies, in part because of their small size and the availability in large numbers of many species such as the Japanese medaka and the common guppy. The development of tumor-predisposed strains of zebrafish and swordtails (Xiphophorus) has resulted in useful fish models in the study of cancer. Ecological studies of aquatic environments need to evaluate fish, and some species are serving as valuable monitors in pollution studies. In addition, there has been a long tradition for the use of fish species in basic physiologic, biochemical, and molecular research. The availability of laboratory fish is reasonably good, but by no means is the distribution network as extensive as exists for small rodents. Specialized fish strains can sometimes be obtained from individual laboratories that have developed the strains if collaborators or colleagues are aware of the work. However, the vast majority of research fish are obtained from animal wholesalers that specialize in aquatic life-forms. A few species, particularly freshwater tropical and ornamental species, are captive-reared in production facilities, but generally with no specialized containment or disease screening. The majority of species, particularly marine and estuary fish, are caught from the wild and undergo little or no acclimation to captive conditions prior to being shipped to laboratories. Prescreening for health problems or conditioning of these species is virtually unknown. The pressure for large numbers of inexpensive animals is what drives this predominance of nonconditioned animals in the teleost research world. More than unfortunate, it is an unacceptable situation that affects the quality of research that can be accomplished with fish models. It is interesting to consider where rodent research would be if the primary source of laboratory rodents was from extensive trapping in Midwestern granaries. Although the fish species commonly used in research are not directly threatened in the wild, it behooves the researcher and the laboratory animal veterinarian to seek out and demand captive-reared and acclimated fish from facilities with reasonable disease screening. Researchers and veterinarians should not be seduced to ignore massive interindividual variability due to disease, variable conditioning, or even species and subspecies differences on the basis of low individual animal cost or the ability to use large numbers of animals at low cost. Fish models should be selected carefully, and all aspects of the three Rs (reduction, refinement, and replacement) should be applied to studies using these models. II.

FACILITY DESIGN

The source of fish and their conditioning, which are covered in more detail later in this chapter, are critically important to the

20. BIOLOGY AND HEALTH OF LABORATORY FISHES

success of an aquatic laboratory animal facility; however, the best-conditioned fish will not thrive in a poorly designed or improperly operated facility. The sophistication of laboratory animal facility design for fishes has not yet reached that for mammals, but it should not surprise the laboratory animal veterinarian that the same principles are key to success in either type of facility design.

A.

General Ergonomic and Safety Considerations

Human ergonomics is a major consideration in all laboratory animal facility design and certainly in the design of aquatic facilities. The controls and monitors for tank environmental systems must be easily accessible. Good visibility into the tanks from a comfortable position is critical. Tanks made entirely of glass usually provide excellent visibility, but larger tanks built with opaque materials often have large blind spots that thwart frequent censusing and daily observation of each animal. Technicians should be able to see all areas of the tank with ease from a safe position. Mirrors can be used to facilitate this in some complex installations. Piscean ergonomics, or the consideration of the fish's needs, is also important in fish facility design. The life-support system should allow provision of appropriate light cycles, thermoregulation, space, and cover for the species being held. These factors are every bit as important for fishes as they are for mammalian laboratory species, and these conditions can be quite variable among fish species. Proper attention to the biological needs of the species being held will be possible in a multipurpose facility only if the controls and the tank configurations are sufficiently flexible to allow easy adjustment and modification. A major design challenge is to provide adequate overhead clearance. It is tempting to pack as much water as possible into a vertical space. However, the minimum overhead clearance for any tank should exceed the depth of the tank. This allows the use of nets without impediment and facilitates handling of fish. Also, considerable time and irritation can be saved by making the access to the top of a tank complete. This usually means having completely removable tops, as well as lighting systems that do not interfere or are easily moved to allow complete access. Good tank covers are also an important safety design feature. They prevent fish from jumping out of tanks unobserved and stop dropped objects from accidentally finding their way into the tanks. The major safety hazard in aquatic facilities is electrocution. Ground-fault circuit breakers should be used throughout the facility, and all grounds should be properly wired. Electrical outlets should be positioned high on the wall ( 3 - 4 ft above the floor), to avoid their being shorted out in the event of flooding or inattentive use of hoses. Rubber gaskets to seal out moisture from outlets not in use help prolong the life of the outlets by reducing corrosion of the internal metal components. Employees

887

should be trained in the safe use of electricity near water, and the floors and drains of the facility should not add to the hazard by retaining standing water. Nets with nonconducting handles should be used, and any form of exposed wiring should be avoided. Another safety concern that may not be obvious to individuals inexperienced in the management of fish systems is the great weight of water (1 liter weighs approximately 1 kg). Even a relatively small 100-liter tank (approximately 25 gallons) will weigh 100 kg (over 200 lb) when full. It is important to examine the structural strength of any facility to ensure that it can safely support the cumulative weight of the water systems planned for it. It is also common for fish tanks and header tanks to be elevated above the floor. The support stands for these tanks must be able to safely support the weight of completely filled tanks with an adequate safety margin, and they should be secure from accidental tipping. All systems should be designed so they can be drained completely without bailing buckets, mops, or sponges. Unfortunately, many bulkhead systems for installing bottom drains have a significant lip, which results in a tank that cannot be drained completely dry passively. The bottoms of these tanks may be built to minimize the amount of mopping and sponging required, or a tank design that does not require a bulkhead fitting may be used. The general rule in plumbing fish facilities is to use gravity as much as possible and to minimize the use of power pumps. This strategy reduces power consumption but means that careful attention must be paid to how water will move when a power failure occurs. It would be devastating for laboratory animals if accidental loss of power or equipment failure resulted in water draining from the holding system. The plumbing should be designed so that adequate water remains in the system for fish to survive when the system stops running for any reason. In routine operations, this means that the process of removing water from the tank should be limited by a standpipe or other device that protects the tank water level. If a pump fails, no siphons should allow the tank to continue to drain without return flow. Pumps and compressors make a great deal of noise. This is particularly true of those used to manage large-water-volume systems. Fish holding facilities where the pumps and equipment are housed adjacent to the water systems often sound like an industrial factory. Technicians cannot be heard without raising their voices, and the general din makes it uncomfortable to remain in the facility for prolonged periods. This environment increases the likelihood of mistakes and minimizes the probability of good observation of the fish. Fish have complex sensory systems designed to gather information from waveforms in the water. They are quite sensitive to sound. Placing large pumps, compressors, and other life-support apparatuses in a separate room or enclosure to isolate the animal facility from their sound is an important design consideration. A good rule of thumb is

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that if you find that being in a room for prolonged periods is uncomfortable because of the noise levels, you can be certain that the fish are being affected by the noise. Air handling and sources are often less carefully considered in the design of research fish facilities than are water sources and handling, which will be covered in some detail in Section II,B. This circumstance is unfortunate because an improperly placed air intake can have serious consequences for a fish facility. In most fish systems, water is limited and aeration is used extensively to maintain reasonable oxygen concentrations and reduce carbon dioxide accumulation in the available water. In addition, airlifts are often used to effect the movement of water through components of the system. Large quantities of air are used in these processes, and even very low amounts of impurities in the air can result in accumulations that can affect fish health or experimental results. Even more care should be exercised in the placement of air intakes for fish systems than is used in design of facilities for mammalian laboratory animals. In addition, care in handling the air, including the implementation of redundant systems, moisture traps, and constantly circulating loops, is very important to the long-term safety of the facility. Regenerative blowers are being used more commonly to supply central air-delivery systems for fish facilities. Regenerative blowers have fewer moving parts than compressors and require much less service and maintenance. Although they deliver relatively low pressures of air, high pressures are not needed for most fish facility applications, and regenerative blowers deliver much higher volumes of air than similarly sized compressors. If the tanks in the facility are not going to be any deeper than 1 m water depth, the use of a regenerative blower rather than an air compressor should be strongly considered. Aeration can be critical to the viability of the fish in many systems, so duplication of the device driving the air supply is a wise investment. The need for water changes in fish holding systems means that consideration of reserve water sources must have high priority in design. Reserve water systems should be thermally tempered and preferably designed to allow delivery of water of appropriate pH and hardness for an immediate 50% water change of every tank and system they serve.

B.

Water Sources and Handling

1. Recirculating Systems The literature is replete with references to "closed systems." In truth, closed systems, in which no additional water or air are provided from external sources after the initial establishment of the system, are extremely rare and are not particularly useful for most research situations. What is more commonly meant by these references is a recirculatling system in which water is filtered through various means and reused. The selection and management of balanced filtration systems appropriate for the

research being conducted are critical to the success of such systems and the health of the fish maintained in them. 2.

Filter Selection

Three major types of filtration are used in aquatic d e s i g n m mechanical, biological, and chemicalmeach with its own purpose and application. Mechanical filtration, sometimes referred to as primary filtration, removes suspended particles from water by passing it through a medium that obstructs the particles. The mesh of the medium is selected based on the size of the particulates that need to be removed and the amount of resistance that can be placed on the pump. As the mesh of the filter medium becomes finer, more pumping effort is required. Also, very fine-meshed filters occlude quickly and require frequent backwashing or replacement. A uniform medium with an effective size of 0.3 mm will remove about 95% of particles down to 6 ~tm in diameter. A coarser medium, 0.45 mm in diameter, will retain 15 ~tm particles. For complete retention of all bacteria, a mechanical filter must exclude particles down to 0.2 ~tm in diameter, a goal that is rarely practical in fish systems of any size. Chemical filtration covers a wide range of methods for removing molecular contaminants from water, including ion exchange, both specific (resins) and nonspecific (activated carbon), and oxidative systems (ozone). Foam fractionation by protein modification and ultraviolet filtration can also be considered methods of chemical filtration, because they rely on basic modifications of chemical structure to remove contaminants. Activated carbon filters are the most commonly applied form of chemical filtration. This is sometimes described as absorption filtration but is actually a relatively nonspecific exchange filter. A finite number of binding sites are available on the carbon, which are capable of binding cations and anions with binding strengths that vary with the ion being bound. These ions undergo constant exchange with ions in the water at a rate inversely proportional to their binding strength to the carbon sites. Ions with strong binding affinity are effectively removed from the water. This works well until all of the binding sites are saturated and competitive binding between the more toxic compounds reaches a point where not all toxic ions can be bound simultaneously. At this point, some ions must be released by mass action. The filter also fails when a very strongly binding ion is introduced into the system and displaces more weakly bound toxic compounds from the binding sites. In either of these cases, the filter designed to remove toxic compounds can become a point source of toxic compounds. Deciding when to change an activated carbon filter can be challenging. Variations in the contaminant load of the water being filtered can make mass-action calculations based on infrequent sampling unreliable. One good strategy is to routinely monitor both the influent and the effluent water at the filter for a relatively common contaminant that is found in the influent water and has relatively low binding affinity for the carbon (usually total organic carbon [TOC]).

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20. BIOLOGY AND HEALTH OF LABORATORY FISHES

When the effluent water begins to show a consistent spillover of the contaminant being monitored, while other factors such as flow rates, pH, and temperature remain stable, the carbon in the filter should be changed. The term biological filtration refers to the biological fixation of wastes into less toxic compounds or forms. Bacterial fixation of nitrogen is the most common form employed, but other forms of biological filtration are used, including the concentration of metals and certain toxic organics in algal scrubbers. A bacterial biological filter must maintain enough heterotrophic bacterial colonies to process the solid nitrogenous wastes of fish into soluble wastes such as ammonia. The ammonia from this process and that directly excreted by the fish are then converted by autotrophic bacteria to nitrite and then to nitrate. The surface area for bacterial growth is usually the limiting factor in biological filters, along with the ability to circulate the waste-laden water into contact with the bacteria responsible for nitrogen fixation. This is the principle behind so-called undergravel filters. Containers filled with plastic-fluted spheres and cylinders that maximize surface area while minimizing flow impedance are often used to provide additional surface area for bacterial growth and easier water contact and are commonly referred to as biofilters. Biofilters need to provide adequate water turnover to avoid accumulation of toxic wastes in the system and to supply suffcient oxygenated water to maintain the aerobic processes of the filter. However, it is also important to provide enough contact time between the filter and waste-laden water to allow effective waste metabolism by the bacteria. An imbalance results in an ineffective biofilter. 3.

Flow-Through Systems

Flow-through systems are also referred to as open systems. They are characterized by continuous addition of new water, with equal volumes being removed. Obviously the amount of flushing that occurs in the system is dependent on the rate of water addition and removal. There are several limitations, the first being the volume delivery limitations of the water source. A 2 hr turnover time for a modest 1000-gallon system will require constant delivery from a source of 12,000 gallons per day, or just over 8 gallons per minute. Municipal water costs can easily be calculated for a 7-day week, and the expense can be quite surprising. Frequently wells are used to reduce the maintenance costs through capital outlay on the well. It is important to have any wells tested for capacity by pumping down at least 24 hr before taking the flow measurements. The decision to draw water from a municipal system or from a well is a complex one. Other factors that need to be figured into the cost equations, beyond the simple cost per gallon of water delivered, include the ongoing expense of removing municipal disinfectants and, for wells, the risk of well failure. If sufficient water is available, the next bottleneck for flowthrough systems is the tempering and conditioning of the water.

Adjustments of water temperature take time and energy. The time can be reduced by increasing the energy, but this invariably increases costs. Finally, the fish themselves can provide limitations to the extent of flow-through operation that is acceptable. Rapid currents generated by high water turnover can force fish to swim more actively to maintain their position in the water column. It is possible to increase this effort to the point where the fish cannot eat enough food to maintain body condition and health over time. This is particularly a problem with sedentary species evolved to live in still waters. Unfortunately, many of the fish species prized for their ability to withstand fluctuations in oxygen tension, pH, and water quality in general have evolved these abilities to exploit still, calm waters. Disposal of effluent water from fish systems, especially when large quantities of water are involved, requires special consideration. This is true for recirculating systems as well as for flowthrough systems. Certainly, if pathogens or toxins are being used in experiments, extra precautions must be taken to avoid discharging the pathogen or toxin with the water effluent. Even if no hazardous substances or pathogens are being used, very stringent nitrogen and other water-quality discharge limits may be enforced that require complex postuse modification of the water before discharge. If municipal waste facilities are intended to receive the discharged effluent, it will be important to establish the water quality, volume, and timing requirements for such discharges in order to avoid heavy fines and/or loss of permission to discharge through that mechanism. Similarly, settling and evaporating ponds and tanks are frequently heavily regulated by local as well as federal or state authorities and require specific attention to detailed design issues if they are employed.

4.

Isolation

The same principles of isolation that have been successful in mammalian laboratory animal medicine apply to aquatic systems. All-in/all-out animal movements may be even more important with fish species because of the poor level of stock screening that currently exists. Any conjoined water system or air handling system should be considered a bridge across isolation barriers. Separation of species and other basic principles of laboratory animal medicine related to disease isolation all apply in aquatic systems. The importance of the air systems deserves some further emphasis. Although it is intuitive to veterinarians that spread of infection through direct contact would occur in fish, and that fomite spread through use of implements shared between tanks or systems would be as much a problem as a shared water system, the importance of the air system in disease spread from fish tank to fish tank is often overlooked. Ideally, each system or tank would be housed in its own room. This is rarely practical but is the most effective way to eliminate aerosolization transfer

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of diseases. Fish don't sneeze, but the fine bubbles coming to the surface of tanks from airstones and airlifts cast millions of small aerosol droplets into the air. These droplets carry bacteria, viruses, and fungal spores. All tanks should have tops, and preferably tops that minimize the chances for direct aerosolization into the room. If possible, tanks should be placed far enough apart to prevent the aerosolized droplets of one tank from settling on another. This distance is dependent on the type of top employed, the location of holes in the top, and the degree of aeration and spray used in the tank system. It is never a good idea to place a tank above another tank, but because of space considerations, it is often a necessity. When this occurs, every precaution should be taken to avoid splash or spill contamination of the lower tanks. If you have only tiered tank systems, fish with infectious disease should be kept in lower aquaria, not in top aquaria. Disinfection of the complex surface areas involved in mechanical and chemical filter media is a false economy. The potential for transmitting disease from one group of fish to the next through incompletely disinfected media is high. In general, biological filters by their very nature cannot be disinfected easily. For larger facilities, it may be practical to build a contact chamber for delivery of ozone or ultraviolet filtration to various aquarium systems in series to achieve a degree of disinfection. Before investing in this technology, it is best to arrange for a knowledgeable consultant. It will be critical that the contact chamber provide complete disinfection before returning the water to the fish holding tanks, or else disease transmission will be a major problem. Systems adequate to kill bacteria may not be adequate to kill protozoa or fungi. Clearly, each system should have its own set of implements (nets, feeding sticks, tongs, etc.), which are used only on the fish in that system. Although disinfection of implements may seem a more economical approach initially, in the actual day-to-day rush of caring for the facility, disinfection often becomes impractical. In most applications, implements do not remain in contact with disinfectants long enough to be effective and hurried rinsing of "disinfected" implements can expose fish to the disinfectant. Disinfection protocols can be effective if sufficient investment is made in redundant nets, buckets, and other implements to allow adequate exposure time to disinfectant and extensive effective rinsing of the tools. A major consideration in maintaining a fish facility is the disinfection and decontamination of the tanks and filter systems themselves between experiments. Each time a tank and a filter are recycled for a new set of fish, they should be cleaned and sanitized to the degree appropriate, considering the known health status of the previous occupants. Most commonly, the water in a system is drained and replaced, perhaps after cleaning any visible dirt from the tank sides. Proponents of this approach argue that it saves time, presents less risk of accidental poisoning with disinfectants, and is cost-effective.

Spraying the walls of the tank (first cleaned of visible debris) with 70% ethanol or 2-propanol and allowing the alcohol to evaporate is another level of disinfection that has been used successfully for most bacterial diseases and enveloped viruses. This approach will not disinfect the plumbing or filtration systems. If disposable filters are being used, this may not be a major problem, but safer disinfection is accomplished by circulating disinfectant throughout the tank system. This process is time-consuming and usually reserved for systems that have held fish with highly infectious problems. The tank and system should be drained and cleaned of visible debris. Then the tank is refilled with a dilute chlorine or quaternary ammonium solution and circulated for an hour or more. The tank is drained, and the system rinsed. A major concern is to be sure that all disinfectant is removed from the system before it goes back on line. Using removable or disposable filtration systems greatly aids in this process as disinfection, and removal of disinfectant from the complex surfaces of filters takes a great deal of time and is unreliable. If chlorine was used, the water can be tested for residual chlorine and thiosulfate added after the tank is refilled. Dilute vinegar solutions have also served as disinfectants of hospital and home aquarium systems. Considerable disinfection can be accomplished through merely altering the pH of the water running through the system and then discarding the water. 5.

Facility Retrofits

As fish species become ever more popular research models, retrofitting of mammalian facilities to house fish is much more common than the construction of new dedicated aquatic facilities. Retrofitting presents several challenges in addition to those faced in the design of new facilities. The same principles as for new facility design, outlined in Section II,B,6, apply to the retrofitting of mammalian facilities for holding fishes. However, the constraints of the existing facilities usually dictate which fish species are appropriate, how many can be held, and, in some cases, the configurations of the water systems. Preliminary assessment of facilities being considered for retrofitting to hold fish must include careful evaluation of all potential water sources and their reliability as well as quality. This includes an assessment of the existing water transport infrastructure to identify potential sources of heavy metals and other toxic substances that may need to be dealt with by using specialized preuse filtering. As a related issue, the drain system of the facility needs assessment. There must be somewhere for all of the water to go when it is disposed of, and the disposal ideally is accomplished in an ergonomic and cost-efficient way while preserving isolation principles. Similarly, the electrical supply of the facility must be judged in light of the relatively high power consumption of aquatic laboratory animal space relative to that of routine mammal space. Electric resistance-based heaters, pumps, and lighting can, and usually do, double or triple the power requirements of the space

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being retrofitted. Besides increasing the power requirements, the addition of water to the space usually dictates an upgrade of the wiring to all ground-fault receptacles and to location of receptacles above splash points. Power-failure alarms and emergency power generators are a necessity for aquatic facilities and should support air supply pumps and generators as a first priority, along with emergency personnel safety lighting. Water recirculation is a second priority, followed by temperature maintenance if at all possible. In most retrofits, air handling units are a fixed item beyond the scope of the budget, but it is important to recognize that aquatic facilities will load large amounts of moisture into the airhandling equation that may exceed the capabilities of the original equipment. This impact can be minimized by careful selection of the system design, including the configuration of tanks and the structure of tank tops. The effects in the retrofitted space can also be reduced by careful selection of wall, floor, and ceiling surface coatings and the minimization of use of metal fixtures in the space. Sealed lights are a good investment, as are exterior, sealed light switches and receptacle boxes. Plasticlaminate wall systems that provide a high degree of water seal will extend the life of the underlying superstructure of the building and are a cost-effective investment. 6.

New Facility Design

The design of new facilities needs to balance flexibility with specialized concern for the immediate intended purpose of the facility. Important early considerations include whether the facility will hold freshwater species, marine species, or both. This will have a major impact on water system design and layout, which in turn greatly affects capitalization costs. It is also important to determine the temperature range of the fish intended for the facility. This range allows you to establish the limits of temperature variability as well as the preferred temperature setting of the facility and will also affect the feasibility of humidity control, an issue of significant concern in aquatic facilities. Finally, it is useful to know the size and, if possible, the species of fishes intended for the facility, as well as the number to be held. Clearly; this helps determine the number and size of primary enclosures as well as the scale of the water handling required. Small tanks offer great flexibility for keeping large numbers of animals in a relatively small space. They are also economical if expensive drugs or chemicals are going to be used as water treatments. Unfortunately, small tanks are also inherently more volatile and must be watched much more carefully to avoid environmental problems that can be detrimental to research resuits. Large tanks offer a greater latitude with water quality and are less confining to the fish. Unfortunately, besides taking up space, large tanks take much more time to drain and refill for disinfection, and research treatments delivered in the water can become prohibitively expensive. Round tanks allow continuous swimming patterns in pelagic

species and can be manipulated to direct flow around the periphery of the tank, minimizing incidences of fish colliding with tank walls or rubbing in corners of the tank. Rectangular tanks fit more neatly into rectangular rooms. If you choose rectangular tanks, remember, not all rectangles are created equal. The depth of the tank and the width-to-length ratio will be important. A fish must be able to turn around freely in its tank. It must also be able to make its normal vertical movements within the water column. From a husbandry standpoint, it is always best to have a working depth that allows the aquarist to reach the bottom of the tank with ease. Certainly, the issue of surface area for air exchange is also important. 7.

Temperature Management

Many holding facilities for fish are designed to control temperature through regulation of the room air temperature. This design is energy efficient but requires careful attention to the dynamics of the heating and cooling system of the building. The human comfort range shifts seasonally. Although this change is not very large, it does occur on the edge of the temperature tolerance ranges of many tropical fish species. The difference between 20~ and 23~ can seriously affect research outcomes, and the lower temperatures can even be life-threatening to some fish. Many tropical fish need temperatures in the range of 24~176 and temperatures of 27~ are sometimes used when treating protozoal infections. By convention, zebrafish for embryologic studies are commonly maintained at 28~ during development, well above normal human indoor comfort zones. Facilities using air heating should be maintained at room temperatures in the 24~176 range. Supplemental heaters should be available to boost temperatures in case of failure of the airhandling system. Glass-encased heaters are safer than metalenclosed heaters, particularly in marine systems. Selection of heater wattage is based on the volume of water that must be heated. A small heater in a large volume will be on a greater proportion of the time. A rule of thumb is 50-100 watts of heater for every 40 liters of water in the system. For larger systems, fused silica heaters may be required. Longer-barreled heaters have lower surface temperatures than shorter heaters of the same wattage and are, therefore, less liable to cause burns to fish caught up against them. Protective guard fences can be placed around heaters to keep fish away. Placing the heater horizontally near the bottom of the tank improves the convection dynamics of the heater but requires a sealed, submersible heater. Submersible heaters are easier to disinfect and may be preferable. If a facility has a large number of tanks, a worthwhile investment is a heater calibration bucket--a bucket or tank of water maintained at a desired temperature by a thermostatic heater. Other heaters can be placed in the tank and calibrated to roughly the temperature of the bucket, before being placed in a tank with fish. The other side of temperature control is the cooling of water

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to maintain temperate or cold-water species. Refrigeration systems are expensive. They are a necessity if you are going to house cold-water fish. The most common refrigeration systems are heat exchangers driven by a compressible refrigerant. Heat exchangers constructed of a variety of materials are available. Titanium is the only metal proven suitable for heat exchangers used in marine systems. Coatings on coils of other metals are susceptible to scratching and other damage, resulting in toxicity problems from leached metals and serious corrosion problems in the chiller. These chillers can be used with some impunity in freshwater systems but are too dangerous for marine systems. Graphite exchange blocks are available and may prove useful in marine systems. Occasionally, a clever designer will propose the use of temperature mixing valves in the design of the water supply system for a fish holding facility. This can be an important consideration and provides a means to temper replacement or makeup water to the temperatures the fishes are experiencing in their tanks. The selection and proper installation of temperature mixing valves are complex and not to be taken lightly. Many mixing valves require comparable water pressure from both hot and cold water systems in order to function properly. This state of balance rarely occurs without careful modification of the plumbing designs for the facility. Seasonal changes in water supply temperatures can also wreak havoc on automated tempering systems. Experienced plumbers and engineers are the best defense against a nonfunctional or even dangerous water tempering system. 8.

Materials Selection

Fish holding systems can be constructed of anything that will retain water. However, only a few materials and designs are suitable for research fish facilities. For small systems, all-glass tanks are probably an ideal choice. They afford excellent visibility and are relatively chemically inert. Glass has certain advantages over plastic. It is harder and less easily scratched during maintenance than acrylic plastics, although plastic-coated safety plate is also readily scratched. Plastics require solvent sealing and cannot be properly sealed with silicone sealants, making construction and repair more difficult. Glass is also less expensive than high-quality clear acrylics. However, glass is more easily broken than most plastics and is difficult to drill for some plumbing applications. Only high-grade or medical-grade silicone should be used in glass tank construction or repair. Low-grade, inexpensive silicone caulks contain heavy metals, cyanide, and organic toxins, which can kill fish. Systems larger than 200 liters require extremely thick glass. Complex plumbing is also more common in these systems, making plastic construction a major benefit. Plastics cover a wide range of materials with diverse properties. Clear plastics used as glass substitutes are usually highly specialized acrylics. They are expensive, relatively soft, and

susceptible to scratching. Acrylics have the advantage over glass in that acrylics can be molded in panes to fill very large spans. They are not subject to shattering with the same forces that affect glass, but they can be broken. Acrylic panes are subject to melting from the heat of photographic lights and can catch fire. Little is known about their interaction with chemicals and drugs used in the treatment of fish. Opaque plastics are used as structural components of systems. New or unknown materials should be tested for toxicity with a bioassay before they are used in facility construction. Materials graded as acceptable for foodstuffs are usually acceptable for aquarium systems. Recycled plastics should be avoided or certainly tested rigorously, examining all lots being used. Fiberglass has the highest tension loading capacity of the plastics. It is relatively inexpensive and probably the most commonly used structural plastic in aquarium construction. It is not suitable for construction of toxicological evaluation facilities, because of the variability of toxin leaching from the hardening resins. Newly constructed tanks incorporating fiberglass should be treated carefully to rid the system of polymerizing agent and trapped metals in the fiberglass resin. This can be accomplished by alternately running the filled system for a day with freshwater of pH 3.0 or lower, followed by freshwater of pH 11 or higher and, finally, by fresh water at pH 3.0, discarding the water after each pH shift. A final leaching with salt water for an additional day should be performed if the tank is destined to be a marine aquarium. The entire treatment process is facilitated by the use of warm water (37~176 A bioassay is still advisable before using such a system. Vinyl is an inexpensive flexible plastic used in swimming pool liners. It can find its way into makeshift holding facilities. It is easily damaged and can have large residuals of toxic plasticizer and heavy metals trapped in the polymerization process. These leach out into the water. Dioctyl phthalate is a common contaminant, and although 10 days of soaking and etching is recommended for its removal, this is far from a certain procedure. Vinyl is a poor choice in any fish system. High-density linear polyethylene and polypropylene tanks are relatively inert and can be stripped with the same protocol described for fiberglass, making them essentially free of heavy metal and plasticizer contaminants. They are expensive, opaque materials but are quite suited to use in research fish systems. Polyvinyl chloride generally is used not in tank construction but in the construction of the plumbing systems that operate tanks. Polyvinyl chloride is basically inert to salt water; however, it comes in several types or schedules, which have different properties. High-impact or unplasticized polyvinyl chloride is most commonly used for plumbing applications but can contain trace amounts of metals, particularly lead, which can be leached in acid water systems. Acrylonitrile butadiene styrene pipes are less likely to cause subtle toxicity problems and are recommended in construction of research systems. Concrete is widely used for very large systems because of its

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20. BIOLOGY AND HEALTH OF LABORATORY FISHES

durability, low cost, and formability. It is a consideration when constructing a facility for large fish and is used by aquaculture and display aquarium facilities. Concrete has a strong resistance to compression but lacks tensile strength and shear resistance, which must be provided by metal reinforcement buried in the concrete. Concrete is very alkaline because of free lime .produced by hydration of the surface of the cement. It also contains small amounts of foreign materials, including chromates, which can leach out slowly over a long period after tank construction. Concrete structures should be thoroughly washed or leached with dilute muriatic acid, and then several coats of sodium silicate or other sealant should be applied before the structures are used for fish. A soaking period of several weeks, adjusting pH and discarding water, is recommended. In many instances, metals are required for the completion of a system where no other material will serve. The major problem with metals and water, particularly seawater, is corrosion. Corroded metals lose structural integrity and strength, and the metal being lost through corrosion is toxic to fish. Stainless steel is considered the most resistant metal to seawater corrosion, but its resistance is only relative. The most available stainless steel, AISI (American Iron and Steel Institute) type 316, is a highmolybdenum alloy, resistant to pitting and crevice corrosion. It is not a high-strength steel, and it will corrode. Where strength and maximal corrosion resistance are needed, titanium is preferred over stainless steel. Other common metals used in tank construction include galvanized fittings and brass or copper. Unfortunately, the galvanized coat placed on iron contains enough zinc to be lethal to fish within very short periods, even when calcium protection is in effect in seawater. Bronze can be a fatal source of zinc and copper. In fish research facility design, it is important to avoid the sublethal and lethal effects of heavy metals on fish behavior and physiology.

C.

III.

MANAGEMENT AND HUSBANDRY

A.

System Startup

It takes about 6 weeks to restart a disinfected biofilter and to have it working at full capacity. This is much too long for any practical research system to be down. One way to avoid the problem of downtime is not to use biofilters. Another is to have portable biofilters that are growing in a reserve system designed to keep the filters healthy. Strings of biorings or other plastic support media, suspended in the water, are a common approach. In a well-established reserve system, reseeding may require no more than placing the biofilter into the system. It may be beneficial to keep the disinfected biofilter downstream of and in close contact with well-seeded biofilters. It will take a minimum of 3 weeks to reseed the disinfected biofilter to a level that will be of any value in the research tank. This can be hastened somewhat by keeping the biofilter reserve system warmer; however, it is difficult to say whether this is a long-term benefit or not. The bacteria working on the biofilters are susceptible to environmental changes, and a large drop in fixation efficiency can occur with just the careful transfer of a biofilter from the reserve system to a tank. This loss is greater if the environmental conditions, including the water temperature, of the research tank and the reserve system are widely disparate. It is important to feed biofilters while they are in the reserve system. This can be accomplished in a number of ways. Some aquarists maintain fish or invertebrates in the reserve system to provide waste material to feed the bacteria. This minimizes the work involved in operating the reserve system but has the obvious disadvantage of having potential reservoirs of infection in the reserve system itself. Another approach is to introduce ammonia into the system on a periodic or continuous basis. In marine systems, ammonium chloride solutions are often used for this purpose.

MONITORING

Fish systems can be designed to minimize most catastrophic events, but a program of careful environmental monitoring is necessary in any aquatic system. Three critical factors should be monitored essentially continuously: temperature, water level, and power failure. Electronic tank monitoring systems capable of measuring temperature, water level, and interruption in electrical supply are available commercially. Systems designed for monitoring incubators and laboratory equipment can be adapted to use in fish tanks. They can also be built from parts available in most electronics stores carrying burglar or fire alarm components. These systems can be programmed to call a prescribed telephone number and notify whoever answers that a problem exists. Many of such systems can even communicate what the problem is. These systems are extremely valuable security in lieu of staffing a fish facility 24 hours a day.

B.

Water M a n a g e m e n t

The most critical issue in water management after certification and monitoring of the quality of the source is the water change procedure. Flow-through systems are constantly undergoing water change--water is removed and new water enters the system continuously. The positioning of inflow and outflow pipes in these systems will have a major impact on the efficacy of this exchange. Ideally the flow dynamics and convection patterns of the tank would be such that only old water leaves the system and all new water remains, but this is rarely achieved. Some degree of mixing of newly added water to old water nearly always occurs, reducing the effective water change. It is important to avoid significant shunting of new water to the outflow, and it is advantageous to have a pattern of mixing in the

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tank that avoids "dead spots," or volumes of water that are never or only very slowly exchanged. In recirculating tanks the challenge of optimizing water mixing is not the primary concern. What is critical is to remove a volume of water prior to introducing the new water. A frequent failure in recirculating systems is due to the misconception that "topping up," or the practice of adding water to compensate for evaporative losses, is equivalent to conducting water changes. Toxic compounds do not evaporate at the same rate as water, and most tend to accumulate in the system water if topping up is allowed to substitute for a true water change. The required rate of water changes is dependent on the configuration of the tanks and the system, as well as the bioload of fish being maintained. A general rule of thumb of 0.75-1% per day is effective as a routine water exchange in a wide variety of systems. This approach of frequent small changes has the advantage of reducing the potential impact of temperature, pH, ionic, or other shocks that can occur if improperly tempered and conditioned water is used in larger volume exchanges. The issue of properly conditioning makeup water is particularly crucial when making 25-50% water changes, which are an excellent therapeutic and mitigative tool in fish health management.

C. Feeding The problem of feeding is as complex for the laboratory animal veterinarian who manages fish as is the provision of suitable space and water. The breadth of laboratory fish species diversity contributes to the difficulty, but this difficulty is compounded by a lack of fundamental nutritional knowledge. Although diets that maintain and even support growth and reproduction are known for common laboratory fish species, these empirically derived diets are usually relatively unrefined. Trace nutrient balance is rarely considered, and quality control, including component selection for these diets, is minimal. Protein sources can vary dramatically from lot to lot, as can processing and storage procedures. Relatively little is known about the natural diets of many commonly kept fish or about the effects of trace imbalances on fish physiology. These problems are all compounded by the relative plasticity of fish growth and development in adapting to nutrition availability. A basic rule that would seem absurd in laboratory mammal management if it weren't so often ignored is that fish should be fed. Because fish can survive for relatively long periods without food, not feeding fish is sometimes seen as a way to circumvent the variability in food lots, difficulties in documenting individual intake in group-housed fish, or time constraints in acclimating fish to new diets. As a result, a large amount of knowledge of fish physiology and a considerable amount of disease model research are based on a catabolic animal. Fish entering a new system should be allowed time to acclimate to new diets before experiments are initiated. In experiments that depend on accu-

rate assessment of individual food intake for interpretation of the results, fish should be housed and fed in a manner that makes this assessment feasible. Development of better diets, including certified standardized diets for laboratory fish, should be encouraged. Feeding patterns should try to mimic the natural feeding patterns of the fish (e.g., constant grazers versus opportunistic predators; crepuscular versus diurnal feeders; etc).

D.

Social Grouping Enrichment

Cover and substrates must allow a fish to be comfortable in the tank but not hinder capture or observation. Plastic piping can often be used to provide hiding places, and clear piping is often accepted by a fish, especially if tank lighting is kept subdued except during observation. Animals that need to burrow in sand can be easily observed through clear glass gravel placed in patches, in removable containers, or covering the entire bottom of the tank. Most laboratory fish are held in species isolation, which can simplify this issue. However, multiple-species housing in a primary enclosure is common in fish management, and interspecific and intraspecific interactions among individuals must be managed appropriately. Not all fishes are cooperative, schooling species. Size differences, time-in-residence territoriality, gender interactions, and other social complexities can have important impacts on the physiology of the fishes being managed and the results of research.

IV.

MEDICAL P R O T O C O L S

A.

Health Screening

Considerable research is wasted every year through the use of diseased fish. The best solution to this problem is to deal with known and reliable suppliers of screened stock, but such suppliers are extremely rare. Random-source fish should be screened for disease prior to inclusion in research protocols. On-site screening at the source has a major advantage of reducing the probability of introducing a disease into the laboratory water systems, but few suppliers provide this service. Some research facilities send their own diagnostic specialists to screen stocks prior to purchase, but most facilities do not have the ability to accomplish this degree of security. Consultant companies are available to conduct screenings and are being used more frequently as investigators and laboratory animal managers realize the true costs of losing experiments to preventable diseases. Alternatively, facilities with good quarantine capabilities are employing more in-house screening for diseases after fish arrive at the facilities. Sometimes in-house laboratories are capable of

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20. BIOLOGY AND HEALTH OF LABORATORYFISHES the screening, but more frequently, private fish health screening consultants are contracted. Sampling for screening is always problematic, because essentially all currently used fish health screening techniques are lethal. Screening panels vary considerably, depending on the fish species in question and the research being conducted. Mycobacteriosis is an important problem and should be screened for in most widely used small tropical fish species. Microsporidian parasites of the brain and other tissues are a common problem in some of the marine species, including Fundulus spp., and can interfere with neurology and behavior studies. Common ciliate, flagellate, and trematode ectoparasites can cause unexpected mortality when fish experience stressful conditions, confounding experimental results. Viral diseases have not been well studied in their ability to affect experimental results, and relatively few validated tests are available for screening, but this situation will change as the popularity of fish continues to grow among researchers. Sampling efficacy for fish health screening is ruled by the same statistical principles that dictate mammalian screening methods. For diseases with relatively high prevalences in infected populations, including Ichthyophthirius multifilis, Ichthyobodo spp., Gyrodactylus spp., and Dactylogyrus spp., relatively few fish need to be screened to establish, with reasonable confidence, whether a population is infected. Other problematic diseases such as mycobacteriosis can require sampling of 30 or more animals, depending on the size of the population being sampled, to detect low prevalences of the disease. Future development of nonlethal testing methods for the important laboratory fish diseases will greatly improve the acceptance of fish health screening in laboratory animal medicine.

B.

Quarantine

The issue of quarantine for fish is closely coupled with the common use of random-source fish and the lack of validated specific screening tools. Quarantining and conditioning fish for 14-21 days avoids many problems, although longer isolation times may be appropriate in circumstances where all-in/all-out management is not practiced. Quarantines of 4 - 6 weeks are recommended for concerns about mycobacterial infections. Fish in quarantine should be held in good water with filtration that maintains appropriate pH and nitrogen waste product levels. Fish should be given time to acclimate after arrival. Prophylactic treatments can be administered if the disease status of the supplying source is known, but generally a diagnostic workup of a subsample of the fish is a better approach. Lethal testing is the most common sampling approach used and allows bacterial cultures of internal organs and histologic examination of all organs. Nonlethal sampling procedures are used with extremely valuable fish and include skin impression smears and gill and fin biopsies taken under anesthesia and examined for

the presence of protozoal and metazoal parasites. In larger fish, blood samples may be drawn for complete blood counts and selected serum chemistry determinations. Treatments are then instituted for the entire population, depending on the findings of the diagnostic screening. Quarantine duration is based on daily assessments of the condition and behavior of the fish population and the success of implemented therapies.

C.

Pain

Equating pain in animals with that of humans is subjective. It is suggested that three stages of suffering should be recognized in animals: discomfort, stress, and pain. Discomfort may be characterized by such negative signs as poor condition, torpor, and diminished appetite. Stress is defined as a condition of tension or anxiety predictable or readily explicable from environmental causes or from or including physical causes. Finally, pain itself is recognized by more positive signs such as struggling, screaming or squealing, convulsions, or severe palpitations. Attempts to assess pain in animals are made more difficult by adaptive responses. It is well known that the fight-or-flight mechanism can override pain perception. Individual differences exist among various species in their ability to tolerate or react to pain. One theory for the perception of pain in higher animals involves the modulation of sensory cues that are normally controlled by specific cells in the spinal cord that operate a gating mechanism. The sensory input is relayed to central receptors by fast myelinated and slow unmyelinated fibers up the spinothalamic tract. An imbalance of input between these two sets of fibers is thought to be the cause of pain. There is a wealth of evidence that interruption of this tract, either by injury or disease, results in a loss of ability to feel pain. A British Veterinary Association Fish Sub-Committee meeting in 1984 concluded that although the scientific evidence shows that fish do not have a spinothalamic pathway, there seems to be no way of showing whether or not another part of the brain has adapted to take over the function of the thalamus (Brown, 1985). In humans, parts of the nonspecific or association cortex have been shown to be concerned with nociceptive input. There are marked differences in cortical development among mammals, birds, reptiles, amphibia, and cartilaginous fishes, the simplest of which have no recognizable cerebral cortex, but there is a paucity of information available on centrally mediated reception of sensory cues in fish. Fish physiologists argue that pain is probably not experienced as a strong sensation by fishes, though forceful or noxious physical or chemical stimuli evoke violent reactions. This contention still does not relieve laboratory animal professionals of the responsibility of giving animals the benefit of the doubt. The main sensory cues to which fish respond in the aquatic environment are chemical, hydrodynamic, acoustic, thermal, electrical, light, and mechanical. The receptor sites for these stimuli vary

MICHAEL K. STOSKOPF

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from species to species. Physical changes in heat flow or touch are recognized by skin receptors, and visual cues are observed as changes in light intensity and/or quality. The inner ear and lateral line receive acoustic input, whereas chemical reception occurs in smell and taste organs located all over the body. To summarize, we are not able to say if fish perceive pain based on theoretical definitions, but we do know that sensory receptors are present for external environmental cues. Although the central reception of these sensory inputs is unclear in fish, clinical signs of acute and chronic stress can be observed in fish, and we are able to determine physiological stress by assaying serum cortisol. Fish do act to remove themselves from an adverse stimulus, albeit in an apparently reflex manner. However, it is reasonable to postulate that fish need to be cognizant of their surroundings to react to stressors in their environment, which would suggest that until more is known, fish should be given the benefit of the doubt in situations that might be considered either stressful or painful.

D.

Anesthesia

Anesthesia should be offered to fish for any painful or extremely stressful procedure and is required in fish for procedures that do not require it in other domestic animals. Immobilization, prolonged transport, and minor surgery for topical lesions all require some degree of tranquilization. General anesthesia is required for more complex surgery and sometimes even for injections. As fish are lifted out of the water, an attempt should be made to protect their eyes. Light and vibrational stimuli, including sound, should be reduced to a minimum during the examination. Stressors should be removed from the environment before a fish is anesthetized. Fish should not be harassed prior to anesthesia. For smooth induction and recovery, anesthetic tanks for immersion anesthesia, and recovery tanks, should be prepared ahead of time. Anesthetic solutions should be properly buffered, and careful calculation of the anesthetic agent is important. Anesthetized fish should be handled carefully to avoid damage to the delicate epidermis. The hands of the operator should always be wet. For long procedures, a recirculating anesthesia machine can be used to deliver anesthetic for maintenance. When anesthetizing a group of fish of a new species, a small number should always be anesthetized first, and their recovery carefully monitored. This allows the dose to be assessed before all fish are involved. After anesthesia, the fish should be constantly monitored until the righting reflex returns and the fish is able to swim unaided in a coordinated manner. Intermittent monitoring should be continued for up to 24 hr to make sure that no long-term side effects occur. When fish are under several of the anesthetic drugs, their blood oxygenation falls. Keeping the

anesthetic water well aerated is critical but is not necessarily sufficient to ensure reasonable oxygenation of the fish's tissues. Considerable study is ongoing concerning the mechanism of the apparently hypoxic effects of drugs such as MS-222 and eugenol. Most anesthetic agents for fish are administered by immersion. Other routes include parenteral intramuscular or intraperitoneal injection and oral ingestion. Although fish can survive for periods of several minutes in air and recover well in water, such an experience is physiologically stressful for them. If long operations are to be performed, constant recirculation systems must be used and the fish must be given access to oxygenated water at all times. Monitoring of anesthetic depth in fishes has been described following the stages and planes commonly used to describe depth of anesthesia in mammals (Stoskopf, 1993). As a fish becomes deeply sedated (stage I, plane 2), voluntary swimming stops and the respiratory rate (opercular movements) becomes slightly slower. The transition to stage II, plane 1, or light narcosis, may be preceded by a brief excitement period and an increase in respiratory rate. The fish loses equilibrium but works to right itself in the water. Deep narcosis (stage II, plane 2) is marked by a decrease in respiratory rate back to normal and total loss of equilibrium. This plane is suitable for external sampling, such as taking fin or gill biopsies. Light anesthesia (stage III, plane 1) is characterized by a further decrease in the respiratory rate and near total loss of muscle tone. Minor surgeries are often performed at this stage and plane. Surgical anesthesia (stage III, plane 2) in fishes is usually characterized by a bradycardia, markedly low respiratory rate, and total loss of reactivity to manipulation. Stage IV, or medullary collapse, is characterized by total loss of gill movement followed in time by cardiac arrest.

1.

Tricaine Methane Sulfonate (MS-222)

Tricaine methane sulfonate, also called tricaine or MS-222, is the only anesthetic fully licensed for use in fish in the United States and is one of the most widely used anesthetic agents for poikilotherms worldwide. A derivative of benzocaine, it has an additional sulfonate radical, rendering it more water-soluble and more acidic than its parent compound. Tricaine has no effect on ciliary action, but its effect on muscular activity is rapid. Recovery is also rapid. In weak solutions no long-term toxic effects have been reported in fish, so it is used for transporting fish long distances. Tricaine is a popular anesthetic agent because of its solubility in water. However, because tricaine is acid in aqueous solution, it should be buffered. Tricaine has been noted by many authors to be a hypoxic agent. This characteristic is associated with several physiological changes, including bradycardia, an increase in resistance to blood flow through the gill lamellae, and erythrocyte swelling that impedes blood passage

20. BIOLOGYAND HEALTH OF LABORATORYFISHES through the gills. Further physiological effects due to hypoxia during tricaine anesthesia can include increased concentrations of blood glucose, lactate, potassium, magnesium, hemoglobin, and hematocrit; increased urinary output; and electrolyte loss. These changes may persist up to 4 - 7 days after anesthesia. In general, a tricaine solution of 100 mg/liter can be used for surgical anesthesia, whereas solutions of 2 0 - 3 0 mg/liter are used for tranquilization and transport. Individual variation from fish to fish and between species can be very wide with this agent, so caution should be used when this dose is administered to species of fish the anesthetist has not had experience anesthetizing. 2.

Metomidate

Metomidate hydrochloride is an imidazole-based nonbarbiturate hypnotic agent with no analgesic properties in humans. Metomidate is effective in subduing fish, but long recovery times are common with this drug. Metomidate reduces plasma cortisol and glucose concentrations and increases fish pigmentation, presumably because of increased production of melanocyte-stimulating hormone on the same primary protein as adrenocorticotropic hormone (ACTH). Small adult rainbow trout can be tranquilized with metomidate at 5 mg/liter. At these doses, total loss of reflexes does not occur. Channel catfish fingerlings have been tranquilized at 5 mg/liter, although this dose may have been too high, because recovery times were reported to exceed 24 hr. Muscle fasciculations are common with this drug, precluding its use in delicate procedures. Dosages generally used in freshwater and marine tropical species of fish vary from 2.5 to 5 mg/liter. Doses reported for tropical marine fish for tranquilization or transportation are in the range of 0.06-0.20 mg/liter. 3.

Carbon Dioxide

Carbon dioxide is soluble in water at a dilution of 1:1.2 by volume at standard temperature and pressure. In humans, a CO2 concentration above 7% causes headache, dizziness, mental confusion, palpitations, hypertension, and dyspnea. Concentrations above 10% cause unconsciousness. Excessive CO2 in the blood can depress respiratory drive, so maintenance of sufficient aeration to sustain correct Po2 levels in the fish is important. Bubbling CO2 into the water is difficult to control, and Po2 levels must be kept high to avoid severe acidosis and hypoxia. 4.

Quinaldine

Quinaldine must be dissolved in acetone or ethanol in order to make it miscible with water. Quinaldine sulfate, however, is readily soluble in water but is acidic and should be buffered with sodium bicarbonate (0.45 g NaHCO3-1 g quinaldine sul-

897

fate). Quinaldine is more potent in water with high pH. It irritates the gills and causes increased branchial mucous secretion. Although it produces a loss of equilibrium and depression of medullary centers in stage III anesthesia, the fish do not lose all reflex response, making it less than satisfactory for some delicate surgical procedures. The required solvents produce a noxious vapor that irritates the eyes of the surgeon. Analgesia with quinaldine is thought to be minimal, but quinaldine is popular for collecting fish from tidal pools and small lagoons. For warm-water species, generally 15-70 mg/liter is used. The dose required to reach stage III anesthesia in many fish species is 16 mg/liter. Doses of 50-1000 mg/liter have been used in tilapia. 5.

Euthanasia

Concerns about appropriate end points in experiments on fish are similar to those with mammals. Ideally euthanasia is achieved by appropriate application of anesthetic overdoses followed by cranial detachment or exsanguination. Many scientists are concerned about potential effects of anesthetic agents on their research data. These investigators should be encouraged to conduct pilot studies to document these effects. Ideally, quantifiable time-associated assessments of physiologic, behavioral, or other appropriate responses should be used to judge the suitability of a proposed euthanasia methods for fishes. Though fish, as ectothermic vertebrates, are not covered by the U.S. Animal Welfare Act, they are covered under the U.S. Public Health Service "Guide for the Care and Use of Laboratory Animals." The American Veterinary Medical Association (AVMA) also publishes specific guidelines for euthanasia that are widely accepted as authoritative by many institutional animal care and use committees (IACUCs). The AVMA euthanasia guidelines have recently undergone extensive review by an expert panel. Several methods of euthanasia of fishes that are popular with some investigators, such as dewatering (removal from the water and subsequent asphyxiation) or hypothermia, are not considered humane or appropriate methods of euthanasia for fishes by the AVMA. The physiologic impacts of dewatering with no adjunct procedure to hasten death should be obvious. Concerns about the use of ice or cold shock relate to the ability of neurons of cold-adapted poikilotherms to fire coherently at temperatures much lower than would cause loss of sensation in mammals. Decapitation without prior anesthesia is currently accepted by the AVMA as a humane method of euthanasia of fishes. Direct immersion of unanesthetized fish into fixatives is not considered appropriate, because of the reactions of the fish to the immersion and the prolonged times that fish can continue to gill in the fixatives.

898

MICHAEL K. STOSKOPF V.

ZOONOTIC CONSIDERATIONS

Although the number of reported outbreaks of fish-related diseases in the United States is increasing, the prevalences of fish-related diseases in humans remain relatively low. Increased awareness of disease symptoms and the increased prevalence of immunocompromising conditions appear to contribute to the increasing number of reported outbreaks. Nevertheless, zoonotic disease should be considered in the management of laboratory fish.

A.

Bacterial Z o o n o s e s

Bacterial diseases of fish with zoonotic potential are generally opportunistic. Development of disease in a human usually requires a compromised immune system. Exposures often result in mild gastroenteritis or localized infections of the skin and underlying tissues, but a few organisms are highly pathogenic and can produce high mortality in infected individuals. Members of the genus Streptococcus have caused disease outbreaks in freshwater and saltwater fish in the southern United States and Japan. Many fish isolates are of the Lancefield's group B and D serotypes. Staphylococcus spp. occasionally produce disease in fish and have been isolated from aquarium water. The potential for human infection exists. Erysipelothrix rhusiopathiae (formerly E. insidiosa) appears not to cause disease in fish but is present in the external mucus of many fishes. Human infections with E. rhusiopathiae are prevalent among persons handling fish. Three forms of the disease are described: localized skin infections (erysipeloid, or "fish rose"), usually involving the fingers or hands; diffuse cutaneous disease, when a localized infection spreads to adjacent tissues; and septicemia. Fish or shellfish are believed to be the source of infection in about a quarter of the reported cases in the United States in the past century. Mycobacterium fortuitum, M. chelonei, and M. marinum are recognized pathogens of fish. Human infections with M. chelonei or M. marinum may not be detected, because incubation of human isolates is commonly done at 37~ and the organisms generally do not grow well above 30~ Mycobacterium fortuiturn has a wider temperature tolerance. In contrast to M. avium infections, few human infections with the three fish pathogens have been reported. Immunocompromised individuals may develop disseminated or respiratory disease, but immunocompetent patients more commonly develop circumscribed cutaneous lesions at sites of penetrating wounds. Humans with M. marinum infections usually have a single granulomatous nodule, usually on the hands or fingers. However, a "sporotrichoid" form of the disease, in which localized infection is followed by spread to nearby lymph nodes, can oc-

cur. The nodular form frequently resolves in weeks to months without treatment. The lack of spread to adjacent tissues is thought to be a result of the organism's intolerance of higher temperatures. When the organism occasionally spreads to adjacent tissues, a T-cell unresponsiveness is suspected. Individuals infected with human immunodeficiency virus (HIV) should be advised to avoid cleaning fish holding systems. It is important to keep in mind that human epidemics of granulomatous skin disease have occurred from swimming in infected waters and that this mode of human infection is much more common than infections from exposure to infected tropical fish tanks. Granulomas from M. marinum usually occur at sites of minor skin abrasions that become apparent 2 - 3 weeks after exposure. Nocardia asteroides and N. kampachi have been isolated infrequently from tuberculoid lesions of fish and humans. These infections can be misdiagnosed as mycobacterial disease because of the similarity of clinical signs and the positive reaction to acid-fast staining. Gram-negative bacteria have been incriminated in fish associated food-borne disease in humans, including Vibrio cholerae 0 group 1, V. cholerae non-01, V. parahaemolyticus, and V. vulnificus. None of these diseases should be considered a large risk in a facility that handles laboratory fish, but human infections with V. vulnificus have been the object of some study because of the high mortality associated with infection. Two clinical syndromes have been described. Most prominently described is a primary septicemia syndrome usually associated with eating raw oysters. Signs include fever, changes in mental status, ecchymotic hemorrhages, bulla formation, and pain in the lower extremities. The mortality rate is approximately 50%, even with prompt diagnosis and treatment. A prominent predisposing factor is preexisting liver disease, especially cirrhosis, which is thought to adversely affect leukocyte migration. The second clinical syndrome is more important in laboratory animal facilities and is a wound infection characterized by cellulitis, edema, hemorrhages, bulla formation, and extensive tissue necrosis. Despite prompt and aggressive treatment, the mortality rate ranges from 25 % to 30% among affected individuals. In the majority of cases, infection is acquired by introduction of contaminated seawater into skin wounds, but cases with relatively freshwater exposure have been reported recently. Aeromonas salmonicida, A. hydrophila, A. sobria, A. caviae, A. schubertii, and A. veronii are currently considered members of the motile Aeromonas complex and produce septicemia in infected fish. They are routinely found in the aquatic environment. The most commonly isolated species, A. hydrophila, is found worldwide in tropical and temperate freshwater and is considered to be part of the normal intestinal microflora of healthy fish. Despite the ubiquity of these organisms, motile aeromonads produce few human infections. Signs are variable in infected individuals, but gastroenteritis and localized wound infections are the most common manifestations. Wound infec-

899

20. BIOLOGY AND HEALTH OF LABORATORYFISHES

tions can be superficial or can progress to cellulitis, deep muscle necrosis, or septicemia. The primary concern is with immunodeficient individuals who might acquire an aeromonad infection as a result of wound contamination. Klebsiella spp. are found in aquatic environments, and one case of K. pneumoniae septicemia has been reported as a consequence of handling contaminated fish. Edwardsiella tarda is a well-documented fish pathogen, and infections in humans through ingestion of contaminated water or fish or from contamination of a wound are known but are rare. Persons with serious preexisting illnesses are predisposed to infection with E. tarda, and the mortality rate is high (44%) in those individuals. Yersinia ruckeri is highly pathogenic to fish, but human infection with this organism is rarely reported. Three cases of Leptospira icterohaemorrhagiae infection in British fish farmers in 1981 caused concern that fish harbor leptospirosis and transmit the disease to humans. Studies of leptospirosis in aquatic species have concluded that fish can harbor the organism, but considerably more field and experimental data are needed to accurately determine the risk of human infection.

B.

Parasitic Zoonoses

In the United States the prevalence of parasitic zoonoses attributable to fish is low, and most reported cases involve consumption of fish that serve as intermediate hosts for parasites of fish predators. Human infections with Eustrongyloides generally cause an anisakiasis-like syndrome of acute abdominal pain. Infection has occurred after ingestion of live bait minnows and sushi, but of more interest to laboratory animal workers is an unusual case that involved the migration of a dracunculoid nematode (Philometra sp.) into an open hand wound (Deardorff et al., 1986).

C.

D.

Toxins

A large number of marine organisms produce toxic substances that can cause illness and death in humans. In most instances, however, poisonings caused by marine species occur as isolated events. The toxicology and clinical syndromes produced by these agents have been comprehensively reviewed (Halstead, 1988).

VI.

A.

DISEASES

Mycobacteriosis

Etiology.

The mycobacterial species that infect fish are classed in Runyon group IV. They can be cultured successfully on standard tryptone soy agar and brain-heart infusion agar. More commonly, more specialized media, including Petragnani, Lowenstein-Jensen, Middlebrook 7H10, and Dorset egg media are used for isolation. Mycobacterium fortuitum and M. marinum are the most common mycobacterial isolates from affected laboratory fishes. Mycobacterium chelonei is also occasionally isolated from infected fish. Whether fish can be infected with other species of mycobacteria, including M. tuberculosis, is controversial. An interesting note is the apparently successful but unreplicated experimental infection of perch with M. leprae in 1951. "Mycobacterium anabanti" and "M. platypoecilus" are obsolete synonyms for M. marinum. "Mycobacterium salmoniphilum" is an archaic synonym for M. fortuitum. Mycobacteriumpiscium is in doubt taxonomically and is not currently accepted as a species.

Clinical signs. Mycobacteriosis is a chronic progressive disease that can but does not necessarily take years to develop into a clinically apparent illness. Clinical manifestations include lethargy, anorexia, fin and scale loss, exophthalmia, emaciation, skin inflammation and ulceration, edema, peritonitis, and nodules in muscles that may deform the fish.

Fungal and Viral Zoonoses

Epizootiology and transmission.

No human infections with fish fungal pathogens have been well described. However, Candida albicans has been cultured from skin lesions of mullet, and human infection is considered possible. No human infections with fish viruses have been reported (Wolf, 1988). However, San Miguel sea lion virus, a calicivirus known to produce vesicular disease in both marine mammals and pigs, has been shown to elicit antibody production in humans and vesicular lesions in primates. The virus is believed to be transmitted by various marine fish, and it has been suggested that humans may become infected during handling of fish vectors.

Mycobacteriosis is worldwide in distribution. All fish species should be considered susceptible. Mycobacteria are widespread in most waters. Mycobacterium marinum has been cultured from swimming pools, beaches, natural streams, estuaries, tropical fish tanks, and city tap water. Infection rates can be quite high in contaminated freshwater tropical fish production facilities. Transmission may be from ingestion of contaminated food. Transovarian transmission has been established in Mexican platyfish and other viviparous fishes. Vertical transmission through eggs is not thought to occur in ovoviviparous fish, but more research is needed.

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MICHAEL K. STOSKOPF

B. Ichthyophthirius multifiliis Infestation

Necropsy findings.

Postmortem examination usually reveals gray or white nodules in the liver, kidney, heart, or spleen. Skeletal involvement can cause deformities. Diagnosis is usually based on clinical signs and the presence of acid-fast bacteria in tissue sections or smears.

Pathogenesis.

Little information is available on the pathogenic mechanisms of mycobacterial infections in fish. Exotoxins are not thought to play a role. The possibility of a role for endotoxins is being investigated. The organisms' resistance to host defense mechanisms is thought to be the primary factor in the pathogenesis of infection.

Differential diagnosis.

Mycobacteria are gram-positive pleomorphic rods that are acid-fast and nonmotile. A major differential disease is nocardiosis, which shares some of the histologic characteristics of mycobacteriosis in fish.

Prevention.

No bacterins are available for prevention of the mycobacterial infection. Prevention is focused on careful lethal screening of incoming fish and effective isolation procedures.

Control. Control is based on avoidance of excessive density or poor water conditions, removal of affected fish, and effective quarantines for periods of 4 or more weeks for incoming fish. Treatment. Usually mycobacteria that infect fish are resistant to commonly used antimycobacterial drugs such as isoniazid. Chloramine-B or chloramine-T, cyclosporin, doxycycline, ethambutol, ethionamide, isoniazid, kanamycin, minocycline, penicillin, rifampicin, streptomycin, sulfonamides, and tetracycline are all drugs that have been reported in therapeutic attempts. Multiple-drug therapy is generally more successful against these organisms than single-drug therapy. Doxycycline and rifampin are the drug combination frequently cited as effective against M. marinum. Many clinicians believe that the difficulty of treatment of this disease, and the risk of spread to other fishes or humans, are sufficient reasons to preclude attempts at therapy. Treatment probably should be reserved for particularly valuable animals in situations and facilities where isolation can be maintained. The principle of multidrug therapy is important in managing infections in humans. Research complications.

Usually a slowly developing disease, mycobacteriosis can be devastating to experiments that must maintain fish for prolonged periods. Granuloma formation can affect almost any organ, potentially affecting organ function. In addition, the granulomas can be mistaken for early neoplastic nodules in carcinogenesis studies.

Etiology.

Ichthyophthirius multifiliis is a holotrichous ciliate that has a worldwide distribution and affects all freshwater fishes. A closely related organism, Cryptocaryon irritans, affects marine fishes. Clinical signs. Predominant signs include small white spots widely distributed over the body and fins. In some cases, infestation is limited to the gills. A diagnosis can be confirmed by microscopic examination of biopsy material from skin or gills. Epizootiology and transmission.

These parasites have a complex life cycle that includes stages on the host as well as in the environment. The white spot observed on the affected fish is called the trophont. It is the encysted feeding stage. Eventually, the trophont enlarges, breaks through the epithelium, and drops to the bottom of the aquarium, where it attaches to any object, such as gravel or tubing. At this point the organism is referred to as a tomont. The time taken for development on the fish is temperature-dependent and requires 3 - 4 days at 22~ up to 11 days at 15~ and nearly 30 days at 10~ The tomont attaches to bottom substrates or plants and begins to undergo mitosis (binary fission). Within 18-21 hr at 23 ~ 25~ this mitosis will result in hundreds of ciliated theronts that are released into the water. Theronts actively swim and, when they encounter a host fish, attach and actively penetrate skin or gill epithelium, where they enlarge until they are visible as a white spot. Free-swimming newly excysted ciliated theronts have only about 48 hr in which to find a host before they die. The disease is usually observed several days after introducing new fish to an aquarium.

Necropsy findings.

The characteristic white trophonts may not be visible on a dead fish. Routinely they excyst and drop from the fish very shortly after death of the fish. Theronts appear round to oval and may be from 30 to 1000 ~tm in diameter. The organism moves slowly by means of cilia observable with a high-power objective. The motion is typically a rolling motion in which the parasite rotates across the epithelial surface. The horseshoe-shaped nucleus is often visible and assists identification.

Pathogenesis.

The disease need not be fatal. Heavy infestations are thought to interfere with osmoregulation, but this is speculative and not based on solid research investigations.

Differential diagnosis.

Ichthyophthirius multifiliis is one of the few fish parasites with cilia surrounding the entire organism. The free-swimming infective ciliated theronts are usually pearshaped, actively motile, and about 3 0 - 4 5 ~tm in diameter.

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20. BIOLOGY AND HEALTH OF LABORATORY FISHES

Prevention.

Prophylactic treatments with saline baths or dilute copper are used on incoming fish during quarantine in some facilities. Maintenance of excellent water quality and minimization of stress are thought to reduce the likelihood of a clinical outbreak. Adequate water changes and cleaning of substrates are thought to help prevent accumulation of high numbers of infective tomonts.

Control. Heavy filtration with diatomaceous earth or membrane filters will reduce the number of circulating theronts. Transferring fish to clean aquaria every day for 7 days will limit the infection by keeping one step ahead of theront reinfestation. Removal of theronts from the water can also be accomplished by making large daily water changes. This method, while efficacious, may stress fish excessively unless attention is paid to makeup water temperature and pH. Alternatively, fish can be removed from a system, and the parasites will eventually die for lack of a host. Elevating the temperature several degrees Celsius over normal temperatures accelerates this process. To ensure that all theronts are eliminated in a system, make at least one complete water change, along with removing debris from the gravel before returning fish to the system after leaving a tank or system fallow. Treatment. Currently available medications do not penetrate the encysted trophonts. All treatment is directed toward preventing reinfection of fish by killing free-swimming trophonts. Formaldehyde at 25 ppm (1 ml/10 gallons) is effective if administered 3 times on alternate days. Water changes of up to 75% should be done 4 - 8 hr after treatments. In addition to chemotherapy, management adjustments serve to control infestations. Elevating water temperatures several degrees Celsius over normal temperatures for 5 - 7 days will limit the infection by adversely affecting the heat-sensitive theronts as well as enhancing the immune response of the host. Research complications.

The most common complication associated with this disease is mortality. Certainly, affected fish become inappetent and lethargic, which can also affect experimental results.

C.

Ichthyobodo necatrix

Infestation

Etiology. Ichthyobodo necatrix, formerly known as Costia necatrix, is a small flagellated protozoal parasite with wide distribution in freshwaters. It has been documented to survive and cause disease in marine fish as well, although similar marineadapted organisms are also known.

Clinical signs. Fish affected with Ichthyobodo are often depressed, anorectic, and in respiratory distress. A whitish film

from excess mucus production is commonly seen on the body surface. Some fish die without visible external signs. Diagnosis is confirmed by microscopic examination of wet mounts of skin or gills. The organisms are actively motile, small (7-15 ~tm long), and somewhat comma-shaped. They can be seen as freeswimming forms or as forms attached to cells by their flagellae. When attached, the parasites move in a characteristic circular fashion.

Epizootiology and transmission.

These flagellates reproduce by simple binary fission. Transmission appears to be by direct contact or exposure to water that has held infected fish within several hours. The disease is found both in winter and summer months but is more serious in warmer water. Infestations are seen most frequently in fish that have recently been shipped from a primary producer. The organism survives only an hour or so off of the fish host.

Necropsy findings.

Fish can die from ichthyobodiasis without showing characteristic lesions at necropsy. Epithelial sloughing, spongiosis, and hyperplasia and increased mucous cell production can be seen in other cases. The parasites leave a dead host quickly after death, making identification of the disease challenging unless affected fish are sacrificed. Clinical diagnosis with skin or gill biopsies is generally more rewarding than necropsyin diagnosing this disease.

Pathogenesis.

The Ichthyobodo organism feeds directly on epithelial cells by penetrating within its gullet. The parasite can destroy gill and skin epithelium. Disease in older fish is usually associated with some sort of stress, often due to temperature fluctuations or transport.

Differential diagnosis.

Identification of low numbers of these parasites does not necessarily mean they are the cause of morbidity observed in the fish. Ectocommensal bobonid flagellates that are nonpathogenic can also be found on the skin and gills of fish and can be confused with Ichthyobodo.

Prevention.

Prophylactic treatments with formalin baths or dilute copper are used on incoming fish during quarantine in some facilities. Maintenance of excellent water quality and minimization of stress are thought to reduce the likelihood of a clinical outbreak. Adequate water changes and cleaning of substrates are thought to help prevent accumulation of high numbers of infective free-swimming stages.

Control. No vaccines are available, and prolonged prophylaxis is not generally recommended in laboratory fish. The disease is more severe in younger fish and is commonly associated with temperature drops, so efforts to isolate young fish from older animals and careful attention to stable water temperatures appear to help control the disease.

902

MICHAEL K. STOSKOPF

Treatment. This parasite is susceptible to most common antiprotozoal therapies. One treatment of 25 ppm of formaldehyde followed by a water change up to 75% in 4 - 8 hr is usually effective in killing attached parasites, as well as those in the water column. Research complications.

This disease is of most concern to researchers studying early development of fish. It can be rapidly fatal, with high mortality rates, if introduced to naive animals or animals undergoing any stress.

D.

Dactylogyridiasis

Etiology.

Dactylogyrid flukes are monogenean parasites common in freshwater fish and can infest species of all major fish groups. Pond-reared fish can be heavily infested.

Clinical signs. Fish in ponds or in the wild rarely exhibit clinical signs. Clinical disease is more common in small closed systems. Clinical signs include rapid respiratory movements, clamped fins, flashing, or rubbing. Fish may also become inactive and sit at the bottom of the aquarium. Death can result from heavy infestations. Diagnosis is confirmed by biopsies of the gills, where worms are readily visible. Dactylogyrid flukes have a four-pointed anterior end, a sucker near the anterior end, and four anterior eyespots. The caudal end has a fixation apparatus, or haptor, that consists of 1 or 2 large hooks surrounded by up to 16 smaller hooklets. The worms are approximately 400 gm long and have both testes and ovaries. Epizootiology and transmission.

Dactylogyrid flukes reproduce by mutual fertilization followed by release of eggs that develop off of the host. Eggs from some species hatch into ciliated forms as early as 60 hr after being released. Other species require 4 - 5 days before hatching. The ciliated larvae attack suitable hosts, lose their cilia, and develop into adult trematodes. Transmission is greatly enhanced by overcrowding of fish. The parasite load per fish in a single aquarium, even within a single host species, can be quite variable. Immunocompetence may play a role in this variability.

Necropsy findings.

These parasites disrupt the epithelial barrier of the fish and cause local disruption of tissues and variable inflammatory responses.

Pathogenesis.

Dactylogyrid flukes are usually found on the gills but can be found on the body. If present in sufficient numbers, they can cause hyperplasia, destruction of gill epithelium, and clubbing of gill filaments, which can lead to asphyxiation. It has been postulated that their feeding habits may aid in the introduction of pathogenic bacteria, in addition to their own impact on host physiology.

Differential diagnosis.

Of the many genera of monogenean trematodes that infest freshwater tropical fishes, the species of the genus Dactylogyrus are the most important. However, several related genera that are challenging for the average clinician to differentiate are also egg-laying monogenean trematodes of freshwater fish capable of causing pathology.

Prevention.

No vaccines are available. Prevention involves careful prescreening and treatment of infected fish prior to introduction into the facility.

Control. Control centers around careful management of water quality and avoiding overcrowding. Treatment.

Specific treatments include long-duration exposure to formaldehyde or short-term baths. Saltwater baths have also been used. Organophosphates are used for freshwater fishes, but extended use of organophosphates can develop resistant strains of flukes. Praziquantel effectively removes monogenean trematodes from gills and body surfaces when administered as a bath at 6 ppm. Low levels of formaldehyde (25 ppm) are effective.

Research complications.

Affected fish can be energetically compromised through increased respiratory effort and may display erratic behavior related to attempts to scrape off the parasites. Heavy infestations seem to make fish more susceptible to other infections and toxic substances.

E.

Gyrodactylidiasis

Etiology.

Many species of the genus Gyrodactylus are described. They infect a broad range of hosts, including most freshwater tropical fishes. Their distribution as a genus is most likely worldwide.

Clinical signs. Inapparent infections are common. The parasites feed on blood and epithelium by scraping and sucking. Lesions can include localized hemorrhagic areas, excessive mucus, and localized ulcerations. Infected fish may have a raggedappearing tail from localized hyperplasia, necrosis, and loss of epithelial cells on the fins. Epizootiology and transmission.

Gyrodactylids are viviparous, and embryos with prominent hooks are commonly seen in adult parasites.

Necropsy findings.

These parasites disrupt the epithelial barrier of the fish and cause local disruption of tissues and variable inflammatory responses.

Pathogenesis. Secondary infections with bacteria (Aeromonas, Flexibacter) are common. Aeromonas hydrophila has been

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20. BIOLOGY AND HEALTH OF LABORATORY FISHES

isolated from gyrodactylids removed from goldfish, suggesting that the worms may actively transmit bacteria.

Differential diagnosis.

Gyrodactylid trematodes are usually found on the skin but may occasionally be found on the gills. They can be up to 0.8 mm long, with two points at the anterior end. An anterior sucker is present, but no eyespots. An attachment organ, or haptor, with two large hooks surrounded by up to 16 hooklets is located at the caudal end.

Prevention.

No vaccines are available. Prevention involves careful prescreening and treatment of infected fish prior to introduction into the facility.

Control.

Control centers around careful management of water quality and avoiding overcrowding.

Treatment. Praziquantel at 3 ppm in aquarium water will effectively remove gyrodactylid trematodes. Older treatments include addition of formaldehyde to aquaria at 25 ppm, saltwater baths (2.5-3%), or organophosphate baths. Extended use of organophosphates can result in development of resistant trematodes. Because these worms are live-bearing flukes, drugresistant ova are not a problem. Single treatments can clear the infestation. Research complications.

Affected fish can be energetically compromised through increased flashing behavior related to attempts to scrape off the parasites. Secondary bacterial infections can result in mortality.

F.

N e m a t o d e Infections

Necropsy findings.

Adult or larval nematodes can be found within the lumen of the intestine, as free migratory forms in the peritoneal cavity or as encysted forms in internal organs or musculature. Larval Eustrongyloides spp. are usually found as encysted red worms in the muscles or peritoneum of the fish. Occasionally, cysts located close to the skin are confused with neoplasias.

Prevention.

Feeding insect larvae or free-swimming copepods to fish should be avoided, because these may carry immature stages of nematodes. Feeding of live foods may allow continuance of infestations, which would otherwise be self-limiting in an aquarium environment.

Treatment. Adult nematodes can be treated with common nematicides in the food. Fenbendazole mixed with commercial food enhanced with cod liver oil and bound with gelatin controis Camallanus spp. Preparations such as Panacur can be used in food at the rate of 0.25% (250 ppm). Because fish may not accept medicated food immediately, but most will begin feeding after a few days, Panacur (equine formulation, containing 100 mg of active drug per milliliter) can be used at 2 ppm in aquarium water. This treatment should be repeated 3 times at weekly intervals. Carbon filters should be removed, and passing medication through undergravel beds should be avoided. A partial water change should be made 2 - 3 days after treatment, and then filtration should be resumed. Because fish are quick to refuse medicated food, withholding food for a few days prior to feeding medicated food may be beneficial. Piperazine in food is also effective against some intestinal nematodes. Ivermectin as a bath has been used to treat nematodes successfully; however, the margin of safety is low for many fish species. Research complications.

Fish farmers believe that Capillaria infections can reduce reproductive potential and growth rates.

Etiology.

A number of genera of nematodes infect teleost fishes. Larval forms of Eustrongyloides spp. and adult forms of Capillaria sp. and Camallanus sp. affect a wide variety of freshwater fishes.

Clinical signs. Clinical signs vary with the species of worm and the species of fish infected. Syndromes range from emaciation and failure to thrive, with various intestinal parasites, to swellings and tumors caused by subcutaneous worms, particularly those using the fish as an intermediate host. Infections by camallanids are usually initially noticed when a red worm protrudes from the anus of the fish.

6.

Microsporidiosis

Etiology. Members of the microsporidian genera Glugea, Pleistophora, and Spraguea (synonym Nosema) contain species that are highly pathogenic to teleost fishes.

Clinical signs. Often no clinical signs are observed. Any signs are usually associated with the mechanical occupation of space in infected organs or disruption of the normal formation of organs and structures. Epizootiology and transmission.

Epizootiology and transmission.

The life cycle of these parasites usually involves an intermediate host such as an aquatic insect that harbors the larval stage of the nematode. Some forms use the fish as an intermediate host to reach a predatory final host.

Microsporidiosis can be fatal and highly contagious. Autogamy appears to initiate spore formation at the end of schizogonic activity. Oral ingestion is the mode of transmission. However, intermediate hosts, such as rotifers and planktonic crustaceans, may be required in the life cycle of the parasites.

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M I C H A E L K. STOSKOPF

Necropsy findings.

The parasites are found in the intestines, pyloric ceca, bile ducts, liver, mesenteric lymph nodes, muscles, neural ganglia, subcutaneous tissues, testes, and ovaries. The microsporidian spore is ovoid and has a thick wall without any opening. In heavy infections, the gut wall can be largely supplanted by cysts. The intestine appears chalk white and pebbled and has a rigid, thickened, hard wall. The epithelium of the intestine is denuded and the lumen of the cecum may be almost occluded.

Pathogenesis.

The life cycle of a microsporidian species consists of two distinct phases: a multiplicative stage (schizogony) and a spore-forming stage (sporogony). The infection is invasive, with diffuse infiltration of tissues. The infective germ, the sporoplasm, is extruded to the exterior through a hollow, coiled polar tube that is everted after the spore has been ingested by a specific host. The sporoplasm is injected into the host cell and undergoes multiple binary fission, producing an enormous number of cells. The parasites do not cause host cell degeneration but stimulate hypertrophy and abnormal development into a xenoparasitic complex, or xenoma. Mechanical distension of the intestinal tissue and starvation are thought to be the cause of death.

Differential diagnosis.

Occasionally the presence of masses of the parasites has been mistaken for neoplasia.

Clinical signs. The function of the gallbladder can be impaired.

Epizootiology and transmission.

Nearly all marine tropical fish collected from the wild harbor species of myxospores from the genera Ceratomyxa, Myxidium, or Leptotheca in their gallbladders.

Necropsy findings.

In heavily infected fish, the bile appears cloudy and opaque, often with an amorphous cheeselike substance.

Pathogenesis.

These parasites generally infect the gallbladder and urinary tract (coelozoic) or are intercellular or intracellular parasites of muscle or connective tissue (histozoic). Trophozoites of coelozoic species attach to the transitional epithelium of the gallbladder and urinary bladder during their reproductive cycles, usually with no apparent damage to the host and without initiating a host reaction. Infected muscle fibers become enlarged and replaced by cysts filled with mature spores that may be encapsulated by the host's connective tissue. Muscular liquefaction is due to a proteolytic enzyme released by the parasites after the death of the host.

Prevention.

The life cycle of the myxosporidians has not been fully elucidated and requires future investigation. Existing evidence indicates that an intermediate host is needed.

Prevention.

Removal of ill or dying fish before cannibalism by other fish in the tank can occur greatly reduces the chance of transmission in closed systems not being fed live food.

Control and treatment. No effective chemotherapeutics have been identified for the treatment or control of these diseases.

Control and treatment. Satisfactory drugs to treat or control these infections are not available. Prescreening of entering fish and culling are the common approaches to control these infections, in addition to elimination of potential intermediate hosts. Research complications.

Research complications.

A common problem is the discovery that large portions of the organ system being studied have been displaced in infected fish. This is particularly a problem in studies of the central nervous system in which asymptomatic fish may be missing up to 50% of their brain tissue.

The most obvious impact on research is the loss of fish unexpectedly during experiments, but potentially far more hazardous is the probable impact on physiologic function of inapparently infected fishes.

I. H.

Myxosporidiosis

Etiology. Classification of myxospores is based on the number of shell valves and the position of the polar capsules in the spore: Members of the order Bivalvulida have two spore wall valves, and Multivalvulida members have three or more valves in the spore wall. There are more than 1100 species of myxosporidia reported in literature, but only a few are described in laboratory fish species. All species of Multivalvulida and a few of Bivalvulida are important histozoic parasites that inflict serious injuries to the hosts.

Lymphocystis

Etiology. Lymphocystis is caused by an iridovirus. The disease is recognized worldwide, occurring in at least 125 species of teleosts belonging to 34 families and 9 orders. The disease occurs in warm-, cool-, and cold-water fish species from freshwater, estuarine, and marine environments.

Clinical signs. Lymphocystis is a chronic but seldom fatal disease. Fish with lymphocystis develop macroscopic nodules (0.3-2.0 mm or more in diameter) that occur primarily on the body surface but can also develop on the internal organs. The nodules appear cream-colored to pink or gray, depending on

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20. BIOLOGY AND HEALTH OF LABORATORY FISHES

the condition of the overlying epithelium and the degree of vascularity of the lesion. They take a week to a year or more to develop, depending on the host species and the environmental conditions. The lesions eventually heal, leaving little scar tissue.

Epizootiology and transmission.

The disease occurs with equal frequency in both sexes and can occur in fish of any age, although the prevalence appears higher in young fish. Lymphocystis can be experimentally transmitted with relative ease within a genus but with difficulty between families. This limited degree of host specificity may indicate that various types of lymphocystis virus exist. In nature, lymphocystis virus appears to be transmitted by exposure of surface wounds to waterborne virus or by ingestion of virus or virus-infected cells. Lymphocystis can be transmitted experimentally by cohabitation, exposure to water containing virus, feeding lesion and lesion homogenate, and applying lesion homogenate to gills or scarified skin. The disease can also be experimentally transmitted by subcutaneous or intraperitoneal lesion implantation or by subdermal or intramuscular injection of lesion homogenate or medium from virusinfected cell cultures.

Necropsy findings.

Lymphocystis viral infection causes cellular hypertrophy. Infected cells do not divide, but the cytoplasm and nucleus become very large. Chromatin condensation and fragmentation are evident, and nucleoli are distorted or indistinct. Feulgen-positive inclusion bodies can be seen in the cytoplasm associated with icosahedral virus particles 150-250 nm in diameter. A thick hyaline capsule forms at the periphery of the cell, and proliferating fibroblasts isolate the infected cell. Plasma cells, lymphocytes, macrophages, and polymorphonuclear leukocytes accumulate at the periphery of the cell.

whereas the epithelial cells affected by epitheliocystis show distinctly peripheral nuclear placement.

Prevention.

No vaccines are available. Careful screening of fish stock sources is the only known prevention. Reduction of fish trauma through appropriate social and behavioral management and reduction of abrasions from harsh substrates or rough handling may help prevent the infection.

Control. Lymphocystis virus is remarkably stable under a variety of storage conditions. Significant levels of infectivity were recovered after 15 years from infected tissue dried over P205 at 4~ Lymphocystis virus is inactivated when exposed to ether or chloroform, heat (56~176 or pH 3.0. The virus is stable to multiple cycles of freezing and thawing. Treatment. Lesion-bearing fish should be removed. The antineoplastic drug 6-mercaptopurine inhibits virus-specific synthesis and the appearance of virus-induced cytopathic effects in cell culture and has been used experimentally to control lymphocystis in fish. In cases where individually valuable fish are severely affected, surgical removal and cauterization of the wounds with dilute iodophor solution can be effective. Care should be taken to avoid burning the surrounding skin by prolonged exposure to the iodophor. Research complications.

Lymphocystis is a chronic, nonfatal disease. The lesions are unsightly and can affect fish energetics if severe enough to affect swimming dynamics and/or food prehension or ingestion.

J.

Infectious P a n c r e a t i c Necrosis

Etiology. Pathogenesis.

The course of the disease is more rapid at warmer water temperatures. A month's time course is usual at 25~ and 11 developmental stages have been described. Viral inclusion bodies generally appear about 8 days after infection of a susceptible cell, and virus is detectable as early as 15 days postinfection. The enlarged nucleus appears to be polyploid. Reinfection is possible, but lesions in second and third infections are usually smaller. Cell-mediated and humeral responses to the infection have been demonstrated late in the course of infection. Regression of lesions begins when precipitin reactions between host serum and lesion homogenates become demonstrable.

Infectious pancreatic necrosis (IPN) is caused by IPN virus (IPNV), an aquatic birnavirus.

Clinical signs. Clinical signs vary with the species of fish affected. IPN and IPN-like viruses are often isolated from fish that show no clinical signs. Acute infection occurs in very young fish of some species and can result in cumulative mortality approaching 100%, particularly in salmonids. Older fish often develop subclinical or inapparent infection. Disease outbreaks in older fish are usually stress-activated in carrier animals. Affected fry and fingerlings swim by rotating on their long axis, or whirling. They are dark, often with exophthalmia, abdominal distension, and mucoid fecal pseudocasts. Anemia is a clinical feature of the disease.

Differential diagnosis.

Lymphocystis is usually differentiated from epitheliocystis on the basis of the cell type affected and the position of the nucleus of affected cells. The dermal fibroblasts affected by lymphocystis usually display a central nucleus,

Epizootiology and transmission.

IPN and IPN-like viruses have been recovered from at least 65 species of fish and shellfish distributed essentially worldwide, including North America,

906 South America, most of eastern and western Europe, and Asia. Horizontal transmission occurs with infected feces, urine, and sex products. Other animals can be mechanical vectors. IPN virus has been recovered from bird and mammals feces and from experimentally infected crayfish. Infectivity persisted in two protozoal species (Miamiensis ovidus and Tetrahymena sp.) that were fed virus-infected cells. Vertical egg-associated transmission is suspected, and iodophor egg treatments are ineffective. The virus has been isolated from eyed eggs and transmitted to zebrafish eggs. The virus may be carried inside the egg with the sperm. The virus can be recovered from the shells of eyed eggs more than 3 weeks after infection and can be recovered from eggshells after hatching. Brood stock can possibly be infected with IPNV by injection of virus-contaminated pituitary extracts used to induce spawning. Experimentally, IPNV can be transmitted by injection, immersion, and feeding. The virus probably gains access by contact with the gills, ingestion with food, or passage through the sensory pores of the lateral line system. Some survivors of infection become virus carriers and are reservoirs of infection. The carrier prevalence can exceed 90% of the survivors of an epizootic. The carrier state can continue for many years. The prevalence of the carrier state varies among species. Cross-species transmission occurs. Stress factors and temperature affect IPNV infection. High, rapid mortality occurs between 10~ and 14~ in salmonids, whereas high, protracted mortality occurs at lower temperatures. At higher temperatures, mortality is reduced. Similar patterns have not been identified in warm-water fishes. Recrudescence of infection in apparently healthy carrier fish stressed by transport, crowding, poor nutrition, increased temperature, or low oxygen concentration has been reported.

Necropsy findings.

At postmortem, the liver and spleen are pale, and the stomach and intestines are empty of food and filled with mucoid fluid. Diffuse petechial hemorrhages throughout the pyloric and pancreatic tissues are characteristic. There is massive necrosis of pancreatic acinar cells and occasionally islet tissue with prominent intracytoplasmic inclusions. The pylorus, pyloric ceca, and anterior intestine also show extensive necrosis. Degenerative changes in renal and hepatic tissues are seen. Pancreatic and hepatic tissues are infiltrated by macrophages and polymorphonuclear leukocytes, and viral particles can usually be demonstrated by electron microscopy.

MICHAEL K. STOSKOPF neutralization is the standard. Internal organs and sex products are the clinical samples of choice. Specimens should be assayed within 24 hr of sampling. A variety of salmonid and nonsalmonid cell lines support viral replication. The serological relations among the IPN and IPN-like viruses are complex, and multiple cross-reacting serotypes occur.

Prevention.

Water from surface water supplies should be disinfected, and wells should be protected from exposure to surface waters and from contamination by birds or mammals. Fish introduced into the facility should be assayed and determined to be specific pathogen-free. Eggs should originate from virusfree brood stock. There is no evidence of maternally transferred immunity, but fry can be protected by passive transfer of antibody or interferon. Inactivated IPNV vaccines elicit a protective response when administered by injection and immersion but not by hyperosmotic infiltration or feeding. Live-virus vaccines present diagnostic and regulatory problems. There are efforts to develop subunit vaccines by using cloned components from virulent strains of IPNV and from avirulent strains that cross-react with virulent strains, but care will need to be exercised in administering these vaccines across fish species.

Control. Infectious pancreatic necrosis is most effectively controlled by preventing contact between the host and the virus. The incidence of acute IPN can also be reduced by controlling factors that promote physiological stress (e.g., high density, inappropriate feeding protocols, poor hygiene). IPNV is not inactivated by exposure to ether, chloroform, or glycerol but is rapidly inactivated when exposed to chlorine, iodophor, ozone, or ultraviolet irradiation. With increasing water hardness, progressively higher concentrations and longer contact times are needed to inactivate the virus with chlorine or ozone. The virucidal activity of chlorine and iodophor is reduced by organic matter and at pH levels above 8.0. Exposure to ultraviolet irradiation (254 nm) causes rapid loss of infectivity at an intensity of 2000 ~tW/cm2. The virus is inactivated by prolonged exposure to [3-propiolactone, formalin, drying, heating at 60~ or pH 2 or 9. Residual infectivity persists with exposure to low concentrations (1:4000) of formalin, but higher concentrations (1:200) at warm temperatures can completely inactivate the virus in 4 days. A contaminated facility can be disinfected by treatment with chlorine (200 mg/liter for 1 hr). Ozone and ultraviolet irradiation can be used to decontaminate large volumes of water.

Differential diagnosis.

Diagnosis is based on clinical signs and history. Confirmed diagnosis requires isolation and identification of the virus based on serological reactivity in neutralization, fluorescent antibody, immunoperoxidase, complement fixation, immunoelectrophoresis, coagulation, or enzyme-linked immunosorbent assay (ELISA) tests. Infectivity

Treatment. No effective chemotherapeutic treatment is known for IPN. Virazole (1-o-ribofuranosyl- 1,2,4-triazole-3-carboxamide) inhibits IPNV replication in cell culture and may reduce mortality in infected fish. Interferon induction with tilorone is not protective. Polyvinylpyrrolidone-iodine, e-aminocaproic

20. BIOLOGY AND HEALTH OF LABORATORY FISHES

acid, and t r a n e x a m i c acid c h e m o t h e r a p y appears to r e d u c e mortality but was not effective in e x p e r i m e n t a l challenges. T h e o b v i o u s r e s e a r c h p r o b l e m of mortality in y o u n g fish is p e r h a p s the m o s t easily dealt with t h r o u g h careful screening of fish sources. T h e m o r e subtle impacts of i n a p p a r e n t virus infection in carriers with the potential for r e c r u d e s c e n c e in stressful conditions can be devastating for a researcher.

Research complications.

REFERENCES

Austin, B., and Austin, D. A. (1987). "Bacterial Fish Pathogens: Disease in Farmed and Wild Fish." Wiley, New York. Brown, L. A. (1985). Pain in fish. In "Pain in Animals." British Veterinary Association, Animal Welfare Foundation Symposium. 7 Mansfield St., London WlM OAT, United Kingdom. Brown, L. A. (1988). Anesthesia in fish. In "Tropical Fish Medicine" (M. Stoskopf, ed.). Vet. Clin. North Am. Small Anim. Pract. 18(2), 317-330. Deardorff, T., Overstreet, R., Okihiro, M., and Tam, R. (1986). Piscine adult nematode invading an open lesion in a human hand. Am. J. Trop. Med. Hyg. 35, 827-830.

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Ferguson, H. W. (1989). "Systemic Pathology of Fish." Iowa State Univ. Press, Ames. Halstead, B. (1988). "Poisonous and Venomous Marine Animals of the World." Darwin Press, Princeton, New Jersey. Hawkins, A. D. (1981). "Aquarium Systems." Academic Press, New York. Noga, E. (1996). "Fish Disease; Diagnosis, and Treatment." Mosby, New York. Pickering, A. D. (1981). "Stress and Fish." Academic Press, New York. Roberts, R. J. (1982). "Microbial Diseases ofFish." Academic Press, New York. Roberts, R. J. (1989). "Fish Pathology." Bailliere Tindall, London. Robins, R. C., Bailey, R. M., Bond, C. E., Brooker, J. R., Lachner, E. A., Lea, R. N., and Scott, W. B. (1991). "Common and Scientific Names of Fishes from the United States and Canada." American Fisheries Society Special Publication 20. Bethesda, Maryland. Spotte, S. (1979). "Seawater Aquariums: The Captive Environment." Wiley and Sons, New York. Stoskopf, M. K. (1993). "Fish Medicine." Saunders, Philadelphia. Stoskopf, M. K., ed. (1988). "Tropical Fish Medicine." Vet. Clin. North Am. Small Anim. Pract. 18. Wheaton, E W. (1977). "Aquacultural Engineering." Wiley and Sons, New York. Wolf, K. (1988). "Fish Viruses and Fish Viral Diseases." Cornell Univ. Press, Ithaca, New York.

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Chapter 21 Design and Management of Animal Facilities Jack R. Hessler and Steven L. Leary

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Facility Planning and Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Basic Concepts and Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Programmatic Areas: Function and Interrelationships . . . . . . . . . . . . C. Architectural and Engineering Features . . . . . . . . . . . . . . . . . . . . . . . D. Communications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Security and Controlled Access . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Environmental Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cage Sanitation and Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Animal Watering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Caging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Biological Safety Cabinets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Vacuum Cleaners . . . . . . . . . . . . . . . . . . . . . . ". . . . . . . . . . . . . . . . . . . E Miscellaneous Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Commissioning and Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Knowledge of Laboratory Animal Science . . . . . . . . . . . . . . . . . . . . . B. Personnel Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Standard Operating Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Crisis Management and Disaster Plan . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

INTRODUCTION

A p r i m a r y objective of l a b o r a t o r y a n i m a l science and m e d i cine is to control variables in the r e s e a r c h animal's e n v i r o n m e n t (Vessel, 1967; Vessel et al., 1973, 1976; L a n g and Vessel, 1976; L i n d s e y et al., 1978; B a k e r et al., 1979; Flatt, 1980; Pakes et al., 1984; Baker, 1998; C h a p t e r 29). T h e m a n y e n v i r o n m e n t a l variables, including genetic, microbial, c h e m i c a l , and physical, are LABORATORY ANIMAL MEDICINE, 2nd edition

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illustrated in F i g u r e 1. C o n t r o l of genetic variables is p r i m a r i l y a m a t t e r of biology, but control of other variables is d e p e n d e n t to a significant d e g r e e on the design and m a n a g e m e n t of the research a n i m a l facility (Hessler, 1999). C o n t r o l l i n g e n v i r o n m e n tal variables is critical b e c a u s e the reliability o f r e s e a r c h data is no better than the least reliable link in the chain of p r o c e d u r e s u s e d to derive the data. It is the role of laboratory a n i m a l specialists to assist the scientist with controlling a n i m a l - r e l a t e d variables in order to m a k e the animal link in the chain as reliable Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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Fig. 1. Conceptual view of the laboratory animal, illustrating how its biology is not only determined by genetic factors but is also influenced by numerous environmental factors, which may in turn influence experimental data derived from the animals. (From Baker et al., 1969.)

as possible. This requires facilities designed to standardize effective management and consistent day-to-day animal care. This chapter considers the salient design features of contemporary research animal facilities, with an emphasis on those features required to effectively control environmental variables and allow for the maintenance of high-quality animal care and use standards while minimizing operational costs.

II.

FACILITY PLANNING AND DESIGN

Planning and designing research animal facilities is a dynamic, creative process. Each facility must be designed to meet its programmatic requirements, that is, the type of activity the facility will support and what will be required to do so effectively. For example, commercial rodent production (Foster et al., 1963), safety testing (Balk, 1980), and basic biomedical research facilities clearly have different programmatic requirements. Within these broad categories, there are many specific programmatic issues to be considered. For example, one biomedical research institution may require a transgenic rodent

barrier facility but not nonhuman primate or canine housing space, whereas another institution may need exactly the opposite. In spite of the many possible differences, the general requirements have evolved to become well defined, having been the subject of numerous publications and symposia starting from the early days of organized laboratory animal science and medicine (Brewer, 1952, 1961; Thorp, 1960; Dolowy, 1961; Runkel, 1964a, b; Jonas, 1965; ILAR, 1978; Hair, 1968; Lang, 1969, 1981, 1983; Lang and Harrell, 1972; Simmonds, 1973; Poiley, 1974; Goldstein, 1978; Otis and Foster, 1983; Hessler and Moreland, 1984; Veterans Administration, 1991; Ruys, 1991b; CCAC, 1993; Hessler et al., 1999a; Rahija, 1999). See Hessler (1999) for a review of the developmental history of laboratory animal facilities.

A.

Basic Concepts and Considerations

1. Goals and Objectives

Although goals and objectives for individual animal research facilities will include many differences, there are many common features. Examples include some or all of the following.

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21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

Design and construction features and operational protocol philosophy should meet all applicable codes and regulations. These could include the "Guide for the Care and Use of Laboratory Animals" (ILAR, 1996), the "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching" (FASS, 1999), "Biosafety in Microbiological and Biomedical Laboratories" (CDC-NIH, 1999), the Animal Welfare Act (Code of Federal Regulations, Title 9, Subchapter A, Parts 13), 1993), the Good Laboratory Practices Act (Code of Federal Regulations, Title 21, Chapter 1, Part 58, 1994), and state and local codes (NABR, 1991). Management considerations should be consistent with design criteria whenever possible. The ability to manage a facility efficiently is to a large extent reliant on the design of the facility. Management procedures may be able to compensate for poor design, but often at a high operational cost. Cost-effective design features combined with appropriate architectural and engineering technology should be incorporated to facilitate efficient management and minimize facility maintenance. Life-cycle costs should be evaluated as compared with the initial costs to provide true value engineering. Facility design should be as flexible as possible to accommodate changing research objectives and species utilization while balancing the objectives of cost-effective construction and efficient facility management. Sufficient animal procedure space should be provided to reduce or even eliminate the need to work with the animals outside the facility. In the case of rodents, the procedure space should be either in the animal room or immediately adjacent to the animal rooms. Uninterrupted maintenance of the animal's physical environment is essential. Maintenance of relative air pressures in containment and barrier facilities is especially critical. This will require redundancy in the heating, ventilating, and air conditioning (HVAC) systems and sufficient emergency powergenerating capacity to maintain normal operation of the animal facility. Controlled access utilizing automated technology should be provided at all key points of entry to the facility to provide effective security, and to areas and rooms within the facility as required to facilitate management of the facility. A safe, efficient, and healthy working environment should be provided for personnel working in the facility. Amenities that make for a quality work environment and enhance the recruitment and retention of staff should be provided. Such amenities include adequate locker and shower facilities, break rooms, and training facilities. 2.

Programming: Getting Started

Programming is the most challenging and important phase of the entire facility development process. There are many programming styles, but all have the intent of serving as a means

for the owners/users to communicate with the facility designer (Cole, 1991). The final product of the programming phase is a written facility program that describes what types of activities the facility is to support and details of what will be required to support those activities. A key component is the space allocation summary that lists all required spaces and the amount of space devoted to each. Determining the space requirements is relatively easy when the objectives are clear in terms of the quantity of each species that the facility will need to house. The numbers are typically expressed in terms of the number of each type of species in the case of larger animals and the number of racks or cages in the case of smaller animals such as mice and rats. This approach may be relatively objective for production and safety testing facilities; however, the process for determining the amount of space in a basic biomedical research facility is usually less defined. Many approaches have been used to determine the required size of the animal facility. A crude estimate sometimes used is to design the animal facility space equal to 15% of the total research space in the building. More customized approaches include basing the size of the animal facility on the historical experience of the institution that is planning the facility m for example, using the ratio between the existing animal facility space and wet biomedical research laboratory space or animal facility space and the number of biomedical research faculty/staff scientists (Cole and Hessler, 1991; Tyson and Corey, 1999). Of course, such ratios must be adjusted according to whether or not the existing animal facility space is considered adequate, excessive, or inadequate. In addition it is important to consider projected changes in the research program that could affect the need for animal facility space. For example, the expanding use of transgenic and targeted gene knockout technology in mice has drastically increased the need for animal facility space at many biomedical research institutions. 3.

Location and Arrangement

Public health, public relations, security, human comfort, animal health, and animal husbandry considerations all dictate that areas for animal care and use be isolated from other areas of the building. Allergies to animals are common. For this reason, exposure to animals, dander-laden air, equipment soiled by animals, and animal waste products must be limited to personnel who have an occupational requirement to be exposed. Careful planning must reconcile the necessity for isolating the animal facility with the desirability of locating animal facilities as near as possible to the research laboratories. With rare exceptions, a single-story, centralized facility with direct access to ground-level transportation is the most efficient facility to operate (Jonas, 1978). Alternative arrangements include a central facility with dedicated elevators to gain access to ground-level transportation; a central facility on multiple floors arranged around dedicated elevators; multiple autonomous units

912

J A C K R. H E S S L E R AND STEVEN L. LEARY

that contain all the necessary animal care and use support services; and satellite facilities that rely to varying degrees on a primary facility for some support services. Often, the spatial arrangement of the existing research space, along with space availability, dictates the arrangement of the animal facilities. If properly planned and managed, almost any arrangement can be made to work, but the more an arrangement varies from the single-floor facility with direct access to ground-level transportation, the greater the facility and operational costs will be for the overall animal care and use program. 4.

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Circulation patterns within the facility and access-egress patterns for research staff, supplies, animals, and trash need to be carefully planned to facilitate efficiency, reduce contamination between animal rooms, and prevent unnecessary exposure of personnel to animals and animal waste products. The focus of traffic flow in an animal facility revolves around the cage sanitation facility and the flow of cages between it and the animal rooms. This may include both horizontal and vertical circulation. The horizontal circulation pattern is one of the early decisions to be made in the facility planning process. The two basic options are single-corridor or dual-corridor. The objective of the dual-corridor circulation pattern is to decrease the potential for cross contamination, especially with airborne contaminants, between animal rooms by maintaining separation between clean and soiled cages and supplies. In theory, dual corridors should be superior in terms of reducing cross contamination; however, as compared with single corridors, they come at a high cost in terms of the ratio of animal housing space to circulation space. Figure 2 illustrates this point. Whether or not dual corridors are cost-effective is a complex issue, and the answer will vary according to the relative weight assigned to each of the many pros and cons. Hessler (1991 a) discusses the advantages, disadvantages, and limitations of single and dual corridors and alternatives to dual corridors for contamination control. Few would disagree that a dual-corridor plan is the best choice if cost and space are not an issue. It is also true that single-corridor systems have proven effective in all types of animal facilities, including barrier and biocontainment facilities. Vertical circulation in facilities without direct access to ground-level transportation and multilevel animal facilities should include a minimum of two dedicated freight elevators, one for transport of clean cages and supplies, and one for soiled cages, trash, and potentially contaminated items. Even more important, two elevators are essential, so that one is available when the other is out of service; therefore, the elevators must have independent mechanical and electrical systems. 5.

Animal Cubicles

Animal cubicles provide maximum flexibility for animal isolation within minimal space by dividing animal rooms into mul-

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Fig. 2. Four types of circulation patterns, focusing around the cage sanitation area: single-corridor bidirectional pattern (A), single-corridor unidirectional pattern (B), dual-corridor pattern with relatively large animal rooms (C), and dual-corridor pattern with relatively small animal rooms (D). The arrows indicate the direction of cage traffic. All four are drawn within the same footprint to illustrate the relative "cost" of the different circulation patterns in terms of the percentage of the footprint devoted to corridors (A = 17%, B = 32%, C = 26%, and D = 44%), and the number and size of animal rooms. The actual percentages serve only to accent the significance of choosing a circulation pattern and do not necessarily apply to a particular plan. (From Hessler and Moreland, 1984.)

tiple small spaces, typically each large enough to hold one or two cage racks. Dolowy (1961) was the first to describe the concept. Animal cubicles have been variously identified as Illinois cubicles, because those described by Dolowy were at the University of Illinois at Chicago; modified Horsfall cubicles, after isolators first described by Horsfall and Bauer (1940); and ani-

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

913

Fig. 3. Side-by-sideanimal cubicles, each approximately6 feet wide x 4 feet deep. Features of the cubicles in this figure include a pair of hinged full-panel glass doors with guardrail, air supply from ceiling (not seen in photo) and exhaust at floor level via triangular stainless steel ducts mounted in each corner, and fluorescent light fixtures vertical-mountedon the air ducts in each back corner.

mal cubicles. The most common cubicle sizes are approximately 4 feet deep x 6 feet wide (Figure 3), although larger cubiclesme.g., 7 feet X 7 feet--that can hold two racks and/or in which a person could perform simple tasks with the doors closed are useful. Typically animal cubicles are used to house smaller animals in cages on mobile cage racks. The animal cubicle concept has been expanded into "large-animal cubicles" for housing larger species typically housed in large cages or floor pens (Hessler 1991b, 1993). Cubicles help to solve the problem of what to do when a facility has plenty of animal housing space but too few spaces to provide the necessary separation of species, source, microbiological status, project, and experimental hazards. They have been used extensively since 1961, especially for specialized housing areas where isolation of small groups of animals and containment of hazardous or potentially hazardous agents are a priority, e.g., quarantine, biohazard, chemical, and radioisotope containment areas. Most cubicle rooms are designed with the air pressure in the service aisle between the cubicles positive to the cubicles. Even so, when a cubicle door is opened, air from that cubicle enters the aisle and then other cubicles in the room. It is therefore reasonable to question the effectiveness of animal cubicles for controlling airborne contaminants. Extensive experience over many years and at least one published study (White et al., 1983) have documented that cubicles do effectively prevent airborne infectious agents from spreading between cubicles in the same room. The reason is probably related to the brief window of opportunity for cross contamination when a cubicle door is open and substantial dilution of the contaminant with large volumes of air ventilating the aisle and cubicles. The usefulness of animal cubicles has decreased with the advent of microisolation cages and various types of ventilated

rodent racks that provide other options for isolating rodents; however, cubicles continue to be useful for conventional housing of rodents and other species. Animal cubicles can be built in place or commercially prefabricated. Prefabricated cubicles typically come complete with lighting and internal ventilation, with and without HEPA filtration and the ability to switch between positive and negative relative air pressures. There are many options regarding architectural and engineering features for animal cubicles and animal cubicle rooms. Many of the options along with pros and cons have been described in detail (Hessler and Moreland, 1984; Ruys, 1988; Hessler, 1991b, 1993; Curry et al., 1998).

B.

Programmatic Areas: Function and Interrelationships

Functionally, animal facilities are divided into animal housing space and support space. The ratio of support space to animal housing space varies considerably from facility to facility, depending on the programmatic requirements, but typically ranges between 30:70 and 70:30. In general, the smaller the facility the higher the percentage of space devoted to support.

1. Support Areas Programmatic requirements will determine which and how much of the following support functions will be required for a given facility. a.

Administrative~Training Suite

Managing an animal facility is an increasingly complex business that requires the coordinated effort of a variety of staff.

914

JACK R. HESSLER AND STEVEN L. LEARY

This is best facilitated by providing office and appropriate support space in a well-designed suite. Preferably the suite would be located adjacent to animal housing areas, but outside the security perimeter. Office space is required for professional, management, supervisory, training, and clerical staff, along with space for office equipment, storage of office supplies, conferencing, training, and storing library and training materials. Animal technician supervisor offices may be scattered throughout animal housing areas, depending on the size of the facility. Office space for veterinary technicians may be located in the facility or adjacent to laboratory space or the surgery suite.

b.

Diagnostic Laborato~'ies and Necropsy

Diagnostic laboratory facilities are an essential component of an adequate veterinary care program. The size and complexity of the laboratory space may be adequate for a simple wet laboratory used to process samples for delivery to a comprehensive diagnostic laboratory, may be adequate to support a comprehensive diagnostic laboratory, or could be anything in between. It is both efficient and convenient for diagnostic laboratory space to be immediately adjacent to or a part of the administrative/training suite. A necropsy laboratory is required in most facilities by both the veterinary and the investigative staffs. Ideally, this should be located in a relatively isolated area adjacent to refrigerated space used for storing animal carcasses.

c.

Imaging and Special Research Support Facilities

Imaging equipment such as radiographic and ultrasound equipment is sometimes included as part of a veterinary care and research support program. In addition, MRI, CT, and PET scanners and rodent whole-body irradiators are also often used as animal research tools. Often, space for imaging equipment is included as part of a surgery suite. If such equipment is to be used with animals housed in a barrier facility, consideration should be given to including space for the equipment inside the barrier. This eliminates the disease hazard inherent in returning animals to the barrier. Optimally, space for the equipment could be arranged so as to be directly accessible from inside as well as outside the barrier. Properly managed, this arrangement would increase access to the equipment without compromising the barrier.

d.

Aseptic Surgery

In accordance with accepted standards (ILAR, 1996), major survival surgical procedures on nonrodent mammalian species must be conducted in facilities designed and managed for that purpose. Therefore, most facilities housing nonrodent mammalian species will require a surgical suite. The design of the surgery facility will depend on the species and the number and complexity of procedures likely to be performed. In addition to operating rooms, the surgery suite should include areas or rooms for preparation and storage of sterile supplies, surgeon

preparation, animal preparation, postsurgical recovery, and perhaps equipment and supply storage. Ideally, these are separate rooms, but at a minimum it is essential to limit activities in the surgery room to those required to conduct the surgical procedure and to separate "clean" and "dirty" activities. Depending on the size of the surgery suite, office space for veterinary technicians and/or veterinarians may be required. It is essential that there be no unnecessary traffic in the surgery suite. Because use of the surgery facility will most likely involve nonrodent mammalian species, the relationship of the surgery suite to the housing area for these species should be considered. Hessler (1991c) provides a detailed sample program description of a surgery facility. In general, standards for conducting survival surgical procedures on rodents are less stringent, but aseptic procedures are still required. If a large number of major survival surgical procedures is anticipated, it is desirable to include a surgical room for this purpose. It need not necessarily be a part of the surgery suite or be dedicated to this purpose, but it should be designed so that it can readily be sanitized prior to use as a surgery room. Cunliffe-Beamer (1993) and Brown (1994) describe surgical facilities and management procedures for rodents.

e.

Animal Procedure Laboratories

Research animal facilities are more than a place to house and care for animals. In recent years the trend has been toward discouraging or even prohibiting the removal and return of animals to the facility. The reasons for discouraging the removal of animals from the animal facility are multifaceted, including concerns for personnel and public health, public relations, and potential impact on experimental results; the reasons for not allowing the return of animals to the facility are related to animal health. Therefore, it is often necessary for investigative staff to perform all required animal procedures within the facility, which necessitates devoting an increased percentage of facility space to procedure laboratories. For most species, shared animal procedure laboratories are very useful. Typically there will be a procedure room convenient to each animal room with one per four to eight animal rooms. During the transition to using only "clean" rodents, it became apparent that the use of shared procedure laboratories facilitated the spread of infectious agents throughout the facility. Avoiding this problem meant doing procedures in the animal room or in dedicated animal procedure space adjacent to each room housing rodents. Around the same time, the practice of housing rodents in a microisolation caging system evolved as the "gold standard" for rodent housing and care. The system includes housing rodents in microisolation cages and opening cages only in HEPA-filtered mass air displacement cabinets (typically mobile clean benches or biosafety cabinets, henceforth referred to as animal transfer cabinets). This naturally evolved into using the cabinets as the place for performing most of the animal procedures. Even though most routine procedures

915

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

on rodents can be performed in the animal transfer cabinets in the animal room, rodent facilities do require a considerable amount of procedure space for performing more complex procedures than can practically be performed in the animal room, including ones that involve extensive equipment. Examples of animal procedure space in a rodent barrier facility include surgery laboratories, laboratories for diagnostic/experimental imaging, a laboratory for whole body irradiation, and transgenic/knockout (TG/KO) animal procedure laboratory. Hessler (1991 c) presents detailed sample program descriptions and layouts of various types of animal procedure space.

f

Facilities for Personnel Health and Hygiene

Consideration for personnel health and hygiene is a high priority that has been documented extensively and detailed in a recent Institute of Laboratory Animal Resources publication (ILAR, 1997). The primary considerations are animal allergens, infectious agents, and chemical and physical hazards. The basic principle is that potential risks must be limited to personnel who have an occupational requirement to be exposed, and then that exposure must be minimized. A combination of architectural and engineering features, along with appropriate equipment and management, is required to minimize the risk to personnel (Hessler, 1991c; Rahija, 1999). To reduce the potential for transporting infectious agents between home and the animal facility, animal care technicians should be required to wear work uniforms, and other personnel working in the facility should be required to wear protective outer garments prior to entering animal rooms. All uniforms and protective outerwear should be provided and laundered by the facility. In addition, eating and drinking should not be permitted in animal housing areas or in most support areas. To accommodate these needs, support facilities should include lavatory, shower, locker room, and a break area. In addition, a laundry room for laundering uniforms and surgical linens will be useful, even if a commercial laundry service is to be used. g.

Cage Servicing: Sanitation, Storage, and Preparation and Bedding Storage

The cage servicing area determines the traffic flow pattern of cages to and from animal rooms. Mobile cages are typically transported between animal rooms, and this area from 1 to 3 times per week; therefore, it is essential that it be conveniently accessible to all animal rooms. As one of the most important areas, its design can significantly impact the operational efficiency of the entire animal facility. Single-room cage sanitation facilities are discouraged because of the increased potential for cross contamination between soiled and clean cages and equipment. Typically, cage sanitation consists of a suite of two rooms, one the soiled side and the other the clean side, with the clean side also being used for clean cage storage and cage setup. A wall and pass-through

cage washer(s) separate the clean and soiled sides. A small personnel door may be provided in the w~ill separating the two sides. The type of cage sanitation equipment (described in Section III,A) and the amount of space required in the cage sanitation area depend on the species housed, cage types, cage rack capacity of the facility, and cage sanitation program (Leary et al., 1998). The soiled side of cage sanitation is typically a single room. It needs to be large enough to accommodate the sanitation equipment, most of which is located on the soiled side or inside a mechanical closet separating the two sides. In addition, the soiled side requires sufficient space to store at least 10% of the mobile cage rack capacity at 1.1 m 2 (12 ft 2) per rack, plus space for dumping soiled bedding from cages and cage rack pans and for prerinsing excessively soiled cages and racks. As noted previously, the clean side of cage sanitation is often designed as a single room where clean cages are stored and set up before being taken to the animal rooms. However, a significantly better arrangement is to divide the clean side of cage sanitation into two rooms to separate the clean cage storage and cage setup function from the cage washers with a clean side vestibule. Using this arrangement, cage traffic flows from the soiled side through the cage washers to the clean side vestibule and then into the clean cage storage and preparation room. This arrangement best contains the heat and moisture emanating from the washer equipment. In addition, if an automatic bedding dispenser is used in line with the tunnel washer, this arrangement helps to contain bedding dust generated by the dispenser and facilitates daily cleaning of the area. Even if an automatic bedding dispenser is not used, the clean side vestibule may be the preferred site to fill cages with bedding as opposed to the clean cage storage and setup area. The clean cage storage and setup room should be immediately adjacent to the clean side vestibule. It needs to be large enough to store 10-30% of the mobile rack capacity of the facility, depending on the types of racks and frequency of sanitation, at 1.8 m 2 (20 ft 2) per rack, and it should include adequate space for cage preparation. Depending on the types of racks to be used, cage preparation may involve storing and filling water bottles, storing and filling shoebox cages and cage pans with bedding, and assembling the cage racks.

h.

Feed and Bedding Storage and Services

The bedding storage room should be located immediately adjacent to the clean side of cage sanitation, preferably connected to the room where bedding will be placed in the cages or pans. A similar location next to the clean cage and rack setup area is a good location for feed storage. Both areas should be sized to hold a minimum 2- to 4-week supply or more, depending on delivery frequency. In large facilities, the dimensions of the rooms and door size and location should be arranged to accommodate the handling of feed and bedding bags on pallets, using mechanical equipment. Consideration should also be given to the

916

JACK R. HESSLER AND STEVEN L. LEARY

fact that the bags must be stored off the floor and spaced 6 inches away from the wall. The feed storage area should also be readily accessible to the clean cage setup area and to animal rooms. Two types of feed storage are usually required: room temperature and refrigerated. Most laboratory animal feeds are made from natural ingredients supplied as dry pellets in sturdy bags weighing up to 23 kg (50 lb each). The maximum recommended storage temperature for natural ingredient feed is 21 ~ (70~ (ILAR, 1996). Stored at this temperature, it should be used within 3 - 6 months of the milling date. Guinea pig and nonhuman primate feed must be used within 3 months of the milling date because of the vitamin C requirement of these species and the labile nature of vitamin C in the feed. Storage of all feed, including dry feeds, at 4~ (39~ offers the advantage of significantly retarding the degeneration of nutritional quality to the point of not changing significantly even after 6 months (Fullerton et al., 1982), and it has the added benefit of helping to control vermin. Purified and chemically defined diets, though dry, are often less stable; thus their shelf life may be significantly less than that of naturalingredient diets unless stored at 4~ (39~ (Fullerton et al., 1982). Refrigerated storage space is also required for fresh meats, fruits, and vegetables. Refrigerated storage for dry feeds must be designed to maintain relative humidity at a level that will not support the growth of mildew and molds. The need for food preparation capabilities ranges from none to complex, depending on the research being supported. Safety testing laboratories that administer test compounds in feed require highly specialized preparation areas that allow for safe mixing of potentially hazardous compounds.

i.

Housekeeping

Housekeeping facilities are required to allow for the sanitation of animal rooms, support areas, and corridors. These include storage rooms for sanitation equipment and supplies and janitorial closets or rooms strategically located in corridors and self-contained areas such as the surgery suite, biocontainment areas, and rodent barriers.

j.

Receiving and Shipping

A dedicated, strategically located, and well-designed receiving and shipping area is critical for all animal facilities. In a medium to large facility, this is a busy area where large volumes of animals, feed, bedding, sanitation supplies, and other supplies are received, much of it on pallets. Shipping animals is often necessary, especially in an animal production facility. In addition, a large volume of trash needs to be transported out of the facility. Ideally, a separate dock or at least an isolated portion of the receiving/shipping area should be provided for trash disposal. At a minimum, the receiving/shipping area should in-

clude a dock, an enclosed receiving room immediately adjacent to the dock, and a room for short-term housing of animals in shipping containers until they can be delivered to an animal room or picked up for shipment. The dock should be designed to accommodate a wide variety of delivery vehicle sizes by inclusion of levelers or lifts. An overhang extending at least 6 feet out from the vehicular edge of the dock is required to prevent animals and supplies from getting wet in inclement weather. Consideration should be given to enclosing docks that are exposed to a high volume of public traffic and/or are located in cold climates. In addition to a standard hinged door for personnel entrance, automatic roll-up doors equipped with flyinginsect air shields should be provided. k.

Waste Storage and Removal

A large amount of waste material, including soiled bedding, general trash, and animal carcasses, is typically generated in animal facilities. Waste storage space and the transport path for disposal of the waste requires careful consideration. For example, direct access to ground-level transportation for removal of the waste materials without transporting it through areas outside the animal facility is desirable, and depending on the local situation with regard to waste removal, space for storing waste and/or mobile waste storage containers should be provided. Space adjacent to the dock through which trash is to be removed may be the best location for storing waste and waste removal containers. The need for refrigerated animal carcass storage was noted previously in Section II,B,l,b. 2.

Animal Housing Areas

Animal housing areas may be programmatically divided into categories such as conventional housing, quarantine, biocontainment, and barrier. Each category may be represented in a conventional animal housing area by using special equipment and management procedures or may be a physically distinct area of the animal facility. Following is a description of a generic animal room and functional descriptions of the various categories of animal housing spaces, some or all of which may be required for any given facility. a.

Animal Room

The ideal animal room shape, size, and features will depend to a large degree on the intended use of the room, and even when that is known, the ideal could still be debatable. To deal with the highly unpredictable nature of research, one strategy is to design a generic animal room suitable for housing any of the common laboratory animal species for any type of study. Some consider this strategy too expensive, in terms of both construction costs and management costs, resulting in animal rooms that are only marginally adequate for some species, too labor-intensive for maintaining other species, and ideal for none. The answer

21. DESIGNAND MANAGEMENTOF ANIMALFACILITIES for many animal facility planners is to separate animal rooms into two types, one for species that require large quantities of water for routine daily sanitation (e.g., dogs, pigs, and nonhuman primates) and those that don't (e.g., rodents and rabbits). Generally, animal rooms should not contain any built-ins other than a sink, and even that could be relatively mobile. Stationary cabinets and other stationary equipment impede sanitation and provide harborage for vermin. b.

Conventional Housing

Conventional animal housing areas are generally considered to be anything that is not specifically designed to be used for one of the following categories but could be managed so as to be useful for many of them. c.

Quarantine

Animals of unknown pathogen status are typically isolated during a quarantine period to allow for evaluation of health status and to provide time for the animals to recover from the stress of shipping (Lowe, 1980). The type of facility required varies according the species involved but should be isolated from rodent barrier facilities. Rodents from most commercial vendors are not typically quarantined; however, a period of stabilization prior to use may be required to improve the reliability of the research data (Lang and Vessel, 1976; Davis, 1978; Dymsza et al., 1963; Flynn et al., 1971; Landi et al., 1982). The increase in transgenic and knockout (TG/KO) animals and the sharing of these unique rodents (usually mice) among research institutions have generated the need for high-level rodent quarantine facilities, equipment, and containment practices for holding animals until they are documented to be "clean" or the genetic line can be rederived (Rehg and Toth, 1998; Durfee and Faith, 1999; Hessler et al., 1999b). Animal cubicles are an adequate option for quarantine areas, even for rodents housed in microisolation cages. d.

Barrier

In the jargon of laboratory animal science, the term barrier refers to facilities and management procedures designed to isolate animals from infectious agents. Barrier facility most often refers to a facility used to produce and/or maintain rodents, and the names rodent barrier and barrier facility are used interchangeably. From a management perspective, a barrier could be created at the room level in a conventional facility by using appropriate equipment (e.g., cages and racks), or it could be a specially designed facility. From the design perspective, a barrier may be a specialized area within a research animal facility or to the entire animal housing portion of the facility. The concept of barrier facilities evolved from the animal production industry's interest in maintaining specific pathogen-free (SPF) animals

917

(Foster et al., 1963). However, it became apparent that research facilities required barriers for in-house production of unique rodents and for maintaining disease-free animals for study (Trentin, 1966; Brick et al., 1969; Simmons et al., 1967a; Christie et al., 1968). Since the advent of immunosuppressed and TG/KO rodents, barriers have come to be considered an essential component of most research animal facilities. Barriers are managed at various levels of microbiological control. The highest level requires facilities designed, equipped, and managed to provide for one or more double-door passthrough autoclaves through which all sterilized cages and most supplies enter the facility, and vestibules with interlocking doors used to introduce packaged sterile supplies by chemically sanitizing the exterior of the package in the vestibule. Animals can be introduced into the containment facility through the vestibule by sanitizing the exterior of the shipping container. A pass-through dip tank filled with strong disinfectants or sterilants can be used to introduce sterile items packaged in watertight containers. Before entering, personnel may be required to shower and change clothes or to wear sterile outer garments over street clothes or uniforms, and to wear head and shoe covers, face mask, and gloves. Air showers are sometimes used at the entrance to barrier facilities. Depending on the intended use of the barrier, space for laboratories, animal procedures, and TG/KO laboratories or space for specialized imaging equipment may be required. Large rodent barriers may include a cage sanitation facility inside the barrier, in which case all cages may not need to be autoclaved unless a disease outbreak occurs. A research rodent barrier may require a quarantine area inside the barrier. This is especially important for a TG/KO facility because quarantine is highly recommended for all foster mothers coming out of a TG/KO laboratory until the young are weaned and the mother's health status is determined. Animal cubicles are useful for this purpose, even if the animals are housed in microisolation cages. As noted previously, "barriers" can be created in conventional animal rooms by using various types of cages and equipment, including microisolation caging systems, mass air displacement racks with HEPA (high-efficiency particulate air) filters, and flexible-film isolators of the type used for maintaining germfree animals. e.

Biohazard Containment

Containment refers to facilities and management procedures intended to prevent the escape of hazardous infectious agents. "Biosafety in Microbiological and Biomedical Laboratories" (CDC-NIH, 1999) a publication of the Centers for Disease Control and the National Institutes of Health, classifies microbiological agents into four biosafety levels (BSL), according to the degree of risk to humans (BSL-1 to BSL-4, with BSL-4 being the highest risk level). This publication describes combinations

918

JACK R. HESSLER AND STEVEN L. LEARY

of laboratory practices and techniques, safety equipment, and facilities required for working with agents and animals in each classification level. When planning an animal facility, a decision must be made as to the level of biocontainment required. Animal studies with BSL-2 agents are relatively common and recently have become more so with the use of viral vectors for gene therapy studies (Webber and William, 1999; Evans and Lesnaw, 1999). The demand for studies with BSL-3 agents is less common; however, many facilities could benefit from having an animal biosafety level three (ABSL-3) facility. While studies with ABSL-2 agents can be conducted in conventional animal rooms using appropriate equipment and ABSL-2 practices, they are more efficiently and consistently conducted at a higher level of safety in an ABSL-3 facility. In addition, an ABSL-3 facility is highly desirable for quarantine of rodents infected with overt and/or adventitious agents or that are of unknown health status. Detailed descriptions of ABSL animal facilities and practices are given in other texts (Barkley, 1979; Barkley and Richardson, 1984; Richmond, 1991, 1996; Hessler, 1995; White, 1996; CDC-NIH 1999; Hessler et al., 1999a; King et al., 1999). In addition, Chapter 24 of this text covers control of biohazards associated with the use of experimental animals.

f

Helfrich-Smith 1980; Fox et al., 1980). Ideally, the chemical and radioisotope containment area should be near the dirty side of the cage sanitation area to minimize transport of contaminated cages through corridors. Also recommended is a separate room where contaminated bedding can be removed from cages or pans inside a laminar airflow cabinet in which the aerosolized contaminant is drawn away from the operator into a HEPA filter (Baldwin et al., 1976) (Figure 4).

g.

Nonhuman Primate Housing

Nonhuman primates are sometimes housed in conventional animal rooms adjacent to rooms housing other laboratory animals. However, because of their relatively "dirty" microbial status as compared with laboratory rodents, their potential for carrying zoonotic diseases (see Chapter 25), and the high level of noise they generate, the ideal arrangement is to house nonhuman primates in an isolated area under ABSL-2 standards.

Chemical and Radioisotope Containment

As with biohazard containment, the objective is to contain the chemical agent or radioisotope as close to the source as possible, preventing cross contamination. Appropriate equipment and management practices are critical, but the physical characteristics of the facility certainly influence the level of safety that can be attained and the consistency at which it can be maintained (Newberne and Fox, 1978; Henkel, 1978; NIH, 1981, 1999). Work with chemicals and radioisotopes in animals may often be carried out safely in conventional animal rooms; however, there are some exceptions. For example, HEPA filtering of exhaust air may be required for working with concentrated levels of especially potent carcinogens, or special shielding may be required for working with large quantities of certain radioisotopes. It is desirable and sometimes essential to isolate such studies to help prevent cross contamination. When small numbers of animals are required, it is inefficient to use an entire animal room for a single study. Animal cubicles, semiridged isolators, microisolation cages, and so forth can provide the isolation necessary to prevent cross contamination while housing multiple studies involving small numbers of animals within a relatively small area as compared with using conventional animal rooms for each study. An area planned for supporting chemical and/or radioisotope studies may utilize one or more rooms with cubicles or equipped with other containment devices and one or more procedure rooms equipped with radioisotope/ chemical fume hoods. Decontamination of cages can usually be accomplished safely with the use of conventional mechanical cage washers (Fox and

Fig. 4. Soiled-beddingdumping station that uses mass air displacement to draw bedding dust awayfrom the operatorwhile dumpingbedding from a cage inside the cabinet. The dust is drawn into the back of the cabinet, first filtering it througha coarse filterand then a HEPAfilterbefore returning the air back into the room. (Courtesyof AllentownCaging SystemsCo., Inc.)

919

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

Clearly, rooms housing nonhuman primates must be arranged and located to avoid the necessity of transporting animals or cages and equipment soiled by the animals through corridors or on elevators outside the animal facility. The objective is to avoid exposing individuals who do not have an occupational requirement to be exposed to nonhuman primate-associated diseases. Special features for a nonhuman primate housing area or room may include additional security and an entry vestibule, typically made of chain-link fence, that prevents animals that get out of their primary enclosure from escaping from the room. Lights and any other fixtures in the animal room must be designed and secured so that animals free in the room cannot damage them and so that they do not impede capturing the animals.

h.

Housing for Canines and Small Agricultural Mammals

As with nonhuman primates, the ideal housing area for canines and small agricultural mammals (e.g., swine, ovine) is one isolated from other animal housing and human occupancy, because these animals also have a relatively "dirty" microbial status and generate high noise levels. The zoonotic disease concern, though present, is not as great as with nonhuman primates. The exception is Q fever in sheep, which in certain situations should be maintained under ABSL-2 standards. Animal procedure space should be provided in this area. Because these animals are commonly used as surgical research models, they should be housed near the surgical suite. Rooms may be provided for postoperative recovery and intensive care of surgical. patients. Generally, these species are housed in mobile doubletiered cages, mobile single-tiered pens, or fixed pens. Although dry bedding systems can be used to house these species, it would be unwise to plan a facility to house these species without floor drains. The location of the floor drain is critical to efficient cleaning. Ideally, it should be in an open floor trough located against the sidewalls of the room so that the cages or pens back up to the drain trough. If pens are used, the trough should be outside the pens, leaving a minimum 18-inch access aisle between the pens and the wall, with the drain trough in the floor of the trough. The room floor should be sloped at a minimum of 3//16 inch per foot from a crown in the center of the room to the floor trough on each side of the room. The bottom of the trough should slope a minimum of V4inch to the foot toward a 6-inch-diameter drain with a trap flush. There could also be a water source at the high points of the floor of the trough, controlled with the same ball-type valve that controls the flow of water to the drain trap flush. C.

Architectural and Engineering Features

Architectural and engineering specifications must be planned to provide a sanitizable, functional, controlled environment. These features include some of the most important details relevant to the design of research animal facilities, most of which are applicable to both new construction and renovations.

1.

Heating, Ventilation, and Air Conditioning (HVAC)

The primary function of the heating, ventilation, and air conditioning (HVAC) system is the stabilization of the research animal's physical environment by supplying year-round ventilation with clean, conditioned air. Many of the animal facilityrelated references cited earlier in this chapter include HVAC systems. A few publications have specifically addressed HVAC systems for animal facilities (Kohloss, 1976; Windman and Ziogas, 1978; Henkel, 1978; Neil and Larsen, 1982; Hessler and Roberts, 1988; White, 1991). The American Society of Heating, Refrigerating, and Air Conditioning Engineers (ASHRAE) has recognized the unique design requirements of HVAC systems for research animal facilities and includes a separate section entitled "Laboratory Animal Rooms" in its handbook (ASHRAE, 1999). To view the matter from a different perspective, the function of the HVAC system is to maintain temperature and humidity and to remove heat, particulate, and gaseous contaminants generated in animal rooms (Sullivan and Songer, 1966; Briel et al., 1972; Kacergis et al., 1996). Clearly, the HVAC system is a critical component for controlling the laboratory animal's physical macroenvironment (the room) and microenvironment (the cage) and for maintaining a healthy work environment for personnel.

a.

Air Quality

The source and degree of filtration determine the quality of the air supplied to a facility. Special care must be taken to assure that supply air is not contaminated by exhaust air from other buildings or parts of the same building, such as the animal facility, incinerator smokestacks, and vehicle exhaust fumes. The degree to which incoming air is filtered varies with the type of animal facility and the judgment of the engineers. For example, the air being delivered to rodent barrier facilities and surgery rooms may be filtered with high-efficiency particulate air (HEPA) filters, whereas the air to other areas of the facility may be filtered with 85% or 95% efficient filters. Using HEPAfiltered air has become increasingly common over the past 20 years for rodent barrier facilities, but its cost-effectiveness is not well documented. Task-directed HEPA filteringme.g., using HEPA filters on ventilated racks and in cage-change cabinetsm may be more cost-effective than HEPA-filtering all the air coming into the facility. HEPA filters have such an important role in animal facilities and equipment used in the care and use of research animals that they deserve special note, including a brief review of their history and technology. The official definition of a HEPA filter is a filter that filters 99.97% of particles measuring 0.3 ~tm in diameter. Current HEPA filter technology collection efficiency is 99.99%. The efficiency actually increases with particle size both larger and smaller than 0.3 ~tm. The first HEPA filters were developed in the 1940s by the Arthur D. Little Company under a classified United States government project to manufacture filters that would capture microscopic radioactive contaminants

JACK R. HESSLERAND STEVEN L. LEARY

920

for the Manhattan Project. After World War II, HEPA filter technology was declassified, and commercial manufacturing started in 1950 (APC Filtration, 1999). Most filters are sieves that filter smaller particles as the size of the sieve pores becomes smaller. Smaller pore sizes increase the resistance to flow across the filter, making it impractical to filter large quantities of air. HEPA filters rely on a totally different principle that allows for high particle collection efficiency and relatively low airflow resistance. To be effective, air velocity through the filter must be low. To filter large volumes of air and maintain low velocity, a very large surface area of filter medium per unit volume of air is required. This is accomplished by deep pleating the filter media in a filter module and then providing as many filter units as required to effectively filter the volume of air required. As the air passes through the filter media, inertial forces bring larger particles into contact with charged filter fibers and Brownian motion brings the very small particles, which behave much like gases, into contact with the filter fibers, where they become entrapped. HEPA-filtering mass quantities of air within animal rooms was considered the ultimate answer to animal disease control from the early-1970s to the mid-1980s. Such systems were referred to as mass air displacement (MAD) rooms and clean rooms. Many MAD rooms are still in use, but their popularity in terms of new installations has waned. In MAD rooms, air is recirculated within the room through HEPA filters at volumes sufficient to change the air 150-600 times per hour, depending on the type of system and clean room class desired (Hessler and Moreland, 1984). Fresh air exchanges are superimposed over the recirculated air. MAD rooms work exceptionally well to control the animal's airborne microbial environment, thereby reducing cross contamination (McGarrity et al., 1969; Beall et al., 1971; van der Waaij and Andreas, 1971; McGarrity and Coirell, 1976). However, they have not prevailed in the laboratory animal industry, arguably because microisolation cages and ventilated racks have proven to be more cost-effective. b.

Ventilation

Air handling units for both supply and exhaust should be dedicated to the animal facility. Air supplied to animal rooms should be 100% outside air. The ventilation rate recommended for animal rooms, expressed in terms of fresh air changes per hour, varies between 10 and 20, with 15 being a commonly used rate. There is no hard-and-fast rule regarding a minimally acceptable ventilation rate, because the amount required depends on the heat load as well as microbial, particulate, and gaseous contaminants generated in the room (McPherson, 1975; Woods, 1975). Control of the heat load in the room is the most critical concern, for excessive temperatures can be lethal. Room temperatures above 29.4~ (85~ can be life-threatening to laboratory rodents unadapted to such temperatures (Gordon, 1990) and almost certainly will stress rodents to the point that studies

will be compromised (Garrard et al., 1974; Gordon, 1993). It is important to keep in mind that if microisolation caging is used, the temperature in the animal's microenvironment can be several degrees higher than the macroenvironment. The prominent gaseous contaminant is ammonia, which is generated by ureasepositive bacteria from the feces splitting each urea molecule from urine into two ammonia molecules. Ammonia production depends on many factors, including the species and density of animals, the sanitation level, and the relative humidity in the room and cage (Briel et al., 1971; Kruckenberg, 1971; Hasenau, et al., 1993; Memarzadeh, 1998). As a general rule, a ventilation rate that adequately controls the heat load when air is delivered to the room at 12.8~ (55~ is adequate to control the gaseous and particulate contaminants. Heat loads for various species of animals are listed in the ASHRAE handbook (ASHRAE, 1999). Variable air volume (VAV) systems for animal rooms designed to automatically maintain established set points for room temperature, relative humidity, and air quality by adjusting airflow based on actual loads should theoretically function well in animal rooms. Having variable air volume capabilities would allow using performance standards as suggested in the "Guide" (ILAR, 1996), as opposed to engineering standards as currently relied on in the industry. The most significant effect would be energy conservation any time the density of animals housed in a room is less than maximum design load. The same applies to other rooms in the facility, e.g., the cage sanitation area, where loads range from very high when the sanitation equipment is being used to very low when it is not. As newer, more reliable control and monitoring systems are tested and proven acceptable, VAV for animal rooms may become common, perhaps even standard. Room Ventilation Patterns and Computational Fluid Dynamics (CFD)

Computational fluid dynamics (CFD) is the use of highly complex mathematical models to predict air circulation patterns in a space. Air temperature, flow rates, heat generation in the space, types and locations of air inlets and outlets, objects in the space, and so forth are considered in the model. It is a powerful design tool for predicting how effectively a particular ventilation system design will function to meet the desired room conditions. Until recently, it has been well accepted that the most effective animal room ventilation pattern is to supply air at the ceiling and exhaust it near the floor; however, there have been suggestions that other options are more cost-effective (Nevans and Miller, 1972; Neil and Larsen, 1982). Data from recently published CFD studies also give cause to reconsider this and other possible dogmas regarding animal facility HVAC systems (Hughes and Reynolds, 1994; Reynolds, 1994; Hughes et al., 1996; Curry et al., 1998; Memarzadeh, 1998). One CFD study suggests that a more efficient way to ventilate an animal room,

21. DESIGNAND MANAGEMENTOF ANIMALFACILITIES in terms of removing airborne contaminants (heat, gases, and particulates), is to supply and exhaust air at the ceiling in all four corners or, better, directly above each cage rack (Hughes, et al., 1996). CFD data from the same publication suggest that an even more efficient configuration is to supply and exhaust room air from a soffit mounted in the center of the ceiling and extending the full length of the long axis of the room. In this CFD model, supply air is directed from radial diffusers in the bottom of the soffit toward the floor. Exhaust inlets located along both sides of the soffit capture the air as it curls from the floor, up the wall parallel with the soffit, across the ceiling, and into the soffit, where it is removed from the room. A full-scale test model of an animal room fitted with this type of soffit is reported to have performed better than predicted by the model (Hughes et al., 1996). In contrast to the CFD studies just cited, CFD studies conducted by the NIH Division of Engineering Services suggest that low returns are superior to ceiling returns (Memarzadeh, 1998). The NIH studies did not evaluate the soffit model. The NIH study predicted that increasing cage temperature would reduce ammonia generation by decreasing the relative humidity in the cage (Memarzadeh, 1998). These CFD studies used different assumptions for key features; thus, additional study will be required to clarify this important issue. Clearly, high returns in each corner or the soffit configurations are tempting options in that they are less costly to construct than low returns and do not take up floor space. A CFD study modeling animal cubicles indicates that air supplied at the floor and exhausted at the ceiling more effectively removes airborne contaminants from animal cubicles than when air is supplied at the ceiling and exhausted at the floor (Curry et al., 1998). The very important question regarding "ideal" or "minimal effective" ventilation rates has been addressed (Hughes et al., 1996; Memarzadeh, 1998), but the answers are complex at best, and the definitive answer, if there is one, has yet to be determined. Certainly, CFD is a valuable, albeit currently expensive, design tool that will likely be used more commonly in the future. d.

Special Ventilation Requirements for Fixed and Mobile Equipment

Fume hoods and certain types of biosafety cabinets are common pieces of fixed equipment with special ventilation requirements that are not unique to animal facilities. Autoclaves also are not unique to animal facilities, but materials routinely autoclaved in animal facilities may be. These specialized materials often result in exceptionally high odor levels. Because of this, particular attention is required to provide exhaust system canopies configured and designed with sufficient airflow to capture the heat, moisture, and odors emanating from the autoclave chamber when the door is opened. Cage sanitation equipment (see Section III,A) and the cage sanitation area, especially the clean and soiled sides of the cage washroom, have ventilation requirements even more unique to animal facilities. Such

921 equipment uses water at a temperature of 82~ (180~ or higher, generating large quantities of heat and moisture inside the equipment and in the room. As with autoclaves, the objective is to capture and exhaust the heat and moisture as directly as possible, which requires exhaust connections connected directly to the washing equipment. Depending on the type of washer, canopies may be required at the entry and, more important, exit ports of the washers. Because of the large amount of moisture exhausted from the equipment and from the room, the cage sanitation area should have an independent, rust-resistant exhaust system. Exhaust ducts should be sealed and drained and should be constructed with water- and acid-resistant materials. Of course, the overall ventilation requirements of the room must take into consideration the enormous heat and moisture load in the room that may include a significant mass of stainless steel at temperatures of 82~ (180~ or higher. Ventilated racks are an example of mobile equipment that may benefit from a direct connection to the ventilation system, although it is not required. Ventilated racks are commonly used as freestanding equipment with blower/filter units that supply HEPA-filtered room air to the cages. They may also be equipped with blower/filter units that capture air coming from the cages and HEPA-filter it before blowing it back into the room (Figure 5, option 1, and Figures 18 and 21). The blower/filter units can be mounted on top of the cage racks but ideally are mounted on wall shelves and connected to the racks with flexible ducting (Figure 6). HEPA-filtering the exhaust air from the cages removes particulate contaminants but does not remove gaseous contaminants and heat. This is best accomplished by coupling the rack exhaust directly to the room exhaust. There are many strategies for integrating both supply and exhaust air of ventilated racks with the ventilation system (Lipman, 1993). Regardless of which strategy is selected, it is important to decide early in the planning process, because the design of the room ventilation system must be matched with the equipment to gain maximum benefit. Not only does the decision affect the physical couplings, but it also affects the cubic feet of air per minute (cfm) of supply air that will be required in the room. For example, rooms with self-contained blower units that return rack exhaust air to the room require higher air exchange rates than rooms that directly exhaust a large portion of the heat load generated by the animals and blower units directly out of the room. A common airflow requirement for ventilated racks is 0.3 cfm per mouse cage sized for a maximum of 4 - 5 mice (approximately 80 in-cage changes per hour). However, the actual amount required may vary, depending on the rack manufacturer. The numbers of cages per mobile rack typically range up to 140, but fixed racks may have more. There are four basic strategies for exhausting air from the rack directly to the room exhaust (Figure 5). Each requires the installation of exhaust ducts in the ceiling, typically 4 inches in diameter and one per rack, with rack and room exhaust coupled via flexible duct. (1) The first strategy requires the ventilated

922

JACK R. HESSLER AND STEVEN L. LEARY

Option #2

Option #1 Exhaust to Room

(

Room Air

Thimble or capture hood

o

IJl I ill a

o

Cage rack w~ manufacturer's supply & exhaust fans 9High first cost. 9Least desirable in terms of effect on macroenvironment. Animal heat load, odors, and gaseous effluent from cages pass through HEPA filter to room. 9Fans produce noise, vibration, heat, and add to the electric load. 9Fans require maintenance and an electrical outlet for each rack.

Room Air

:1 I I

I

I

~ To redundant v building exhaust fans

t

o

Cage rack w~ manufacturer's supply & exhaust fans + thimble connection to building exhaust system 9Highest first cost. Improved macroenvironment (reduced room heat load, odors, and gaseous effluent). 9Fans produce noise, vibration, heat, and add to the electric load. 9Fans require maintenance and an electrical outlet for each rack. 9Thimbles, capture hoods require balancing & may use excess room air. Rack exhaust may go to room if there is a problem with the static pressure in the room exhaust duct.

Option #3 r

Option #4 To redundant

To redundant v building exhaust fans

)

bui~ing exhaust fans

Room Air J

II Ir II

iF

D

II

Ei

I! [I

II I1 II

II

D

II

Rack supply fan and self-balancing Venturi valve hard-ducted to room exhaust Mid-range first cost.

9

9Improved macroenvironment (reduced noise, heat load, odors, gaseous effluent). 9Supply fan: -

-

produces noise, vibration, heat. requires maintenance.

- requires an electrical outlet for each rack. 9Venturi valve: - Does not require power. - Automatically balances rack and room flows when rack(s) removed from system.

Fig. 5.

Conditioned room air

I

Single fan providing filtered, conditioned room air to multiple racks in same room.

Self-balancing Venturi valves hard-ducted to common HEPA filtered air supply fan for all racks in room, and to room exhaust 9Lowest first and operating cost when multiple racks are connected to a single supply fan. 9No supply and exhaust fan noise, vibration, heatload in room. 9No power requirements for each rack. 9Automatically balances rack and room flows when rack(s) removed from system. 9Energy savings: allows for greatest reduction in room ventilation rate. 9Quietest operation.

Options for coupling ventilated racks to the animal room or the room ventilation system. (Courtesy of Phoenix Controls Corporation.)

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

923

Fig. 6. Wall-mountedshelf for holding the blower/filterunits for ventilated racks.

rack to be equipped with both supply and exhaust blowers that balance the rate of airflow to and from the rack and cages independent of the room ventilation system. Instead of the exhaust air being blown into the room, it is blown directly into the room exhaust via a capture hood or thimble connection in the ceiling (Figure 5, option 2). This strategy may be considered the easiest to set up and balance because the rack air balance is independent of the room air balance. Although this is the simplest method, it is the costliest of all four for equipment and operation because it requires two rack fans and uses additional room air for exhaust. (2) A second strategy is to hard-duct the exhaust from the rack directly to the building exhaust with an airtight connection, replacing the rack exhaust blower unit with a selfbalancing, pressure-independent constant airflow device to control the airflow and assure a constant flow of air from the room (Figure 5, option 3). The self-balancing device is required to assure a constant flow of air from the rack regardless of pressure fluctuations in the building exhaust such as may occur because of increasingly dirty filters, slipping fan belts, or disconnection of other racks from the system. It is important to have redundant building exhaust blowers on the duct serving the racks to assure uninterrupted rack exhaust. Constant-flow, pressureindependent, self-balancing Venturi valves have been used by research laboratories for a number of years to balance fume hoods but have been only recently introduced to the laboratory animal science industry. Reportedly, these nonelectronic devices are capable of maintaining constant airflow, independent of air pressure, indefinitely without requiring recalibration or routine maintenance. The advantages of this rack-to-buildingexhaust approach are reduced equipment cost, considerably lower noise levels, reduced maintenance costs, and better control of room pressurization. (3) A third strategy is to direct air from the rack exhaust fan via a hard-ducted airtight connection

into the facility exhaust. This strategy is not generally recommended, because it complicates balancing the exhaust system for the entire facility and has the potential to pressurize the exhaust system, resulting in backflow into other rooms. (4) A fourth strategy is to hard-duct the room and rack exhaust together with a sealed connection without a rack exhaust fan or self-balancing Venturi valve, relying solely on the room exhaust system to draw air from the rack. This is the least reliable strategy for maintaining room and rack air balance. The third and fourth strategies both require a balancing damper on each exhaust port in the room. In addition, when a rack is not attached to the exhaust duct, both the third and fourth strategies require inserting a device into the open exhaust duct that has an airflow resistance similar to that of a rack in order to maintain room and overall system air balance. With any of the strategies for exhausting air directly into the building exhaust, HEPA filtration of the air coming from the rack is generally not required, but some degree of filtration is highly recommended in order to keep bedding dust from littering the building exhaust system. Hard-ducting the facility air supply directly to the ventilated rack is generally not practical because of the cost required to provide dependable temperature control of air being supplied to the rack. The most practical approach is to employ a strategy that draws already conditioned air from the animal room into blower units that supply HEPA-filtered air to each cage. This typically involves a supply air blower/filter unit for each rack. Alternatively, a single blower/filter unit may supply filtered air to all the racks in the room. This requires a pressureindependent constant-airflow device in the supply duct to each rack to assure proper flow regardless of the rack's airflow resistance or the number of racks connected to the supply air source at a given time, once the system is balanced (Figure 5, option 4; and Figure 7). The advantages of this alternative are

924

J A C K R. H E S S L E R AND S T E V E N L. L E A R Y

Fig. 7. Three single-sided ventilated racks along one wall ventilated similarly to the system described in Figure 5, option 4, with air supplied from a common blower/HEPA filter unit located above the ceiling that delivers air to all the racks in the room. Note the air supply and exhaust hookups at the ceiling for each rack. (Courtesy of Dr. Robert Faith, Center for Comparative Medicine, Baylor College of Medicine.)

reduced noise in the holding rooms and fewer blower/filter units to maintain. Ideally, a backup supply blower/filter unit should be installed to automatically provide air to the racks in the event that the primary unit fails. At a minimum, a backup unit should be readily available for immediate replacement. e.

Air Balancing

Another critical function of the ventilation system is the control of airborne contaminants by the maintenance of appropriate relative air pressures throughout the facility (Hessler, 1991a; Hessler et al., 1999a). This involves balancing supply and exhaust to maintain predetermined relative air pressures between adjoining spaces, typically between the room and the corridor. Table I summarizes various options, depending on facility type and circulation pattern. Proper balancing is highly dependent on two things: proper sealing of the room envelope, and installation of mechanical equipment designed to maintain the appropriate volumetric offset between supply and exhaust air to achieve adequate differential pressures, typically between .08 and 0.2 cm (0.03 and 0.075 inches) of water. Proper air balance is important in controlling contaminants, but it does have limitations. Most significant, the relative air pressure may become zero in the spaces on either side of an opened door, allowing airborne contaminants to move freely between the spaces (Keene and Sansone, 1984). Relative air pressures in animal rooms of a single-corridor fa-

Table I Relative Air Pressure between the Corridor and the Animal Rooms a Dual corridor c Managed as

Single corridor b

Clean

Soiled

Conventional facility Barrier facility Containment facility

+ or + or -

+ + +

+ or -

a+, corridor positive to animal room; - , corridor negative to animal room. b 4- o r -- S i n g l e - c o r r i d o r c o n v e n t i o n a l : In a conventional facility, the air

pressure in the corridor is generally maintained positive to the animal rooms. The exceptions are facilities with mixed conventional and clean (relative to animal health status) rooms, where the air pressure in the clean rooms is maintained positive to the corridor and in the conventional rooms is maintained negative to the corridor. The air pressure in rooms being managed to contain hazardous agents must be negative to the corridor. 4- o r - S i n g l e - c o r r i d o r barrier: Both options are used. Following is a rationale for each. C o r r i d o r n e g a t i v e to a n i m a l r o o m s - - T o keep airborne contaminants out of the room. C o r r i d o r p o s i t i v e to a n i m a l r o o m s - - ( 1 ) Infectious agents of concern are not ordinarily present in a barrier facility, so the rationale of keeping airborne contaminants out of the room does not apply as it does in a mixed facility. (2) A "break" will occur in an animal room at some time, and the management objective is to contain the infectious agent, as in a biocontainment facility, until it can be detected and eliminated from the room and the facility. Keeping air pressure in the corridor positive to the animal room has the added benefit of reducing animal allergens and odors in the corridors and throughout all areas of the facility. c 4- o r - D o u b l e - c o r r i d o r c o n t a i n m e n t : Both options are used, with negative being more common, but positive may be preferred in some situations.

21.

cility are dependent on how the facility is to be managed: conventional, containment, or barrier. In a single-corridor conventional facility, the animal room air pressure may be balanced positively or negatively to the corridor. Typically, rooms are balanced negative to the corridor to reduce animal allergens and odors in the corridor, thereby creating a more pleasant work environment and reducing exposure of personnel to animal allergens. In a conventional facility housing both "clean" and "dirty" animals, rooms with "clean" animals may be managed as a barrier, in which case those rooms would be balanced positive to the corridor. For this reason, the ability to automatically reverse room air pressure relative to the corridor without having to rebalance the entire system is a highly desirable feature in a single-corridor conventional animal facility. In a single-corridor containment facility, where the objective is to contain airborne contaminants, the relative air pressure in the animal rooms will be balanced negative to the corridor. The opposite does not necessarily hold for a single-corridor barrier facility, where the choice depends more on management philosophy. One philosophy calls for balancing animal rooms positive to the corridor in an effort to keep airborne contaminants out; the other calls for balancing animal rooms negative to the corridor on the assumption that if a break were to occur, the infectious agent would be contained in the room in which it occurred until detected and eliminated. Both management philosophies have merit, and neither is clearly right or wrong. However, one advantage to the latter is that it maintains corridors relatively free of animal allergens, which are well documented as a serious and common occupational hazard (ILAR, 1997; ReebWhitaker and Harrison, 1999). In dual-corridor facilities, regardless of facility type, relative air pressures are typically balanced with the clean corridor positive to animal rooms and animal rooms positive to the soiled corridor; however, in some instances both corridors may be positive to the animal rooms.

f

925

DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

Temperature and Relative Humidity Control

Each animal room requires individual temperature control to adjust for the wide variability in heat loads due to species differences and/or animal density. The standard design temperature range for animal rooms is 18~176 (65~176 A narrower range may be acceptable for facilities designed for a single purpose, e.g., rodent production. Room temperatures as low as 18~ (65~ are desirable for some commonly used species, e.g., rabbits, but occasions for room temperatures over 26.6~ (80~ are rare and usually involve the maintenance of relatively exotic species (Weihe, 1965). Most critical is the ability to maintain a steady-state temperature in the animal room. The temperature control system should be capable of maintaining temperature +__ 1~ (+__ 2~ around any set point selected from the designed temperature range (ILAR, 1996). Control of relative humidity (RH) is of equal importance to

temperature control, but the degree of acceptable variability is much wider. The generally accepted range for RH control is between 30% and 70%. Usually, RH can be controlled within this range without individual room controls. Zonal control may be desirable in some situations: rooms likely to house only animals using dry bedding/litter systems could be zoned separately from those likely to house animals using large amounts of water for daily sanitation, e.g., dog and nonhuman primate rooms. As noted previously, ammonia production in rodent cages is directly related to the room RH (Hasenau et al., 1993; Memarzadeh, 1998). From this point of view, maintaining RH in the range of 3 0 - 5 0 % is highly desirable. Because low humidity can cause dehydration of young animals, especially newborn rodents, it is important to avoid RH below 30%. In cold climates, this requires extensive humidification, and even in many warmer, moist climates, some degree of humidification is required in cool weather. Clean steam should be considered for humidification in order to avoid the potential confounding effects of chemical additives routinely used in steam boilers. Boiler additives are generally considered safe, without any known health effects at the levels present in air humidified with boiler steam. However, the degree to which the chemicals may or may not alter the research animal's biological response to an experimental variable is impractical to document for the wide array of animal models that may be used in the facility. In addition, the seasonal variation in the level of chemical additives in the air, being present in relatively large quantities in the winter and absent in the summer, is in itself an unnecessary environmental variable. For these reasons, chemically treated boiler steam is best avoided.

g.

Redundancy

Redundancy is essential to maintain the research animal environment without significant interruptions for repairs or routine maintenance of the HVAC system. This requires designing redundancy into critical HVAC system components such as air handlers, pumps, chillers, heat sources, and building automation systems. Among the many system options are parallel or N + 1 air handling systems; dual chillers; boilers and pumps installed as parallel or N + 1 systems; cross connections with other, lower-priority sources to access available chilled water or steam; and spare parts available for quick replacement. Central energy plant utilization is another option; however, if the central plant does not have redundancy and emergency power, dedicated chillers and boilers must be available to back up the animal facility.

h.

Energy Conservation

There are numerous systems designed to recover energy from exhaust air, including runaround coils, lithium chloride entropy wheels, and direct heat-transfer loop systems (Gorton, 1975).

JACK R. HESSLERAND STEVEN L. LEARY

926

Choosing a system that avoids contaminating incoming air with outgoing air should be a prime consideration. Another method of conserving energy is recirculation of animal facility air. This requires cleansing recycled air of gaseous and particulate contaminants by using combinations of HEPA filters and absorbents such as alumina pellets impregnated with potassium permanganate or scrubbers (Jeszenka et al., 1981). Such systems have not been widely used, because of intensive maintenance requirements and the potential for cross contamination in the event of a malfunction; however, newer technology may enhance their desirability. Another energy-saving candidate may be variable-volume ventilation control systems. These provide only the amount of conditioned air required to maintain preset environmental conditions. This is useful not only for animal rooms but also for areas of the facility where ventilation requirements may vary, e.g., the cage sanitation area.

2.

Power and Lighting

a.

P o w e r and E m e r g e n c y P o w e r

Duplex receptacles with waterproof covers are required to provide 110-volt power for equipment commonly used in animal rooms, e.g., ventilated racks, data processing equipment, scales, HEPA-filtered mass air displacement cabinets, and sanitation equipment. A generic distribution of outlets in a smallto medium-sized room might call for one in the center of each wall mounted 1.2 m (4 ft) off the floor. Larger rooms should have two or more outlets along the longest walls. If the room is being planned for a specific use--e.g., housing of rodents in ventilated racks--the location of the outlets can be planned for the specific location of racks and cage changing stations. Ground fault interrupters (GFIs) should be used for every circuit in areas of the facility where water will be routinely used, which is most of the facility. Emergency power to maintain all essential services should be available in the event of a main power failure. At a minimum, emergency power should include HVAC at 100% capacity; any animal housing equipment that relies on power to maintain airflow; all environmental control and monitoring systems; at least one light fixture per animal room and other life-safety lighting as required by code; the security system; the surgery room; and freezers. Ideally, the emergency generator is sized and set up to provide uninterrupted power automatically for the entire animal facility. Considering the expense of separate wiring systems to selected equipment and locations, this may also prove to be cost-effective. b.

Lighting

The standard source of light in animal rooms is lighting from recessed or surface-mounted fluorescent ceiling fixtures with watertight, gasketed lenses. Fixtures should be arranged to pro-

vide uniform lighting throughout the room. Computer modeling should be considered to predict light distribution and intensity in relationship to equipment and especially in front of cages. Photoperiods influence many of the animal's biological functions (Hause et al., 1967; Haus and Halberg, 1970; Haus et al., 1970; Bellhorn, 1980; Tucker et al., 1984; Brainard et al., 1986; Cherry, 1987). Therefore, maintaining constant photoperiods is a critical component of controlling the research animal's environment. Because windows preclude maintaining constant light levels and photoperiods, windows are considered undesirable. One exception may be nonhuman primate rooms in which windows could be considered a form of environmental enrichment, providing that the lack of year-round consistency will not interfere with studies conducted in the rooms. A typical light cycle is 12 hr on and 12 hr off. A central microprocessor system is generally preferred over timer controls located at each room, where they can more easily be tampered with and/or damaged, but each room requires separate light control because certain studies may require alternative cycles, including reverse cycles. Although lights should not be turned on during the dark cycle, some routine tasks may need to be performed during this time. In these instances, dark room safety lights would be useful. They are considered to have minimal affect on the animal's circadian rhythm and can be controlled with a maximum 1 hr timer switch located immediately outside the room. The proper spectrum for animal room lighting remains an open question. Current general practice uses broad-band fluorescent white light (cool white) tubes. Full-spectrum lighting for research animals has been suggested (Mulder, 1971; Burns et al., 1976; Saltratelli and Coppola, 1979; Brainard et al., 1986) but is not yet widely accepted as necessary or costeffective. Light-emitting diodes (LEDs) have been suggested as an alternative source of animal room light. LEDs induce a normal circadian pattern with regard to melatonin production and do not cause retinal damage in albino rats (Heeke et al., 1999). Light intensity has received a great deal of attention since it was shown that high light levels caused retinal damage to albino animals (Noell et al., 1966; Kuwabara and Gorn, 1968; O'Steen and Anderson, 1971; O'Steen et al., 1972; Robinson and Kuwabara, 1976; Kuwabara and Funahashi, 1976; O'Steen and Donnelly, 1982; Robinson et al., 1982; Semple-Rowland and Dawson, 1987; Darren et al., 1999). In recognition of the phototoxic effect of light on the retina of albino animals, the "Guide" (ILAR, 1996) recommends that rooms housing albino animals be approximately 325 lux (30 foot-candles) at 1.0 m (3.3 ft) above the floor. This level appears to be sufficient for animal care, does not cause clinical signs of phototoxic retinopathology in albino rats after 90 days (Bellhorn, 1980), and causes only "minimal retinal lesions" after 790 days (Weisse et al., 1974). Out of concern for phototoxicity, two-staged high-intensity/ low-intensity (800/325 lux) lighting has become a common feature of animal rooms (Balk, 1980), especially for rooms hous-

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES ing albino rodents. However, this may not be cost-effective, because, as noted previously, 325 lux (30 foot-candles) appears to be adequate for routine animal care (Bellhorn, 1980). Detailed work with individual rodents can be performed in a lighted animal transfer/procedure cabinet. There is also concern that even relatively brief periods of increased light levels can cause retinal damage. If dual lighting levels are provided for rodent rooms, the low level should be the default level and the high level should be controlled with a timer switch located near the door, either inside or immediately outside the animal room. This should allow for a variable time period of up to 1 hr, after which the lights should automatically change back to the low level. The high-level timer switch should be wired to render it inoperable during the programmed dark cycle. One useful application of a dual lighting system is the ability to vary the light level according to the species housed in the room. In this instance the light levels would be centrally controlled and would not require a timer switch located at the room. It should be noted that phototoxicity is usually limited to albino animals. Permanent retinal damage does not appear to occur in animals with normally pigmented eyes at typical indoor lighting levels. Therefore, it would be acceptable and even desirable to provide light levels of 800-1100 lux (75-100 foot-candles) for animal rooms designed to house only dogs, nonhuman primates, or other animals that normally have pigmented eyes. 3.

Plumbing and Drainage

Floor drains in rodent or rabbit rooms are neither essential nor recommended. In these rooms, adequate sanitation can be maintained with wet mops and/or wet vacuums. However, in the pursuit of program flexibility, drains are often provided in every animal room. Rarely does this prove cost-effective. Drains used only intermittently should be capped and sealed to prevent the trap from drying, allowing the escape of sewer gases into the room. Open drains used only periodically should be equipped with automatically controlled rim flushes or at least with wallmounted manual flush valves near the drain. Providing "in case of need" drains in animal rooms adds to the drain placement/ floor slope dilemma. The center of the room is probably the least desirable location for a drain. The "ideal" drain location depends on its intended use; however, if this is not known, a location in a corner of the room may prove to be a useful option. Too little floor slope makes the drain useless for most purposes, while a slope that provides adequate drainage may be problematic in a room housing rodents in mobile racks. Most floor drains should be at least 10.2 cm (4 inches) in diameter. In rooms where animal wastes may routinely be flushed down the drain, drains of 15.3 cm (6 inches) in diameter are preferred. (Floor slopes and drain locations in rooms where daily sanitation involves large volumes of water are discussed in Section II,B,2,h.) Housing for aquatic animals should have strate-

927 gically located floor drains; however, a room without floor drains can be used for housing aquatic animals as long as it has a sink with a drain. Areas using hoses for routine cleaning and sanitation require strategically placed hose bibs, and hose reels are a highly desirable option to increase efficiency. Hot- and cold-water mixing valves can be used to control water temperature. If multiple rooms will involve the routine use of hoses me.g., certain animal rooms and cage sanitation a r e a m i t is highly desirable to provide a separate temperature- and pressure-controlled recirculating system to supply water to these locations. This provides maximum safety with regard to water temperature and can be designed and regulated to deliver water at higher pressures (e.g., 100 psi) than the house system, which also increases efficiency. It is highly desirable to have a sink in every animal room or suite of animal rooms. A stainless steel, wall-mounted mop sink works well in the animal room or suite. It is useful for hand washing, is easy to clean, keeps the floor open, and is ideal for dumping the mop bucket. Hands-off controls are desirable. A cold-water hose bib-mounted on the wall under or near the mop sink at a height suitable for filling mop buckets is very useful. Mobile sinks in animal rooms are also an option. The method of providing drinking water for each species needs to be determined early in the planning process so that the appropriate plumbing requirements can be provided. Options for supplying water include water bottles, which will require a bottle filling station and equipment for cleaning bottles, and automatic watering, which involves many options that need to be carefully considered. Treatment of animal drinking water also needs to be decided. Treatment options could include none, acidification, chlorination, and/or reverse osmosis. Safety eyewash and shower stations are required in any place where caustic chemicals may be used. This includes both sides of the cage sanitation, near animal water bottle filling equipment, near the reverse osmosis water production unit, and in most laboratories, especially those containing chemical fume hoods. Plumbing should be provided for specialty gases such as medical oxygen and carbon dioxide, compressed air, and vacuum. Areas requiring such services may include laboratories, animal procedure rooms, surgery rooms, and necropsy areas. In a large facility, it is highly desirable to have carbon dioxide and oxygen piped in from a central location near the receiving dock. Detergents, acid, and neutralizing agents may be piped to cage sanitation areas from vats or barrels located at or near the receiving dock. 4.

Interior Surfaces

Interior surfaces must be durable and, above all, sanitizable (ILAR, 1996; Thibert, 1980). Although life-cycle costs should always be considered, this is an area where life-cycle costs

928

JACK R. HESSLER AND STEVEN L. LEARY

deserve very careful consideration. Less expensive interior surfaces could easily cost many times more than more expensive surfaces over the life of the facility. The room envelope, with the exception of the door, should be completely sealed. All junctions and penetrations, including utility boxes, should be airtight to facilitate air balancing and vermin control.

a.

Floors

The ideal floor is monolithic; is chemical- and stain-resistant; is slip-resistant even when wet, yet relatively smooth and easy to sanitize; does not require sealers or waxes to maintain an acceptable appearance; and must be capable of supporting equipment without gouging, cracking, or pitting. The most popular flooring materials today include a variety of seamless troweledor broadcast-applied polymer composites, ranging in thickness from V8to 1/4 inch, the most popular of which are various types of epoxy aggregate (Novak, 1982). Methyl methacrylates, polymer flooring materials that were introduced to the laboratory animal industry in the late 1980s, appear to be gaining in popularity. Many flooring materials work well in a rodent room, but few materials work consistently well in hard-use, high-moisture areas such as cage sanitation. Tile, especially ceramic tile with epoxy grout top dressing (Figures 10 and 11), has proven to be a relatively maintenance-free flooring choice in cage sanitation areas where seamless polymer floors frequently fail. Grouted tile floors are not recommended for corridors because of the excessive noise generated by cage racks rolling across the grouted joints. Floor coverings should have a V2-inch radial-coved base a minimum of 10 cm (4 inches) high to form a watertight seal at the floor-to-wall junction, which facilitates sanitation.

animal facility areas. However, the relatively recent availability of extremely durable mineral-fiber composite materials either directly on metal studs or in combination with fiberglassreinforced gypsum board on stud walls is a viable alternative in many areas of the facility, especially in rodent rooms. Such walls are especially useful in earthquake-prone locations where concrete block cannot be used and cement plaster may not be practical because of its propensity to crack easily. Such composite panels, laminated to concrete block walls or fiberglassreinforced gypsum on metal-stud walls, may be the wall surface of the future. Composite panels are often a viable solution for refurbishing high-maintenance walls that will not hold a coat of paint. Protective wall curbs or guardrails are essential in corridors but may also be cost-effective in animal rooms and other areas where wall damage from caging and other equipment is likely. Curbs should be approximately 6 inches high and sloped to the floor 4 - 6 inches out from the wall. The seamless flooring material should cover the curb and be joined to the wall to avoid a shelf that collects dirt. Guardrails should be sturdy, sanitizable, and constructed to avoid providing harborage for cockroaches and other pests. Extruded solid-aluminum rails fastened to the wall with 5.3-10.6 cm (1.5-3 inch) 1-beam standoffs have proved very useful in animal facilities (Figure 8). Guardrail height should be carefully matched to the equipment used in the facility. A double row of guardrails may be provided (Figure 8); however, if there is to be only one row, it may be advantageous to place it near the floor at a height where it can protect the wall from platform carts and similarly constructed equipment. This height generally works to protect walls from cage racks and other types of carts commonly used in an animal facility. c.

b.

Walls

Walls should be reasonably smooth and capable of withstanding scrubbing, cleaning, disinfecting agents, and impact from high-pressure water. Concrete blocks coated with block filler of sufficient quality and texture to eliminate pits and sealed with epoxy paint have been the most widely used wall material for many years. This material consistently performs well in most areas of the facility with the exception of high-moisture areas, where coatings tend to peel from the surface. To some extent, there has been a revival of the structural glazed facing blocks commonly used in the 1940s and 1950s with the addition of an epoxy or other moisture-impervious resin top-dressing over the conventional grout. This proves to be a maintenance-free wall that performs exceptionally well in high-moisture areas such as animal rooms sprayed daily with water and cage sanitation areas. Ceramic tile over a water-resistant foundation such as concrete block provides an alternative that is nearly as functional for use in these high-moisture areas (see Figures 10 and 11). Gypsum board on studs has rarely proved suitable for most

Ceilings

Like walls, ceilings should be resistant to cleaning and disinfecting agents and capable of withstanding impact from highpressure water. Gypsum board ceilings sealed with epoxy paint are adequate for relatively dry areas of the facility, including rodent rooms, but are generally inadequate for high-moisture areas like cage sanitation areas. A drop ceiling with lay-in panels is not generally recommended for animal housing rooms because the panels impede sanitation and vermin control. If drop ceilings are used, the panels and T bars should be waterresistant, and there should be a seal between the panel and the T bar. In recent years, moisture-resistant composite panels sealed with gaskets and clamps to suspended fiberglass T bars have proved to be a virtually maintenance-free choice for ceilings and particularly cost-effective for use in high-moisture areas such as cage sanitation and dog and nonhuman primate housing rooms. In all cases, the ceiling-to-wall junction should be sealed. The minimal recommended ceiling height is 2.7 m (9 ft) and may need to be higher in a rodent facility planning to stack rodent cages higher than 2.1-2.4 m (7-8 ft).

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

Fig. 8. Doublerow of guardrails to protect walls and doors. Note how a rail has been formed to protect the wall-mounted automatic watering equipment.

5.

Doors

For many years, the standard animal room door size has been 107 cm (42 inches) wide X 2.1 m (7 ft) high. This size is adequate for many types of facilities. However, nonhuman primate and rodent high-density caging may be require wider and/or higher openings. Doors measuring 122 cm (48 inches) wide X 2.4 m (8 ft) high frequently prove useful for animal rooms. If 8foot high doors are provided for animal rooms, it is important to make certain that all doors in the facility through which the higher cage racks will be transported are also at least 8 ft high. This includes all corridor doors, doors in and out of the cage sanitation area, the rack washer doors, and dock doors. Hollow metal door frames are most commonly used. Stainless steel or fiberglass-reinforced polyester frames perform much better for animal rooms but are more expensive. Hospital stops are preferred to facilitate cleaning. Jamb guards may be mounted on the corridor side. There must be no doorsill, because this seriously impedes the movement of cage racks through the door. Like door frames, hollow metal doors are commonly used, but stainless steel or fiberglass-reinforced polyester doors prove more durable. The latter may be more expensive initially but generally have a lower life-cycle cost. Doors should be sealed and flush, top and bottom, not recessed. Hollow metal doors require plastic or stainless steel kickplates on both sides and stainless steel or plastic edge guards on the strike side. Automatic drop bottoms should be surface-mounted on the animal room side of the door, leaving no gaps larger than V4 inch. A view

929

panel is highly desirable, if not essential, for security and personnel safety. Size and shape of the view panel are a matter of choice, but it should provide a clear view of the room from the corridor. Light control through the view panel may be desirable and can best be provided with carefully selected red laminated glass, e.g., V8-inch clear annealed glass with an inner layer of Opti-Color film No. 5557 (Monsanto Chemical). Other options include a variety of solid blackout view panel coverings attached either with magnets or hinges and latches, most of which are inconvenient and/or high-maintenance. Hospital, lever-type door openers are a good choice. Push/pull plates should be mounted on both sides of the door. Strike plates should have a cup design. If fire codes permit, it may be preferable to eliminate the latch. If access to the animal room is controlled via a security system, magnetic locks are generally found to require less maintenance than electric strikes. Assuming that doors swing into the room, a crash rail extending the width of the door should be mounted just below the door handle on the corridor side and should protrude away from the door enough to protect the door handle. A heavy-duty surface-mounted, positive, selfclosing door closer with variable delays and hold opens is essential. Hinges should be stainless steel, heavy-duty, standard, or continuous. Swing-clear hinges can be used to optimize door width. Door seals of various types may be required to control air movement around the door to facilitate balancing the ventilation system. Automatic sliding or hinged doors are highly desirable in high-traffic areas, such as cage sanitation and dock areas, involving the movement of cage racks and bulk supplies, and for any corridor doors that must remain closed when not in use. Depending on the situation, they may be opened with sensors or switches in the form of wall-plate switches or pull switches suspended from the ceiling. 6.

Vermin Control

Control of vermin and insects, especially wild or escaped rodents and cockroaches, without the use of organic insecticides and baits is a management challenge that can be greatly aided by careful planning and construction. Such chemicals present an unacceptable variable in the research animal's environment. Most are potent hepatic microsomal enzyme inducers that have the potential to change biological baselines and alter the animal's response to experimental studies (Fouts, 1970). The basic approach is to seal vermin out and eliminate hiding and nesting places within. It is essential to seal all cracks, joints, utility penetrations, lights, wall switches, communication, and power outlets. Animal rooms should have a minimal amount of builtins, consisting of little more than a paper towel dispenser, utility hangers, and possibly a sink. These should be sealed to the wall or mounted away from the wall to eliminate hiding places and allow cleaning between the wall and the mounted item. Animal rooms should not have casework. Casework for animal

JACK R. HESSLER AND STEVEN L. LEARY

930

procedure rooms and other laboratory spaces in the animal facility should be of an open design type to reduce hiding places and facilitate cleaning. Boxed-in casework should be avoided. The most important step in the control of cockroaches involves pretreating all hollow and dead spaces in the facility during construction, including inside concrete blocks and studded walls, with amorphous silica or boric acid at a rate of 5 gm/m 2. There should be zero tolerance for eating or drinking in animal areas during the construction phase. High-pressure sodium (not mercury vapor) lamps or dichrome yellow (not incandescent flood) lamps located at exterior doors or vents can reduce the influx of vermin and insects into the facility. Air curtains with a velocity of 490 m (1600 ft) per minute can help reduce the influx of flying insects at frequently used exterior entrances that may be open for extended periods of time, such as loading dock doors.

7.

Noise Control

The research animal's stress response to noise has the potential to influence experimental data (Rails, 1967; Peterson, 1980). Although some noise production is unavoidable in an animal facility, the most significant noise-producing areas can be identified and measures taken to reduce the exposure of animals and people to excessive noise. The primary noise producers are cage sanitation areas, canine housing rooms, and sometimes, depending on the species, nonhuman primate rooms. Design features such as strategically locating these areas and architectural measures that reduce sound transmission should be carefully considered. These features include double entry doors, soundproof walls, corridors and support areas being located around the noise-generating areas, and the noise-generating areas being located next to outside walls or mechanical spaces. Conventional acoustical materials impede sanitation and vermin control and should be avoided. However, sound-attenuating panels that can easily be removed, washed, and sanitized in mechanical cage washers are available and should be considered for use in especially noisy areas of the facility, e.g., dog rooms and the cage sanitation area. All in-room activities, including cage changing, must be conducted in a manner that generates as little noise as possible. The facility ventilation system is a common source of unnecessary noise. The air ducts and outlets must be sized to avoid excessive noise, and the rooms must be balanced to avoid excessive pressure drops across the room door that result in whistling. As noted in the HVAC section, other penetrations in the room envelope should be sealed airtight to facilitate proper air balancing as well as to avoid whistling. Vacuum equipment used to transport bedding generates a large amount of noise and should be either isolated or insulated or both to assure adequate sound attenuation. Sound insulation should extend to the transport tubing. Fire alarms selected for animal housing areas should disturb

the animals as little as possible. Most rodent species cannot hear frequencies below 1000 kHz, although guinea pigs are capable of hearing down to 200 kHz. Fire alarms that operate between 400 kHz and 500 kHz are available and should be selected for use in facilities housing rodents.

D.

Communications

Essential communications design features include telephone lines strategically located throughout the facility to include most rooms (but not animal rooms), computer network lines in most rooms (including animal rooms), and video cable lines in selected rooms (surgical and training rooms). All but the smallest animal facilities require an intercom system to function efficiently. Speakers should be liberally spread throughout the facility (including animal rooms) and two-way communication devices should be strategically located in corridors throughout the facility. Speaker wiring should be grouped according to relative room noise levels so that speaker volume can be adjusted for differences in background noise levels. For example, a relatively low volume level may be appropriate for most animal rooms and corridors, but cage sanitation areas and dog rooms may require a higher volume level. In addition to communication, an intercom system may provide music as white noise for animal rooms. Background white noise, e.g., music, is thought to reduce animal stress produced by routine noise-generating activities. Special consideration should be given to communication between barrier and biocontainment areas and other areas of the facility. Fax capability is another useful way of communicating with these areas, often being the best way to transfer hard copy information out of containment areas and into barrier areas.

E.

Security and Controlled Access

The great value of research animals, combined with increasingly militant activities opposing the use of animals in research and safety testing, requires that all research animal facilities have a sound security program that controls access to the facility at all perimeter ports. All routinely used access points must be equipped with microprocessor-controlled security access devices managed by the institution's security service. Given the importance of security for research animal facilities, security systems that use biometrics--e.g., thumb, palm, or retinal scan, voice recognition, and so o n m s h o u l d be considered. Controlled access within the animal facility provides additional security; however, its primary benefit is as a management tool. Once an individual is inside the facility, access to rooms should be limited. This typically requires controlling and monitoring access to each animal room and other selected areas, e.g., barrier and biohazard. This type of control is important to

931

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

protect personnel and animal health and to maintain the integrity of research. Because of the large number of individuals requiring access and the high personnel turnover, key locks are unmanageable in most research institutions; therefore, automated, microprocessor-controlled security systems are required to effectively manage access control. Some individuals, especially the animal care staff, will require frequent access to controlled rooms or areas. Careful consideration should thus be given to the selection of a convenient-to-use personal identification system. The internal access control system is best managed by animal facility personnel.

III.

Care of laboratory animals at the level required by contemporary research is an equipment-intensive and equipmentdependent business. Planning a research animal facility requires careful consideration and detailed knowledge of all major equipment to be used in the facility, both fixed and mobile.

A. 1.

F.

Environmental Monitoring

Mechanical and electronic equipment can and does fail. When these failures occur, the health and well-being of the animals and the integrity of the research can be seriously compromised. All contemporary research animal facilities must have an automated electronic monitoring and recording system to document that all environmental systems are performing as designed (Small, 1982). There are two approaches to electronic environmental monitoring: one uses data directly from the environmental control system, and the other uses a completely independent system that includes redundant sensors. Obviously, the independent system affords the greatest level of safety, because it provides the opportunity to correct a potentially serious problem that could otherwise be missed should the control and monitoring systems disagree. In terms of physical environment, the primary parameters to be monitored in the animal room include temperature, relative humidity, airflow (not as critical but useful), relative air pressure, and light. The objective is to continuously monitor these parameters and give notice when they are outside preset ranges. An effective system allows for two alarm levels: a red alarm that requires immediate attention, and a yellow alert that requires attention at the next regularly scheduled workday. A yellow alert allows failing systems to be repaired during normal working hours before they become a red alarm. For example, a temperature greater than 1~ (2~ but less than 3~ (6~ over or under the set point may be considered a yellow alert, whereas one that exceeds 3~ (6~ over or under the set point may be considered a red alarm. The system must have the ability to immediately notify appropriate individuals and/or stations when a red alarm occurs and to give reports listing all the red alarms and yellow alerts by parameter, value, location, and time. In addition, the system should be able to prepare reports, including daily summaries documenting environmental conditions in each animal room. Such reports are required for Good Laboratory Practice (GLP) studies but are good management tools for all animal studies. Sometimes the environmental monitoring system is also used to monitor the automatic watering system and to control animal room lights and animal facility security.

EQUIPMENT

Cage Sanitation and Sterilization

Cage Washers

Adequate control of the research animal's microbial environment is dependent to a large degree on having the proper sanitation and sterilization equipment. Hand washing of cages and reliance on chemical disinfectants to achieve sanitation are an option but are not recommended, even for small facilities (Thibert, 1980; Vesell et al., 1973, 1976). Not only is this option labor-intensive, but it is also only marginally effective, increases the potential for leaving unacceptable chemical residues on the cages, and exposes personnel to chemical hazards. Sanitation, which involves destroying vegetative pathogenic organisms, is better achieved by using mechanical washers and water at temperatures in excess of 82.2~ (180~ (ILAR, 1996). The 82.2~ (180~ standard was apparently first published by the National Sanitation Foundation, which set standards for commercial dishwashers (National Science Foundation, 1953). In fact, sanitation can be achieved at temperatures lower than 82.2~ by using longer exposure times, e.g., 1 second at 82.2~ (180~ 15 sec at 71.6~ (161~ and 30 min at 61.7~ (143~ (Wardfip et al., 1994). The control systems on contemporary washers monitor water temperature and will not allow the machine to start or complete the cycle unless the temperature reaches 82.2~ or greater. Sometimes washers are set up such that only the final rinse temperature is 82.2~ or greater. Two basic types of cage washers are commonly used: batch washers and tunnel washers, also called belt or conveyor washers. Ruys (199 l a) provides a detailed description of each type, along with utility requirements. In a batch washer, soiled equipment is placed in a chamber and sprayed with high pressure on all sides with large volumes of hot water through a series of selected detergent/rinse cycles, after which it is removed, cleaned, and sanitized. Batch washers come in a variety of chamber sizes. Cabinet washers are the smallest, with a typical chamber size of 122 cm (48 inches) wide X 79 cm (31 inches) high X 86 cm (34 inches) long (Figure 9). Typically, the bottom of the wash chamber is several feet off the floor. Cabinet washers are suitable for cleaning relatively small numbers of cages and water bottles that can be lifted into the chamber. Cage and rack washers are typically pit-mounted so that the floor of the washing chamber is level with the room floor and cage racks may be easily rolled into the chamber (Figure 10). Chamber

JACK R. HESSLER AND STEVEN L. LEARY

932

Fig. 9.

Cabinet-typecage/bottle washer. (Courtesy of Getinge/Castle,Inc.)

sizes vary from those that hold only a single cage rack to those that hold up to four cage racks at a time. As indicated in Section III,A, double-door pass-through washers are preferred to allow for separation of soiled and clean cages. Water may be fresh for each cycle, except for the final rinse water, which is

saved and used as the prerinse water for the next load. An option for storing water between loads is to have side tanks. For example, one side tank can store alkaline detergent wash water; the other can store acid-treated wash water. Originally, electromechanical timers were used to control cycles, but most modern batch washers are controlled digitally with microprocessors that offer greater flexibility by allowing for a variety of preprogrammed wash and rinse cycles, depending on the type of equipment being cleaned and sanitized. Cycle options include prerinse, acid detergent wash followed by a rinse, alkaline detergent wash followed by a rinse, and final rinse. Depending on the cycle chosen and its length, each load can take between 20 and 30 min, including loading and unloading time. The second commonly used cage washer is the conveyor, or tunnel, washer (Figure 14 illustrates the load end of a tunnel washer). Items to be cleaned and sanitized are placed on a conveyor that moves through a tunnel divided into sections, e.g., prerinse, detergent wash, rinse, and final rinse. Water is usually recirculated: the recirculating rinse water is used for prerinse, which is discarded, and the final rinse water flows into the recirculating rinse water to freshen it. Often a dryer and bedding dispenser section are added to the tunnel washer. Tunnel washers come in a variety of sizes; however, a typical tunnel washer may have a conveyor about waist high, a tunnel opening of 7 6 107 cm ( 3 0 - 4 2 inches) wide • 91 cm (36 inches) high • about 4.6 m (15 ft) long. In addition, it is common to have extensions on the load and unload ends. These could add 46 cm (18 inches) or more on both ends, and a dryer and bedding dispenser could each add another 1.8-2.4 m ( 6 - 8 ft). It is also useful to have a 3 m (10 ft) or longer roller conveyor to collect cages at the end of the conveyor belt. The width and speed of the conveyor de-

Fig. 10. Two cage and rack washers with an enclosed mechanical service space between the washers and a sanitation chemical storage room to the right of the washers. Features include stainless steel doors and frames into the service area and the chemical storage room, ceramic tile floor and walls with epoxy grout, exhaust hoods above the doors to each washer, and the trough drain in front of the cages and the chemical storage room. The floor in the entire room slopes toward the trough drain.

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES termine the washing capacity. The longer the washing tunnel, the faster the conveyor belt can run and with the same amount of exposure time in each section. This type of washer is especially useful for sanitizing shoe-box rodent cages, cage pans, water bottles, and other small equipment. A third, less commonly used type of cage washer is an indexing washer, which is separated into various wash and rinse sections in which the conveyor stops to expose the load for a period of time in each section. Index washers may be either cage and rack washers or tunnel washers. Tunnel washers used in combination with robots for loading and unloading the conveyor are programmed to index-wash. In the case of indexing rack washers, the racks are typically pulled through the washer by a cable attached to the rack. Indexing washers have not been used extensively in the United States.

2. Bedding Disposal Moving soiled bedding from the cage to a final disposal location is a major logistical and labor-intensive challenge. The most common method is to dump the bedding from the cage to a container, move the container to a larger container outside the animal facility, and move it from there to its final disposal location, usually a landfill or an incinerator. The only significant improvement in this basic method has been the use of special bedding disposal cabinets (Rake, 1979). These cabinets use mass air movement to draw the soiled bedding dust away from the operator who is dumping the bedding inside the cabinet, directing it first through a gross filter and then thorough a HEPA filter before discharging it back into the room (Figure 4). This

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provides a safer work environment for personnel and is especially useful, even essential, for dumping bedding cages containing known carcinogens or toxic chemicals. Many different strategies have been tried to reduce the intensity of labor required to perform the essential task of removing soiled bedding from the facility. Some of these are in use, but none has come to dominate. If local codes allow, disposal units that dump the bedding directly into the sanitary sewer system are probably most efficient (Figure 11). Both grinder and hammer types with a hopper and an auger have been used successfully. Drain line blockage can be avoided by properly sizing the drains and by directing water from the cage and rack washers by a short run of drain coming from the disposal unit. Another commonly used disposal strategy includes various types of vacuum systems that deliver the soiled bedding, either directly from the cage or from a hopper, to a receptacle from where it is transported by an auger into an incinerator or dumped directly into a bulk disposal receptacle outside the facility. Another less commonly used strategy, first introduced into research animal facilities in the late 1980s, involves transporting the bedding from a hopper in the cage sanitation area to a bulk disposal receptacle outside the building in a slurry of water. The slurry system separates the water from the bedding at the disposal end and recirculates the water to transport more bedding.

3. Bedding Dispensers Many different types of mechanical bedding dispensing strategies have been tried, typically ones that fill one or two shoebox rodent cages at a time, but have not been widely accepted,

Fig. 11. Soiledside of a cage sanitation facility with a bedding dump/disposal station that dumps bedding directly to the sanitary sewer system. To the left is the load end of a tunnel washer. To the near side of the disposal unit is a wall-mounted stainless steel sink typical of a type often recommendedfor use in animal rooms. The wall and floor finish is ceramic tile with epoxy grout.

JACK R. HESSLER AND STEVEN L. LEARY

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Fig. 12. Beddingdispensing unit attached to the end of a tunnel washer. The dispenser automatically turns over rodent cages and dispenses the appropriate amount of bedding into the cages. Attached to the dispenser is a wall-mounted vacuum systemthat collects bedding dust generated by the dispenser.

perhaps because they are not significantly faster or more convenient than the old reliable handheld-scoop method. For facilities with a large number of bedded rodent cages, bedding dispensers attached to the tunnel washer have been widely used (Figure 12). Such dispensers have a conveyor in line with, but at a lower level than, the tunnel washer conveyor so that cages fall onto the dispenser conveyor. As they fall, they are flipped over and then filled with bedding as the conveyor takes them through the dispenser. Even with the most dust-free bedding, this type of dispenser generates a significant amount of dust, much of which can be collected with a vacuum attached to the dispenser. As noted in Section II,B,l,g, if this type of dispenser is to be used, it is highly recommended that the clean side of the cage sanitation area, where the dispenser will be located, be separated from the clean cage storage area to contain the dust to an area as small and as easily cleaned as possible.

4.

Robotic Cage Washing

The use of robots is the latest innovation in washing rodent shoe-box cages. The enormous increase in mouse cage census at some institutions has stimulated interest in robotic cage washing (Figure 13). This involves two robots, one on each end of an indexing tunnel washer equipped with a dryer and an automatic bedding disposal system (Klein et al., 1999). A robot on the soiled side latches onto cages from a stack loaded on a hand truck, dumps the soiled bedding into a disposal hopper, and then loads the cages onto the tunnel washer conveyor. On the clean side, another robot moves the clean cages with bedding off the

bedding dispenser conveyor and stacks them on a transport hand truck. Robots that are currently available handle either one or four cages at a time. Ideally, the bedding dispenser is filled from a bulk storage hopper. The initial cost for robots is high, and the cost-effectiveness depends on the number of cages to be processed. The economic feasibility point will vary according to local issues, especially labor costs, but a minimum of 3 6 0 0 - 4 0 0 0 cages per day may be considered a ballpark number at this time.

5.

Sterilization Equipment

The primary method of sterilization, which involves destroying all microbial life, involves using steam under pressure in autoclaves. Autoclaves are routinely used in research animal facilities to sterilize cages and supplies required for housing rodents in germfree isolators and/or under barrier conditions, equipment and supplies contaminated with biohazardous agents, and surgical instruments and supplies. Equipment and supplies in the autoclave chamber are exposed to temperatures in the range of 121~176 (250~176 In rare situations involving very small research animal facilities, it may be possible to get by with a single autoclave, but typically more than one will be required. In large facilities, a separate autoclave may be required for each of these functions. Barrier and biocontainment facilities often require bulk autoclaves that hold from one to four racks of cages (Figure 14). High-vacuum autoclaves are highly recommended because they significantly improve both sterilization effectiveness and efficiency as compared with grav-

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

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Fig. 13. Robot used for loading and unloading shoe-box cages from an indexing tunnel washer with a dryer and in-line bedding dispenser. This robot handles four cages at a time. It removes the cages from a stack of soiled cages on a cart, dumps the soiled bedding into a bedding dispensing unit, and places the cages on the washer belt. At the other end of the tunnel washer a second robot unloads the clean and bedded cages from the belt and stacks them on a cart. (Courtesy of Getinge/Castle, Inc.)

ity autoclaves. Most m o d e r n autoclaves have digital control systems. Bulk autoclaves usually include hinged or sliding doors, the choice of which may depend on the physical layout of the area where the autoclave is to be installed. To avoid exposing animals to chemical additives typically found in boilergenerated steam, clean steam should be considered for autoclaves used to sterilize animal cages, feed, and bedding. Ethylene oxide sterilization is rarely required for animal husbandry support but is frequently required for supporting experimental surgery programs. Ethylene oxide is a carcinogen; therefore, detailed safety requirements governing the installation of ethylene oxide sterilization equipment have been established and must be followed. Newer technology, still in the formative stages, involves the use of hydrogen peroxide or gaseous chlorine dioxide in sealed chambers for sterilization of cages and other hard-surface items.

B.

Fig. 14. High-vacuum double-door pass-through bulk autoclave with a chamber that is 107 cm wide and 213 cm high and deep (42 inches wide, 84 inches high and deep), typical of autoclaves often used in rodent barrier facilities and infections containment facilities. Note the air exhaust hood located above the autoclave opening. This is especially important at the discharge side of the autoclave.

Animal Watering

The animal watering strategy to be used is another of the many important decisions to be made in the process of planning an animal facility and will greatly affect the design and equipment requirements. There are multiple strategies for efficiently delivering clean water to laboratory animals and for maintaining the ever-increasing standards for the quality of water provided

JACK R. HESSLER AND STEVEN L. LEARY

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to laboratory animals (Lempken, 1991; Novak, 1999). Water bottles and bowls continue to be a common means of providing water to research animals, but automatic watering systems are also becoming very common. Depending on the species housed, a facility will often use a combination of bowls, bottles, and automatic watering. 1.

Water Bottles: Sanitizing, Handling, and Filling

When designing facilities that will house large numbers of rodents, it is critical to carefully plan the logistics and equipment for handling, sanitizing, and filling water bottles. For example, a TG/KO mouse facility designed for 10,000 cages will need to process that many 473 cm 3 (16 oz) water bottles per week. Of course, the cages must accommodate the larger bottle. Watering bottles are usually glass or polycarbonate plastic with neoprene corks and stainless steel sipper tubes. Some bottles deliver water through a hole in the side of the bottle in place of a sipper tube. Equipment to facilitate the use of water bottles includes stainless steel cases for holding water bottles during cleaning, filling, and transport; a stainless steel cart to transport water bottle cases; cleaning/sanitizing equipment, which may be a cabinet or tunnel washer for bottles and an ultrasonic cleaner for sipper tubes; and bottle-filling equipment, which usually fills one case of bottles at a time. Often included in this equipment list is an autoclave to sterilize sipper tubes. Many facilities also autoclave the bottle and water for rodents housed under barrier conditions. Water bottles are usually stored and filled in the clean cage storage and setup area. In facilities that process a large number of bottles, it may be useful to provide a separate room for storing and filling them. 2.

Automatic Watering

Automatic watering systems are widely, if not almost exclusively, used for supplying water to dogs, sheep, goats, pigs, nonhuman primates, rabbits, and rats and are available with an array of stainless steel valves specifically designed for each individual species. Automatic watering for mice, while extensively used, is not as universally accepted, because of concern for leaking valves that may flood the cage. When valves were located outside the cage, this was less of a concern. However, valves are usually placed inside microisolation caging, increasing the chance for cage flooding. To maintain the integrity of the microisolation cages and to facilitate sterilization of the watering valves, special in-cage valves have been developed that are attached to the cage and couple to the rack manifold via a quick disconnect. For the most part, the newer in-cage valves address the cage-flooding problem satisfactorily, but occasionally flooding occurs. Of course, even water bottles may completely empty into a cage, but in that case the supply of water is at least limited. The primary motive for using automatic watering is the reduction of animal care costs, because handling water bottles is

significantly more labor-intensive and thus more costly than automatic watering. The cost savings of automatic watering must be weighed against the problems inherent in using automatic watering. The most basic equipment involved with an automatic watering system includes, starting from the source of water, a distribution system to pressure-reducing stations strategically located throughout the facility where water, typically at 0.21 kg/cm 2 (3 psi), is delivered to a selected number of rooms. Within the rooms, flexible recoil hoses with quick connects on both ends attach the distribution system to the rack manifold to which the watering valves are attached. From this basic plan, systems become more complex as design options are added to maintain the quality of water delivered to animals. In the early years of automatic watering use, scant attention was paid to sanitization of the distribution system or the rack manifold, other than sanitizing the rack and manifold in a rack washer at 82~ (180~ As it became apparent that microbial contamination was a significant problem, strategies for sanitizing the system were developed. These included avoiding dead ends in the distribution system and using control systems to periodically flush distribution lines with freshwater. Some systems will periodically flush the rack manifold even during use. In others, the central processing unit that regulates the automatic flush of the distribution system will also monitor the system for leaks. A variety of sanitation equipment strategies have been developed for flushing distribution lines, rack manifolds, and recoil connector hoses with chlorinated water and for equipping rack washers to flush rack manifolds with 82~ (180~ When water conservation is an objective, microbial growth can be controlled by recirculating system water through ultraviolet (UV) lights. The UV light controls bacterial growth, and stagnant pools of water are eliminated. Unfortunately, none of these sanitation strategies proved to be as effective as was originally thought, because the biofilms that line the inside of water pipes are resistant to chemical sanitation (Costerton and Lappin-Scott, 1989; Anderson et al., 1990; Edstrom Industries, 1999). Not surprisingly, autoclaving the manifold on the rack proves to be the most reliable sanitation method (Costello et al., 1998). This serves only to emphasize the usefulness of stainless steel rack manifolds and bulk autoclaves as standard equipment in an animal facility. 3.

Water Quality and Treatment

Clearly, drinking water quality is an important part of the animal's environment and must be controlled (Newell, 1980). The unaltered potable municipal water supply is the most common used water source. Since the late 1950s, however, there has been a growing trend toward water treatment for microbial control and chemical contaminant reduction. In the late 1950s and early 1960s it was reported that mice irradiated with normally sublethal doses were dying, and some that received lethal doses

21. DESIGN AND MANAGEMENTOF ANIMAL FACILITIES died prematurely from Pseudomonas aeruginosa bacteremia (Wensinck et al., 1957; Flynn, 1963). This was a major complication for the many irradiation studies being conducted at the time. Similar findings were reported in the 1970s in chemically immunosuppressed mice (Pierson et al., 1976; Brownstein, 1978). Beginning in the early 1960s, there were numerous reports indicating that hyperchlorination and/or acidification of the animal drinking water would effectively control bacterial contamination and greatly reduce the incidence of Pseudomonas aeruginosa bacteremia in immunosuppressed animals (Beck, 1963; McPherson, 1963; Trentin et al., 1966; Bywater and Kellett, 1977; Urano and Maejima, 1978; Newell, 1980; Homberger et al., 1993). Hyperchlorination was widely used in the past, but today the most common water treatment used for controlling microbial contaminants in animal drinking water delivered via water bottles is acidification to a pH of 2.5-3.0. Both methods have the potential to alter biological data (Fiddler, 1977; Hall et al., 1980; Herman et al., 1982). Generally, water is treated automatically by using titration equipment attached in-line with the bottling filling station. The next most common water treatment is purification. In the 1960s and 1970s, the quality and variability of municipal water supplies received a lot of attention, raising the issue of controlling this important aspect of the research animal's environment. The concern was twofold: first, contaminants at levels that may not pose a danger to public health could potentially influence the results of a long-term study, e.g., a 2-year carcinogen study in rodents; second, there was the need for standardized water quality. The only practical way to address this concern was to use purified water; thus, starting in the 1970s, some facilities started purifying drinking water for laboratory animals. Various water purification strategies have been used, but the one that has come to dominate is reverse osmosis. It is difficult to estimate what percentage of laboratory animals receive purified drinking water today, but it is substantial and increasing. Water quality for aquatic animals is another important consideration. Chlorine normally present in municipal water supplies is often incompatible with life for some aquatic species. Municipal water must be treated to remove all chlorine before being used for housing of aquatic species.

C.

Caging

1. Materials Consideration for animal comfort and adequate sanitation should be the primary factors in cage design and the selection of fabrication materials. Resistance to rust and harsh chemicals is a must, and longevity is highly desirable. Stainless steel is by far the most commonly used metal, although aluminum is occasionally used. The next most commonly used material for cages is polycar-

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bonate (Lexan, General Electric; Makrolon, Bayer). This material, used for molded plastic cages, was introduced in 1962. It is clear and durable, with high impact strength, and holds up well in 82.2~ (180~ wash water. It withstands autoclaving at 120~ (250~ but repeated autoclaving significantly shortens the useful life of the cage, causing the polycarbonate to become opaque and to craze, crack, and generally degenerate. For this reason, transparent amorphous thermoplastics are now being used. Recent examples of this material include polyphtalate carbonate, introduced in 1987 and commonly known as known as high-temperature polycarbonate (Apec, Bayer); polyetherimide, introduced in 1988 (Ultem, General Electric); polysulfone, introduced in 1994 (Udel, Amoco); and polyphenyl sulfone, introduced in 1995 (Radel, Amoco). Expanded metal, metal bars, and wire have long been used as flooring material for housing dogs, pigs, sheep, and goats. Starting in the late 1970s and early 1980s, polyvinyl chloridecoated expanded metal and molded plastic mesh began to replace bare steel floors. In January 1998 the U.S. Department of Agriculture amended Animal Welfare Act standards pertaining to wire flooring used in primary enclosures for dogs and cats. Plastic- or fiberglass-coated wire floors are now required if the wire is less than or equal to V8 inch in diameter (USDA-APHIS, 1998). The use of other materials for cage fabrication that combine the sanitation and durability characteristics of stainless steel with the warmth, comfort, and sound-dampening acoustical properties of wood are currently being introduced as an alternative to stainless steel. Examples include composites of highstrength, fiber-reinforced thermoset and thermoplastic polymer matrix materials, fiberglass-reinforced panels, polyvinyl chloride foamboard, and acrylics (G. A. Heidbrink and W. E. Britz, personal communication, 1999). Molded plastic cages with perforated bottoms have proved to be a very satisfactory cage for housing rabbits and guinea pigs (Figure 15). These newer materials combine with innovative design features to produce cages with thermoneutral sound-dampening surfaces and optimal exposure for ventilation and visualization. These cages show promise to enhance the animal's microenvironment and reduce stress for the animal, thus enhancing the quality of the research data. 2.

Caging Systems

A wide variety of fixed and mobile caging systems are used to house laboratory animals. The most common fixed cage type is the floor pen, in which animals are housed either directly on the floor or on a raised floor. With species-appropriate modifications, floor pens may be suitable for housing a variety of animals, including dogs, cats, nonhuman primates, sheep, goats, pigs, and adult chickens. Typical pen sizes might vary from 0.9 to 1.8 m (3 to 6 ft) wide and 1.8 to 3.6 m (6 to 12 ft) deep. In comparison to portable caging that can be sanitized in a cage

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Fig. 15.

JACK R. HESSLER AND STEVEN L. LEARY

Molded plastic rabbit cages with perforated floor (A). Close-up of the perforated floor (B). (Figure provided by Allentown Caging Systems Co., Inc.)

21. DESIGNAND MANAGEMENTOF ANIMALFACILITIES and rack washer, fixed caging can be difficult to sanitize. However, most pens are constructed such that they can be disassembled and removed. Fixed caging may also make the space relatively inflexible. These relative disadvantages can be eliminated by using movable pens with raised floors that can be easily removed from the room and sanitized in a cage and rack washer. Most laboratory animal caging systems consist of multiple cages suspended from or sitting on the shelves of a mobile rack. Rabbit, cat, dog, and nonhuman primate cages are sometimes constructed as an integrated nonremovable part of the rack. For planning purposes, a typical rack measures approximately 0.6 m (2 ft) deep, 1.5-1.8 m ( 5 - 6 ft) wide, and up to 2.3 m (7.5 ft) high. Exceptions are dog and nonhuman primate cages that may be up to 0.9 m (3ft) deep, with similar width and height. 3.

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tions to allow animals in adjacent compartments to be blocked from viewing each other or to view each other, or to touch each other while protecting fingers from being bitten. Such features are considered useful for introducing animals to each other and for judging their likely compatibility prior to housing them together. These cages are also useful for housing species that require more vertical space to accommodate their natural behavior. Another primate cage configuration designed to provide environmental enrichment has a common socialization compartment situated between double-stacked cages with common access to all four cages. Other strategies include tunnels for connecting individual cages. Environmental enrichment strategies for other species, including rabbits and rodents, are also being investigated and tested. Examples include group housing of rabbits on shavings on the floor (Gunn-Dore, 1997) and provision of nesting materials and hiding places in rodent cages.

Cage Size and Environmental Enrichment

Minimal cage size requirements are established by Animal Welfare Act (AWA) regulations (CFR, Title 9, Subchapter A, Parts 1-3) and the "Guide for the Care and Use of Laboratory Animals" (ILAR, 1996). A 1985 amendment to the AWA stipulates that dogs must be exercised unless they have continuous access to at least twice the minimal floor space required for the animal. For pair- or group-housed animals, the enclosure must provide at least 100% of the space required to house each dog individually. If the dogs cannot be pair- or group-housed and providing twice the minimal required space is not practical, the animals must be provided the opportunity to exercise on a routine basis as established by the attending veterinarian. Many strategies are used to meet this exercise requirement, including doors periodically opened between pairs of cages or pens to allow animals to share the combined space, thus giving the animals access to twice the minimum required housing area for a period of time; allowing the dogs to run loose in the dog room or putting a floor pen in the room and rotating the dogs through it; devoting entire rooms to dog exercise, sometimes in multiple pens or with a divider panel down the center of the room to separate males and females; and using a gated corridor to contain the dogs in specific areas of the corridor. Environmental enrichment for nonhuman primates, also required by the 1985 amendment to the AWA, spurred many cage innovations (ILAR, 1998). Examples include enrichment devices that could hang on the cage or were made as an integral part of the cage. Such devices commonly involved food because this is generally considered to be the best strategy for primate enrichment (Bayne et al., 1991). Another approach strongly supported by many is pair or group housing whenever possible. The "quad" cage provides for individual or group housing within the same unit (Figure 16). It has four individual cage compartments with movable or removable vertical partitions and removable horizontal partitions. It typically comes with op-

4.

Rodents

Both direct and indirect bedding cage systems have been used for housing rodents for many years. The direct bedding system is used with solid-bottom shoe-box cages; the indirect bedding system is used with wire-bottom cages suspended over a litter pan. Mice are most commonly housed in bedded shoe-box cages. Rats and guinea pigs are housed both in suspended wire-bottom cages and bedded shoe-box cages. An exception is the widespread use of wire-bottom cages for housing mice and rats being used on toxicology studies in the United States, especially for pharmaceutical development. The National Toxicology Program has used solid-bottom bedded cages for rodents since its inception in the early 1980s, and the National Center for Toxicology Research has done so since the early 1970s. Out of animal welfare concerns, there has been a gradual shift away from wire-bottom cages toward solid-bottom bedded cages for all rodents. This trend is supported by the 1996 "Guide" (ILAR, 1996), which states: "Rodents are often housed on wire flooring, which enhances sanitation of the cage by enabling urine and feces to pass through to a collection tray. However, some evidence suggests that solid-bottom caging, with bedding, is preferred by rodents (Fullerton and Gilliatt 1967; GroverJohnson and Spencer 1981: Ortman et al., 1983). Solid-bottom caging, with bedding, is therefore recommended for rodents." The cited references document damage to plantar nerves of the hindfeet of guinea pigs and rats housed on wire-bottom cages (Fullerton et al., 1967; Grover-Johnson and Spencer, 1981; Ortman et al., 1983). Other publications document the preference of rats (Blom et al., 1993a; Manser et al., 1995, 1996) and mice (Blom et al., 1993b) for bedded cages over wire-bottom cages, especially when resting (Manser et al., 1996; Blom et al., 1993b). The process of developing strategies for housing rodents free of adventitious pathogens has been one of the more important

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JACK R. HESSLER AND STEVEN L. LEARY

Fig. 16. Nonhumanprimate quad cage. This caging consists of four individual cage compartments with movable or removable vertical partitions and removable horizontal partitions to allow for pair or group housing and for accommodating species that require more vertical space than their size might otherwise require, in order to accommodatetheir natural behavior. Such cages typically come with options to allow animals in adjacent compartments to be blocked from viewing each other, or to view each other through a clear panel, or to touch each other through two wire mesh panels strategically spaced to allow touching while protecting fingers from being bitten. (Courtesy of Allentown Caging SystemsCo., Inc.)

and dynamic journeys in laboratory animal science and medicine. This process began in the late 1950s when Dr. Lisbeth Kraft published a paper (Kraft, 1958) that documented that housing mice in a filtered cage prevented the spread of epizootic diarrhea of infant mice. Certainly, this was a major milestone in laboratory animal science. Kraft's initial publication was followed by other publications that documented the effectiveness of the filtered cage for controlling the spread of disease in rodent colonies (Kraft et al., 1964; Schneider and Collins, 1966; Poiley, 1967; Flynn, 1968). Interestingly, the mainstay of efforts to maintain rodents free of adventitious pathogens in the research environment is still the filter-top cage, commonly referred to as a microisolation cage. There have been many renditions of cage covers since Kraft's original filtered cage, but the industry has settled primarily on molded transparent plastic

tops with filter-paper inserts. This concept was first developed in the mid-1970s (Sedlachek and Mason, 1977), with commercial production starting in the early 1980s (Figure 17). It is a simple design that has been likened to a petri dish (Sedlachek et al., 1983; R. P. Orcutt, personal communication, 1999) in that the filter cover sits on top of the cage with an overlapping flange but no positive seal, similar to the way the lid fits on a petri dish. An integral part of the cage-level barrier system for housing rodents is the mobile HEPA-filtered mass airflow cabinet developed for changing cages and performing procedures (Sedlachek and Mason, 1977; McGarrity and Coriell, 1973; Sedlachek et al., 1981). Using this cage-level barrier system, cages are opened only in a HEPA-filtered mass air displacement workstation, hereafter referred to as a cage change cabinet (Figure 18). Two basic types of mobile cage change cabinets are in common

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

Fig. 17. Microisolation rodent cage from ventilated rack equipped with quick disconnect for automatic watering valve and air supply.

use; both have open-face fronts and are closed on the other three sides. One type is the "clean bench," which delivers air from the back of the cabinet toward the opening, focusing on protecting the animal in the cabinet. The other type is designed to protect

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both the animals in the cabinet and the environment outside the cabinet. It delivers HEPA-filtered air from the top of the cabinet toward return-air plenums in the front and back of the workbench, while at the same time drawing air from the room into the air-return slot in front of the work surface. Some, but not all, of this type of cage change cabinets are certified class II type A biological safety cabinets (see Section Ill,D). Other types of cage change cabinets are open on two or more sides, blowing HEPA-filtered air down toward the workbench (Figure 19). Of course, the cage change cabinets are also used for performing procedures on the animals, because the microisolation cages are opened only in a clean-air environment. At the same time that cage-level barriers were being developed and becoming more common, other strategies for maintaining "clean" rodents were advanced. These include solidbottom bedded cages suspended from a wire or perforated sheet metal shelf covered by filter paper (Figure 20); mass air displacement cage racks that direct HEPA-filtered air horizontally from the back of the rack, across each shelf, and into the room, thus isolating each shelf, and potentially each cage, with "clean" air (McGarrity and Coriell, 1973); portable HEPA-filtered mass air displacement "clean rooms" and "clean tents" varying in

Fig. 18. Microisolationcaging system. A mobile HEPA-filtered mass air displacement clean-bench cabinet used as part of a microisolation caging system together with a microisolation cage. With this caging system, the microisolation cages are opened only inside the cabinet, e.g., when animals are transferred from a soiled cage to a clean cage. Note the ventilated microisolation cage rack in background. (Courtesy of Lab Products, Inc.)

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JACK R. HESSLER AND STEVEN L. LEARY

Fig. 19. A mobile HEPA-filtered mass air displacement rodent cage change cabinet open on all sides. (Courtesy of Dr. Robert Faith, Center for Comparative Medicine, Baylor College of Medicine.)

Fig. 20. Rodent shoe-box cages suspended from perforated rack shelf normally covered with filter media (filter media not shown in figure). (Courtesy of Lab Products, Inc.)

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21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

rack or room size (Beall et al., 1971; van der Waaij and Andreas, 1971; McGarrity and Coriell, 1976); and a ventilated rack that provides individual ventilation for each enclosed shelf. In addition to reducing cage-to-cage spread of organisms, these systems provided the added benefit of reducing animal allergen loads in the air in the animal room. These systems are all still in use, but microisolation caging systems have come to dominate the field. It has been well documented that top filters on rodent cages create an effective cage-level barrier (Kraft et al., 1964; Rowe et al., 1964; Schneider and Collins, 1966; Poiley, 1967; Simmons et al., 1967b; Flynn, 1968; McGarrity and Coriell, 1973; Wescott et al., 1976; Sedlacheck and Mason, 1977; Sedlachek et al., 1981; Lipman et al., 1987; Whary et al., 2000). However, microisolation cages compromise the animal's microenvironment. In open, bedded shoe-box cages, ammonia, moisture levels, and temperature are increased above room levels (Flynn, 1968; Murakami, 1971; Raynor et al., 1983; Hirsjarvi and Valiaho, 1987). This increase is significant and includes increased levels of carbon dioxide when the cage is equipped with a filter top (Serrano, 1971; Lipman et al., 1987; Corning and Lipman, 1991; Lipman, 1992). The microenvironment is improved in static microisolation cages placed on mass air displacement racks as compared with the same isolation cages placed on a standard rack (Corning and Lipman, 1992). At levels encountered in cage environments, ammonia has been reported to affect the tracheal epithelium of rats significantly and to increase the severity of the rhinitis, otitis media, tracheitis, and pneumonia in rats caused by M y c o p l a s m a p u l m o n i s (Gamble and Clough, 1976; Broderson et aL, 1976; Schoeb et al., 1982). In another study, ammonia levels even higher than those typically found in rodent cages caused no significant changes in blood pH, Pco2, Po2 , blood ammonia concentration, hepatic microsomal enzyme activity, or the histologic appearance of lungs and trachea (Schaerdel et al., 1983). The first ventilated microisolation cages (with filtered air blowing into the cage) were developed by Edwin E Les of the Jackson Laboratory, Bar Harbor, Maine. His initial objective was to control cage-to-cage disease transmission while improving the animal's microenvironment (Les, 1968). By the mid-1970s the objective expanded to saving space by increasing animal housing density (Les, 1975, 1977). Eventually, the system evolved to include collecting air coming out of the cage (Les, 1983). In the late 1980s and early 1990s, ventilated rodent caging systems became widely accepted. To date, ventilated caging systems have met expectations. They have been documented to provide an effective cage-level barrier (Schulhof, 1980; Cunliffe-Beamer and Les, 1983; Wu, 1985; Lipman et al., 1993) while significantly improving the animal's microenvironment with regard to moisture, CO2, and ammonia levels (Keller et al., 1983; Wu et al., 1985; Lipman et al., 1992; Huerkamp and Lehner, 1994; Reeb et al., 1998). In fact, ventilated microisolation caging systems have proven so effective at maintaining

an acceptable microenvironment over long intervals between cage changes that it has become standard practice to change cages 1 time per week, and some facilities even extend the cage change interval to 2 weeks. The result is a considerable reduction in animal care costs when compared with the usual twice weekly changes required for static microisolation cages. There are several ventilation configuration options available with ventilated cages, including supply only; supply and exhaust, with the cage air pressure maintained slightly positive to the room (barrier); and supply and exhaust, with the cage air pressure maintained negative to the room (containment). Because it is very difficult to guarantee that the air pressure in each cage is actually negative to the room, the typical unsealed ventilated cage is probably not the best selection for containment of highlevel infectious agents or carcinogens; however, sealed, ventilated microisolation cages suitable for this purpose are available. Ventilated rodent racks may be supplied with one blower/ filter unit for supply air or one unit for both supply and exhaust (Figures 18 and 21). Blower/filter units typically come mounted on top of the rack, but a better option is to put them on a wall-mounted shelf over the rack (Figure 6). This avoids rack vibration produced by the blower units and eliminates the need to transfer the units from rack to rack. Options also include coupling the rack exhaust to the room exhaust, which was described in detail in Section II,C,1. Lipman (1999) presents a comprehensive description and review of isolator rodent caging systems. Germfree isolators provide the most effective method of controlling the animal's microbial environment (Figure 22). The type most commonly used today consists of clear, flexible, plastic vinyl chambers of various sizes maintained under positive pressure in which animals are housed in cages and operators work inside the isolator through sleeved gloves sealed to ports in the isolator (Trexler, 1963). Earlier models were made of stainless steel with glass view ports (Rayniers, 1957). All supplies and equipment, as well as the air entering the isolator, are sterilized. Germfree isolators are commonly used by commercial breeders to maintain foundation-breeding colonies for production-breeding colonies and for maintaining notobiotic animals in the research environment. The maintenance of animals in isolators is labor-intensive and therefore expensive. Rigid plastic isolators maintained under negative pressure are also used for experimental work with infectious agents.

D. Biological Safety Cabinets Biological safety cabinets (BSCs) are considered a primary containment device for working with infectious agents. For detailed descriptions of BSCs and their installation requirements, see CDC-NIH (1999) and Stuart (1999). There are three classes of cabinets: I, II, and III. Class I and II

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JACK R. H E S S L E R AND STEVEN L. LEARY

Fig. 21. Ventilated microisolation mouse cage rack with two blower/HEPA filter units. One unit filters room air before blowing it into the cages, and the other filters air captured from the cages before returning it to the room. (Courtesy of Allentown Caging Systems Co., Inc.)

21.

DESIGN AND MANAGEMENT OF ANIMAL FACILITIES

Fig. 22.

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Germfreeisolator. (Courtesyof Harlan.)

BSCs have inward airflow at velocities of 75-100 linear feet per minute through an open front to protect personnel and the room environment from the hazardous agent being worked with. All exhaust air from the cabinet passes through HEPA filters before being discharged into the room or the laboratory exhaust system. Class II BSCs have the additional benefit of protecting objects in the cabinet from extraneous microbial contamination by bathing them with a high volume of vertical laminar-flow HEPA-filtered air coming down from the top of the cabinet and across the work surface to returns at the back and front of the work surface. Class I and II BSCs are suitable for working with infectious agents up to BSL-3. A class III BSC cabinet is a totally enclosed, gas-tight, ventilated cabinet that offers the highest degree of personnel and environmental protection from infectious aerosols, as well as protection from extraneous microbiological contaminants for the materials in the cabinet. All operations in the cabinet are performed through attached rubber gloves. Supply air is HEPA-filtered, and exhaust air is filtered through two HEPA filters. Class III BSC cabinets provide the highest level of containment and are suitable for working with infectious agents classified at the highest biosafety level, BSL-4.

Class I BSCs are rarely used and are not routinely manufactured. Class II BSCs are the most commonly used in research animal facilities. They come in two types, A and B. Type A is suitable for containing particulate hazardous agents only and may be exhausted into the room through HEPA filters or to the outside via a thimble connection to the building exhaust ductwork. For example, portable class II type A BSCs are often used as rodent cage change cabinets. Class II type B BSCs are suitable for containing infectious agents, volatile chemicals, and radionuclides. They have a face velocity of 100 linear feet per minute and are hard-ducted to the exhaust system. Class II type B BSCs are further subdivided into types B 1, B2, and B3 depending on multiple features, including the degree of air recirculated within the cabinet versus that discarded--i.e., B 1, 70% recirculation; B2, 30% recirculation; and B3, 0% recirculation, 100% exhausted. The higher the percent exhausted, the greater the control of volatile hazards. The placement of BSCs in the laboratory, especially class II cabinets, requires careful consideration to avoid activities around the cabinet that could significantly disturb the airflow into the open face of the cabinet, such as heavy traffic and swinging doors.

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JACK R. HESSLER AND STEVEN L. LEARY E.

Vacuum Cleaners

Built-in central vacuum systems in animal facilities fade in and out of favor because of their many advantages and disadvantages. The most significant advantage of a central vacuum system is that it allows for the odiferous and dust-laden air from the vacuum generator to be discharged outside the animal facility. The most significant disadvantages are related to maintenance problems. Portable vacuum cleaners used inside animal facilities should be equipped to HEPA-filter the discharge air, which effectively reduces the particulate discharge if the HEPA filter is properly maintained. Unfortunately, the HEPA filter does not filter out odors. If portable vacuum cleaners are to be used for vacuuming in animal rooms, it is useful to install vacuum hose connections through the corridor wall into the room for connecting the vacuum in the corridor to the cleaning implement in the room.

F.

Miscellaneous Equipment

A large variety of miscellaneous movable equipment is required to operate an animal facility. Besides vacuum cleaners, this includes floor scrubbers; power washers; carts for moving cages; cases for handling water bottles; carts for moving water bottles in cases; and equipment for handling and storing feed and bedding, which may include forklifts, powered and/or hand-operated pallet trucks, hand trucks, and scales. The primary point is to emphasize the importance of deciding what type and how much portable equipment will be required and then planning the facility accordingly. For example, floor scrubbers may require power to charge the batteries, a water source to fill the tanks, and a drain to drain the tanks. Ideally, this would all be done where the scrubber is stored. If the facility has a dual clean/dirty corridor layout, a separate floor scrubber may be required in both the clean and dirty corridors, and storage and maintenance space will be required for both.

IV.

C O M M I S S I O N I N G AND VALIDATION

Some of the most important aspects of planning, building, and opening a new facility involve commissioning and validation. This is a detailed intense process that is best facilitated by experienced professionals independent of the architectural and engineering firms and the institution. Commissioning starts early in the design phase with a view toward final commissioning and validation of the structural, mechanical, electrical, and plumbing systems and equipment, especially built-in equipment. Commissioning involves challenging, verifying, and documenting the building structure, utilities, systems, and equip-

ment before routine operation is authorized. Validation involves establishing documented evidence that provides a high degree of assurance that a specific test process will consistently produce results at predetermined specifications and quality attributes. Detailed protocols should be prepared and tested for every aspect of the facility to document that every critical component functions as intended. This may even include housing animals in the facility for a period of time to test for any possible toxic environmental effects, which is a routine part of validation for a toxicology/safety testing facility.

V.

MANAGEMENT

Controlling the research animal's physical environment is the objective. Clearly, this requires having adequate facilities. However, a well-managed "marginal" facility will better achieve this objective than a poorly managed "ideal" facility. Is management more important than facilities? Yes, with the following caveats: (1) The facility must meet a minimal level of quality. (2) It must be understood that the definition of "minimal level of quality" evolves toward ever finer control of the environment as research technology evolves toward higher degrees of sophistication that require better control of the environment, which means better facilities and better management. (3) The more a facility design varies from the ideal, the higher the cost of maintaining the animals and, more important, the more difficult it becomes to assure the maintenance of minimal environmental standards. Human nature dictates that the more difficult it is to do a routine task, the greater is the chance that it will not be routinely done. A.

Knowledge of Laboratory Animal Science

Managing research animal facilities is a complex business with many options and successful approaches. It requires detailed technical knowledge of laboratory animal science, including all the factors that make up the animal's environment and how best to control those factors, plus an understanding of and ability to implement sound management principles. A detailed description of how to manage a facility is well beyond the scope of this chapter. The most critical point is to understand that almost everything that is done in the routine care of research animals has the potential to significantly influence the animal's environment and potentially its response to experimentally induced variables. Examples include the quality of feed (Newberne, 1975; NIH, 1978; Newberne and Fox, 1980) and bedding (Seeger et al., 1951; Ferguson, 1966; Vesell, 1967; Port and Kaltenbacvh, 1969; Sabine et al., 1973; Schoental, 1973, 1974; Vesell et al., 1973, 1976; Vlahakis, 1977; CunliffeBeamer et al., 1981). Any changes in animal care procedures must be carefully considered and often should involve input

21. DESIGN AND MANAGEMENTOF ANIMAL FACILITIES from scientists using animals in the facility before the changes are implemented.

B.

Personnel Training

The foundation of a sound management program is adequately trained personnel provided with the proper tools to carry out their duties in a safe and comfortable environment. Providing training for personnel caring for and using laboratory animals not only is sound management but also is required by federal law (Animal Welfare Act, CFR, Title 9, Subchapter A, Parts 1-3). The Institute for Laboratory Animal Resources publication titled "Education and Training in the Care and Use of Laboratory Animals: A Guide for Developing Institutional Programs" (ILAR, 1991) provides guidelines and resources needed to coordinate and implement a quality training program in compliance with Federal law. In addition, the American Association for Laboratory Animal Science (AALAS) offers excellent training materials in laboratory animal science for training animal care technicians and administers a program that certifies knowledge and understanding of laboratory animal science at three levels. Documenting and recognizing achievement is a strong motivator for personnel to grow in their knowledge and competence. Clearly, knowledge improves job performance, and improved job performance improves animal welfare, and increased animal welfare is a key to good science, which is the ultimate goal. Tying AALAS certification to automatic promotions is an even stronger motivator and establishes the basis for a career ladder that empowers personnel to advance based on their own initiative as opposed to having to wait for a leadership position to open and then being one of the lucky few to be chosen. Such a career development ladder will improve employee retention, and maintaining an experienced well-trained staff is vital to maintaining a high-quality animal care program. Regarding personnel safety and health, a sound occupational health and safety program must be high priority. Adequate facilities, safety training, and an occupational health program are all critical components of an occupational health program. An excellent resource for establishing a program is the National Academy Press publication titled "Occupational Health and Safety in the Care and Use of Research Animals" (ILAR, 1997).

C.

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ment, including the HVAC system, cage washers, autoclaves, and watering equipment. Equipment performing outside of ranges specified in the SOPs should be adjusted or repaired to bring it within compliance with the SOP. Of course, SOPs are worthless if not followed. Ideally, SOPs should always be followed to the letter and revised before practices are changed. Failure to follow SOPs is considered a violation of Good Laboratory Practice standards (CFR, Title 21, Chapter 1, Part 58). To maintain practice in line with SOPs requires a routine review of each SOP, preferably every 6 months but no less than once each year. Any divergence from an SOP discovered during the review process provides an opportunity either to revise the SOP to reflect the procedures being followed if such is deemed desirable or, if it is not deemed desirable, to return to performing the procedures as specified in the SOP. Reviews should include all personnel involved or potentially involved with performing the procedures covered by the SOP. Such reviews constitute a valuable continuing education format. Going over relevant SOPs with new employees is a vital part of introductory training. Training records should document that all personnel should sign off on every SOP relevant to their jobs.

D.

Crisis Management and Disaster Plan

A crisis and disaster management plan is another important component of a sound management program. This should consist of a written document that covers a wide variety of emergency situations varying from the very serious--e.g., longterm utility failures and massive physical damage to the animal facility from natural disasters--to less catastrophic but nonetheless important crises--e.g., critical personnel shortages due to winter storms, bites or scratches, accidental biological exposure to hazardous agents, and the arrival of unexpected animals. It should include a list of things personnel should know or do to be ready, the line of authority for various types of crises and disasters, when to call for emergency help, information to be given when calling for emergency help, emergency telephone numbers, and an animal evacuation plan. Just like SOPs, this is a flexible document that should be reviewed periodically, no less than annually, and the review should serve as a training opportunity for all involved staff.

Standard Operating Procedures

Another important feature of a sound management program is having written standard operating procedures (SOPs) outlining every routine procedure conducted in the facility. Every procedure in the animal facility is relevant to controlling the animal's environment. Of particular relevance to facilities and equipment are SOPs related to environmental quality control. These include routinely validating and documenting the performance of all equipment involved with controlling the animal's environ-

VI. Conclusions

The maintenance of research animals in a comfortable, stressfree, controlled environment, which is essential to conducting contemporary biomedical research, requires both adequate facilities and sound management. Each must complement the other. Clearly, the facility must be designed and constructed to

JACK R. HESSLER AND STEVEN L. LEARY

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include environmental control systems, equipment, and structural features required to adequately control the environment. Just as clearly, the better the facility is designed to facilitate sound management and efficient animal care, the lower the cost of animal care and the more likely the environmental variables involved with routine daily care will be adequately controlled. The more completely the environmental variables can be controlled, the more reliable and reproducible the research data will be, which in turn reduces the number of animals required to achieve the research goals. Properly designed and managed research animal facilities are essential for high-quality science, sound research economics, and, most important, humane care and use of laboratory animals.

REFERENCES

Anderson, R. L., Holland, B. W., Carr, J. K., et al. (1990). Effects of disinfectants on pseudomonads colonized on the interior surface of PVC pipes. Am. J. Public Health 80, 17-21. APC Filtration, Inc. (1999). HEPA and ULPA Filters: History. ASHRAE (American Society of Heating, Refrigerating, and Air-Conditioning Engineers) (1999). Laboratory animal facilities. In "ASHRAE Handbook: Heating, Ventilation, and Air-conditioning Applications," pp. 13.1313.19. Atlanta. Baker, D. (1998). Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin. Microbiol. Rev. 11, 231-266. Baker, H. J., Lindsey, J. R., and Weisbroth, S. H. (1979). Housing to control research variables. In "The Laboratory Rat" (H. J. Baker, J. R. Lindsey, and S.H. Weisbroth, eds.), Vol. 1, "Biology and Diseases," pp. 169-192. Academic Press, New York. Baldwin, C. L., Sabel, E L., and Henke, C. B. (1976). Bedding disposal cabinet for containment of aerosols generated by animal cage cleaning procedures. Appl. Environ. Microbiol. 31,322-324. Balk, M. (1980). Animal-facility design criteria for a toxicologic testing laboratory. Pharm. Technol. 4, 59-64. Barkley, W. E. (1979). Abilities and limitations of architectural and engineering features in controlling biohazards in animal facilities. In "Symposium on Laboratory Animal Housing" (Institute of Laboratory Animal Resources, ed.), pp. 158-163. National Academy of Sciences-National Research Council, Washington, D.C. Barkley, W. E., and Richardson, J. H. (1984). Control of biohazards associated with the use of experimental animals In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, E M. Loew, eds.), pp. 595-602. Academic Press, Orlands, Florida. Bayne, K., Mainzer, H., Campbell, G., et al. (1991). The reduction of abnormal behavior in individually housed rhesus monkeys (Macaca mulatta) with a foraging/grooming board. Am. J. Primatol. 23, 23-35. Beall, J. R., Torning, E E., and Runkle, R. S. (1971). A laminar flow system for animal maintenance. Lab. Anim. Sci. 21,206-212. Beck, R. W. (1963). The control of Pseudomonas aeruginosa in a mouse breeding colony by the use of chlorine in the drinking water. Lab. Anim. Care 13 (Part 2), 41-45. Bellhorn, R. W. (1980). Lighting in the animal environment. Lab. Anim. Sci. 30, 440-448. Blom, H. J. M., Van Tintelen, G., Baumans, V., et al. (1993a). Comparison of

sawdust bedding and wire mesh as cage flooring in preference tests with laboratory rats. In "Evaluation of Housing Conditions for Laboratory Mice and Rats: The Use of Preference Tests for Studying Choice Behavior" (H. J. M. Blom, ed.) pp. 71-80. Univ. Utrecht, Fakulteit Diergeneeskunde Proefschrift Rijksuniversiteit Utrecht, Met samenvatting in het Nederlands, Utrecht. Blom, H. J. M., Witkam, A. C. P., Schlingmann, F., et al. (1993b). Demonstration of preference of clean vs. soiled cages as expressed by laboratory mice. In "Evaluation of Housing Conditions for Laboratory Mice and Rats: The Use of Preference Tests for Studying Choice Behavior (H. J. M. Blom, ed.), pp. 81-99. Univ. Utrecht, Fakulteit Diergeneeskunde Proefschrift Rijksuniversiteit Utrecht, Met samenvatting in het Nederlands, Utrecht. Brainard, G. C., Vaughan, M. K., Reiter, R. J. (1986). Effect of light irradiance and wavelength on the Syrian hamster reproductive system. Endocrinology 119, 648-654. Brewer, N. R. (1952). Some problems in animal quarter construction in urban areas. Proc. Anim. Care Panel 3, 64-69. Brewer, N. R., and Penfold, T. W. (1961). Thoughts concerning the design of animal quarters. Proc. Anim. Care Panel 11, 4-18. Brick, J. O., Newell, R. F., and Doherty, D. G. (1969). A barrier system for a breeding and experimental rodent colony: description and operation. Lab. Anim. Care 19, 92-97. Briel, J. E., Kruckenberg, S. M., and Besch, E. L. (1972). "Observations of Ammonia Generation in Laboratory Animal Quarters." IER Publication 72-03, Kansas State Univ., Manhattan. Broderson, J. R., Lindsey, J. R., and Crawford, J. E. (1976). The role of environmental ammonia in respiratory mycoplasmosis of rats. Am. J. Pathol. 85, 115-127. Brown, M. J. 1994. Aseptic surgery for rodents. In "Rodents and Rabbits: Current Research Issues" (S. M., Niemi, J. S. Venable, and H. N. Guttman, eds.), pp. 67-72. Scientists Center for Animal Welfare, Bethesda, Maryland. Brownstein, D. G. 1978. Pathogenesis of bacteremia due to Pseudomonas aeruginosa in cyclophosphamide-treated mice and potentiation of virulence of endogenous streptococci. J. Infect. Dis. 137, 795-801. Burns, E. R., Schering, L. E., Pauly, J. E., and Tsal, T. H. (1976). Effect of altered lighting regimens, time-limited feeding, and presence of Ehrilich acities carcinoma on the circadian rhythm in DNA synthesis of mouse spleen. Cancer Res. 36, 1538-1544. Bywater, J. E. C., Kellett, B. S. (1977). Inhibition of bacteria in mouse drinking water by chlorination. Lab. Anim. 11, 215-217. CCAC (Canadian Council on Animal Care) (1993). Approaches to the design and development of cost-effective laboratory animal facilities. In "CCAC Proceedings," pp. 123-152. CCAC, Ottawa, Ontario. CDC-NIH (Centers for Disease Control-National Institutes of Health) (1999). "Biosafety in Microbiological and Biomedical Laboratories," 4th ed. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health, stock no. 017-040-00547-4. U.S. Goverment Printing Office, Washington, D.C. Cherry, J. A. (1987). The effect of photoperiod on development of sexual behavior in fertility in golden hamsters. Physiol. Behav. 39, 521-526. Christie, R. J., Williams, F. P., Whitney, J. R., and Johnson, D. J. (1968). Techniques used in the establishment and maintenance of a barrier mouse breeding colony. Lab. Anim. Care 18, 544-549. Cole, M. N. (1991). Laboratory animal facility planning. In "Handbook of Facilities Planning" (T. Ruys, ed.), Vol. 2, "Laboratory animal facilities," pp. 199-214. Van Nostrand Reinhold, New York. Cole, M. N., and Hessler, J. R. (1991). Space standards, In "Handbook of Facilities Planning" (T. Ruys, ed.), Vol. 2, "Laboratory animal facilities," pp. 67-75. Van Nostrand Reinhold, New York. Corning, B. F., and Lipman, N. S. (1991). A comparison of rodent caging systems based on microenvironmental parameters. Lab. Anim. Sci. 41, 498503.

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES Corning, B. E, and Lipman, N. S. (1992). The effects of a mass air displacement unit on the microenvironmental parameters within isolator cages. Lab. Anim. Sci. 42, 206-210. Costello, T., Watkins, L., Straign, M., et al. (1998). Effectiveness of rack sanitation procedures for elimination of bacteria from automatic watering manifolds. Contemp. Top. Lab. Anim. Sci. 37, 50-51. Costerton, J. W., and Lappin-Scott, H. M. (1989). Behavior of bacteria in biofilms. AMS News 55, 650-654. Cunliffe-Beamer, T. L. (1993). Applying principles of aseptic surgery to rodents. AWIC Newsl. 4(2), 3-6. Cunliffe-Beamer, T. L., and Les, E. P. (1983). Effectiveness of pressurized individually ventilated (PIV) cages in reducing transmission of pneumonia virus of mice (PVM). Lab. Anim. Sci. 33, 495. (Abstract). Cunliffe-Beamer, T. L., Freeman, L. C., and Myers, D. D. (1981). Barbiturate sleeptime in mice exposed to autoclaved or unautoclaved wood beddings. Lab. Anim. Sci. 31, 672-675. Curry, G., Hughes, H. C., Loseby, D., et al. (1998). Advances in cubicle design using computational fluid dynamics as a design tool. Lab. Anim. 32, 117127. Davis, D. E. (1978). Social behavior in a laboratory environment. In "Symposium on Laboratory Animal Housing" (Institute of Laboratory Animal Resources), pp. 44-63. National Academy of Sciences-National Research Council, Washington, D. C. Dolowy, W. C. (1961). Medical research laboratory of the University of Illinois. Proc. Anim. Care Panel 11, 267-290. Durfee, J. W., and Faith, R. E. (1999). Biological security: quarantine and testing policies for protecting transgenic colonies. Lab. Anim. 28, 45-48. Dymsza, H. A., Miller, S. A., Maloney, J. E, et al. (1963). Equilibration of the laboratory rat following exposure to shipping stress. Lab. Anim. Care 13, 60-65. Edstrom Industries. (1999). Evans, M. E., and Lesnaw, J. A. (1999). Infection control in gene therapy. Infect. Control Hosp. Epidemiol. 20, 568-576. FASS (Federation of Animal Science Societies) (1999). "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching." Savoy, IL. Ferguson, H. C. (1966). Effect of red cedar chip bedding on hexobarbital and pentobarbital sleep time. J. Pharm. Sci. 55, 1142-1148. Fiddler, I. J. (1977). Depression of macrophages in mice drinking hyperchlorinated water. Nature (Lond.) 120, 735-736. Flatt, R. E., ed. (1980). Defining the laboratory animal and its environment: Setting the parameters. Proceedings of the Fourth Charles River International Symposium on Laboratory Animals, Boston, MA, Oct. 1979. Lab. Anim. Sci. 30(2), 277-480. Flynn, R. J. (1963). Pseudomonas aeruginosa infection and radiobiological research at the Argonne National Laboratory: effects, diagnosis, epizootiology, and control. Lab. Anim. Care 13, 23-35. Flynn, R. J. (1968). A new cage cover as an aid to laboratory rodent disease control. Proc. Soc. Exp. Biol. Med. 129, 714-717. Flynn, R. J., Poole, C. M., and Tyler, S. A. (1971). Long distance air transport of aged laboratory mice. J. Gerontol. 26, 201-203. Foster, H. L., Foster, S. J., and Pfau, E. S. (1963). The large scale production of caesarean-originated, barrier-sustained mice. Lab. Anim. Care 13, 711718. Fouts, J. R. (1970). Some effects of insecticides on hepatic microsomal enzymes in various animal species. Rev. Can. Biol. 29, 377-389. Fox, J. G., and Helfrich-Smith, M. E. (1980). Chemical contamination of animal feeding systems: evaluation of two caging systems and standard cagewashing equipment. Lab. Anim. Sci. 30, 967-973. Fox, J. G., Donahue, P. R., and EssigmannJ. M. (1980). Efficacy of inactivation of representative chemical carcinogens utilizing commercial alkaline and acidic cage wash compounds. J. Environ. Pathol. Toxicol. 4, 97-106.

949 Fullerton, E M., and Gilliatt, R.W. (1967). Pressure neuropathy in the hind foot of the guinea pig. J. Neurosci. Neurosurg. Psychiatry 30, 18-25. Fullerton, E R., Greenman, D. L., and Kendall, D. C. (1967). Pressure neuropathy in the hind foot of the guinea pig. J. Neurol. Neurosurg. Psychiatry 30, 18-25. Fullerton, E R., Greenman, D. L., Kendall, and D. C. (1982). Effects of storage conditions on nutritional qualities of semipurified (AIJN-76) and natural ingredient (NIH-07) diets. J. Nutr. 12, 567-573. Gamble, M. R., and Clough, G. (1976). Ammonia build-up in animal boxes and its effect on rat tracheal epithelium. Lab. Anim. 10, 93-104. Garrard, G., Harrison, G. A., and Weiner, J. S. (1974). Reproduction and survival of mice at 23~ J. Reprod. Fertil. 37, 287-298. Goldstein, S. J. (1978). A theory of architecture: the orchestration of information. In "Symposium on Laboratory Animal Housing" (Institute of Laboratory animal Resources), pp. 16-24. National Academy of SciencesNational Research Council, Washington, D.C. Gordon, C. J. (1990). Thermal biology of the laboratory rat. Physiol. Behav. 47, 963-991. Gordon, C. J. (1993). "Temperature Regulation in Laboratory Animals." Cambridge Univ. Press, New York. Gorton, R. L. (1975). Energy conservation in water heating and HVAC systems. In "Symposium on Laboratory Animal Housing" (Institute of Laboratory animal Resources), pp. 179-183. National Academy of Sciences-National Research, Council, Washington, D.C. Grover-Johnson, N., and Spencer, P. S. (1981). Peripheral nerve abnormalities in aging rats. J. Neuropathol. Exp. Neurol. 40, 155-165. Gunn-Dore, D. (1997). Comfortable quarters for laboratory rabbits. In "Comfortable Quarters for Laboratory Animals" (V. Reinhardt, ed.), 8th ed., pp. 46-54. Animal Welfare Institute, Washington, D.C. Hall, J. E., White, W. J., and Lang, C. M. (1980). Acidification of drinking water: its effects on selected biological phenomena in male mice. Lab. Anim. Sci. 30, 643-651. Hare, R., and O'Donoghue, P. N., eds. (1968). "Laboratory Animal Symposia: The Design and Function of Laboratory Animal Houses." Laboratory Animals LTD, Ashrord Kent, England. Hasenau, J. M., Baggs, R. B., and Kraus, A. L. (1993). Microenvironments in microisolation cages using BASK/c and DC-1 mice. Contemp. Top. Lab. Anim. Sci. 32, 11-16. Haus, E., and Halberg, E (1970). Circannual rhythm in level and timing of serum corticosterone in standardized inbred mature C mice. Environ. Res. 3, 81-106. Haus, E., Lakatua, D., and Halberg, E (1967). The internal timing of several circadian rhythms in the blinded mouse. Exp. Med. Surg. 25, 7-45. Heeke, D. H., White, M. P., Mele, B. D., et al. (1999). Light-emitting diodes and cool white fluorescent light similarly suppress pineal gland melatonin and maintain retinal function and morphology in the rat. Lab. Anim. Sci. 49, 297-304. Henkel, C. B. (1978). Design criteria for animal facilities. In "Symposium on Laboratory Animal Housing (Institute of Laboratory animal Resources), pp. 142-157. National Academy of Sciences-National Research Council, Washington, D.C. Herman, L. M., White, W. J., and Lang, C. M. (1982). Prolonged exposure to acid, chlorine, or tetracycline in the drinking water: effects on delayed-type hypersensitivity, hemagglutination titers, and reticuloendothelial clearance rates in mice. Lab. Anim. Sci. 32, 603-608. Hessler, J. R. (1991a). Single versus dual-corridor systems: advantages, disadvantages, limitations, and alternatives for effective contamination control. In "Handbook of Facilities Planning" (T. Ruys, ed.), Vol. 2, "Laboratory Animal Facilities," pp. 59-67. Van Nostrand Reinhold, New York. Hessler, J. R. (1991b). Animal cubicles. In "Handbook of Facilities Planning (T. Ruys, ed.), Vol. 2, "Laboratory Animal Facilities," pp. 135-154. Van Nostrand Reinhold, New York. Hessler, J. R. (1991 c). Facilities to support research. In "Handbook of Facilities

950 Planning" (T. Ruys, ed.), Vol. 2, "Laboratory Animal Facilities," pp. 3554. Van Nostrand Reinhold, New York. Hessler, J. R. (1993). Animal cubicles: questions, answers, options, opinions. Lab. Anim. 22, 21-32. Hessler, J. R. (1995). Methods of biocontainment. In "Current Issues and New Frontiers in Animal Research" (K. A. L. Bayne, M. Greene, M. E. D. Prentice, eds.) pp. 61-58. Scientists Center for Animal Welfare, Greenbelt, Maryland. Hessler, J. R. (1999). The history of environmental improvements in laboratory animal science: caging systems, equipment, and facility design. In "Fifty Years of Laboratory Animal Science" C. McPherson and S. Mattingly, eds.). American Association for Laboratory Animal Science, Memphis. Hessler, J. R., and Moreland, A. E (1984). Design and management of animal facilities., In "Laboratory Animal Medicine," G. Fox, B. J. Cohen, F. M. Loew, eds.) pp. 505-526. Academic Press, and Orlando, Florida. Hessler, J. R., and Roberts, J. (1988). Full scale modeling of air and air contaminant distribution in spaces designed for housing research animals. In "Building Systems: Room Air and Air Contaminant Distribution" (L. L. Christianson, ed.), pp. 185-190. American Society of Heating, Refrigeration, and Air-Conditioning Engineers, Atlanta. Hessler, J. R., Broderson, R., and King, C. (1999a). Animal research facilities and equipment. In "Anthology of Biosafety 1. Perspectives on Laboratory Design" (J. Y. Richmond, ed.), pp. 191-217. American Biological Safety Association, Mundelein, Illinois. Hessler, J. R., Broderson, R., and King, C. (1999b). Rodent quarantine: facility design and equipment for small animal containment facilities. Lab. Anim. 28, 34-40. Hirsjarvi, P. A., and Valiaho, T. U. (1987). Microclimate in two types of rat cages. Lab. Anim. 21, 95-98. Homberger, E R., Zsuzsanna, P., and Thomann P. E. (1993). Control of Pseudomonas aeruginosa infection in mice by chlorine treatment of drinking water. Lab. Anim. Sci. 43, 635-637. Horsfall, E L., and Bauer, J. H. (1940). Individual isolation of infected animals in a single room. J. Bacteriol. 40, 569-580. Huerkamp, M. J., and Lehner, N. D. M. (1994). Comparative effects of forcedair, individual cage ventilation or an absorbent bedding additive on mouse isolator age microenvironment. Contemp. Top. Lab. Anim. Sci. 33, 58-61. Hughes, H. C., and Reynolds, S. (1994). The use of computational fluid dynamics for modeling airflow designs in a kennel facility. Contemp. Top. Lab. Anim. Sci. 34, 61-64. Hughes, C. H., and Reynolds, S., and Rodrigues, M. (1996). Designing animal rooms to optimize airflow using computational fluid dynamics. Pharm. Eng. (March-April), 44-65. ILAR (Institute of Laboratory Animal Resources) (1978). "Laboratory Animal Housing." National Academy of Sciences-National Research Council, Washington, D.C. ILAR (1991). "Education and Training in the Care and Use of Laboratory Animals: A Guide for Developing Institutional Programs." Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. National Academy Press, Washington, D.C. ILAR (1996). "Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. National Academy Press, Washington, D.C. ILAR (1997). "Occupational Health and Safety in the Care and Use of Research Animals." Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. National Academy Press, Washington, D.C. ILAR (1998). "The Psychological Well-being of Nonhuman Primates." Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. National Academy Press, Washington, D.C. Jeszenka, E. V., White, W. J., Lang, C. M., et al. (1981). Evaluation of a chemical scrubber for the removal of gaseous contaminants from recycled air. Lab. Anim. Sci. 31, 489-493.

JACK R. HESSLER AND STEVEN L. LEARY Jonas, A. M. (1965). Laboratory animal facilities. J. Am. Vet. Med. Assoc. 146, 600 -606. Jonas, A. M. (1978). Centralized versus dispersed animal care facilities. In "Symposium on Laboratory Animal Housing" (Institute of Laboratory Animal Resource), pp. 11-15. National Academy of Sciences-National Research Council, Washington, D.C. Kacergis, J. B., Jones, R. B. Reeb, C. K., et al. (1996). Air quality in an animal facility: particulates, ammonia, and volatile organic compounds. Am. Ind. Hyg. Assoc. J. 57, 634-640. Keene, J. H., and Sansone, E. B. (1984). Airborne transfer of contaminants in ventilated spaces. Lab. Anim. Sci. 34, 453-457. Keller, G. L., Mattingly, S. E, and Knapke, E B. (1983). A forced-air individually ventilated caging system for rodents. Lab. Anim. Sci. 33, 580-582. King, C., Hessler, J. R., and Broderson, R. (1999). Small animal research facilities management. In "Anthology of Biosafety 1. Perspectives on Laboratory Design" J. Y. Richmond, ed.), pp. 219-231. American Bilogical Safety Association, Mundelein, Illinois. Klein, H., Frankenberg, L., Conboy, T., et al. (1999). Development, design, and operation of a robotic rodent cage washing and waste handling system. Lab. Anim. 28, 34-37. Kohloss, E H. (1976). Energy sources and costs for building systems. In "Laboratory Animal Housings: Proceedings of a Symposium Held at Hunt Valley Maryland, September 22-23, 1976," pp. 184-190. Institute of Laboratory Animal Resources, National Academy of Sciences, Washington, D.C. Kraft, L. M. (1958). Observations on the control and natural history of epidemic diarrhea of infant mice. Yale J. Biol. Med. 31, 121-137. Kraft, L. M. (1980). The manufacturing, shipping and receiving, and quality control of rodent bedding materials. Lab. Anim. Sci. 30, 366-374. Kraft, L. M., Pardy, R. F., Pardy, D. A., et al. (1964). Practical control of diarrheal disease in a commercial mouse colony. Lab. Anim. Care 14, 16-19. Kruckenberg, S. M. (1971). Control of odoriferous and other gaseous components in laboratory animal quarters. In "Proceedings of the Symposium on Environmental Requirements for Laboratory Animals," pp. 83-100. IER Publication 71-02, Kansas State Univ., Manhattan. Kuwabara, T., and Gorn, R. A. (1968). Retinal damage by visible light: an electron microscopic study. Arch. Ophthalmol. 79, 69-78. Kuwabara, T., and Funahashi, M. (1976). Light damage in the developing rat retina. Arch. Ophthalmol. 94, 1369-1374. Landi, M. S., Kreider, J. W., Lang, C. M., and Bullock, L. P. (1982). Effects of shipping on the immune function in mice. Am. J. Vet. Res. 43, 1654-1657. Lang, C. M. (1981). Special design considerations for animal facilities. In "Proceedings of the National Cancer Institute Symposium on Design of Biomedical Research Facilities" (D. G. Fox, ed.), pp. 117-127. Cancer Research Safety Monograph Vol. 4, National Institute of Health Publication No. 81-2305. Bethesda, Maryland. Lang, C. M. (1983). Design and management of research facilities for mice. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 37-50. Academic Press, New York. Lang, C. M., and Harrell, B. T. (1969). An ideal animal resource facility. Am. Inst. Archit. J. 52, 57-61. Lang, C. M., and Harrell, B. T. (1972). Guidelines for a quality program of laboratory animal medicine in a medical school. J. Med. Educ. 47, 267-271. Lang, C. M., and Vessel, E. S. (1976). Environmental and genetic factors affecting laboratory animals: impact on biomedical research. Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 1123-1124. Leary, S. L., Majoros, J. A., and Tomson, J. S. (1998). Making cagewash facility design a priority. Lab. Anim. 27, 28-31. Lempken, B. (1991). Drinking water. In "Handbook of Facilities Planning," Vol. 2. "Laboratory Animal Facilities (T. Ruys, ed.), pp. 174-180. Van Nostrand Reinhold, New York. Les, E. P. (1968). Ventilated cages for mice. National meeting of American Association for Laboratory Animal Science Las Vegas, Nevada. (Abstract 67).

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES Les, E. P. (1975). Saving space with individual cage ventilation for mice. National meeting of American Association for Laboratory Animal Science, Boston. (Abstract 67). Les, E. P. (1977). Pressurized, individually ventilated caging and its effect on reproduction of A/J and DBA/2J mice. National meeting of American Association for Laboratory Animal Science, Anaheim, California. (Abstract 63). Les, E. P. (1983). Pressurized, individually ventilated (PVI) and individually exhausted caging for laboratory mice. Lab. Anim. Sci. 33, 495. (Abstract). Lindsey, J. R., Connor, M. W., and Baker, H. J. (1978). Physical, chemical, and microbial factors affecting biologic response. In "Laboratory Animal Housings: Proceedings of a Symposium Held at Hunt Valley Maryland, September 22-23, 1976," pp. 31-43. Institute of Laboratory Animal Resources, National Academy of Sciences, Washington, D.C. Lipman, N. S. (1992). Microenvironmental conditions of isolator cages: an important research variable. Lab. Anim. 21, 23-26. Lipman, N. S. (1993). Strategies for architectural integration of ventilated caging systems. Contemp. Top. in Lab. Anim. Sci. 32, 7-10. Lipman, N. S. (1999). Isolator rodent caging systems (state of the art): a critical review. Contemp. Top. Lab. Anim. Sci. 38, 9-17. Lipman, N. S., Newcomer, C. E., and Fox, J. G. (1987). Rederivation of MHV and MEV antibody positive mice by cross-fostering and use of the microisolator caging system. Lab. Anim. Sci. 37, 195-199. Lipman, N. S., Corning, B. E, and Coiro, M. A. (1992). The effects of intracage ventilation on microenvironmental parameters in isolator-type cages. Lab. Anim. 26, 206-210. Lipman, N. S., Corning, B. E, Saifuddin, M. D. (1993). Evaluation of isolator caging systems for protection of mice against challenge with mouse hepatitis virus. Lab. Anim. 27, 134-140. Loew, E M. (1980). Considerations in receiving and quarantining laboratory rodents. Lab. Anim. Sci. 30, 323-329. Manser, C. E., Morris, H., and Broom, D. M. (1995). An investigation into the effects of solid or grid cage flooring on the welfare of laboratory rats. Lab. Anim. 29, 353-363. Manser, C. E., Elliott, H., Morris, T. H., et al. (1996). The use of a novel operant test to determine the strength of preference for flooring in laboratory rats. Lab. Anim. 30, 1-6. McGarrity, G. J., and Coriell, L. L. (1973). Mass airflow cabinet for control of airborne infection of laboratory rodents. Appl. Microbiol. 26, 167172. McGarrity, G. J., and Coriell, L. L. (1976). Maintenance of axenic mice in open cages in mass airflow. Lab. Anim. Sci. 26, 746-750. McGarrity, G. J., Coriell, L. L., Russell, W., et al. (1969). Medical applications of dust-free rooms: 2. Use in an animal care laboratory. Appl. Microbiol. 18, 142-146. McPherson, C. W. (1963). Reduction of Pseudomonas aeruginosa and coliform bacteria in mouse drinking water following treatment with hydrochloric acid or chlorine. Lab. Anim. Care 13, 737-744. McPherson, C. W. (1975). Why be concerned about ventilation requirements of experimental animals? ASHRAE Trans. 81, 539-541. Memarzadeh, F. (1998). "Ventilation Design Handbook on Animal Research Facilities Using Static Microisolators." Animal Facility Ventilation Handbook Vols. 1 and 2. National Institutes of Health, Division of Engineering Services, Bethesda, Maryland. Mulder, J. B. (1971). Animal behavior and electromagnetic energy waves. Lab. Anim. Sci. 21, 389-393. Murakami, H. (1971). Differences between internal and external environments of the mouse cage. Lab. Anim. Sci. 21, 680-684. NABR (National Association for Biomedical Research) (1991). "State Laws concerning the Use of Animals in Research." NABR, Washington, D.C. National Science Foundation (May 1953, amended April 1965 and November 1977). NSF standard No. 3 for commercial spray type dishwashing machines. National Sanitation Foundation, Ann Arbor, Michigan.

951 Neil, D.H., and Larsen, R. I. (1982). How to develop cost-effective animal room ventilation: build a mock-up. Lab. Anim. 11, 32-37. Nevins, R. G., and Miller, P. L. (1972). Analysis, evaluation, and comparisons of room air distribution performancemA summary. ASHRAE Trans. 78, 235-242. Newberne, P. M. (1975). Influence on pharmacology experiments of chemicals and other factors in diets of laboratory animals. Fed. Proc., Fed. Am. Sco. Exp. Biol. 34, 209-218. Newberne, P. M., and Fox, J. G. (1978). Chemicals and toxins in the animal facility. In "Symposium on Laboratory Animal Housing" (Institute of Laboratory Animal Resources), pp. 118-138. National Academy of SciencesNational Research Council, Washington, D.C. Newberne, P. M., and Fox, J. G. (1980). Nutritional adequacy and quality control of rodent diets. Lab. Anim. Sci. 30, 352-363. Newell, G. W. (1980). The quality, treatment, and monitoring of water for laboratory rodents. Lab. Anim. Sci. 30, 377-383. NIH (National Institutes of Health) (1978). "Nutritional Requirements of Laboratory Animals." National Academy of Sciences, Washington, D.C. NIH (1981). "Guidelines for the Use of Chemical Carcinogens." National Institutes of Health Publication 81-2385, Bethesda, Maryland. NIH (1999). "Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines)." U.S. Dept. of Health, Education and Welfare, Public Health Serv., NIH, Bethesda, Maryland. Noell, W. K., Walker, V. S. Kang, B. S., et al. (1966). Retinal damage by light in rats. Invest. Ophthalmol. 5, 450-473. Novak, G. R. (1982). Survey results: floors and flooring materials. Lab. Anim. 1, 48-49. Novak, G. (1999). Selecting an appropriate watering system for your facility. Lab. Anim. 28, 43-46. Ortman, J. A., Sahenk, J., and Mendell, J. R. (1983). The experimental production of Renaut bodies. J. Neurol. Sci. 62, 233-241. O' Steen, W. K., and Anderson, K. V. (1971). Photically evoked responses in the visual system of rats exposed to continuous light. Exp. NeuroL 30, 525534. O'Steen, W. K., Shear, C. R., and Anderson, K. V. (1972). Retinal damage after prolonged exposure to visible light: a light and electron microscopic study. Am. J. Anat. 134, 5-21. O'Steen, W.K., and Donnelly, J. E. (1982). Chronologic analysis of variations in retinal damage in two strains of rats after short-term illumination. Invest. Ophthalmol. Vis. Sci. 22, 252-255. Otis, A. P., and Foster, H. L. (1983). Management and design of breeding facilities. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, p. 20. Academic Press, New York. Pakes, S. P., Lu, Y.-S., and Meaner, P. C. (1984). Factors that complicate animal research. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 649-665. Academic Press, Orlando, Florida. Peterson, E. A. (1980). Noise and laboratory animals. Lab. Anim. Sci. 30, 422439. Pierson, C. L., Johnson, A. G., and Feller, I. (1976). Effect of cyclophosphamide on the immune response to Pseudomonas aeruginosa in mice. Infect. Immun. 14, 167-177. Poiley, S. M. (1967). The development of an effective method for control of epizootic diarrhea in infant mice. Lab. Anim. CarelT, 501-510. Poiley, S. M. (1974). Housing requirements--general considerations. In "Handbook of Laboratory Animal Science" (E. C. Melby Jr. and N. H. Altman, eds.), Vol. 1, pp. 21-60. CRC Press, Cleveland. Port, C. D., and Kaltenbacvh, J. P. (1969). The effect of corncob bedding on reproductivity and leucine incorporation in mice. Lab. Anim. Care 19, 46-49. Rahija, R. J. (1999). Animal facility design. In "Occupational Medicine: State of the Art Reviews," pp. 407-422. Hanley and Belfus, Philadelphia. Rake, B. W. (1979). Microbiological evaluation of a biological safety cabinet modified for bedding disposal. Lab. Anim. Sci. 29, 625-632.

952 Rails, K. (1967). Auditory sensitivity in mice: Peromyscus and Mus musculus. Anim. Behav. 15, 123-128. Raynor, T. H., Steinhagen, W. H., and Hamm, T. E. (1983). Differences in the microenvironment of a polycarbonate caging system: bedding vs. raised wire floors. Lab. Anim. 17, 83-89. Reeb, C., Jones, R. B., and Berg, D. W. (1998). Microenvironment in ventilated animal cages with differing ventilation rates, mice populations, and frequency of bedding changes. Contemp. Top. Lab. Anim. Sci. 37, 43-49. Reeb-Whitaker, C. K., and Harrison, D. J. (1999). Practical management strategies for laboratory animal allergy. Lab. Anim. 28, 25-30. Rehg, J. E., and Toth, L. A. (1998). Rodent quarantine programs: purpose, principles, and practice. Lab. Anim. Sci. 48, 438-447. Reyniers, J. A. (1957). Control of contamination in colonies of laboratory animals by the use of germ-free techniques. Animal Care Panel 7, 41-49. Reynolds, S. (1994). CFD modeling optimizes containment elimination. Eng. Syst. 11, 35-77. Richmond, J. Y. (1991). Hazard reduction in animal research facilities. Lab. Anim. 20, 23-29. Richmond, J. Y. (1996). Animal biosafety levels 1-4: an overview. In "Proceedings of the Fourth National Symposium on Biosafety: Working Safely With Animals" (J. Y. Richmond, ed.), pp. 5-8. Centers for Disease Control, Atlanta. Robinson, W. G., Jr., and Kuwabara, T. (1976). Light-induced alterations of retinal pigment epithelium in black, albino, and beige mice. Exp. Eye Res. 22, 549-557. Robinson, W. G., Jr. Kuwabara, T., and Zwaan, J. (1982). Eye research. In "The Mouse in Biomedical Research" H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 69-95. Academic Press, New York. Rowe, W. P., Hartley, J. W., and Hubner, R. J. (1964). Mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection of mice. Proc. Soc. Exp. Biol. Med. 112, 161-165. Runkel, R. S. (1964). Laboratory animal housing, Part I. Amer. Inst. of Architects, March 1964. Runkel, R. S. (1964). Laboratory animal housing, Part II. Amer. Inst. of Architects, April 1964. Ruys, T. (1988). Isolation cubicles: space and cost analysis. Lab. Anim. 17, 25 -23. Ruys, T. (1991 a). Cage, rack, and tunnel washers. In Ruys T., ed. "Handbook of Facilities Planning" (T. Ruys, ed.), Vol. 2, "Laboratory Animal Facilities," pp. 267-279. Van Nostrand Reinhold, New York. Ruys, T. (1991b), ed. "Handbook of Facilities Planning," Vol. 2, "Laboratory Animal Facilities," pp. 267-279. Van Nostrand Reinhold, New York. Sabine, J. R., Horton, B. J., and Wicks, M. B. (1973). Spontaneous tumors in C3H-Avy and Cch-A vyga mice: high incidence in the United States and low incidence in Australia. J. Natl. Cancer Inst. 50, 1237-1242. Saltratelli, C. G., and Coppola, C. P. (1979). Influence of visible light on organ weights of mice. Lab. Anim. Sci. 29, 319-322. Schaerdel, A. D., White, W. J., Lang, M. C., et al. (1983). Localized and systemic effects of environmental ammonia in rats. Lab. Anim. Sci. 33, 40-45. Schneider, H. A., and Collins, G. R. (1966). Successful prevention of infantile diarrhea of mice during an epizootic by means of a new filter cage unopened from birth to weaning. Lab. Anim. Care 16, 60-71. Schoeb, T. R., Davidson, M. K., and Lindsey, J. R. (1982). Intracage ammonia promotes growth of Mycoplasma pulmonis in the respiratory tract of rats. Infect. Immun. 38, 212-217. Schoental, R. (1973). Carcinogenicity of wood shavings. Lab. Anim. 7, 47-49. Schoental, R. (1974). Zearalenone in the diet, podophyllotoxin in the wood shaving bedding, are likely to affect the incidence of "spontaneous" tumors among laboratory animals. Br. J. Cancer 30, 181. Schulhof, J. (1980). Keeping clean with filter-top and ventilated cages. Lab. Anim. 19, 23-36. Sedlachek, R. S., and Mason, K. A. (1977). A simple and inexpensive method for maintaining a defined flora mouse colony. Lab. Anim. Sci. 27, 667-670.

JACK R. HESSLER AND STEVEN L. LEARY Sedlachek, R. S., Orcutt, R. E, Suit, H. D., et al. (1981). A flexible barrier at cage level for existing colonies: production and maintenance of a limited stable anaerobic flora in a closed inbred mouse colony. In "Recent Advances in Germfree Research" (S. Sasaki et al., eds.), pp. 65-69. Tokia Univ. Press, Japan. Sedlacek, R. S., Suit, H. D., and Rose, E. E (1983). Cost effective practical gnotobiotics at the cage level. In "The Contribution of Laboratory Animal Science to the Welfare of Man and Animals" (J. Archibald et al., eds.) Eighth ICLAS/CALAS Symposium, Vancouver 1983, pp. 261-266. Gustav Fisher Verlag, Stuttgart. Seeger, K. C., Tomhave, A. E., and Lucas, W. C. (1951). A comparison of litters used for broiler production. Del. Agric. Exp. Stn. Bull. 289. Semple-Rowland, S. L., and Dawson, W. W. (1987). Retinal cyclic light damage threshold for albino rats. Lab. Anim. Sci. 37, 289-298. Serrano, L. J. (1971). Carbon dioxide and ammonia in mouse cages: effect of cage covers, population, and activity. Lab. Anim. Sci. 21, 75-85. Simmonds, R. C. (1973). Selected topics in laboratory animal medicinemthe design of laboratory animal homes. Aeromed. Rev. 7-73, 1-45. Simmons, M. L., Wynns, L. E I., and Choat, E. E. (1967a). A facility design for production of pathogen-free, inbred mice. ASHRAE J. 9, 27-31. Simmons, M. L., Richter, C. B. Franklin, J. A., et al. (1967b). Prevention of infectious diseases in experimental mice. Proc. Soc. Exp. Biol. Med. 126, 830-837. Small, J. D. (1982). Environmental and equipment monitoring. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 3, pp. 83-100. Academic Press, New York. Stuart, D. G. (1999). Primary containment. In "Anthology of Biosafety 1. Perspectives on Laboratory Design" (J. Y. Richmond, ed.), pp. 45-61. American Biological Safety Association, Mundelein, Illinois. Sullivan, J. E, and Songer, J. R. (1966). Role of differential air pressure zones in the control of aerosols in a large animal isolation facility. Appl. Microbiol. 14, 674-678. Thibert, E (1980). Control of microbial contamination in the use of laboratory rodents. Lab. Anim. Sci. 30, 339-348. Thorp, W. T. S. (1960). The design of animal quarters: A symposium on laboratory animals mtheir care and their facilities. J. Med. Education 35, 4-14. Trentin, J. J., Van Hoosier, G. L., Jr., Shields, J., et al. (1966). Establishment of a caesarean-derived, gnotobiote foster nursed inbred mouse colony with observations on the control of Pseudomonas. Lab. Anim. Care 16, 109118. Trexler, E C. (1963). An isolator system for control of contamination. Lab. Anim. Care 13, 572-581. Tucker, H. A., Petitclere, A. D., and Zinn, S.A. (1984). The influence of photoperiod on body weight gain, body composition, nutrient intake and hormone secretion. J. Anim. Sci. 59, 1610-1620. Tyson, K. W., and Corey, M. A. (1999). Facility design: forecasting space requirements. Lab. Anim. 28, 28-31. Urano, T., and Maejima, K. (1978). Provocation of pseudomoniasis with cyclophosphamide in mice. Lab. Anim. 12, 159-161. USDA-APHIS (U.S. Department of Agriculture, Animal and Plant Health Inspection Service). 1998. Wire flooring and temperature standards amendment. Anita. Care Q. Rep. (Winter 1997/Spring 1998), 3. van der Waaij, D., and Andreas, A. H. (1971). Prevention of air-borne contamination and cross-contamination in germfree mice by laminar flow. J. Hyg. 69, 83-89. Vessel, E. S. (1967). Induction of drug-metabolizing enzymes in liver microsomes of rats and mice by softwood bedding. Science 157, 1057-1058. Vessel, E. S., Lang, C. M., White, W. J., et al. (1973). Hepatic drug metabolism in rats: impairment in a dirty environment. Science 179, 896-897. Vessel, E. S., Lang, C. M., White, W. J., et al. (1976). Environmental and genetic factors affecting the response of laboratory animals to drugs. Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 1125-1132. Veterans Administration. (1993). "Veterinary Medical Unit (VMU) VA Design

21. DESIGN AND MANAGEMENT OF ANIMAL FACILITIES Guide." Department of Veterans Affairs, Veterans Health Administration, Office of Construction Management, Office of Architecture and Engineering, Standards Service, Washington, DC. Vlahakis, B. (1977). Possible carcinogenic effects of cedar shavings in bedding of C3H-A vytBmice. J. Natl. Cancer Inst. 58, 149-150. Wardrip, C. L., Artwohl, J. E., and Mennett, B. T. (1994). A review of the role of temperature versus time in an effective cage sanitation program. Contemp. Top. Lab. Anim. Sci. 33, 66-68. Webber, D. J., and William, A. R. (1999). Gene therapy: a new challenge for infection control. Infect. Control Hosp. Epidemiol. 20, 530-532. Weihe, W. J. (1965). Temperature and humidity climatograms for rats and mice. Lab. Anim. Care 15, 18-28. Weisse, I., Stotzer, H., and Seitz, R. (1974). Age- and light-dependent changes in the rat eye. Virchows Arch. A 362, 145-156. Wensinck, E, van Bekkum, E W., and Renaud, H. (1957). The prevention of Pseudomonas aeruginosa infection in irradiated mice and rats. Radiat. Res. 17, 491-499. Wescott, R. B., Malczewski, A., and Van Hoosier, G. L. (1976). The influence of filter top caging on the transmission of pinworm infections in mice. Lab. Anim. Sci. 26, 742-745. Westwood, J. C. N., Mitchell, M. A., and Legace, S. (1971). Hospital sanitation: the massive bacterial contamination of the wet mop. Appl. Microbiol. 21, 693-697.

953 Whary, M. T., Cline, J. H., King, A. E., et al. (2000). Containment of Helicobacter hepaticus by use of husbandry. Comp. Med. 50, 78-81. White, B. (1991). Mechanical systems (HVAC). In "Handbook of Facilities Planning," (T. Ruys, ed.), Vol. 2, "Laboratory Animal Facilities," pp. 308320. Van Nostrand Reinhold, New York. White, W. J. (1996). Special containment devices for research animals. In Richmond, J.Y., ed. "Proceedings of the Fourth National Symposium on Biosafety: Working Safely with Research Animals, Atlanta, Georgia" (J. Y. Richmond, ed.), pp. 109-112. CDC, Atlanta. White, W. J., Hughes, H. C., Singh, S. B., et al. (1983). Evaluation of a cubicle containment system in preventing gaseous and particulate air-borne crosscontamination. Lab. Anim. Sci. 33, 571-576. Windman, A. L., and Ziogas, A. L. (1978). Engineering objectives for laboratory animal housing. In "Symposium on Laboratory Animal Housing" (Institute of Laboratory Animal Resources), pp. 84-88. National Academy of Sciences-National Research Council, Washington, D.C. Woods, J. E. (1975). Influence of room air distribution on animal cage environments. ASHRAE Trans. 81, 559-571. Wu, D., Joiner, G. N., and McFarland, A. R. (1985). A forced-air ventilation system for rodent cages. Lab. Anim. Sci. 35, 499-504.

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Chapter 22 Preanesthesia, Anesthesia, Analgesia, and Euthanasia M. Michael Swindle, George A. Vogler, Linda K. Fulton, Robert P. Marini, and Sulli Popilskis

I. II.

III.

IV.

V.

LABORATORYANIMALMEDICINE,2nd edition

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preoperative Assessment and Preparation . . . . . . . . . . . . . . . . . . . . . .

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Anesthetic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Intraoperative Monitoring and Support . . . . . . . . . . . . . . . . . . . . . . . .

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Analgesic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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G. Species Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Euthanasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preoperative Assessment and Preparation . . . . . . . . . . . . . . . . . . . . . .

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Intraoperative Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intraoperative Monitoring and Support . . . . . . . . . . . . . . . . . . . . . . . . Special Anesthetic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . .

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E Acute and Chronic Analgesic Therapy . . . . . . . . . . . . . . . . . . . . . . . . Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Preoperative Assessment and Preparation . . . . . . . . . . . . . . . . . . . . . . C. Intraoperative Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Special Anesthetic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Intraoperative Monitoring and Support . . . . . . . . . . . . . . . . . . . . . . . . E Postoperative Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Acute and Chronic Analgesic Therapy . . . . . . . . . . . . . . . . . . . . . . . . H. Euthanasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Swine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Preoperative Assessment and Preparation . . . . . . . . . . . . . . . . . . . . . .

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Intraoperative Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Intraoperative Monitoring and Support . . . . . . . . . . . . . . . . . . . . . . . .

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Special Anesthetic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . Postoperative Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acute and Chronic Analgesic Therapy . . . . . . . . . . . . . . . . . . . . . . . .

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Euthanasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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M. M I C H A E L S W I N D L E , E T AL. VI.

VII.

I.

Small Ruminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Preanesthetic, Anesthetic, and Recovery Considerations . . . . . . . . . . C. Postoperative Recovery, Pain Assessment, and Analgesic Agents . . . N o n h u m a n Primates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Preoperative Assessment and Preparation . . . . . . . . . . . . . . . . . . . . . . C. Intraoperative Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Intraoperative Monitoring and Support . . . . . . . . . . . . . . . . . . . . . . . . E. Special Anesthesia Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . E Postoperative Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Euthanasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION

The purpose of this chapter is to provide practical advice for administering anesthetics and analgesics to the most commonly used laboratory animal species. It is not meant to be an exhaustive review of all anesthetic and analgesic protocols used in these species. An entire textbook in this American College of Laboratory Animal Medicine (ACLAM) series has been dedicated to this subject (Kohn et al., 1997), and readers are referred to that source for more complete information. The most commonly used rodent species, rabbits, dogs, pigs, small ruminants, and nonhuman primates were the selected species for inclusion in this chapter. These species encompass the overwhelming majority of laboratory animals that are currently utilized. References for in-depth discussion of anesthetic and analgesic protocols for each species are included within its particular section.

II.

A.

RODENTS

Introduction

Rodents, especially rats and mice, are the most numerous and arguably the most important group of animals used in research. Among the factors that differentiate anesthetic techniques used in rodents from those more commonly employed in larger species are the small size of the animals, the perceived and often real difficulties of vascular and airway access, and the frequent need to anesthetize large numbers of animals in a relatively short time. Trends that have combined to focus more attention on safe and appropriate anesthetic and analgesic delivery and monitoring for rodents include humane concerns; scientific recognition of the effects of pain, anesthesia, and analgesia on experimental design; and the expense of generation or purchase of genetically altered rodents. This section will briefly discuss common anesthetic techniques for mice and rats, with comments on hamsters,

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gerbils, and guinea pigs. Drugs, techniques, and approaches to anesthesia and analgesia for rodents are rapidly evolving. The reader is urged to consult laboratory animal anesthesia texts (Wixson and Smiler, 1997a; Flecknell, 1996a; Flecknell and Waterman-Pearson, 2000) and journals for more complete and timely information.

B.

Preoperative Assessment and Preparation

1. Preanesthetic Evaluation

Anesthetic protocols that are satisfactory for healthy animals can be fatal or can severely compromise unhealthy ones. A vigilant rodent health surveillance program is the first line of defense but cannot be expected to detect alterations in husbandry or experimental manipulations that would affect anesthesia outcomes. Preanesthetic considerations should include an assessment of appearance, behavior, and weight for evidence of abnormality as well as for establishing doses for injectable agents. The history of the group should be examined to identify experimental, housing, and strain-specific features that might affect choice of anesthesic agents and techniques. In any case, it is important to allow adequate time for newly arrived animals to become acclimated to changes in housing, diet, and husbandry conditions and to recover from shipping stress. An acclimation period of at least 3 days has been recommended for rodents (Landi et al., 1982). No anesthetic regimen is universally safe or appropriate. Changes in strain, sex, age, and specific procedures may be expected to alter the effects of anesthesia, often to a surprising degree. Unless the anesthetic regimen is known to be safe and effective in the animals to be used, a pilot study should be conducted to assess and verify the effects of the proposed anesthetic and analgesic protocol (Flecknell, 1996b). 2.

Choice of Anesthetic Techniques

All anesthetics have undesirable properties, so a primary goal must be to select an anesthetic technique that has the least ad-

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22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA

verse effect on the animal and on the research. Thus, the goals and limitations of the research must be taken into account in formulating an anesthetic protocol. For example, when prolonged stable anesthesia is required, the choice might favor an inhalation or continuous infusion method to avoid the variations that tend to occur with repeated bolus administration of drugs. At the same time, although it is essential that an adequate depth of anesthesia is reached and maintained for the needed time, a prolonged recovery period can further stress the animal and the resources of the facility, favoring selection of agents that have rapid recovery characteristics or agents that can easily be reversed, such as a2-agonists, benzodiazepines, and some opioids. Finally, some techniques that meet all of the preceding criteria are too technically demanding or simply too expensive for the proposed research. Inhalation anesthesia with modern agents requires the proper equipment and training. Precise intravenous infusion methods may require an infusion pump, as well as vascular access and experience with the technique. In many cases, only brief anesthesia may be needed, as for procedures that may not be extremely painful but that require adequate restraint for the safety of the animal and the operator (e.g., bleeding, injections, or sampling of small amounts of tissue). Brief exposure to an inhalation agent or to carbon dioxide is often selected to meet these needs. In summary, selection of a suitable anesthetic technique must include professional and humane considerations, scientific requirements and restrictions, and recognition of technical and personnel limitations. 3.

Preanesthetic Medications

Preanesthetic drugs are less frequently used in rodents than in larger species. However, the advantages of sedation, analgesia, and reduced doses of the general anesthetic agent or agents apply equally well to rodents. The principal disadvantage is probably the necessity for handling the animals twice rather than once for the induction of anesthesia. Drugs commonly used as preanesthetic agents in other species are frequently incorporated into anesthetic cocktails for rodents. However, if postprocedural analgesia is needed following very short procedures, or if preemptive analgesia is desired, then some means of preanesthetic administration must be used. Preanesthetic administration of tranquilizers, including the phenothiazine derivatives, benzodiazepines, and potent analgesics will all tend to substantially reduce the required dose of the principal anesthetic agent or agents.

C.

Anesthetic Agents

A summary of commonly used drug doses is provided in Tables I-V. These dosages should be used as guidelines, not as firm recommendations. Although guidelines are useful starting

points, final determination of optimum doses depends on careful observation of the animal's responses, consideration of experimental needs, and judicious fine tuning. 1.

Injectable Anesthetics

Injectable agents and combinations of agents are the most common choice for rodent anesthesia. Their ease of use and apparent simplicity tend to conceal complex actions and side effects, but when correctly used, injectable agents are safe, effective, and convenient. After a short discussion of injectable drug preparation and administration for rodents, the general characteristics of the agents will be described, followed by a brief description of typical uses in mice, rats, hamsters, and guinea pigs. Many drugs used in rodent anesthesia are formulated for use in larger species, with the result that their concentration is often too high for accurate and convenient administration to rodents. Measurement errors that might be inconsequential in larger patients can have large effects in small ones. Further, many drug mixtures are compounded extemporaneously by the user, sometimes without taking into account issues of measurement and dosing. These difficulties are addressed by diluting drugs to easily measurable concentrations. At the same time, final volumes can be adjusted to yield convenient dose rates, such as 0.2 ml per 100 gm body weight for a rat, or 0.10 ml per 10 gm body weight for a mouse. Thoughtful dilution and concentration adjustment increase not only the convenience but also the safety of drug administration in small rodents. Suitable diluents are water for injection, U.S.P. ("United States Pharmacopeia") or saline for injection, U.S.P. In the authors' experience, diluents containing preservatives should be avoided. Drug cocktail compounders should be sure that the mixtures are compatible, stable, and safe. Examples can be found in Flecknell (1996a). Intraperitoneal (IP) injection is commonly the route of choice because of the relatively large potential space for injection and the ease and rapidity with which it can be carried out. Even with reduced volumes, intramuscular injection is unreliable and usually painful in conscious rodents, and intravenous injection is often difficult, with the possible exception of rat, and perhaps mouse, tail veins. Subcutaneous injection is also relatively straightforward and useful for some drugs and for perioperative fluid support. a.

Barbiturates

Once the dominant drugs for rodent anesthesia, barbiturates are waning in popularity. At sufficient doses these sedative hypnotics produce general anesthesia with muscle relaxation, loss of consciousness, and failure to respond to noxious stimuli. As a class, they have minimal analgesic effect independent of their ability to affect consciousness. They also cause dose-related respiratory and cardiovascular depression, which worsens with

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time. As in other species, repeated doses of short-acting barbiturates are cumulative and will result in prolonged recovery. Barbiturates tend to have a narrow therapeutic window. In some species, such as hamsters and gerbils, the window is so narrow as to be a crevice, and considerable experience and finesse are required for safe use. Sodium pentobarbital is the most frequently used barbiturate for rodent anesthesia, and it is usually given IP. If suitably diluted, it can be given intravenously (IV) in intermittent boluses or as a continuous infusion. Whereas dose requirements are somewhat predictable for rats, the effect of a given dose of pentobarbital in mice can be highly variable, depending on strain, sex, age, and housing conditions (Lovell, 1986a,b,c). Recovery can be lengthy, especially with longer surgical procedures, and is further prolonged by hypothermia. Sodium thiopental provides brief anesthesia following a single dose. It is probably best given IV, as in larger species, in which case other and safer agents would ordinarily be preferred. Methohexital is an ultra-short-acting barbiturate that is sometimes used for its rapid induction and recovery characteristics. It has been given by both IP and IV routes. Duration of anesthesia is relatively brief and usually inadequate for surgery (Wixson and Smiler, 1997b; Flecknell, 1996b; Dorr and WeberFrisch, 1998). Inactin (EMTU, or ethylmalonyl urea) has a longer duration of effect than pentobarbital and is usually reserved for procedures requiring more than 3 hr in the rat (Buelke-Sam et al., 1978). The primary advantage of inactin is a greater duration of stable anesthesia. Propofol, an alkylphenol derivative, should be administered IV. It produces respiratory and cardiovascular depression if administered too rapidly or if used as the sole means of achieving surgical anesthesia (Glen and Hunter, 1984; Brammer et al., 1993). The vehicle supports microbial growth well, so careful aseptic technique is essential if an opened vial or bottle is to be kept for more than a brief time. In use, propofol resembles rapid-acting barbiturates, such as thiopental, with the important exception that recovery from propofol is relatively fast, even following repeated boluses or infusion. b.

Tribromoethanol (Avertin)

Once a nearly defunct sedative-hypnotic, tribromoethanol has not been commercially available under the proprietary name Avertin for many years. It has gained renewed life as a very popular anesthetic for the many brief surgical procedures needed in transgenic mouse production. Used properly, tribromoethanol is an adequate anesthetic for short surgical procedures in mice when given IP. For safety, it is essential that it be prepared, stored, and used properly. Tribromoethanol is not stable if stored at room temperature or if exposed to light. The products of decomposition are irritant and toxic, resulting in significant morbidity and mortality (Papaioannou and Fox, 1993).

c.

Dissociative Agents

Ketamine and tiletamine are the two representatives of this class, for which phencyclidine is the parent compound. Ketamine produces a degree of analgesia and immobility without muscle relaxation. Used alone, it is generally considered to be adequate for restraint but not for anesthesia. For this reason, and because of its wide margin of safety, ketamine is almost always combined with various tranquilizers and sedatives to make an almost infinite variety of anesthetic cocktails. Ketamine can be given orally, intramuscularly (IM), IV, and subcutaneously (SC) but, for operator convenience and animal comfort, is usually given IP. Typical ketamine mixtures are given in the dose tables and mentioned under the specific species discussions following this section. Tiletamine (Telazol) is available only in combination with zolazepam, a benzodiazepine. Telazol has been used alone or in combination with xylazine or butorphanol to produce anesthesia in rats (Silverman et al., 1983; Wilson et al., 1992, 1993). Aside from duration of effect, there seem to be few reasons to favor this more expensive combination in rodents over the various ketamine-based formulations. d.

Neuroleptics

These drugs are combinations of potent opioids and butyrophenone tranquilizers. The two that have seen widespread use are Innovar-Vet and Hypnorm. Innovar-Vet is no longer produced, and Hypnorm is available only as an investigational drug in the United States. Both products use fentanyl as the opioid analgesic, albeit at different concentrations. Whereas InnovarVet contained droperidol as the butyrophenone, Hypnorm contains fluanisone, apparently to better effect. In human practice these agents used alone would be termed neuroleptanalgesics. To produce neuroleptanesthesia, additional agents, such as nitrous oxide, would be added. In fact, the recommendations for Hypnorm in rats include the addition of a benzodiazepine, either diazepam or midazolam. The combination of Hypnorm and a benzodiazepine is reported to reliably produce adequate to good anesthesia in mice, rats, and hamsters (Green, 1975; Flecknell and Mitchell, 1984). e.

a2-Adrenergic Agonists

Members of this group used in rodent anesthesia include xylazine and medetomidine. These drugs are anxiolytic and analgesic and provide muscle relaxation. They also may cause hyperglycemia, bradycardia, peripheral vasoconstriction, hypothermia, and diuresis of variable severity in different species and with varying doses and drug mixtures (Hsu et al., 1986; Wixson et al., 1987a). Even so, the combination of ketamine and xylazine is generally reliable, safe, and convenient in rats and some other species and enjoys wide popularity (Green et al.,

959

22. PREANESTHESIA,ANESTHESIA,ANALGESIA,AND EUTHANASIA 1981; Wixson et aL, 1987). Ketamine can also be combined with medetomidine to provide similar effects (Nevlainen et al., 1989). The sedation produced by a2-adrenergics can be effectively antagonized. The sedation, diuresis, and cardiovascular effects produced by xylazine can be reversed by yohimbine (Hsu et al., 1986). Atipamezole was developed to antagonize a2-adrenergics, especially medetomidine, more specifically and rapidly (Flecknell, 1997). The ability to reverse the effects of these drugs can be used to advantage to shorten recovery time, as well as to rescue the occasional patient that displays severe adverse reactions to the drugs.

f.

Miscellaneous Injectables

a-Chloralose. As a sedative hypnotic that provides minimal analgesia, a-chloralose is often used for studies involving autonomic reflexes. It is recommended that painful manipulations, such as surgical procedures, should be carried out under a more effective anesthetic, after which a-chloralose can be substituted to produce prolonged stable study conditions (Wixson and Smiler, 1997c; Flecknell, 1996c; Silverman and Muir, 1993). It is not considered suitable for survival procedures. Alphaxolone-alphadolone (Saffan, Althesin). Alphaxolonealphadolone is a seldom used mixture of two steroidal anesthetic agents that can produce brief anesthesia. Anesthesia can be prolonged by intravenous infusion or intermittent IV bolus (Green et al., 1978). Alphaxolone-alphadolone is available in the United States only as an investigational drug. Chloral hydrate. As a sedative hypnotic, chloral hydrate at doses of 400 mg/kg IP can produce anesthesia for about 1-2 hr in rats, causing respiratory, cardiovascular, and thermoregulatory depression. Chloral hydrate has been associated with peritonitis and adynamic ileus, apparently related more to concentration than to total dose. It is suggested that the concentration not exceed 5% w/v (Silverman and Muir, 1993; Field et al., 1993; Vachon et al., 1999). Metomidate and etomidate. Metomidate and etomidate are imidazole 5-carbonic acid derivatives. Given IV, they produce rapid loss of consciousness, minimal analgesia, and good cardiovascular stability. Only etomidate (Amidate) is available in the United States. When used in rodents, they are usually combined with a potent opioid (Wixson and Smiler, 1997a; Flecknell, 1996a). Urethane. Ethyl carbamate, or urethane, provides greater analgesia than a-chloralose, as well as prolonged and relatively stable anesthesia in rats (Field et al., 1993). It is sometimes used in conjunction with a-chloralose, to gain the advantage of increased analgesia. Urethane is carcinogenic and is unsuitable for recovery anesthesia.

2.

Inhalation Anesthetics

Inhalation anesthesia circumvents many of the difficulties associated with injectable agents. Because the agents are used to effect, issues of dose calculation and variations in response do not arise. These agents are not controlled substances and also escape the burden of detailed record keeping required for barbiturates, opioids, benzodiazepines, and ketamine. Available agents include enflurane, halothane, isoflurane, sevoflurane, and desflurane. Methoxyflurane is not currently produced in the United States. Ether is sympathomimetic and is not recommended because of increased respiratory secretions as well as storage and safety concerns. In general, the remaining agents are characterized by rapid induction and recovery. To varying degrees, all inhalation anesthetics cause dose-related cardiovascular and respiratory depression, but these effects are frequently less severe than equipotent doses of injectable agents. No currently produced inhalation agents are analgesic at subanesthetic doses. Currently, isoflurane probably possesses the best combination of properties in terms of expense and safety of personnel and patient. Halothane is a direct myocardial depressant and undergoes somewhat greater metabolic degradation than isoflurane or desflurane, presenting a greater risk in terms of personnel exposure. Sevoflurane is expensive but can provide a smoother and more rapid induction and recovery than isoflurane. Desflurane is also expensive, requires a specialized vaporizer, and has minimal advantages over isoflurane. The equipment needed for rodent anesthesia is relatively simple, comprising a flowmeter, a vaporizer, an induction chamber, a delivery circuit, and some means of waste gas disposal. Face masks are commonly used in rodents, although endotracheal intubation, if mastered, permits assisted or controlled ventilation. The means of waste gas disposal varies with the facilities available. Because of increased interest in inhalation anesthesia for rodents, new equipment and innovative designs that more specifically address the needs of rodent anesthesia are becoming commercially available. Basic equipment and techniques for administration, including endotracheal intubation, are described in Vogler (1997) and Flecknell (1996a). Bell jar or anesthetic chambers may be used for induction of anesthesia with inhalant agents. However, this type of administration is dangerously imprecise and requires careful monitoring. Fume hoods or scavenging systems must be used to prevent environmental contamination with this technique (Flecknell, 1996b). Nitrous oxide is sometimes used to reduce the amount of potent inhalation anesthetic agent needed and to minimize respiratory and cardiovascular depression, but it is inadequate as a sole anesthetic. As with all inhalation agents, adequate scavenging is essential to avoid potential human health hazards. Scavenging systems that rely on activated charcoal are not effective for removing nitrous oxide. Carbon dioxide is used for momentary anesthesia, by chamber

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exposure to concentrations of 7 0 - 8 0 % COz, with the balance being oxygen. The effect is extremely brief but can be adequate for procedures such as orbital bleeding. The mixture is unsuitable for more than momentary applications.

wound. With care to assure adequate general anesthesia and to avoid toxicity, both short-acting (lidocaine) and longer-acting local anesthetics are useful. e.

3. Anesthestic Adjuvants a.

Phenothiazine Tranquilizers

Acepromazine and, to a lesser extent, chlorpromazine and promazine are the most common representatives of the large group of phenothiazine tranquilizers. They produce sedation and potentiate the effects of anesthetics but do not provide analgesia. They also cause vasodilation, with some depression of blood pressure, and should be used with great caution in dehydrated or hemorrhaging patients.

Neuromuscular Blocking Agents

Paralytics may be required for specific experimental protocols, but their use should be justified, because no analgesia is provided by these agents, and once the animal is paralyzed, it is difficult to assess anesthesia. For this reason the anesthetic protocol to be used should be demonstrated to be adequate to perform the study procedures humanely without the use of neuromuscular blocking agents. Their use in research is reviewed by Hildebrand (1997).

D. b.

Benzodiazepines

Diazepam and midazolam are often used as adjuvants to anesthesia. They have similar effects, including anxiolysis, sedation, and muscle relaxation but are not analgesic. Midazolam is shorter-acting and is water-soluble, making it preferable for mixtures intended to provide short-term anesthesia. The effects of these agents can be quickly terminated by a specific reversal agent, flumazenil. c.

Opioids

As anesthesia adjuvants, opioid agonists provide sovereign analgesia and permit the use of lower doses of anesthetic agents. Although a number of synthetic opioid agonists are available, fentanyl and alfentanil are probably the most frequently used in rodent anesthesia. Both provide analgesia with good cardiovascular stability, usually accompanied by bradycardia and respiratory depression, which may in some degree be balanced by the lower doses of anesthetics needed. Alfentanil is a relatively short-acting opioid and is often preferred for continuous infusion techniques. Atropine or glycopyrrolate can be used to counteract bradycardia but should be used with caution, if at all, when medetomidine is also a component of the anesthesia protocol, because of the occurrence of urinary retention in the mouse (Flecknell, 1997). Opioid agonists can be effectively reversed by specific antagonists, such as naloxone, as well as by some partial agonists and mixed-function agonistantagonists, such as butorphanol, nalbuphine, and buprenorphine, which are usually used for postoperative analgesia. d.

Local Anesthetics

Lidocaine, bupivicaine, and others are often used to supplement light levels of general anesthesia, as well as to provide some degree of postoperative analgesia. In rodents they are usually given by local infiltration or topical application to the

Intraoperative Monitoring and Support

Monitoring anesthesia in rodents is often limited to observation of respiratory rate and character, color (if the animal is an albino), and response to surgical stimulus. Depth of anesthesia may be estimated by pedal withdrawal response and eye reflexes but is probably most reliably indicated by response to surgical stimulus (Whelan and Flecknell, 1992). Heart rate and oxygen saturation can be monitored in larger animals, such as rats and guinea pigs, by pulse oximetry, using newer veterinary instruments, but this is generally not feasible in smaller rodents. Research instruments can measure inspired and expired carbon dioxide, but monitors suitable for routine use in rodents are not available. Electrocardiography is usually used only as a research tool. Hypothermia is a reliable feature of anesthesia and surgery in rodents. Temperature can be monitored by using small rectal and surface probes, and surgical support should include measures to maintain normothermia. Approaches to thermal support such as circulating hot-water pads or heating lamps are discussed in Wixson and Smiler (1997a) and Flecknell (1996a). Fluid support in the form of warmed subcutaneous fluids is useful and simple, especially with prolonged procedures. Another reliable feature of anesthesia in rodents is modest to severe hypoxia and hypercapnia. Moderate hypercapnia can be tolerated by healthy animals for reasonable periods of time and may be difficult to correct without recourse to mechanical ventilation. Hypoxia, on the other hand, is often easily and inexpensively corrected by providing a low flow of oxygen by mask during surgery, even when multiple animals are undergoing surgery simultaneously.

E.

Special Anesthetic Considerations

1. Anesthesia for Pregnant Rodents Apart from the widespread and successful use of tribromoethanol in transgenic mouse production, information is lim-

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA

ited. Suitability of an anesthetic protocol will depend to some extent upon the stage of pregnancy and the species to be used. General principles and approaches are given in Wixson and Smiler (1997a) and Flecknell (1996a). 2.

Neonatal Anesthesia

Pentobarbital, fentanyl-droperidol, and hypothermia and inhalation agents have all been used successfully for neonatal anesthesia (Wixson and Smiler, 1997a; Parks et al., 1992; Danneman and Mandrell, 1997). Of these methods, hypothermia and inhalation techniques were the most consistently successful. Hypothermia is the preferred alternative if inhalation equipment is not available. Handling and manipulation of the neonate may result in cannibalism. 3.

Stereotactic and Neurosurgery

Anesthesia for stereotactic surgery has usually relied on the same anesthetics used for other purposes. Because placement of ear bars for fixation of the head is often more painful than the surgery itself, a relatively deep plane of anesthesia is needed. Removal of the ear bars following surgery can result in an abrupt increase in the apparent depth of anesthesia (Gardiner and Toth, 1999). Face-mask adapters, used in place of the incisor bar, are now commercially available, allowing more convenient use of inhalation techniques. For nonsurvival studies involving measurement of autonomic reflexes, a-chloralose, with or without urethane, is often used. Control of blood gas values may be required and will usually entail mechanical ventilation. 4.

Anesthesia for Imaging Procedures

Magnetic resonance imaging (MRI) of rodents presents difficulties in terms of administration methods, thermal support, and monitoring. For short procedures, any injectable method compatible with the study can be used. For longer procedures, infusion techniques or inhalation anesthesia is usually more satisfactory. Although infusion pumps and conventional inhalation equipment must be kept at a safe distance, both can be effectively used. Long, small-bore infusion lines can be used to deliver injectable agents. For inhalation techniques, rodents can be placed in plastic cylinders with fresh gas entering at one end of the cylinder and exiting at the opposite end to the scavenging system. Monitoring may be limited to intermittent visual observations, but with larger rodents, MRI-compatible pulse oximeters may be used. Methods for thermal support also require innovation. Wrapping the animal in insulation might interfere with imaging and visual monitoring. Passive warming devices, such as warmed fluid bags can be useful. If infusion techniques are used, a commercial or homemade warm-air system can be very effective (Wong, 1997).

961 F.

Analgesic Therapy

Recognition and relief of pain in rodents have received increasing attention. The ethical and scientific value of relieving pain and distress in experimental animals is well accepted in principle but remains a challenging and often controversial topic in practice. 1.

Assessment of Pain/Discomfort

Species-specific signs of acute and chronic pain and approaches to pain recognition and to providing and monitoring analgesia are described in Danneman (1997), Flecknell (1996a), and elsewhere. Carefully constructed scoring systems, specifically adapted to the conditions of the protocol, are particularly useful in recognizing pain and monitoring analgesia in rodents. 2.

Nonpharmacologic Methods

Almost invariably, the term analgesia is associated with drugs. But drugs cannot be substituted for meticulous surgical technique, nor for excellent nursing and husbandry. Comfortable bedding, easy access to palatable food and water, and attention to wound care and to issues of social deprivation will greatly improve the quality of postoperative recovery. 3.

Pharmacologic Methods

The major drugs used for analgesia include opioids and nonsteroidal anti-inflammatory drugs (NSAIDs). As with all drugs used in anesthesia and analgesia, these agents are generally safe and effective but are not completely free of adverse effects. a.

Opioids

For severe pain, pure opioid agonists, such as morphine, are preferred, despite a relatively brief period of effect (Wixson and Smiler, 1997a; Flecknell, 1996a). For pain of moderate or lower intensity, partial opioid agonists and mixed-function agonistantagonists, such as butorphanol, nalbuphine, and buprenorphine are often used. Buprenorphine is popular because of its greater duration of effect and relatively low incidence of adverse effects. However, in the rat, pica and possibly long-term weight reductions have been reported (Clark et al., 1997; Jacobsen, 2000). Although usually given as an injection, buprenorphine can also be delivered orally (Flecknell, 1999a). b.

NSAIDs

Newer drugs, such as carprofen and ketoprofen, are potent and effective in many cases, and their use has been reviewed and reported (Liles and Flecknell, 1992; Flecknell, 1999b). Acetaminophen was shown by Cooper et al. (1997) to be effective in the rat when delivered parenterally but had little demonstrable effect when administered in drinking water.

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962 c.

Local Anesthesia

Infiltration of incision sites can be used to decrease postoperative pain, but the duration of effect is relatively brief, and with currently available drugs the technique may be more useful intraoperatively than in the postoperative period. Spinal and epidural administration of analgesics has not been reported for clinical analgesia in rodents.

G.

ceptable anesthetic protocol with new techniques or agents. Published studies concerning anesthetic techniques in hamsters, gerbils, and guinea pigs are few compared with those for mice and rats, reflecting their relative numbers as research animals. To an even greater degree, information concerning analgesia is scant. Commonly suggested agents and doses are given below and in the dose tables, but the phrase "to effect" assumes particular significance with these species (Tables I-V). 1.

Species Considerations

Even within species, rodents manifest surprising variability in response to "standard" doses of injectable agents and even inhalation agents. A pilot study is the best way to establish an ac-

Mice

Table I lists selected anesthetics and analgesia doses for mice. Tribromoethanol is usually preferred in transgenic production facilities, yielding safe and effective surgical anesthesia for at least 15 min when properly used (Papaioannou and Fox, 1993).

Table I

Mice: SelectedAnesthetics and Analgesic Dosesa Drug Tranquilizers Acepromazine Diazepam Midazolam Anesthestics Barbiturates Thiopental Methohexital Pentobarbital Dissociative agents and combinations Ketamine + acepromazine Ketamine + medetomidine Ketamine + xylazine Neuroleptanesthetics Fentanyl-fluanisone + (Hypnorm) midazolam Inhalant agents CO2/O2 Halothane Isoflurane Other agents Alphaxolone-alphadolone Propofol Tribromoethanol Analgesics Buprenorphine Butorphanol Morphine Acetaminophen Miscellaneous Atropine

Dose

Route

Duration

2-5 mg/kg 5 mg/kg 5 mg/kg

SC, IP IP IP

? min ? min ? min

25-50 mg/kg 8-16 mg/kg 30-50 mg/kg

IV IV IP

10 min 2-5 min 20-40 min

100 mg/kg 5 mg/kg 75 mg/kg 1 mg/kg 80-100 mg/kg 10 mg/kg

IP

20-30 min

IP

20-30 min

IP

20-30 min

10.0 ml/kg b

IP

30-40 min

70 - 80%/20 - 30% 1-4%, to effect 1-4%, to effect

Inhalant Inhalant Inhalant

10-15 mg/kg 12-26 mg/kg 240 mg/kg

IV IV IP

5-10 min 5-10 min 15-45 min

0.05-0.1 mg/kg 1-5 mg/kg 2.5 mg/kg 200 mg/kg

SC SC SC PO

8hr 4hr 2-4hr ?hr

0.04 mg/kg

SC

? min

aDoses adapted with modificationsfrom Wixson and Smiler (1997a) and Flecknell (1996a). bSee Flecknell (1996a) for mixing instructions.

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA Combinations of ketamine and xylazine, or ketamine and medetomidine, given IP, may produce surgical anesthesia for up to 30 min, although depth of anesthesia may be variable (Erhardt et al., 1984; Voipio et al., 1990). Reversal of xylazine or medetomidine with atipamezole can significantly shorten the recovery period. If available, fentanyl-fluanisone plus midazolam is often the preferred choice; details of preparation and use are described in Flecknell (1996a). Pentobarbital can be used with suitable precautions (Wixson and Smiler, 1997a; Flecknell, 1996a). Carbon dioxide and oxygen will provide brief restraint.

963

With proper equipment and training, inhalation anesthesia is effective, safe, and simple. Endotracheal intubation is difficult. C o m m o n l y used analgesics include buprenorphine or NSAIDs. 2.

Rats

Ketamine with xylazine, or fentanyl-fluanisone with midazolam, if available, are the most c o m m o n l y used and perhaps the most reliable for routine use with rats (see Table II) (Wixson and Smiler, 1997a; Flecknell, 1996a). Pentobarbital is less reliable

Table II

Rats: Drug Dosesa Drug Tranquilizers Acepromazine Diazepam Midazolam Anesthestics Barbiturates Thiopental Methohexital Pentobarbital EMTU (inactin) Dissociative agents and combinations Ketamine + acepromazine Ketamine + medetomidine Ketamine + xylazine Neuroleptanesthetics Fentanyl-fluanisone + (Hypnorm) midazolam Inhalant agents

C02/02

Halothane Isoflurane Other agents a-Chloralose Alphaxolone-alphadolone Chloral hydrate Propofol Urethane Analgesics Buprenorphine Butorphanol Carprofen Ketoprofen Morphine Miscellaneous Atropine Atipamezole

Dose

Route

2-5 mg/kg 5-15 mg/kg 5 mg/kg

SC, IP SC IP

? min ? min ? min

20-40 mg/kg 10-15 mg/kg 40-60 mg/kg 80 - 100 mg/kg

IV IV IP IP

5-10 min 5-10 min 20-60 min 60-240 min

75 mg/kg 2.5 mg/kg 75 mg/kg 0.5 mg/kg 40-75 mg/kg 5-10 mg/kg

IP

20-30 min

IP

20-30 min

IP

20-40 min

2.7 ml/kg b

IP

30-40 min

70-80%/20-30% 1-4%, to effect 1-4%, to effect

Inhalant Inhalant Inhalant

55-65 mg/kg 10-15 mg/kg 300-450 mg/kg 7.5-10 mg/kg 1000-1500 mg/kg

IP IV IP IV IP

8-10 hr 5-10 min 60-120 min 5-10 min 8-24 hr

0.01-0.05 mg/kg 2 mg/kg 5 mg/kg 5 mg/kg 2.5 mg/kg

SC SC SC SC SC

8-12 hr 4hr ?hr ?hr 2-4hr

0.04 mg/kg 0.1-1 mg/kg, varies with a2 dose

SC IP, SC

? min

aDoses adapted with modifications from Wixson and Smiler (1997a) and Flecknell (1996a). bSee Flecknell (1996a) for mixing instructions.

Duration

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964

but remains useful, especially for nonrecovery applications. For long-duration anesthesia using injectable drugs, infusion techniques with propofol, alphaxolone-alphadolone, or other agents are more satisfactory than repeated intermittent injections. Carbon dioxide and oxygen are often used for brief restraint, achloralose and urethane are useful in limited circumstances with the precautions described in Section II,C,l,f. Inhalation anesthesia has many advantages in the rat and is increasingly popular. Endotracheal intubation is feasible with practice (Tran and Lawson, 1986; Cambron et al., 1995; Flecknell, 1996a). Buprenorphine is commonly used and safe, although there are some reports of pica and reduced long-term weight gain following use (Clark et al., 1997; Jacobsen, 2000), as well as adverse reactions when used in combination with medetomidine (Hedenqvist

et al., 2000). NSAIDs, including carprofen and ketoprofen, are also useful (Flecknell, 1999b). 3.

Hamsters

As with rats, ketamine with xylazine, or fentanyl-fluanisone with midazolam, appears to be safe and reliable for procedures of moderate duration with hamsters (see Table III) (Wixson and Smiler, 1997a; Flecknell, 1996a). A combination of Telazol and xylazine given IP was reported to produce surgical anesthesia (Forsythe et al., 1992). Pentobarbital is reported to have a narrow margin of safety in hamsters and should be used with caution (Flecknell, 1996a). Inhalation anesthesia is safe and effective with the equipment and techniques used for other rodents.

Table III

Hamsters: Drug Dosesa Drug Tranquilizers Acepromazine Diazepam Anesthestics Barbiturates Methohexital + diazepam Pentobarbital Dissociative agents and combinations Ketamine + acepromazine Ketamine + medetomidine Ketamine + xylazine Tiletamine-zolazepam + xylazine Neuroleptanesthetics Fentanyl-fluanisone + (Hypnorm) midazolam Inhalant agents CO 2/ O 2 Halothane Isoflurane Other agents a-Chloralose (hypnosis only) Alphaxolone-alphadolone Urethane Analgesics Buprenorphine Butorphanol Morphine Acetaminophen Miscellaneous Atropine

Dose

Route

Duration

5 mg/kg 5 mg/kg

IP IP

? min ? min

15-30 mg/kg 2.5-5 mg/ml 50-90 mg/kg

IP

15-30 min

IP

30-60 min

150 mg/kg 5 mg/kg 100 mg/kg 0.25 mg/kg 100-200 mg/kg 10 mg/kg 20- 30 mg/kg 10 mg/kg

IP

45-120 min

IP

30-60 min

IP

30-60 min

IP

10-30 min

4.0 ml/kg ~

IP

20-40 min

70 - 80%/20 - 30% 1-4%, to effect 1-4%, to effect

Inhalant Inhalant Inhalant

80-100 mg/kg 120-160 mg/kg 1000- 2000 mg/kg

IP IV IP

3-4hr 40-60 min 6-8hr

0.01-0.5 mg/kg (?) 1-5 mg/kg 2.5 mg/kg 200 mg/kg

SC SC SC PO

?hr 4hr 2-4hr ?hr

0.04 mg/kg

SC

?hr

aDoses adapted with modifications from Wixson and Smiler (1997a) and Flecknell (1996a). bSee Flecknell (1996a) for mixing instructions.

965

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA

Endotracheal intubation is somewhat easier than in rats and can be accomplished using the same techniques. 4.

Gerbils

Reliable anesthesia in gerbils is produced using a combination of fentanyl and metomidate, with fentanyl-fluanisone and ketamine plus medetomidine being less dependable in producing a surgical plane of anesthesia (see Table IV) (Flecknell, 1996a). Alternatively, Telazol given IP also resulted in surgical anesthesia but with a prolonged recovery time (Hrapkiewicz et al., 1989). Pentobarbital should be used with caution. Inhalation anesthesia using halothane or isoflurane may be the safest and most reliable means of anesthetizing these small rodents. 5.

Guinea Pigs

Guinea pigs are considered to be among the more difficult laboratory animals to safely anesthetize. Although rarely aggres-

sive, guinea pigs are very apprehensive in novel surroundings, have few accessible peripheral vessels and unusually tough skin, and can be difficult to intubate. Successful anesthesia and recovery require careful monitoring and perioperative support, as well as appropriate agents, dose routes, and doses (see Table V). Intramuscular administration of Innovar-Vet and of ketamine with acepromazine has been reported to cause self-mutilation, and the IM route is probably best avoided (Leash et al., 1973; Latt and Ecobichon, 1984). Ketamine with xylazine given IP will usually result in an adequate surgical plane for about 30 min but may require supplementation with an inhalant (Radde et al., 1996). Fentanyl-fluanisone with midazolam is also effective for a similar period of time (Flecknell et al., 1984). Judicious use of local anesthetics can reduce the risks of deep anesthesia and the dangers associated with repeated doses of injectable agents. Endotracheal intubation is difficult in the guinea pig, so inhalation anesthesia is usually maintained by mask, using halothane or isoflurane. In the authors' experience, laboratory guinea pigs often have a considerable amount of pasty feed in their mouths,

Table IV

Gerbils: Drug Dosesa Drug

Dose

Route

Duration

Tranquilizers

Midazolam Medetomidine

5 mg/kg 0.1-0.2 mg/kg

IP IP

? min ? min

36-100 mg/kg

IP

50-60 min

75 mg/kg 3 mg/kg 75 mg/kg 0.5 mg/kg 50 mg/kg 2 mg/kg

IP

60-90 min

IP

20-30 min

IP

20-50 min

8.0 ml/kg b

IP

20 min

70 - 80%/20- 30% 1-4%, to effect 1-4%, to effect

Inhalant Inhalant Inhalant

80-120 mg/kg 75 mg/kg 0.5 mg/kg 250-300 mg/kg

IP IP

75 min 20-30 min

IP

15-30 min

0.01-0.5 mg/kg (?)

SC

?hr

0.04 mg/kg

SC

?hr

Anesthestics

Barbiturates Pentobarbital Dissociative agents and combinations Ketamine + acepromazine Ketamine + medetomidine Ketamine + xylazine Neuroleptanesthetics Fentanyl-fluanisone + (Hypnorm) midazolam Inhalant agents CO2/ O2 Halothane Isoflurane Other agents Alphacloxone-alphadolone Fentanyl + medetomidine Tribromoethanol Analgesic

Buprenorphine Miscellaneous

Atropine

aDoses adapted with modifications from Wixson and Smiler (1997a) and Flecknell (1996a). bSee Flecknell (1996a) for mixing instructions.

M. MICHAEL SWINDLE, ET AL.

966

Table V Guinea Pigs: Drug Dosesa Drug Tranquilizers Acepromazine Midazolam Anesthestics Barbiturates Pentobarbital Dissociative agents and combinations Ketamine + acepromazine Ketamine + medetomidine Ketamine + xylazine Neuroleptanesthetics Fentanyl-fluanisone + (Hypnorm) midazolam Inhalant Agents Halothane Isoflurane Other agents a-Chloralose Urethane Analgesics Buprenorphine Codeine Morphine Miscellaneous Atropine

Dose

Route

Duration

2.5-5.0 mg/kg 5 mg/kg

IP

? min ? min

15-40 mg/kg

IP

60-90 min

125 mg/kg 5 mg/kg 40 mg/kg 0.5 mg/kg 40 mg/kg 5 mg/kg

IP

45-120 min

IP

30-40 min

IP

30 min

8.0 ml/kg b

IP

45-60 min

1-4%, to effect 1-4%, to effect

Inhalant Inhalant

70 mg/kg 1500 mg/kg

IP IV, IP

3-10 hr 5-8hr

0.01-0.05 mg/kg (?) 25-40 mg/kg 5-15 mg/kg

SC SC SC

8hr 4hr 2-4hr

0.05 mg/kg

SC

?hr

aDoses adapted with modificationsfrom Wixson and Smiler (1997a) and Flecknell (1996a). bSee Flecknell (1996a)for mixing instructions.

which can contribute to airway obstruction when they are anesthetized. This residue can be removed or reduced by gently rinsing the mouth with 1 0 - 2 0 ml of tap water before induction. H.

Euthanasia

Euthanasia methods must meet both experimental and humane criteria. Anesthetic overdose, using conventional anesthetic agents or specially formulated products, such as highly concentrated pentobarbital, are useful in selected circumstances. Carbon dioxide is the most common means of euthanasia for small rodents, although some controversy exists regarding administration rate and method (Andrews et al., 1993; Danneman et al., 1997; Hackbarth et al., 1999). Although death is produced relatively rapidly in adults, it is much slower in neonates. Cervical dislocation and decapitation are humane and rapid in small rodents if carried out by experienced operators. Guinea pigs may struggle excessively when exposed to carbon dioxide. In this

case, relatively quick and calm euthanasia has been achieved by passing carbon dioxide first through a small jar containing halothane- or isoflurane-impregnated cotton before introducing it into the euthanasia chamber (M. C. LaRegina, personal communication, 1985). This practice would result in levels of inhalant agent well in excess of those needed for anesthesia.

Ill.

A.

RABBITS

Introduction

The rabbit remains an important research animal by virtue of its convenient size, ease of handling, definition in a number of models, and availability at various ages and reproductive status. Rabbits have been considered difficult to anesthetize safely, a

22. PREANESTHESIA,ANESTHESIA,ANALGESIA,AND EUTHANASIA reputation earned when pentobarbital was in wide usage and rabbits were of questionable health status. With the development of newer anesthetic drugs and techniques, and with the availability of rabbits with defined health status from quality vendors, this reputation has been dispelled. Although rabbits continue to be an anesthetic challenge because of individual variation in drug response and their timid nature, there now exist numerous methods for safe and effective induction and maintenance of anesthesia in the rabbit (Wixson, 1994; Flecknell, 1996; Lipman et al., 1997). The purpose of this section is to provide a basic guide to commonly used techniques.

B.

Preoperative Assessment and Preparation

1. Preoperative Evaluation

Rabbits should be purchased specific pathogen-free for Pasand other infectious agents that might influence research. The two breeds most commonly used for research in the United States are the New Zealand White and the Dutch Belted rabbits. Rabbits that specifically model certain diseases, such as the Watanabe heritable hyperlipidemia rabbit, a model for familial hypercholesterolemia, may present additional challenges to the anesthetist. Rabbits within a particular study should be purchased from a specific vendor only, because genetic differences will exist among stocks maintained by different vendors. Rabbits should be allowed stabilization periods of a minimum of 72 hr prior to use in a research project. Testing during the stabilization period will be determined by the health status of the vendor colony, the duration of use, and the demands of the specific protocol. There is no consensus on the requirement for overnight food withdrawal for rabbits (Flecknell, 1996). Proponents argue that there is more consistent anesthesia and that the reduction in intragastric volume allows more effective diaphragmatic excursion with consequent improvements in ventilation. Opponents argue that coprophagia precludes complete gastric emptying and that the inability of the rabbit to vomit makes food withdrawal unnecessary. Rabbits under 3 kg should not have more than 12 hr of food withdrawal; these rabbits may develop metabolic acidosis and a decline in blood glucose concentration (Bonath et al., 1982). teurella multocida

2.

Choice of Anesthetic Technique

The choice of anesthetic technique is determined by the desired duration of anesthesia, the nature of the surgical stimulus, the physiologic effects of the technique, their potential impact on the variables studied, and the age and preoperative status of the animal. The hyperglycemic effect of a2-agonists such as xy-

967

lazine, for example, should be considered in studies where glucose concentration is important (Gleed, 1987). 3. Preoperative Medications

The use of preoperative medications may reduce the anesthetic requirement for maintenance agents, relieve patient apprehension, and facilitate handling. These agents suffice as agents of restraint when subjecting animals to noninvasive procedures like phlebotomy or mask induction with inhalants. Anticholinergics may be used to prevent vagal reflexes and to reduce salivary and tracheobronchial secretion that may compromise the airway during anesthesia. Atropine use may be ineffective in some rabbits because of the existence of atropinesterase, an enzyme that degrades atropine into inactive products (Ecobichon and Comeau, 1974). The enzyme may be present in up to 50% of rabbits and is heritable. Consequently, some authors have recommended very high doses of atropine, such as 1-2 mg/kg (Hall and Clarke, 1991), with frequent redosing, such as every 15-20 min (Sedgwick, 1986). Glycopyrrolate is an effective parasympatholytic agent in the rabbit and produces 60 min of elevation of heart rate, prevention of ketamine/xylazine-associated bradycardia, and antisialogogue effect when administered at a dose of 0.1 mg/kg IM (Olson et al., 1994). Tranquilizers used in rabbits include phenothiazines (principally acepromazine), benzodiazepines (diazepam and midazolam), and a2-agonists (xylazine and medetomidine). Medetomidine has greater affinity and selectivity for a2-recepters than does xylazine (Hellebrekers et al., 1996). These agents may be used as sole agents in minor procedures such as echocardiography, radiology, physical examination, and phlebotomy. Benzodiazepine and the ct2-agonists have specific antagonists that provide the anesthetist greater control over duration of sedation or anesthesia. Drug dosages are listed in Table VII.

C.

lntraoperative Anesthesia

In this section, the most commonly used anesthetic agents will be described. Dosages are given in Tables VI and VII. 1. Injectable Anesthetics

Injectables are useful for induction of anesthesia in preparation for the use of inhalants or as maintenance agents in short procedures. Increments of the initial dosage may be used to prolong anesthesia. The popularity of injectables may be attributed to their ease of administration, predictability, and reasonable efficacy and safety. Many injectable combinations cause hypoxemia; the use of supplemental oxygen should be considered (Hellebrekers et al., 1996; Peeters et aL, 1988).

968

M. MICHAEL SWINDLE, ET AL.

Table VI Rabbits: Drug Doses a

Anticholinergics Atropine Glycopyrrolate Sedatives/tranquilizers Diazepam Midazolam Acepromazine Xylazine Medetomidine

Barbiturates Thiopental Thiamylal sodium EMTU Methohexital Pentobarbital Dissociatives Ketamine Ketamine + xylazine Ketamine + xylazine (reverse with yohimbine) Acepromazine + ketamine + xylazine (preop. with atropine, 0.04 mg/kg IM) Ketamine + acepromazine Ketamine + diazepam Ketamine + medetomidine Neuroleptanalgesies Fentanyl-fluanisone Diazepam + fentanyl-fluanisone Midazolam + fentanyl-fluanisone Other Propofol Medetomidine + propofol medetomidine + midazolam + propofol

Route

Dosage

Drug

0.04-2.0 mg/kg (0.5 mg/kg commonly recommended) 0.1 mg/kg 5-10 mg/kg 1-2 mg/kg 2 mg/kg 0.75-10.0 mg/kg (0.75-1.0 mg/kg most frequently used)

IM, SC IM, SC IM IM, IV IP, IV IM

0.25 mg/kg 6 mg/kg 15-30 mg/kg 50 mg/kg 15 mg/kg 29 mg/kg 47.5 mg/kg 5-10 mg/kg 20-60 mg/kg

IV (1% solution) GTE b IV (2.5% solution) GTE IV (1% solution) GTE IV (2% solution) GTE IV GTE IV (1% solution) GTE IV

20-60 mg/kg 10 mg/kg 3 mg/kg 22-50 mg/kg 2.5-10 mg/kg 0.2 mg/kg 0.75-1.0 mg/kg 35-40 mg/kg 3-5 mg/kg

IM IV IV IM

75 mg/kg 5 mg/kg 60-80 mg/kg 5-10 mg/kg 25 mg/kg 0.5 mg/kg

IM IM (given 30 min prior to ketamine) IM IM (given 30 min prior to ketamine) IM SC

0.2-0.6 ml/kg 1.5-5 mg/kg 0.2-0.5 ml/kg (administer diazepam 5 min prior to fentanyl-fluanisone) 2 mg/kg 0.3 ml/kg (administer midazolam 5 min prior to fentanyl-fluanisone)

IM, SC IM, IV, IP

7.5-15 mg/kg

IV

0.25 mg/kg followed in 5 min by 4 mg/kg 0.25 mg/kg 0.5 mg/kg

IM IV IM IM

IV SC IM

IM, SC IP, IV IM

(continues)

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA

969

Table VI (Continued)

Drug

Dosage

a-Chloralose + urethane Urethane

Route

2 mg/kg

IV

32 mmol (10 gm)/liter in saline at dose of 258 ~tmol (80 mg)/kg 400-500 mg/kg (5.61 mmol/kg) in 1 liter of saline (2.81 mol/liter) 1-1.6 gm/kg 1.5 gm/kg

Reversal agents

a2-Antagonists Yohimbine Atipamezole Benzodiazepine antagonist: flumazenil Opioid antagonist: naloxone

IV (slowly)

IP IV

0.2 mg/kg

IV

0.001-0.1 mg/kg

IV

aAdapted from Lipman et aL (1997) and Wixson (1994). bGTE, given to effect.

2.

Dissociatives

As a sole agent, ketamine may be used for restraint for noninvasive procedures and for endotracheal intubation, although most investigators prefer using ketamine with another agent for this latter purpose (Lindquist, 1972; Green et al., 1981).

Ketamine has also been used in combination with xylazine, medetomidine, diazepam, and myriad other agents. Ketam i n e / x y l a z i n e may be a u g m e n t e d with acepromazine or butorphanol to provide anesthesia of greater duration and depth (Lipman et al., 1990; Marini et al., 1992). These agents provide approximately 3 0 - 4 5 min of loss of the pedal withdrawal

Table VII

Rabbits: Bolus or Infusion Regimensa Drug a-Chloralose + urethane a-Chloralose + urethane Sedation Ketamine + xylazine Maintenance Ketamine + xylazine Ketamine + xylazine Sedation: propofol Maintenance: propofol Induction Ketamine + xylazine Maintenance Propofol Fentanyl Vecuronium a

Dosage 60 mg/kg 400 mg/kg followed by 1-3 ml 1% a-chloralose q30-50 min 40-60 mg/kg 800 mg/kg, followed by 3-4 ml/hr 1% a-chloralose

Route

IV

35 mg/kg 5 mg/kg

IM IM

1 mg/min 0.1 mg/min 25 mg/kg 5 mg/kg

Continuous IV transfusion

1.5 mg/kg 0.2-0.6 mg/kg/min

IV One-third bolus dose over 1 min, remainder over 4 min IV bolus Continuous IV infusion

25 mg/kg 15 mg/kg

IM IM

0.6 mg/kg/min 0.48 mg/kg/min 0.003 mg/kg/min

Continuous IV infusion

Intermittent bolus or continuous IV infusion regimens. Adapted from Lipman et aL (1997).

M. MICHAELSWINDLE,ET AL.

970

reflex. Physiologic effects of these combinations include depression of respiratory rate, hypoxemia, hypercarbemia, hypotension, and bradycardia. A constant-rate infusion technique has also been described (Wyatt et al., 1989). Procedures of moderate surgical stimulus intensity (such as carotid or iliac endarterectomy) may be performed with ketamine/xylazine. Perineural injections of these combinations may lead to selftrauma. Medetomidine, administered IM with subsequent IV ketamine, produces approximately 20 min of surgical anesthesia. As with ketamine/xylazine techniques, moderate hypoxemia, bradycardia, and respiratory rate reduction occur (Hellebrekers et al., 1996). The combination agent Telazol (Parke-Davis, Morris Plains, New Jersey), which contains the dissociative tiletamine and the benzodiazepine zolazepam, has been shown to be nephrotoxic in the rabbit and ~s best avoided (Doerning et al., 1992).

and preservation of or increase in heart rate (Korner et al., 1968; Warren and Ledingham, 1978; Morita et al., 1987; Borkowski et al., 1990). Pentobarbital may influence variables in research and has been associated with less myocardial damage after coronary artery ligation when compared with halothane or a-chloralose (Chakrabarty et al., 1991) and reduction of plasma potassium ion concentration with consequent elevation of plasma renin and aldosterone concentration (Robson et al., 1981). Tolerance (tachyphylaxis) may be observed if animals are to be anesthetized more than once weekly. Pentobarbital has been used in conjunction with guaifenesin, diazepam, and xylazine. Thiamylal, thiopental, methohexital, and ethylmalonyl urea (EMTU) are other barbiturates that have been used in the rabbit. As with pentobarbital, these agents should be used as dilute solutions and injected slowly. b.

3.

Neuroleptanesthesia-Neuroleptanalgesia

The most useful agent of the neuroleptanesthesia-neuroleptanalgesia class is a combination of fentanyl and fluanisone (Hypnorm; Janssen Animal Health, Oxon, United Kingdom) available in Europe (Flecknell et al., 1983; Flecknell and Mitchell, 1984). It may be used for restraint or for sedation prior to administration of inhalation agents. The use of this agent after IV or IP diazepam or midazolam produces anesthesia of moderate duration; increments of Hypnorm IM or diazepam IV will prolong anesthesia. Premedication with parasympatholytics is important when using opioids. Naloxone, dopram, and various mixed agonist/antagonist opioids may be used to reverse fentanyl-induced respiratory depression. Use of mixed agonist/antagonist opioids for this purpose provides fentanyl reversal while preserving analgesia of various duration (DeCastro and Viars, 1968). When buprenorphine was used in this fashion to reverse Hypnorm, it provided 420 min of analgesia while reversing depression of respiratory rate and effecting normalization of oxygenation and carbon dioxide elimination (Flecknell et al., 1989). a.

Barbiturates

Pentobarbital is less commonly used than in the past. Successful pentobarbital anesthesia requires considerable finesse, because the doses at which surgical anesthesia and respiratory arrest occur are extremely close. Use of manual chest compression or compression of the thorax using an elasticized bandage may reverse pentobarbital-induced apnea. Slow IV injection of one-third the calculated dose of pentobarbital diluted 1:1 with saline enhances safety (Lipman et al., 1997). Physiologic effects may include respiratory depression or arrest, decreased arterial blood pressure, peripheral vasodilation, decreased cardiac output, depression of the vasopressor response to hemorrhage,

Propofol

Propofol has been evaluated in several studies, the sum of which suggests that as a sole agent it is suitable only for induction and noninvasive procedures (Blake et al., 1988; Ko et al., 1992; Aeschbacher and Webb, 1993a,b). Degree of sedation or anesthesia, as well as alteration of physiologic variables, depends upon infusion rate. Ko used medetomidineatropine and medetomidine midazolam atropine premedication prior to propofol induction and found the combination useful for anesthesia induction sufficient to achieve endotracheal intubation. Pedal withdrawal reflexes and preanesthetic levels of heart rate, respiratory rate, mean arterial pressure, and end tidal CO2 were preserved (Ko et al., 1992). Medetomidine administered IM and followed by IV propofol provides approximately 11 min of surgical anesthesia with clinically acceptable preservation of physiologic variables (Hellebrekers et al., 1996). c.

Urethane

Urethane continues to be used alone and in combination with other agents. Among its characteristics are a long duration of action, excellent muscle relaxation, numerous endocrine effects, hemolysis, prolonged recovery, carcinogenic potential, and reduced response of vascular smooth muscle to norepinephrine (Bree and Cohen, 1965; Maggi et al., 1984). These features require its regulation by institutional safety personnel and restrict its use in animals to nonsurvival procedures. Chloralose urethane has historically been used by physiologists because of its reputation for preservation of baroreceptor reflexes (Sebel and Lowdon, 1989). 4.

Inhalational Anesthesia

The inhalant agents are especially useful in rabbit anesthesia because of their reliability, efficacy, ease of manipulation of anesthetic depth, and reduction in recovery time when com-

22. PREANESTHESIA,ANESTHESIA,ANALGESIA,AND EUTHANASIA pared with many injectable agents. Invasive manipulations are best achieved through the use of inhalants. They may be administered via face mask or endotracheal tube. Rabbits should be sedated prior to face-mask inductions because the animals may struggle vigorously in this setting. Endotracheal tubes of 3.0-4.0 internal diameter may be used in most rabbits. Both blind and visual intubation techniques have been described and are summarized elsewhere (Lipman et aL, 1997). Anesthesia in rabbits may be maintained by using a chosen inhalant in balance with oxygen delivered via Bain circuit at 2 - 3 times the minute respiratory volume (Flecknell, 1996; Lipman et al., 1997). Inhalational anesthesia with isoflurane, enflurane, or halothane has recently been shown to protect the ischemic rabbit myocardium from infarction when compared with anesthesia with pentobarbital, propofol, or ketamine/ xylazine (Cope et al., 1997). a.

Isoflurane

Isoflurane is currently the most commonly used inhalant for rabbit anesthesia. Cardiac safety, rapid induction and recovery, minimal hepatic transformation, and attendant reduction in viscerotoxicity are all features of this agent (Blake et al., 1991). Disadvantages of isoflurane include breath holding at first exposure, hypotension, and respiratory depression. The MAC (minimum alveolar concentration) of isoflurane in rabbits is 2.05 ___ 0.18%, and 1.39 ___0.32% for halothane and 2.86 + 0.18% for enflurane (Drummond, 1985). The physiologic effects of 1.3 MAC isoflurane in rabbits include increased heart rate, preservation of hepatic blood flow, and reduction in cardiac output, respiratory rate, Pco2, mean arterial blood pressure and renal blood flow (Blake et aL, 1991). Isoflurane produces significantly less depression of myocardial contractility than does halothane of equivalent MAC concentration (1 MAC) (Marano et al., 1997). Nitrous oxide may be used as a carrier gas at a ratio of 2:1 (N20: O2), but concerns over waste gas pollution and recreational abuse have reduced the use of this agent. To avoid diffusion hypoxia when the technique includes N20, 10 min of pure 02 should be administered at the completion of anesthesia. b.

Other Inhalants

Halothane, enflurane, and methoxyflurane are far less commonly used in rabbit anesthesia. Desflurane and sevoflurane remain infrequently used.

971

scending order of usefulness and accuracy for determination of depth of anesthesia, are pinna, pedal withdrawal, corneal, and palpebral (Borkowski et al., 1990; Hellebrekers et al., 1996). Other indices of anesthetic depth, such as muscle tone, jaw tone, vocalization, and gross purposeful movement in response to surgical stimuli, may be used (Hellebrekers et al., 1996). Supplemental heat sources should be used judiciously both intraoperatively and postoperatively to reduce hypothermia and attendant changes in metabolism of injectable drugs and MAC reduction of inhalants. Drapes, circulating hot-water blankets, hot-water bottles, and use of warm IV and irrigation fluids should be considered, depending on the circumstances. Conventional veterinary monitoring equipment may be used to monitor such cardiopulmonary variables as heart rate and rhythm, direct arterial blood pressure, oxygen saturation, and pulse rate. End tidal CO2 may be evaluated but better reflects Pco2 when measured at the pulmonary tip of the endotracheal tube (Rich et aL, 1990). Arterial blood pressure measurement is facilitated by the presence of two large and percutaneously accessible peripheral arteries, the central auricular and saphenous arteries. Pulse oximetry is best performed using transmission clips on the tongue or reflectance probes intrarectally. Fluid infusion rate for most procedures of short to moderate duration is 10 ml/kg/hr. The rate for neurosurgical procedures is 4 ml/ kg/hr (Hindman et al., 1990).

E.

Special Anesthetic Considerations

1. Spinal Anesthesia Spinal anesthesia of the rabbit has been described in both clinical and research settings (Kero et al., 1981; Hughes et al., 1993). It has been used as a model for evaluating the pharmacology and toxicology of spinal anesthesia and analgesia. Both epidural and subarachnoid catheterization procedures have been described (Langerman et al., 1990; Jensen et al., 1992; Madsen et al., 1993). Although subarachnoid space cannulation requires exposure of the lumbar spinal column and incision of the ligamentum flavum, both surgical and percutaneous techniques have been described for epidural catheterization (Taguchi et aL, 1996; Malinosky et al., 1997). To the authors' knowledge, the use of analgesics administered through these routes has not been evaluated for the clinical setting in the rabbit.

2. Hypnosis D.

Intraoperative Monitoring and Support

Intraoperative monitoring and support of rabbits are similar to those of other animals of similar size. Monitoring of reflexes, body temperature, and cardiopulmonary variables should be performed by trained personnel. The reflexes, ranked in de-

Hypnosis, or the immobility response, describes a constellation of physiologic and behavioral changes in rabbits effected by physical manipulation with or without incantation and reduced lighting (Danneman et al., 1988). Gentle head and neck traction appears to be commonly, but not uniformly, used in this technique. Hypnotized rabbits exhibit miosis, analgesia,

M. MICHAEL SWINDLE,ET AL.

972

increased depth of respiration, and reduced respiratory rate, heart rate, and blood pressure. The immobility response is not inhibited by naloxone. Although fascinating to the experimentalist, variation in response among rabbits limits the usefulness of this technique. It should not be considered a suitable surrogate for analgesia or anesthesia. 3.

Long-term Anesthetic Preparations

Long-term anesthesia in the setting of nonsurvival surgery in the rabbit requires rigorous attention to hydration, body temperature, adequacy of anesthetic depth, and monitoring of physiologic variables (Lipman et al., 1997). For those techniques that include paralytics, guidelines described in the NIH Workshop on Preparation and Maintenance of Higher Mammals during Neuroscience Experiments should be consulted (National Institutes of Health, 1991). A common technique is for surgery to be performed without paralytics, so that adequacy of analgesia may be determined; paralytics are then used in conjunction with anesthesia during the period of data collection. The use of paralytics with a high "autonomic margin of safety" allow the experimentalist to use heart rate and blood pressure as indices of depth of anesthesia. Increases in these variables, suggestive of a sympathoadrenal response to anesthesia, should prompt administration of additional anesthetic. A typical regimen may include the following: induction and initial administration using an injectable technique, followed by maintenance with an inhalant or total IV infusion technique and pancuronium or vecuronium (Mills et al., 1987; Hindman et al., 1990; De Mulder et al., 1997). These techniques have been reviewed by Lipman et al. (1997).

F.

Acute and Chronic Analgesic Therapy

Preemptive administration of analgesia should be considered as part of the complete program of analgesia in rabbits subjected to surgery (see Table VIII). Use of local infiltrative techniques in association with general anesthesia may also be used to enhance postoperative analgesia. Topical and intracameral anesthetics should be considered as adjuncts to general anesthesia in ophthalmic procedures (Zemel et al., 1995; Barquet et al., 1999). EMLA cream (Astra, Westborough, Massachusetts), a mixture of prilocaine and lidocaine that is administered topically, is useful for vasodilation and analgesia during venotomy and arteriotomy procedures (Flecknell et al., 1990; Hellebrekers et al., 1996). 1. Assessment of Pain and Discomfort

The most frequently recognized signs of pain and discomfort in rabbits include inappetance, an unkempt appearance due to a failure to groom, and reduced activity. Postoperative evalu-

ation for pain is greatly assisted by preoperative assessment of the demeanor of individual rabbits and their food and water consumption. 2. Methods of Analgesic Drug Delivery

Parenteral analgesics may be administered IM, IV, or SC (Table VI). Intravenous administration of ~t-agonist opioids has been associated with muscle rigidity, opisthotonus, and oculogyric effects (Borkowski et al., 1990; Marini et al., 1993). Consequently, these drugs are best administered only after sedation with appropriate sedatives or as part of a neuroleptanesthetic or analgesic combination (Lipman et al., 1997). Oral administration, although used in rabbits, has not been fully characterized for efficacy to the authors' knowledge. Patch administration of analgesics has not been described in rabbits. 3.

Opioids

The opioid of choice in rabbits is buprenorphine, a partial agonist/antagonist with a long duration of action (8-12 hr) (Flecknell, 1996; Lipman et al., 1997). This agent has only mild respiratory depressant properties, in contrast to the pure ~t-agonists. A dose of 0.02-0.05 mg/kg IV, SQ, or IM produced 10 hr of analgesia when a thermal stimulus was used (Flecknell and Liles, 1990). Because onset of action is 30 min, this agent should be administered intraoperatively or preoperatively. Other opioid drugs that may be useful include morphine, which provides 2 - 4 hr of potent analgesia at doses of 2 - 5 mg/ kg SC or IM (Wixson, 1994). It may cause sedation, respiratory depression, and moderate histamine release. Butorphanol (0.10.5 mg/kg), a mixed agonist/antagonist agent, is useful in providing short-term analgesia in rabbits (4 hr). Butorphanol may

Table VIII Rabbit: Analgesicsa

Analgesic Aspirin Acetaminophen (with or withoutcodeine) Ibuprofen Buprenorphine Butorphanol Flunixin Piroxicam Meperidine Morphine Fentanyl Nalbuphine Pentazocine

Dosage 100 mg/kg per os 1 ml drug/100 ml drinking water 10-20 mg/kg, IV 4 hr 0.01-0.05 mg/kg SC, IV 6-12 hourly 0.1-0.5 mg/kg IV, 4 hourly 1.1 mg/kg IM, ? 12 hourly 0.2 mg/kg per os, 8 hourly 5-10 mg/kg SC, 2-3 hourly 2.5 mg/kg SC, 2-4 hourly 5-20 ~tg/kg,IV bolus 15 ~tg/kg,continuousinfusion over2 hr 1-2 mg/kg IV, 4-5 hourly 5 mg/kg IV, 2-4 hourly

aAdapted fromLipmanet al. (1997) and Wixson (1994).

22. PREANESTHESIA,ANESTHESIA,ANALGESIA,AND EUTHANASIA be added to acepromazine or xylazine to effect both sedation and analgesia (Lipman et al., 1997). 4.

NSAIDs

Numerous nonsteroidal antiinflammatory drugs (NSAIDs) have been advocated for rabbits, but rigorous data are lacking. Some of the agents that have been advocated are aspirin (10 mg/ kg SQ) and acetominophen-codeine at 1 ml drug/100 ml water (Lipman et al., 1997). In an experimental fracture model, both piroxicam and flunixin were shown to reduce limb swelling, but analgesic action was not independently evaluated (More et al., 1989). The cyclooxygenase inhibitor ketorolac tromethamine (Toradol; Roche Laboratories, Nutley, New Jersey), has been evaluated in the rabbit at various doses because of its antithrombotic effect (Shufflebarger et al., 1996; Delaporte-Cerceau et al., 1998). Unfortunately, analgesic activity independent of investigation of these antithrombotic and anti-inflammatory effects appears lacking. In another study using a rabbit model of acute temporomandibular joint inflammation, intraperitoneal and intra-articular administration of 50 gg of ketorolac decreased the generation of the inflammatory mediators, prostaglandin E:, and bradykinin (Swift et al., 1998). The drug is well absorbed with no untoward effects related to ophthalmic, IM, or intranasal administration (Rooks et al., 1985; Santus et al., 1993).

IV.

A.

DOGS

Introduction

The wealth of data on agents and techniques for the management of anesthesia of dogs results from the accumulation of clinical experience with this species and from the long-standing popularity of dogs as research models (Kohn et al., 1997; Thurmon and Benson, 1996). Customizing anesthetic techniques for a specific research project is possible through the application of this information. The goal of this section is to summarize the use of most common anesthesia and analgesia agents and techniques in a research setting. Dosages are given in Table IX.

B.

Preoperative Assessment and Preparation

1. Preoperative Evaluation

Preoperative assessment is an important starting point that helps formulate the anesthetic plan. It usually includes history of previous use, physical examination, and pertinent laboratory data. A limited physical examination usually includes obtaining

973

body weight and temperature and observing mucous membranes, capillary refill time, and auscultation of heart and lungs. Preanesthetic laboratory data should be customized to the needs of each protocol. Concomitant natural condition or experimental disease may influence selection of appropriate anesthetics. For example, animals with induced heart failure are less tolerant of induction with thiopental and may be better managed with high doses of fentanyl. Elevated progesterone and increased levels of endogenous endorphins reduce requirements for inhaled anesthetics in pregnant animals. 2.

Methods of Anesthetic Delivery

Intramuscular (IM) injections in dogs are commonly/made into the caudal muscles of the thigh. Subcutaneous (SC) sites include the interscapular region, the lateral thoracic region, and the lumbodorsal region. The uptake of drugs from SC, and to lesser degree IM, injection can be variable and influenced by the rate of hydration and local perfusion. Cephalic and, less commonly, saphenous veins can be used for both venipuncture and administration of drugs. In dogs, external jugular veins are easily accessible. Jugular vein catheters allow for a rapid infusion of emergency drugs and fluids and are less subject to positional problems. The femoral artery offers a site for insertion of the indwelling catheter to monitor direct blood pressure. Whenever the femoral artery is being used, manual pressure must be applied to prevent a hematoma formation, especially in anticoagulated animals. 3.

Preoperative Medication

Anticholinergic drugs are used to diminish salivary and bronchial secretions and to prevent bradycardia. Bradycardia is of potential significance in small and young animals because their cardiac output is heart rate-dependent. Atropine can be administered to block the cardiac vagal nerves and prevent excessive salivation. Inhibition of salivary and respiratory tract secretions is the primary rational for using glycopyrrolate as a premedication. It has longer duration of action than atropine ( 2 - 4 hr versus 30 min), and an increase in the heart rate is usually seen only after IV but not IM administration. Inclusion of an anticholinergic as part of preoperative medication is not indicated in cardiac surgery, because of potential for ventricular tachycardia and bigeminal patterns (Popilskis and Kohn, 1997). 4.

Tranquilizers and Sedatives

Acepromazine is a popular phenothiazine derivative tranquilizer for use in dogs. Prolonged tranquilization and hypotension are frequent side effects of acepromazine. A low dose of acepromazine (0.025 mg/kg IV) may offer a protection against arrythmogenic effects of epinephrine under halothane or barbiturate anesthesia (Dyson and Pettifer, 1997). Diazepam,

974

M. MICHAEL SWINDLE, E T AL.

Table IX Dogs: Drug Doses a Drug

Miscellaneous injectable restraint agents Diazepam Midazolam Acepromazine Medetomidine Opioids and neuroleptanalgesics Morphine Oxymorphone Butorphanol Fentanyl Acepromazine/butorphanol Acepromazine/buprenorphine Acepromazine/oxymorphone Midazolam/oxymorphone Specific antagonists a2-Antagonist atipamezole Benzodiazepam: flumazenil Opioid: naloxone Injectable anesthetics Propofol Etomidate Etomidate/medetomidine a-Chloralose Barbiturates Thiopental Pentobarbital Dissociative agents and combinations Ketamine/acepromazine Ketamine/midazolam Ketamine/diazepam Ketamine/diazepam/medetomidine Tiletamine-zolazepam (Telazol) Inhalants Halothane (MAC = 0.87%) Isoflurane (MAC = 1.3%) Sevoflurane (MAC = 2.09%) Analgesics Morphine Oxymorphone Meperidine Fentanyl (Duragesic) Fentanyl Buprenorphine Butorphanol Aspirin Ketorolac Carprofen Bupivicaine Miscellaneous Anticholinergics Atropine Glycopyrrolate

Dosage

Route

0.2-0.4 mg/kg 0.2-0.4 mg/kg 0.05-0.1 mg/kg 0.04 mg/kg

IM, IV IM, IV IM, SC IM

0.2-0.6 mg/kg 0.05-0.1 mg/kg 0.2-0.4 mg/kg 0.002-0.01 mg/kg 0.01-0.08 mg/kg/hr 0.05/0.2 mg/kg 0.05/0.075-0.01 mg/kg 0.05/0.1 mg/kg 0.1-0.2/0.05-0.1 mg/kg

IM, SC IM, IV IM, IV IV IV infusion IM, IV IV IM, IV IM, IV

0.04-0.5 mg/kg 0.2-5.0 mg/kg 0.01 mg/kg

IM IV IV, IM

4 - 6 mg/kg 0.2-0.4 mg/kg/min 1.5-3.0 mg/kg 0.5/0.015 mg/kg 80-100 mg/kg

IV IV infusion IV IV / IM IV

8-17 mg/kg 20- 30 mg/kg

IV IV

2-4/0.2 mg/kg 6/0.2-0.4 mg/kg 6/0.2-0.4 mg/kg 5/0.25/0.005 mg/kg 6-10 mg/kg

IM, IV IM, IV IV IV IM, SC, IV

1-3% 1-3%

0.25-0.5 mg/kg 0.1 mg/kg 0.1-0.2 mg/kg 0.1 mg/kg 2-4 mg/kg 50 ~tg 2 ktg/kg + 100 lxg/hr 0.01-0.02 mg/kg 0.1-0.5 mg/kg 10-20 mg/kg 15-30 mg 2.0-4.0 mg/kg 1.5 mg/kg (0.25% solution)

IV, IM, SC Epidural IV, IM, SC Epidural IM, SC Transdermal Epidural infusion IM, SC IM, IV PO

0.02-0.04 mg/kg 0.02 mg/kg

IM, SC, W IM, SC

PO, IM Intrapleural

(continues)

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA

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Table IX (Continued)

Drug Muscle relaxants Pancuronium Vecuronium Specific antagonist Neostigmine Antiarrhythrnics Ventricular dysrhythmia:lidocaine Supraventricular tachycardia: esmolol Cardiotonic drugs Amrinone (Inocor)

Calcium chloride Dobutamine Dopamine Ephedrine Norepinephrine (Levophed) Phenylephrine (Neo-Synephrine) Nitrovasodilators Sodium nitroprusside Nitroglycerin

Dosage

Route

0.1 mg/kg 0.05-0.1 mg/kg

IV IV

1-3 mg

IV (mix with atropine to prevent bradycardia)

2-4 mg/kg 20-50 ~tg/kg/min 0.25-0.5 mg/kg 50-200 ~tg/kg/min

IV Continuous infusion IV bolus Maintenance infusion

1-3 mg/kg 5-10 ~tg/kg/min 5-10 mg/kg 5-20 ~tg/kg/min 5-20 ~tg/kg/min 1- 3 ~tg/kg/min 2.5-5.0 mg 0.1-0.5 ~tg/kg/min 30-50 ~tgbolus, 1 ~tg/kg/min

IV slowly Infusion IV IV infusion IV infusion IV infusion for oliguria IV (1:10 dilution) IV infusion Infusion

1-10 ~tg/kg/min 1-5 ~tg/kg/min

IV IV

a benzodiazepine, is a useful sedative agent and is a viable alternative to acepromazine administration. It displays minimal cardiovascular depressant effects even at the higher doses. However, intramuscular injection of diazepam is painful and unreliable. In contrast, midazolam, a water-soluble benzodiazepine, is well absorbed after intramuscular injection. It is most frequently given IV in combination with ketamine to induce anesthesia. A specific antagonist, flumazenil, rapidly reverses the effects of diazepam and midazolam. When administered at the dose of 0.2 mg followed by a second dose of 0.3 mg/kg IV or IM, it reverses benzodiazepine-induced respiratory depression in dogs (Heniff et al., 1997). The use of xylazine, an t~2-agonist, has been diminishing in recent years. Marked bradycardia and cardiac arrest accompany xylazine administration in dogs. Medetomidine, a potent a2-agonist, produces a dose-dependent sedation and analgesia. Although hypotension occurs frequently, higher doses of medetomidine can raise the blood pressure because of an effect on peripheral receptors (Alibhai et al., 1996). Decrease in a respiratory rate is commonly observed, with some dogs showing signs of cyanosis. IV administration of medetomidine induces a diuretic effect in dogs that lasts up to 4 hr; therefore the drug should be used with caution in hypovolemic and dehydrated animals (Burton et al., 1998). Sedative and undesirable side effects of medetomidine can be rapidly reversed with atipamezole. Atropine may be used to prevent some of the side effects of the a2-agonists (Table IX).

C. 1.

Intraoperative Anesthesia

Dissociatives

A combination of ketamine and diazepam IV is often used to induce reliable sedation and avoid perioperative excitement. Ketamine and midazolam also provide a smooth induction and chemical restraint of short duration. Cardiovascular effects are minimal except for pronounced tachycardia. Other side effects include salivation and hypertonia. Addition of medetomidine IV improves the quality of anesthetic induction, ease of intubation, and extended duration of analgesia in dogs (Ko et al., 1998). However, respiratory depression is also evident; oxygen supplementation is recommended. Tiletamine-zolazepam (Telazol) is most useful when prolonged restraint is anticipated; the duration can be further extended by increasing the dose or administering supplemental doses. Tachycardia usually accompanies administration of Telazol, and characteristic rough recovery is often seen following Telazol anesthesia in dogs. Telazol, in combination with a2-adrenergic agonists or opioids, produces rapid induction and surgical anesthesia of approximately 1 hr duration. An anticholinergic agent is recommended to prevent vagal-mediated bradycardia (Kohn et al., 1997). a.

Propofol

Propofol provides a smooth induction with adequate muscle relaxation sufficient for procedures of short duration. Because

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976

rapid clearance of propofol contributes to a fast awakening, propofol is usually administered as a constant-rate infusion after induction by intravenous bolus. Apnea and decreased systemic blood pressure are noticeable side effects after propofol induction; accordingly, ventilatory and fluid support should be available. Analgesic properties of propofol can be augmented with an addition of a2-agonists such as medetomidine (Hellebrekers et al., 1998). Because of the potential for bacterial contamination and iatrogenic sepsis, unused portions of propofol should be discarded after use. b.

Etomidate

Etomidate infusion causes a decrease in respiratory function but minimal changes in hemodynamic values of a healthy dog. It is not generally used as a sole agent. However, in dogs with impaired left ventricular function (i.e., cardiomyopathy), there is an increase in left ventricular afterload that further diminishes cardiac performance during etomidate anesthesia. Adequate anesthesia with smooth recovery is produced by supplementing etomidate with medetomidine. Etomidate specifically suppresses endogenous cortisol production. The relative risk or benefit of blocking the stress response is still debatable (Dodam et aL, 1990). c.

Opioids

Opioids are often used in combination with other anesthetic drugs to provide control of surgical and postoperative pain. Morphine administration preoperatively provides sedation but is likely to cause vomiting and defecation in dogs. Oxymorphone in combination with acepromazine or midazolam produces reliable sedation and analgesia that is suitable for mildly invasive protocols (Ilkiw, 1992; Thurmon and Benson, 1996). Availability of specific antagonists offers an advantage of reversal of sedation and other side effects. Atropine is commonly added to this neuroleptanalgesic combination to reduce the incidence of bradycardia. Neuroleptanalgesic combinations (acepromazine/butorphanol, acepromazine/buprenorphine) provide good to excellent sedation, are well tolerated hemodynamically by healthy dogs, and are more appropriate for various radiographic studies (Stepien et al., 1995; Newell et al., 1997). They are also used if nonmanual restraint is desired, to reduce personnel exposure to radiation. The acepromazine/butorphanol combination does not significantly alter renal blood flow and may be useful for studies measuring glomerular filtration rate (Newell et al., 1997). However, it affects gastrointestinal motility and inhibits functional examination of the gastrointestinal (GI) tract (Scrivani, 1998). Fentanyl, sufentanil, and alfentanil, at higher doses, are useful induction agents in dogs with various degrees of heart failure.

a-Chloralose

a-Chloralose has been used as a continuous infusion for cardiovascular physiologic studies because it maintains hemodynamic function and reflexes similar to those of conscious dogs. It is considered to be an ineffective analgesic and is best used for prolonged nonsurvival procedures that require light anesthesia or combined with other agents. Morphine and induction agents such as thiopental have been used to avoid excitement and clonic convulsions associated with a-chloralose (Silverman and Muir, 1993). However, the use of other agents will diminish the cardiovascular sparing effects of the agent, and other anesthetics should be considered. d.

e.

Barbiturates

Thiobarbiturates are best used for induction or for very brief anesthesia. Thiopental is a popular and useful induction agent prior to maintenance with inhalational agents. Methohexital is an ultra-short-acting barbiturate that has been recommended for induction of greyhounds, which typically respond poorly to thiobarbiturates like thiopental. Pentobarbital is less desirable as a general anesthetic because of its narrow margin of safety, poor analgesic properties, and prolonged recovery. It retains its popularity as an anesthetic for nonsurvival surgeries. Animals should be provided with assisted ventilation because of pronounced respiratory depression that accompanies pentobarbital anesthesia (Green, 1979).

2.

Inhalational Anesthesia

The ability to titrate anesthesia to the desired effect and to administer anesthesia safely for prolonged periods of time has popularized inhalant anesthesia. Halothane and especially isoflurane have the widest application in dogs. a.

Isoflurane

Isoflurane provides almost ideal characteristics of rapid induction and recovery with minimal toxicity and acceptable levels of cardiopulmonary depression. In adequately sedated dogs, isoflurane can be used at concentrations of 4% to provide for mask induction. Relatively high oxygen flow rates ( 3 - 4 liter/ min) are needed for effective induction. The disadvantage of isoflurane induction by face mask is the struggling and associated stress that occurs in nonmedicated animals because of the pungent odor of the agent. Hence, induction is most easily accomplished with thiopental or propofol, and isoflurane is administered in concentrations of 0.75-2.25% to maintain general anesthesia. Although isoflurane produces a less pronounced effect on the myocardium, a moderate hypotension is usually associated with the administration of isoflurane. The hypotension is easily corrected with a bolus of phenylephrine 1-2 ~tg/ kg or a continuous infusion to effect. Supplementing isoflurane with 5-10 ~tg/kg of fentanyl may also ameliorate some hemodynamic side effects.

22. PREANESTHESIA,ANESTHESIA,ANALGESIA,AND EUTHANASIA b.

Sevoflurane

The use of newer inhalational anesthetics, such as sevoflurane, has been investigated as an alternative to isoflurane in dogs (Mutoh et al., 1997; Muir and Gadawski, 1998). Because of relatively high costs associated with the use of sevoflurane, it is commonly administered in a low-flow (15 ml/kg) system or in a closed circuit with the fresh gas flow at 3 ml/kg/min. There is a dose-dependent decrease in blood pressure, cardiac output, and stroke volume with the use of sevoflurane in dogs, but at equipotent doses, systemic and coronary vasodilatory effects of sevoflurane are less pronounced than those of isoflurane in dogs (Tomiyasu, 1999). Although sevoflurane does not cause airway irritation, it produces respiratory depression at clinically relevant concentrations. The recovery from sevoflurane anesthesia is fast and free of excitement and delirium in dogs.

c.

Nitrous Oxide

Nitrous oxide (N20) is usually administered with another potent inhalant agent to reduce the concentration of the second agent necessary for anesthesia. However, except in humans, relatively little reduction in other anesthetics is achieved through the use of nitrous oxide.

D.

Special Anesthetic Considerations

1. Cardiac Anesthesia

The dose, speed of induction, and selection of specific anesthetic protocols depend on the animal's normal or altered cardiac physiology and the potential of the anesthetic agent to disrupt physiologic compensatory mechanisms. Although premedication may be useful to reduce excitement and struggling, the use of a2-agonists and anticholinergic drugs should be avoided because of cardiac dysrhythmias associated with their use. Induction is usually accomplished with an IV bolus of thiopental or propofol; however, in animals with impaired cardiac output, induction is done safely with high dosages of fentanyl or other opioid agent supplemented with midazolam or propofol. Oxygen by face mask should be provided to avoid rapid desaturation. Isoflurane is the preferred maintenance agent because of its minimal effect on cardiac parameters and its ability to maintain normal rhythm. Intraoperative fentanyl infusion will attenuate the decrease in blood pressure associated with isoflurane. Placement of a femoral arterial catheter is often needed for continuous assessment of arterial blood pressure and analysis of arterial blood gases. Central venous pressure is obtained by cannulization of the external jugular vein for management of fluid therapy and administration of emergency drugs. IV boluses or continuous infusions of lidocaine may be necessary to treat or prevent ventricular arrhythmias. Phen-

977

ylephrine or norepinephrine are used to treat intraoperative hypotension. 2.

Anesthesia for Thoracic Surgery

Most of the preanesthetic and anesthetic drugs depress respiratory function, a condition that may last into the early postoperative period; their use should be carefully considered. Because controlled ventilation is instituted during thoracic surgery, the use of paralytic agents, such as pancuronium and vecuronium, in adequately anesthetized animals may be necessary for efficient ventilation and to reduce muscle fasciculation during lateral thoracotomy. Continuous monitoring of direct arterial blood pressure and heart rate will determine adequate analgesia. Pulse oximetry is essential to monitor saturation so as to detect arterial hypoxemia. Postoperative considerations following thoracic surgery include postoperative pain control (intercostal nerve blocks, epidural or parenteral opioids), prevention of atelectasis (reexpansion of lungs and suctioning of the trachea and oropharynx), and avoidance of pneumothrax (placement of the chest tube). 3.

Pediatric Anesthesia

Appropriate anesthetic management of pediatric anesthesia must be considered in a context of physiologic and pharmacologic differences between adult and pediatric animals. Because pediatric animals have a limited ability to increase stroke volume, their cardiac output is heart rate-dependent. Therefore, the use of a2-agonists and some opioids is best avoided. Inclusion of atropine in preoperative medication will reduce the likelihood of bradycardia. Airway obstruction and apnea lead to rapid desaturation because of high oxygen consumption. To prevent hypoxemia, especially for prolonged or invasive surgical procedures, puppies should be intubated with a noncuffed endotracheal tube. Maintaining normal body temperature is more difficult in puppies than in adults; therefore, active temperature monitoring needs to be instituted. Using heat lamps, thermal blankets, and warm intravenous fluids will minimize the severity of hypothermia. In general the choice of anesthetics for neonatal and pediatric animals does not differ greatly from that for adults. 4.

Anesthesia for Magnetic Resonance Imaging (MRI)

Anesthetizing an animal in MRI presents several challenging problems related to the physical environment. The constant magnetic field exerts a strong pull on ferromagnetic materials present in anesthesia machines and also interferes with anesthetic monitoring devices. Therefore, IV infusion of propofol or thiopental is administered to provide anesthesia during MRI. Respiratory depression manifested by hypoxemia is one of the potential problems associated with the infusion of IV

M. MICHAEL SWINDLE,ET AL.

978

anesthetics. A special gated pulse oximeter is available to monitor oxygenation and pulse rate of the anesthetized animal within the MRI environment.

E. Intraoperative Monitoring and Support The intensity and invasiveness of monitoring will vary with the protocol design, availability of resources, choice of anesthetic methods, health status of the animal, and anticipated physiologic challenges. Maintenance of an anesthetic record should be a part of perioperative monitoring in that it provides an ongoing record of trends and changes, and it represents a potentially valuable component of the research protocol. Ideally, heart rate, respiratory rate, blood pressure, pulse oximetry, and monitoring of body temperature should be provided. Heart rate and rhythm are evaluated by continuous electrocardiogram (ECG) monitoring. Arterial blood pressure can be estimated by placing an inflatable cuff around an extremity, with the width of the cuff about 40% of the circumference of the leg to which it is applied. The most accurate method to obtain arterial blood pressure in an anesthetized dog is by intra-arterial cannulation of the femoral or cranial tibial artery. This method not only allows for continuous monitoring of blood pressure but also provides an access for sampling of arterial blood gases. For complex surgical procedures that may produce cardiovascular dysfunctions, a pulmonary artery catheter is placed via the right external jugular catheter to assess cardiac function, preload volume, and efficacy of a variety of therapeutic interventions. Because the external jugular vein is easily accessible in dogs, cannulation with the a 18- or 20-gauge indwelling catheter allows for measurement of central venous pressure and rapid infusion of fluids or emergency drugs. Noninvasive and continuous measurement of oxygen saturation to detect arterial hypoxemia can be done with the help of a pulse oximetry finger probe attached to the dog's tongue. End tidal CO2 monitors can be effectively used to obtain information on an animal's airway patency and adequacy of ventilation. Attached between the breathing circuit and endotracheal tube, it displays CO2 levels and can also generate a CO2 waveform called a capnogram.

F. Postoperative Recovery The physiologic instability of an animal recovering from anesthesia and surgery requires close surveillance. Respiratory emergencies, circulatory complications, and postoperative hypothermia are commonly encountered in the immediate postoperative period. The residual effects of most anesthetics produce a depression of ventilation and either the accumulation of secretions or airway obstruction, conditions that may also lead to arterial hypoxemia. These conditions are best diagnosed by obtaining an arterial sample for the analysis of arterial blood gases. Supplemental nasal oxygen should be provided to correct

any inadequate postoperative oxygenation, especially in dogs recovering from cardiac or pulmonary procedures. To avoid airway obstruction, the oropharynx and trachea should be gently suctioned before the removal of the endotracheal tube and extubation should not be attempted until the dog regains a swallowing reflex. Circulatory complications frequently encountered during postoperative care include cardiac dysrhythmias and hypotension. Although tachycardia, as a result of inadequate pain relief, and hypovolemia or hypercarbia are commonly seen during the immediate postoperative recovery period, consecutive packed cell volume (PVC), and particularly ventricular tachycardia are ominous signs that should be given prompt attention. Repeated lidocaine boluses followed by a continuous IV infusion may be administered to suppress potentially lethal ventricular dysrhythmias. Maintaining urinary catheters during the immediate recovery period is important in the early recognition of insufficiently replaced intraoperative fluids or inadequate cardiac output. Providing the dog with a bolus of IV crystalloid solution usually corrects hypovolemia. The use of vasopressors (phenylephrine) or inotropes (dopamine, dobutamine) may be necessary to increase blood pressure or improve cardiac output. Hematocrit evaluation will help rule out a continuous blood loss. Hypothermia in the postoperative period should be treated with heat lamps or heating blankets. Space heaters may be used to preheat the recovery area.

G. Acute and Chronic Analgesic Therapy Pain is a predictable response to surgery as the effects of anesthetics dissipate in the early postoperative period. Effective postoperative analgesia is often dependent on appropriate assessment of pain in a recovering animal. Subjective evaluation of pain can be assessed by various pain scoring systems. A modified visual analog scale that incorporates behavioral indicators of pain (vocalization, changes in ambulation, etc.) provides the basis of subjective pain evaluation (Popilskis et al., 1991; Thompson and Johnson, 1991). Commonly with any pain scoring system, a higher number correlates with the severity of pain and thus provides a rationale for the use of analgesics. The opioids are effective in providing postoperative pain relief. Oxymorphone produces a profound analgesia with mild sedation and minimal respiratory depression. Butorphanol and buprenorphine provide moderate pain relief, which may be limited by a "ceiling effect." Buprenorphine induces a longer period of analgesia, making it an attractive alternative to other analgesic regimens. (Flecknell, 1987). Although opiates are effective in alleviating postoperative pain, conventional methods of opioid administration have some important drawbacks (Short, 1987). Namely, IM administration can result in variable absorption, especially in hypotensive and hypothermic animals, and thus can delay effective analgesia. Continuous IV infusion may carry a greater risk of respiratory

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22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA

depression and sedation, thus requiring close monitoring. The effective and safe alternative to parenteral opiates is the technique of epidural administration. The epidural method of pain relief offers an advantage of producing effective and prolonged analgesia with minimal depression of sympathetic nervous system, no changes in motor function, and absence of sedation. Morphine, being less lipidsoluble than other opiates, provides a twofold benefit for epidural morphine administration. Because morphine has limited uptake from epidural space, it allows more of the drug to penetrate into central spinal fluid and consequently more of the drug to be available at the receptor site in the spinal cord. Widespread distribution of morphine throughout the cerebrospinal fluid also makes lumbar administration as effective as thoracic for pain relief after upper abdominal and thoracic procedures. A single preemptive injection of epidural morphine provides effective postoperative analgesia up to 24 hr with minimal side effects (Popilskis et al., 1993). As the opioids become more soluble, the onset of action is reduced, but there is a decrease in duration of analgesia as well. Epidural oxymorphone produces effective analgesia for up to 10 hr in dogs (Popilskis et al., 1991; Vesal et al., 1996). Sedation and bradycardia are more pronounced with the use of this opioid. The fact that fentanyl is very lipophilic helps explain the short duration of analgesia and provides a rationale to administer epidural fentanyl via continuous infusion (Popilskis et al., 2000). The rationale of transdermal drug application using skin patches is to achieve constant plasma drug concentrations for a prolonged period. In dogs, application of a 50 gg transdermal fentanyl patch results in plasma fentanyl concentrations that are considered analgesic for 24-72 hr after application (Kyles et al., 1996).

caine and bupivicaine, which has a longer duration of activity, are used for local infiltration to produce sensory anesthesia around the incision site. The technique of specific nerve blocks in the dog is rarely used because of the more effective technique of epidural anesthesia. Lumbosacral epidural anesthesia is a safe and effective technique for surgical procedures caudal to the umbilicus in dogs (Skarda, 1989). Ultimately, improvement of postoperative pain management can be achieved with administration of analgesic drugs in combination, with the goal of altering more than one pathway of pain transmission. Balanced analgesic techniques appear to offer several advantages by enhancing and prolonging analgesia and reducing the incidence of side effects associated with administration of specific drug groups. The combination of epidural morphine and systemic ketorolac with addition of bupivicaine or metedomidine to epidural morphine is an example of combinations of analgesic drugs that can lead to considerable improvements in postoperative pain management.

H.

Euthanasia

Pentobarbital overdose (> 100 mg/kg IV) is the preferred form of parenteral euthanasia. Because physical restraint is an added stress to the animal and the technician, sedation prior to euthanasia may be necessary for uncooperative animals.

V.

A.

SWINE

Introduction

1. NSAIDs

Recently, more potent injectable NSAIDs have become available to treat postoperative pain. Carprofen and ketorolac have been shown to produce reliable postoperative analgesia with no sedation or respiratory depression. Preoperative administration of carprofen has also been shown to provide a preemptive analgesia effect in dogs (Lascelles et al., 1998). Caprofen also appears to be safer than other traditional NSAIDs (Fox and Johnston, 1997). Ketorolac inhibits platelet function for at least 24-48 hr, and it should be used with caution in animals with natural or acquired coagulopathies.

Research-oriented textbooks contain complete descriptions of anesthetic techniques and appropriate selection criteria for the various protocols specific to laboratory swine (Flecknell, 1996; Smith et al., 1997; Swindle, 1998). Veterinary-oriented textbooks also contain useful information on anesthesia in swine, but care must be taken to consider the physiologic effects of the anesthetics on the research protocol (Riebold et al., 1995; Thurmon and Benson, 1996). Information on handling and selection of swine for surgical protocols is included in Chapter 15 in this text as well as in a textbook by Swindle (1998). The purpose of this section is to provide an abbreviated guide to the most commonly used techniques in a research setting.

2. Local, Regional, and Epidural Anesthesia

Intercostal nerve blocks provide analgesia and contribute to improved pulmonary function after thoracotomy in dogs (Berg and Orton, 1986). As an alternative, insertion of an intrapleural catheter and administration of bupivicaine provide effective analgesia with fewer side effects than direct nerve blocks (Thompson and Johnson, 1991; Kushner et al., 1995). Lido-

B.

Preoperative Assessment and Preparation

1. Preoperative Evaluation

Swine should be selected from sources with a known health status and standardized preventive health program. They should

M. MICHAEL SWINDLE,ET AL.

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be stabilized and/or conditioned in the research institution for 5 - 7 days prior to performing anesthesia for survival surgery. Stabilization should include a physical exam as a minimum and, depending on the source and purpose of the research, laboratory tests, including a fecal exam, complete blood cell (CBC) count, and blood chemistry determination. Vaccination against common diseases may be appropriate for animals on long-term projects. A judgment should also be made in advance of the protocol on whether a particular breed or age of pig should be selected. The criteria for selection of swine for research projects and the differences between miniature and domestic farm breeds of swine are discussed in Chapter 15, as well as in Swindle (1998). Diseases of swine and their potential complications to research are also discussed in Chapter 15. 2.

Choice of Anesthetic Technique

The choice of anesthetic technique should be based on the physiologic effects of the anesthetic protocol and the potential complications that a particular protocol may have on the research being conducted. Many swine are used in cardiovascular research; therefore, stable hemodynamics, which may be greatly influenced by the anesthetic protocol, are important for many projects. The basic criteria for selection of anesthetics for swine are similar to those for other species. Malignant hyperthermia, discussed in Chapter 15, is a unique genetic condition in certain breeds of domestic swine. Swine herds can be prescreened for this potential complication, which is triggered by many inhalant and injectable anesthetic agents, particularly halothane. Swine may also have congenital heart defects, such as ventricular septal defect (VSD) and patent foramen ovale (PFO) (Swindle et al., 1992). Auscultation of swine prior to anesthesia is useful for detecting these conditions, as well as determining whether the animals have chronic respiratory diseases, which are common in some herds of domestic swine. 3.

Preoperative Medications

Preoperative medications may be appropriate to relieve anxiety and decrease the amount of general anesthetic to be administered. Anticholinergics may also be useful in preventing the vagal reflex that may occur during endotracheal intubation and manipulation of the cardiovascular and pulmonary systems. Both atropine and glycopyrrolate have been utilized successfully as anticholinergics in swine. Atropine is also useful to counteract bradycardia associated with some protocols, such as those that use high-dose opioids. Tranquilizers are mainly used to reduce anxiety, facilitate handling, and reduce the dosage of general anesthetics. The phenothiazine derivatives, especially acepromazine, have been widely used for this purpose. Benzodiazepine agents, such as diazepam and midazolam, are also used in swine for this purpose. Midazolam is used as a sole agent to provide approxi-

mately 20 min of relaxation to perform cardiovascular imaging techniques, such as echocardiography, with minimal effects on cardiovascular parameters (Swindle, 1998; Smith et al., 1991). Drug dosages are provided in Table X.

C.

Intraoperative Anesthesia

In this section, the appropriate selection of anesthetics used in research protocols is discussed. The drug dosages are included in Table X. 1. Injectable Anesthetics

Injectable anesthetics may be useful to induce anesthesia prior to administering an inhalant agent or for short-term procedures. Protocols that infuse these agents may be indicated in cases where inhalant anesthesia is not appropriate or is unavailable. Most injectable combinations only provide 2 0 - 3 0 min of surgical anesthesia. Consequently, it is preferable to administer continuous infusions of these injectable agents when longer anesthetic periods are required. Repeated bolus injections of these agents result in an unstable plane of anesthesia (Smith et al., 1997; Swindle, 1998). a.

Dissociatives

The dissociative agents, when administered alone, are not sufficient to provide surgical anesthesia or relaxation for endotracheal intubation. Ketamine and tiletamine-zolazepam (Telazol) are the two most widely used agents in swine. They are frequently combined with phenothiazine derivatives, benzodiazepines, and a2-agonists. Combinations include ketamine/acepromazine, ketamine/ azaperone, ketamine/diazepam, ketamine/midazolam, ketamine/xylazine, ketamine/medetomidine, and Telazol/xylazine (Thurmon et al., 1988; Swindle, 1998; Smith et al., 1997; Ko et al., 1992; Flecknell, 1996, 1997; Portier and Slusser, 1985). These combinations typically require the addition of other agents, such as barbiturates, or the administration of inhalant agents via a face mask in order to provide enough relaxation for endotracheal intubation. All of the combinations provide 2 0 30 min of restraint when administered IM. The combinations of ketamine/xylazine, ketamine/medetomidine, and Telazol/xylazine are sufficient to provide anesthesia for minor surgical procedures. Ketamine/medetomidine may provide up to 45 min of chemical restraint as a single injection but rapidly induces hypothermia. None of the combinations is suitable for visceral analgesia unless it is provided as a continuous IV infusion. An infusion of ketamine/xylazine/guiafenesin is useful for providing stable hemodynamics for cardiovascular protocols (Thurmon, 1986). Ketamine/medetomidine is preferred when an a2-agonist is indicated for a protocol, because it has less

981

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA

Table X Swine: Drug Dosesa Drug

Dosage

Route

Dissociative agents and combinations

Ketamine Ketamine + acepromazine Ketamine + diazepam Ketamine + xylazine Ketamine + fentanyl-droperidol (Innovar-Vet) Ketamine + azaperone Ketamine + xylazine + oxymorphone Ketamine + climazolam Tiletamine-zolazepam (Telazol) Tiletamine-zolazepam (Telazol) + xylazine

11-33 mg/kg 10-33 mg/kg/hr 22-33 mg/kg 1.1 mg/kg 15 mg/kg 2 mg/kg 20 mg/kg 2 mg/kg 11 mg/kg 1 ml/14 kg 15 mg/kg 2 mg/kg 2 mg/kg 2 mg/kg 0.075 mg/kg 20 mg/kg 5-1.0 mg/kg 4 - 6 mg/kg 4 - 6 mg/kg 2.2 mg/kg

IM, IV IV infusion IM IM IM IM IM IV (2 X dose for IM)

IM IM IM

Barbiturates

Pentobarbital Thiopental Thiamylal

20-40 mg/kg 5-40 mg/kg/hr 6.6-30 mg/kg 3-30 mg/kg/hr 6.6-30 mg/kg 3-30 mg/kg/hr

IV Continuous IV infusion IV Continuous IV infusion IV Continuous IV infusion

2-8 mg/kg 55-100 mg/kg 4-8 mg/kg 0.245 mg/10 kg 0.3 mg/kg 1 ml/kg/hr 100-500 gg/kg 4 mg/kg 1 mg/lb 1 mg/lb followed in 20 min by 10 mg/lb 1 mg/lb 0.83-1.66 mg/kg 12-20 mg/kg/hr

IM IV IV

Miscellaneous injectable restraint agents

Azaperone a-Chloralose Etomidate Etorphine/acepromazine (Immobilon) + diprenorphine (Revivon) Ketamine xylazine glyceryl guaiacolate Midazolam Metomidate Meperidine + azaperone + ketamine + morphine Propofol

See text for mixture IM IV

IV Continuous IV infusion

Analgesics

Fentanyl Sufentanyl Buprenorphine Butorphanol Meperidine Oxymorphone Pentazocine Phenylbutazone Aspirin Carprofen

0.02-0.05 mg/kg 30-100 gg/kg/hr 5-10 gg/kg 10 - 30 gg/kg/hr 0.05-0.1 mg/kg 0.1-0.3 mg/kg 2-10 mg/kg 0.15 mg/kg 1.5-3.0 mg/kg 10-20 mg/kg 10 mg/kg 2.0-3.0 mg/kg

IM q2 hr IV drip IM q2 hr IV drip IM q8-12 hr IM q4-6 hr IM q4 hr IM q4 hr IM q4 hr PO q12 hr PO q4 hr PO BID

(continues)

M. MICHAEL SWINDLE,ET AL.

982

Table X (Continued)

Drug

Dosage

Route

Miscellaneous

Antiarrhythmics Bretylium tosylate Lidocaine Calcium channel blocker: diltiazem Paralytic agents Pancuronium Vercuronium Succinylcholine Coronary vasorelaxant: nitroglycerin Anticholinergic: atropine Malignant hyperthermiatreatment and prophylaxis: Dantrolene

3.0-5.0 mg/kg 2-4 mg/kg 50 ~tg/kg/min 2-4 mg/kg

IV q30 min IV Continuous IV infusion PO TID

0.02-0.15 mg/kg 5-6 ~tg/kg/min 1.0 mg/kg 1.1 mg/kg 200 ~tgdiluted in 2 ml saline 0.05 mg/kg 0.02 mg/kg

IV Continuous IV infusion IV IV Infused slowlyinto coronary sinus IM IV

5 mg/kg

IV

See text for references. Only commonlyused agents are listed here. The reference books cited in Section V,A provide completeinformationon drug dosages and administrationtechniques. a

deleterious cardiovascular effects than xylazine. Telazol and Telazol/xylazine may also be more cardiopressive than other dissociative combinations. Their usage in research protocols in swine should be limited to single-dose administrations for chemical restraint or for protocols in which cardiovascular depression is unimportant. Ketamine/medetomidine and ketamine/midazolam have a protectant effect against cardiacarrhythmias and provide stable hemodynamics when administered as continuous IV infusions (Swindle, 1998; Smith et al., 1997).

b.

Propofol

Propofol is administered as a continuous IV infusion. Its use in research in swine is limited because of its minimal analgesic effects and significant cardiovascular depression in higher dosages. However, it may be useful as a continuous infusion for nonsurvival teaching protocols (Ramsey et al., 1993; Foster et al., 1992).

c.

Barbiturates

The effects of the barbiturates in swine are similar to those in other species. Barbiturates are administered IV as bolus injections to facilitate endotracheal intubation or as continuous IV infusions for general anesthesia. Tranquilizers may be utilized as preanesthetics to reduce the IV dosage by one-third to onehalf. The barbiturates are potent respiratory depressants in swine. Thiobarbiturates, such as thiopental, are less potent than pentobarbital and are shorter-acting; consequently, they are easier and safer to control. The thiobarbiturates are minimally me-

tabolized by the liver, unlike pentobarbital, and are mainly excreted by the kidneys. The recovery time from thiobarbiturates may be as short as 20 min, whereas it may be hours for pentobarbital. For longer protocols the barbiturates should be administered as continuous IV infusions. They are most useful for nonsurvival teaching protocols, but thiobarbiturate infusions may be useful for providing a stable plane of cardiovascular hemodynamics when other agents are contraindicated (Smith et al., 1997; Swindle, 1998). d.

Opioids

Opioids may be used as analgesic adjuncts to other anesthetics to provide balanced anesthesia or may be administered in high-dose infusions for cardiovascular protocols. They have minimal effects on cardiac contractility and coronary blood flow when administered for those purposes. The opioids administered most commonly for cardiovascular protocols are fentanyl, sufentanil, and alfentanil. The latter two agents are more potent than fentanyl and consequently require lesser volumes for infusion; however, their potency may induce muscular rigidity, Which can be controlled by starting the IV infusion first. The opioids induce bradycardia, especially when administered as IV boluses for induction of anesthesia. This bradycardia is transient but may be counteracted by atropine. For invasive surgical procedures, administration of low doses of inhalant agents is necessary during surgical manipulation. The analgesia provided by continuous infusions of these agents is adequate to provide a long-term stable plane of anesthesia and/or chemical restraint for cardiovascular measurements after the surgical manipulations are completed (Swindle, 1998).

22. PREANESTHESIA,ANESTHESIA,ANALGESIA,AND EUTHANASIA e.

Etomidate

Etomidate does not provide any advantage over other agents in research protocols in swine. It is relatively ineffective as a sole agent for any purpose other than short-term chemical restraint. It may be combined with azaperone or ketamine to provide anesthesia suitable for minor surgery (Worek et al., 1988; Smith et al., 1997). f

a2-Agonists

Xylazine, medetomidine, detomidine, and metomidine are the most commonly used az-agonists in swine. These agents are associated with blockage of the cardiac conduction system and with cardiovascular depression. Medetomidine has the fewest cardiovascular effects of this class of agents in swine. These agents are useful in combination with dissociative agents for short-term surgical analgesia or for inclusion in combination IV infusion protocols. They have minimal usage as sole agents for chemical restraint (Smith et al., 1997; Swindle, 1998; Vainio et al., 1992; Vainio and Ojala, 1994; Riebold et al., 1995; Flecknell, 1996, 1997). g.

Miscellaneous Anesthetics

a-Chloralose was promoted in the past as an agent to provide anesthesia for cardiovascular hemodynamic measurements. However, it must be used at a high dosage or in combination with other agents in swine to provide analgesia, which minimizes its effectiveness for this purpose, a-Chloralose may be replaced by other IV infusion protocols, such as high-dose opioids or ketamine/xylazine/guiafenesin, that provide adequate analgesia for these protocols (Silverman and Muir, 1993; Swindle, 1998; Thurmon, 1986).

2. InhalationalAnesthesia Administration of general anesthesia using inhalational agents is the preferred method for swine for most protocols. However, proper administration of these agents requires an investment in equipment both for administration of the agents as well as monitoring of physiological parameters. Personnel must also be properly trained in the techniques involved in this form of anesthesia. Flow rates for gas delivery are variable between units, but as a general guideline 5-15 ml/kg/min is usually sufficient. Nitrous oxide, halothane, and methoxyflurane have potential human health hazards when the vapors are inhaled on a chronic basis. No long-term health effects have been described for the minimally metabolized agents, such as isoflurane, desflurane, and sevoflurane. However, arbitrary limits have been set for environmental exposure to these agents as well. An evacuation system to control waste gases and periodic monitoring of the operating room to check for leaks in the circuits are essential

983

components of a program that uses inhalational anesthetics. Maintenance of equipment, including cleaning and calibration of the vaporizers and flowmeters as well as leak testing of anesthesia circuits, is also essential. a.

Nitrous Oxide

Nitrous oxide is ineffective as a sole agent for anesthesia in swine. However, it may be administered in combination with oxygen to reduce the amount of inhalational agent that must be administered. This reduces the dose-dependent cardiovascular depression associated with inhalational anesthetics in swine. Nitrous oxide is administered as an adjunct to oxygen in a 3 3 66% combination. Nitrous oxide-oxygen (2:1) provides blood gas measurements similar to those in unanesthetized swine and minimizes the amount of inhalant that must be administered (Swindle, 1998; Smith et al., 1997). Diffusion hypoxia and the potential for abuse of the agent by personnel are considerations. b.

Halothane

Halothane has a MAC of 0.91-1.25 % (Tranquilli et al., 1983; Smith et al., 1997). It is more cardiodepressant than the newer agents discussed below. Halothane is metabolized by the liver as well as being eliminated by the lungs. c.

lsoflurane

Isoflurane is the least cardiodepressent of the inhalational agents commonly used in swine. Isoflurane has a MAC value of 1.58% (Smith et al., 1997; Eisele et al., 1985). Less than 1% of this agent is metabolized by the liver. Isoflurane is generally used at concentrations of 2 - 4 % for induction and 0.5-2.0% for maintenance of general anesthesia. When using nitrous oxideoxygen 1:1 or 2:1 for delivery, the isoflurane concentration may be reduced to 0.5-1.0%. Isoflurane in nitrous oxide is commonly used for maintaining long-term anesthesia with physiological measurements. d.

Desoflurane and Sevoflurane

Desoflurane and sevoflurane have physiologic effects similar to those of isoflurane. They are not commonly used in swine, because of the increased expense (Weiskopf et al., 1992).

D.

Intraoperative Monitoring and Support

Intraoperative monitoring and support of swine are similar to those of other large animal species. In order to monitor homeostasis, the following parameters should be monitored: muscular reflexes, ECG, heart rate, blood pressure, blood gas saturation values, and core temperature. Swine are susceptible

M. MICHAEL SWINDLE,ETAL.

984

to hypothermia because of their relatively hairless skin; consequently, the use of circulating hot-water blankets and protection from stainless steel surfaces should be considered. Animals should be completely covered by drapes during surgery to prevent heat loss. Animals may be monitored by a variety of mechanical means. Pulse oximeters can monitor oxygen saturation and pulse rates. Surgical monitors may be obtained that monitor ECG, rectal temperature, and blood pressures. Blood pressure may be monitored either by invasive intravascular techniques or by use of blood-pressure cuffs on the tail, medial saphenous artery, or radial artery. Pulse oximetry finger cuffs can be attached to the tongue, ear, tail, or dewclaw. There will be some variability in the ability of the pulse oximetry cuff to function, depending on skin pigmentation and thickness of the body part. IV fluid maintenance rates are 5-10 ml/kg/hr (Smith et al., 1997; Swindle, 1998).

E.

Special Anesthetic Considerations

1. Cardiac Anesthesia

Anesthesia for cardiac surgery requires that the protocol minimize effects on cardiovascular function. Parameters that need to be considered when selecting a protocol include cardiac contractility, cardiac output, blood pressure, myocardial oxygen consumption, and prevention oi~cardiac arrhythmias. Isoflurane delivered in nitrous oxide-oxygen (2:1) minimizes cardiodepressant effects during surgery. If an inhalant agent is contraindicated, then high-dose opioid infusions may be used, especially if there is a requirement to maintain myocardial contractility and coronary blood flow. The physiologic effects of the various anesthetics should be reviewed prior to performing complex cardiac surgeries (Swindle, 1998). Bretylium and lidocaine infusions may be necessary as preventives for fatal cardiac arrhythmias. Paralytic agents, such as pancuronium and vercuronium, will be necessary to paralyze the diaphragm during cardiac manipulation and may be useful for providing increased exposure when using a lateral thoracotomy. Paralytic agents should not be administered until there is an assurance that adequate analgesia has been obtained. This can be ascertained by performing skin and muscular incisions prior to their administration. Heart rate and blood pressure should be monitored during surgical manipulation in a paralyzed animal to assure adequate anesthesia (Swindle, 1998). Performing cardiopulmonary bypass (CPB) and extracorporeal membrane support (ECMO) procedures in swine is more difficult than in most species. In order to perform these techniques as survival procedures, a multidisciplinary team of personnel competent in these procedures is necessary. It is beyond the scope of this chapter to describe CPB and ECMO procedures; however, they are described elsewhere in a detailed stepwise fashion, which should be adequate for most research facilities to perform them successfully (Swindle, 1998; Smith, 1994).

2.

Pediatric Anesthesia

Neonatal dosage rates are frequently different from dosage rates for adults. The dosage ranges given in this manuscript are safe for swine of all ages in our experience. Control of hypothermia during anesthesia is especially important in the neonate, particularly in the first week postnatally, when they are incapable of controlling their own body temperature adequately (Swindle et al., 1996). 3.

Neuroanesthesia

Swine have not been used very often in neurosurgical research, and specific anesthetic protocols for neurosurgery have not been published. The general principles of neuroanesthesia for other species should be applied. For instance, all inhalant anesthetics increase blood flow to the brain in swine, and swelling of the tissue may result postsurgically after manipulation; therefore, techniques to reduce brain swelling, such as the use of diuretics and hypertonic glucose solutions, should be applied, Ventilation rates and volumes may also influence the oxygen supply to the central nervous system (Swindle, 1998). 4.

Obstetrics and Gynecology

Most anesthetic agents will cross the epitheliochorial placentation of swine to affect the fetus. Transport across the placenta is enhanced if the agent is lipophilic, and some of these agents may reach a higher concentration in the fetus than in the sow. Tocolytic agents, such as terbutaline, may be useful if uterine contractions under anesthesia need to be controlled. Reviews of the special considerations and detailed protocols for performing fetal surgery in swine have been published (Swindle et al., 1996). 5. Anesthesia for Imaging Procedures (MRI and PET)

Specialized imaging procedures require that a long duration of anesthesia be provided without equipment that may be affected by the imaging equipment. Depending on the equipment and duration of the imaging procedure, the short-term (20-30 min) injectable protocols described above may be adequate for the procedure. Complete relaxation without paralysis is necessary to obtain clear images. The combinations of ketamine/ midazolam and ketamine/medetomidine provide the best relaxation for these procedures. Infusion protocols with thiobarbiturates may be used if having an IV infusion is not contraindicated (Swindle, 1998).

F.

Postoperative Recovery

Postoperative recovery procedures for swine are similar to those for other species. A complete description of these proce-

22. PREANESTHESIA,ANESTHESIA, ANALGESIA,AND EUTHANASIA dures and the emergency procedures for cardiopulmonary distress have been published. Extubation can be performed when the pig is moving into a sternal position and struggling against the endotracheal tube. It is best to let the air out of the cuff of the tube first and allow the pig to stabilize prior to removing the tube. Apnea frequently occurs when the tube is removed, and compressive manipulation of the chest or stimulation of the pharynx and epiglottis may have to be instituted to start spontaneous respiration. In some cases, the pig may have to be reintubated and respirated with a handheld respiratory bag. The apnea is more likely to occur with injectable anesthetics, such as the barbiturates, than with inhalants. Monitoring of cardiopulmonary function during recovery can be provided by pulse oximetry and appropriate countermeasures taken when hypoventilation or cardiac emergencies occur. If pulse oximetry is not available, then the pulse and respiratory rates should be monitored either by auscultation or observation (Swindle, 1998; Smith, 1994).

G.

Acute and Chronic Analgesic Therapy

If surgical procedures are performed, it is best to provide preemptive analgesia prior to making the skin incision or at least prior to removing the animal from general anesthesia if intraoperative administration of these agents is not possible (Smith, 1994; Swindle, 1998). 1. Assessment of Pain and Discomfort

Swine are generally sedentary animals that respond to the presence of humans only during manipulation or feeding activity. Pigs that are hyperactive and vocalizing tend to be in distress postsurgically. Incisional pain and abnormal posture are other reliable indicators of pain or distress. Pigs will readily eat, even after major surgical procedures, if they are comfortable. Consequently, swine that are not resting comfortably and responding to feeding are probably in pain or distress postsurgically (Swindle, 1998). 2. Methods for Analgesic Drug Delivery

Parenteral analgesics are usually administered IM or SC in the neck. IV administration is generally given by using indwelling catheters in the ear vein or one of the other cannulated vessels intraoperatively. Swine can readily be induced to take oral medication when the substance is placed in a food treat. Canned dog and cat food, apples, chocolate syrup, and sweets are usually successful when using this technique (Swindle, 1998). 3. NSAIDs

The newer-generation NSAIDs, such as ketorolac, ketoprofen, and carprofen, have been utilized successfully in postoper-

985

ative analgesia protocols either by injection or per os. For some procedures they have been effective in BID dosages as sole agents, but usually they are combined with buprenorphine (see Section V,G,5) in the lower dose range of each agent to maximize the effects of opioids and NSAIDs (Fosse et al., in preparation; Flecknell, 1996; Swindle, 1998). 4. Local, Regional, and Epidural Anesthesia

Local and regional anesthesia has not been commonly used in research settings and is typically used as an adjunct to analgesia rather than as the sole anesthetic (Smith et al., 1997; Thurmon, 1996; St. Jean and Anderson, 1999). The most common areas are dorsal nerve root blocks in the intercostal spaces or lumbar regions as an adjunct to anesthesia and analgesia for dorsalventral surgical incisions, such as thoracotomies. Infiltration of the incision with local anesthetics is also performed prior to making the initial incision as a form of preemptive analgesia. Xylazine (2 mg/kg diluted in saline), xylazine (1 mg/kg) plus lidocaine (10 ml 10% solution), and medetomidine (0.5 mg/kg diluted in saline) have been utilized epidurally to provide analgesia during general surgery (St. Jean and Anderson, 1999; Ko et al., 1992). 5.

Opioids

Buprenorphine is generally considered to be the opioid analgesic of choice postoperatively. Preemptive analgesia with this agent preoperatively or intraoperatively reduces the course of postoperative analgesia and the dosage that may have to be given. There is a wide range of therapeutic effectiveness, depending on the procedure and time of administration. For major surgical procedures, such as thoracotomies or visceral transplantation, a dosage of 0.05-0.1 mg/kg may be needed in the the initial stages. The dosage may be reduced by 50-75% of the initial dosage BID, depending upon the clinical condition of the animal. Buprenorphine may be combined with NSAIDs (see Section V,G,3), such as ketoprofen, carprofen, or ketorolac, to reduce the dosage and get a synergistic effect. Fentanyl and sufentanil have been used for both balanced anesthesia and as high-dose opioid infusions for cardiac surgery. These agents have a short half-life and are not good for postoperative analgesia when administered as bolus injections. Anecdotal accounts indicate that dermal patches of fentanyl may be used in swine, but a controlled study with blood levels of the agent has not been published to date. Effectiveness of fentanyl patches can be variable, depending upon housing conditions and such factors as moisture and heat. In our experience, it is possible to get analgesia using 50-100 ttg patches, but animals may show signs of overdosage and have to be monitored. Until such time as a controlled study is available, the use of these patches should be considered experimental in swine.

M. MICHAEL SWINDLE,ET AL.

986

H.

Euthanasia

Most of the injectable forms of euthanasia utilized in other large animal species are suitable for swine. Pentobarbital overdose (> 150 mg/kg) is the preferred form of parenteral euthanasia. It is acceptable to administer KC1 injections or perform exsanguination while swine are under general anesthesia (Swindle, 1998).

VI.

source of warmth and oxygen. Anesthesia is monitored in much the same way as for other species. Because eye position at both very light and deep levels of anesthesia may be similar, eye position should not be considered as a reliable indicator of the depth of anesthesia. Although lightly anesthetized animals will have a relaxed palpebral reflex and a rotated eye position, both adequately to deeply anesthetized animals may have a centrally positioned eyeball. Most importantly, the animal's response to surgical stimulus and vital signs reflecting the stability of the cardiac and respiratory systems should be assessed (Ewing, 1990; Taylor, 1991; Dunlop and Hoyt, 1997).

SMALL RUMINANTS 2. Injection Sites A.

Introduction

Many of the practices and equipment used for anesthesia and surgery in small animal species can also be used for small ruminants. Often, existing runs, kennels, and transport cages designed for other larger traditional laboratory animals (such as dogs and swine) can be easily modified for small ruminants. The friendly and docile nature of these animals makes them a desirable animal model for research and teaching programs. Comprehensive sources providing information on anesthesia, analgesia, and surgery for small ruminants are available (Benson and Thurmon, 1986; Ewing, 1990; Dunlop and Hoyt, 1997; Smith and Sherman, 1994; Taylor, 1991; Fraser, 1995; Fulton et al., 1994; Williams, 1995; Riebold, 1994, 1996; Thurmon and Benson, 1986). Even though many of the anesthetics, analgesics, and sedative drugs commonly used for small ruminants do not carry a product license specifically for these animals, those listed in this manuscript (Table XI) have been widely tested in clinical and experimental settings (Ewing, 1990; Smith and Sherman, 1994; Taylor, 1991). Specific anesthetic regimens should be devised in consultation with the attending veterinarian, taking into consideration the objectives of the protocol. The following section will outline anesthetic problems not encountered in simple-stomached animals and review commonly used protocols for small-ruminant anesthesia and analgesia.

B. Preanesthetic, Anesthetic, and Recovery Considerations 1. Preoperative Evaluation

Prior to using any anesthetic protocol, a thorough physical examination should be performed, with special attention given to the respiratory system. At a minimum, blood workup should include total protein and packed cell volume values. This is especially important for animals that have been on pasture, because intestinal parasite burden may contribute to blood loss. Supportive therapy during anesthesia should include fluids injected IV for maintenance and replacement needs and as a

Appropriate sites for SC injections are the neck (Williams, 1990), flank, and axillary area. IM injections should use the neck, triceps, and quadriceps muscles in order to prevent temporary or permanent lameness associated with ischiatic nerve irritation. The SC route for injections is preferable over the IM route and should be used whenever possible. The jugular or cephalic veins are accessible for IV injections (Ewing, 1990). Other veins, such as the saphenous, may be used if the animal is restrained properly. 3.

Common Complications

The most common complications encountered with anesthetizing ruminants are associated with the effects of the digestive tract on the respiratory system. These include regurgitation and aspiration, bloat, and inadequate oxygenation (Dunlop and Hoyt, 1997; Ewing, 1990; Hellyer, 1991; Taylor, 1991; Thurmon and Benson, 1986). a.

Prevention of Regurgitation

Active regurgitation during light planes of anesthesia or passive regurgitation under deep anesthesia may lead to aspiration of rumen contents, causing asphyxiation or progressive foreign body pneumonia. Although there will not be a significant decrease in ruminal contents, withholding food for 12-24 hr may decrease ruminal pressure and decrease the risk of regurgitation (Muir et al., 1989). This may also decrease the rate of fermentation and gas production in the rumen, thereby decreasing the risk of bloat (Thurmon and Benson, 1986). Fasting is not recommended for neonatal or pregnant animals (Dunlop and Hoyt, 1997). In our opinion, water should not be withheld for more than 4 - 6 hr for most procedures prior to anesthesia. b.

Prevention of Aspiration: Intubation

To help prevent aspiration of rumen contents, endotracheal intubation is essential. While the animal is in a state of fairly deep anesthesia and positioned in sternal recumbency the animal should be quickly intubated, using a cuffed endotracheal

987

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA Table Xl

Small Ruminants: Drug Dosesa Drug

Dosage

Route

Anticholinergics

Glycopyrrolate

0.005-0.01 mg/kg

SC

0.02-0.1 mg/kg 0.25-0.5 mg/kg

SC or IV IV

0.1-0.2 mg/kg 0.05 mg/kg 0.05 mg/kg 2 mg/kg 0.22 mg/kg 11 mg/kg 0.01 mg/kg 0.03 mg/kg 0.5 mg/kg 2 mg/kg

IM IV IV IV (may be mixed in same syringe) IM IM IV

0.12 mg/kg 0.02 mg/kg or 0.06 mg/kg

IV IV IM

8-14 mg/kg 10-16 mg/kg

IV to effect IV to effect

10 mg/kg BID 0.005-0.01 mg/kg BID 0.002 mg/kg mixed with 0.9% saline to volume of 0.13 ml/kg 1.1-2.2 mg/kg BID 6 mg/kg q6 hr 2.0-4.0 mg/kg BID

IM or SC every 2-4 hr IM or SC every 4-6 hr

Sedative or tranquilizers

Acepromazine Diazepam a2-Agonists and dissociative agents (and combinations)

Xylazine Xylozine Xylazine + ketamine Xylazine + ketamine Medetomidine Detomidine Diazepam followedby ketamine

IV IV

az-Antagonists (reversal agents)

Yohimbine Atipamezole Barbiturates

Thiamylal Thiopental Analgesics

Meperidine Buprenorphine Flunixin meglumine Phenylbutazone Carprofen a

Epidurally IM or SC daily for up to 5 days IM or IV every 24-48 hr IV may provide analgesia for up to 48-72 hr

See text for references and discussion.

tube. Lidocaine (2%) may be dropped on the vocal cords to prevent laryngospasms prior to intubation. Cetacaine should not be used, because there have been reports of resultant methemoglobinemia in some ruminant species (Lagutchik et al., 1992). Because of the long, narrow oral cavity and distant laryngeal opening in these animals, an otherwise difficult intubation may be facilitated by using a laryngoscope with a long, modified Miller blade in order to more clearly visualize the laryngeal opening and the position of the tube. A rubber-tipped stylet may be inserted into the trachea first, and the endotracheal tube passed over the stylet (Muir et al., 1989). The stylet should be removed as soon as the endotracheal tube is passed through the trachea. The cuff of the endotracheal tube should be inflated as soon as the tube is properly positioned (Ewing, 1990; Smith and Sherman, 1994; Taylor, 1991; Thurmon and Benson, 1986).

c.

Prevention of Bloat

Normal eructation in the ruminant is prevented by anesthesia and by dorsal or lateral recumbent positioning. As a result, gas

increases in the rumen, causing ruminal tympany, or bloat; rumen contents put pressure on the diaphragm, causing the residual lung capacity to decrease and interfere with ventilation. Pressure on the major vessels then impedes venous flow returning to the heart. Cardiac output, blood pressure, and tissue perfusion may be compromised, leading to possible pulmonary and ventilation perfusion mismatches. Bradycardia, hypoxemia, and hypercarbia may result, all of which can be life-threatening (Ewing, 1990; Taylor, 1991; Thurmon and Benson, 1986). Passing a stomach tube after intubation can help resolve gaseous distension. An equine uterine flushing catheter made of silicon rubber with a relatively large inflatable balloon at the distal end will facilitate placement of the tube into the gas pocket. The use of this type of tube also reduces drainage of ruminal contents and blockage of the tube (Vogler et al., 1992).

d.

Ventilation and Fluid Management

Airway maintenance is important in these species, which have copious salivary and respiratory secretions. Intermittent positive

M. MICHAELSWINDLE,ETAL.

988

pressure ventilation throughout the course of anesthesia helps to prevent hypercarbia and to maintain a constant plane of anesthesia. At least 30% inspired oxygen should be provided for all anesthetic procedures lasting longer than 10 min (Taylor, 1991). Seven to 10 breaths per minute and a tidal volume of at least 10-22 ml/kg should be allowed. If oxygen is administered by hand bagging, the tidal volume should be adequate to cause chest expansion without exceeding 25-30 cm water pressure. If spontaneous ventilation is being allowed, the animal should be "sighed" at 5 min intervals at a level of 30 cm water pressure (Ewing, 1990; Riebold, 1994). Fluid maintenance can be achieved with an initial flow rate of 10 ml/kg/hr for about the first 30 min, slowed to a flow rate of 4 ml/kg/hr through an intravenous catheter placed in the jugular, auricular, or cephalic vein (Dunlop and Hoyt, 1997). 4. Immediate Postanesthetic Recovery Considerations

The animal must be supervised and monitored until it is completely recovered. The same precautions taken to prevent the consequences of regurgitation (such as aspiration), ruminal distension, and inadequate oxygenation should be followed. If an inhalant anesthetic is used, the vaporizer should be turned off and the rebreathing system flushed with oxygen. The animal should be left connected to the system until it shows signs of recovery, such as an active palpebral reflex, limb movement, chewing, and swallowing. When strong swallowing reflexes are evident, remove the endotracheal tube with the cuff inflated in order to remove any accumulated saliva or regurgitant near the cuff. The animal should be supported throughout recovery in sternal recumbency until able to maintain this position on its own (Taylor, 1991; Thurmon and Benson, 1986). 5.

Preoperative Medications

a.

Anticholinergics

The use of anticholinergics in ruminants is controversial (Reibold, 1994). The present authors do find the use of glycopyrrolate (0.005-0.01 mg/kg SC) beneficial in decreasing salivary secretions for facilitation of endotracheal intubation. b.

Sedatives and Tranquilizers

Sedatives may be incorporated as premedications prior to general anesthesia or in conjunction with regional anesthesia for certain procedures (Taylor, 1991). Premedication using sedatives helps make IV catheterization easier and induction smoother by minimizing stress and anxiety. Although some do not consider acepromazine to be very useful in most small ruminants (Hellyer, 1991), acepromazine (0.02-0.1 mg/kg IV or SC) is useful for providing mild sedation with minimal respiratory depression (Smith and Sherman,

1994; Dunlop and Hoyt, 1997). A total dose of 3 mg of acepromazine should not be exceeded for small ruminants. c.

a2-Agonists and Dissociative Combinations

Xylazine (0.1-0.2 mg/kg IM, 0.015-0.025 mg/kg IV), an a2-agonist, may be used as a premedication to provide sedation without recumbency. Ketamine and xylazine are often used together as a preanesthetic to permit endotracheal intubation of the animal before using inhalation anesthetics. Xylazine (0.05 mg/kg) is given IV, and ketamine (2 mg/kg IV) is given 3-5 min later. Alternatively both the ketamine and the xylazine may be mixed in the same syringe and administered simultaneously. The IV dose of ketamine may be repeated if needed (Ewing, 1990). For a longer affect, administration of xylazine (0.22 mg/ kg) and ketamine (11 mg/kg) IM in combination will give approximately 50 min of anesthesia; a prolonged recovery time of 1.5-2 hr is usual with this combination (Ewing, 1990). Ruminants are more sensitive to the effects of xylazine than are most other species; goats are considered to be more sensitive to the effects of xylazine than are other ruminants. The lowconcentration (20 mg/ml) small-animal formulation should be used. Xylazine has the potential for causing cardiovascular and respiratory depression, rumen atony with bloat, hyperglycemia with resultant diuresis, and abortions in late gestation (Thurmon and Benson, 1986; Riebold, 1996). Xylazine should not be used for animals with depressed cardiovascular function or urinary tract obstruction. The effects of xylazine can be reversed with the a2-adrenergic antagonist yohimbine. The dose of 0.12 mg/kg IV is effective for most ruminants. A higher dose of 1.0 mg/kg IV is needed for sheep (Riebold, 1996). Animals dosed with yohimbine for reversal of xylazine effects should be monitored closely in case redosing is required. Other a2-agonist agents used for ruminants include medetomidine (0.01 mg/kg IV) and detomidine (0.03 mg/kg). Additionally atipamezole (0.02 mg/kg IV or 0.06 mg/kg IM) is an a2-antagonist that has been used for reversal (Dunlop and Hoyt, 1997). Diazepam (0.25-0.5 mg/kg IV) is useful in providing sedation without analgesia in ruminants when slowly given IV (Taylor, 1991). Diazepam may be substituted for the xylazine in combination with ketamine when xylazine is contraindicated. A premedication dose of diazepam (0.5 mg/kg, or one-half this dose if the animal is over 50 kg) is slowly injected IV. Ketamine (2 mg/kg IV) should be given about 3 min later (Ewing, 1990). A review of the references previously cited reveals other ketamine/xylazine/diazepam cocktails that are useful for small ruminants. d.

Barbiturates

Ultra-short-acting barbiturates are often employed as induction agents. Thiamylal (8-14 mg/kg IV) or thiopental (10-

22. PREANESTHESIA,ANESTHESIA,ANALGESIA,AND EUTHANASIA 16 mg/kg IV) allows intubation prior to inhalation anesthesia (Ewing, 1990). The initial one-third of the calculated dose should be given as a bolus, with the remaining dose given slowly to effect. These doses may be decreased if using a premedication sedative. Because of the high alkalinity of these agents, there is a potential for perivascular necrosis if extravasation occurs. It is advisable to administer these agents through an IV catheter placed in the jugular vein. e.

Mask Induction and Inhalant Anesthetics

Mask induction utilizing a large dog mask can be achieved with halothane or isoflurane in oxygen. Initially, the animal should be allowed to breathe 100% oxygen at a flow rate of 4 6 liter/min for several minutes to achieve denitrification (Benson and Thurmon, 1986). The inhalant should be slowly increased by 0.5% increments every 30 sec until a 3-3.5% vaporizer setting is reached. Intubation is likely to be possible within 10 min (Ewing, 1990). Once the animal is intubated, the oxygen flow rate can be decreased to 1-2 liter/min, and the vaporizer setting can be maintained at a level of 0.5-2% for halothane and 1-2% for isoflurane. Oxygen flow rate can be calculated by the following formula: 1.5-2.5 ml/kg body weight • 5 = oxygen flow rate in ml/min (Dr. E P. Elliot, personal communication, 1994). Proper precautions should be used to reduce human exposure to waste gases by ensuring that tightfitting masks, cuffed endotracheal tubes, and proper scavenging systems are used during inhalation anesthesia. f.

Local Anesthetics

Local and regional analgesics are often appropriate for certain procedures, including orthopedic surgeries. Landmarks and procedures are detailed elsewhere (Benson and Thurmon, 1986; Dunlop and Hoyt, 1997). Goats are more sensitive to lidocaine than are other small ruminants. A total dose of 2% lidocaine should not exceed 15 ml in any goat. Administration of diazepam as an anticonvulsant and proper ventilation and fluid support must be provided, should complications arise (Benson and Thurmon, 1986). Drug dosages are listed in Table XI. Co Postoperative Recovery, Pain Assessment, and Analgesic Agents

Many of the small ruminants are often stoic and exhibit only subtle signs of pain. Gas anesthetics provide for rapid recovery without any residual analgesic effects. The incorporation of preemptive analgesic agents in a balanced anesthesia protocol to be administered prior to surgical manipulation is therefore imperative. Not only will this optimize necessary pain management, but it will also allow for a smooth induction, requiring lower doses of anesthetics and allowing for a smooth recovery from anesthesia.

989

1. Assessment of Pain and Discomfort

Postsurgical evaluation of the animal should include close monitoring for overt and subtle signs of pain. Presurgical parameters, including respiratory rate, heart rate, amount of vocalization and activity, and general appearance and attitude, should be recorded, to be used as a basis for postsurgical evaluation. Increased heart rate, rapid shallow respirations, grunting, and grinding teeth might be indications of pain. A depressed appetite, guarding of affected area, vocalization on movement or palpation, and other changes in behavior are further evidence that an animal is in pain (Benson, 1992). A ruminant might also appear depressed and disinterested in its surroundings if experiencing pain. 2. Opioids

The opioids are a class of analgesics often employed as preemptive and postoperative analgesics. Meperidine (10 mg/kg) or buprenorphine (0.005-0.01 mg/kg) may be used as premedications (E wing, 1990). Buprenorphine (0.005-0.01 mg/kg) may have a longer duration of action than others in its class. Although the recommended dosing interval for most species is 6-12 hr, buprenorphine has a shorter duration of action in ruminants and should be given every 4 - 6 hr (Flecknell, 1989; Fujimoto, 1992). The incorporation of buprenorphine (0.005 mg/kg SC) as a premedication, with subsequent dosing of buprenorphine at 0.005-0.01 mg/kg SC every 4 - 6 hr provides excellent postoperative analgesia in most cases (L. K. Fulton, personal experience). The present authors recommend the epidural use of buprenorphine for orthopedic procedures involving the hindlimbs. Buprenorphine (0.002 mg/kg) is diluted with 0.9% saline to a volume of 0.13 ml/kg. If cerebrospinal fluid is aspirated after placement of the spinal needle, one-half of the calculated dose is administered. 3. NSAIDs

NSAID agents such as flunixin meglumine (1.1-2.2 mg/kg) may be incorporated into postsurgical analgesic protocols, especially those involving the musculoskeletal system. Flunixin meglumine should be dosed as needed every 12-24 hr but should not exceed 2 - 4 doses. Higher repeated doses can cause hemorrhagic gastritis (Paddleford, 1992). Phenylbutazone (6 mg/kg IM or IV) provides analgesia for mild to moderate pain (Allen and Borkowski, 1999). The use of carprofen (2.0-4.0 mg/kg IV) may provide analgesia for up to 72 hr. Carprofen has been approved for preoperative use in other species, and it may be considered safer and less ulcerogenic than other NSAIDs (Grisneaux et al., 1999; Dunlop and Hoyt, 1997). The use of the NSAIDs in conjunction with opioids may provide for synergistic analgesic effects, and the drugs should be used together for preemptive and postoperative analgesia when possible (Benson

M. MICHAEL SWINDLE,ET AL.

990

and Thurmon, 1986; Ewing, 1990; Dunlop and Hoyt, 1997; Smith and Sherman, 1994; Taylor, 1991; Riebold, 1994, 1996; Thurmon and Benson, 1986).

VII.

N O N H U M A N PRIMATES

A.

Introduction

Nonhuman primates are important models for a wide variety of biomedical and behavior research because of their phylogenetic proximity to humans. When selecting methods for anesthesia and restraint, the diversity of the order Primates must be considered. The wide range of body size and weight of nonhuman primates plays an important role in selecting not only an appropriate anesthetic but also the dosage of the drug to be administered. Extrapolation of anesthetic and analgesic doses from one primate species to another should be done with caution because of differences in the responses of some species to certain agents. Generally, New World primates require higher doses of injectable anesthetics and tranquilizers (i.e., ketamine, tiletaminezolazepam, diazepam) per kilogram of body weight than Old World primates require. However, the anesthetic dose of alphaxolone-alphadolone for macaques can exceed the lethal dose for the adult Saimiri sciureus (Logdberg, 1988). The overall goal of this section is to provide a general guide to anesthesia and analgesia of nonhuman primates, so that they may be provided with optimal care during their use as research subjects. Drug dosages are listed in Table XII.

B.

Preoperative Assessment and Preparation

ture of the surgical procedure should be taken into consideration. A baseline hematocrit may be desirable for animals undergoing surgical procedures that may produce vascular volume deficiencies. Preoperative fasting is an accepted practice for nonhuman primates undergoing surgical procedures. Although it is conventional practice to fast primates for at least 12 hr, members of the Callitrichidae (marmosets and tamarins) and other small species are fasted only for 4 - 6 hr, to avoid perioperative hypoglycemia and hypotension. In situations requiring emergency surgery or in pregnant animals with delayed gastric emptying, inclusion of histamine 2 antagonists (cimetidine 10 mg/kg, ranitidine 1.5 mg/kg) may reduce the risk of aspirational pneumonia (Popilskis et al., 1992). 2.

Methods of Anesthetic Delivery

IM injection in nonhuman primates is commonly done into the caudal muscle of the thigh. For repeated IM injections, it is advisable to alternate the leg used to reduce the possibility of muscle or nerve irritation by drugs with low pH, such as ketamine. In nonhuman primates, SC injections into lateral thigh or dorsolateral sites in the back can be done with the help of "squeeze-back" cages. The uptake of drugs from SC injection, and to lesser degree IM injection, can be variable and influenced by the rate of hydration and local perfusion. Chemical restraint is usually needed in primates for IV injections or blood withdrawal. Cephalic and less commonly saphenous veins can be used both for venipuncture and administration of drugs. The femoral vein and artery are commonly used for the withdrawal of relatively large blood volumes. The femoral artery also offers a site for insertion of the indwelling catheter (20- to 22-gauge) to monitor direct blood pressure. Whenever the femoral artery is being used, manual pressure must be applied to prevent a hematoma formation, especially in anticoagulated animals.

1. Preoperative Evaluation

3.

Preoperative Medication

Preoperative assessment is an important starting point that helps formulate the anesthetic plan. It usually includes history of previous use, physical examination, and pertinent laboratory data. Despite limitations associated with performing a thorough physical examination in nonhuman primates, signs of illness such as unusual posture, anorexia, and abnormal feces or urinary output can be readily identified. Laboratory animal handlers who have regular contact with individual animals can provide even more insight into an animal's unique characteristics that warrant more detailed evaluation. Routine laboratory tests should be performed while nonhuman primates are in quarantine and later as part of a preventive medicine program. It is usually not necessary nor is it practical to perform preoperative clinical laboratory testing in healthy animals. However, the na-

Anticholinergic drugs are used to diminish salivary and bronchial secretions and prevent bradycardia. Bradycardia is of potential significance in small and young animals because their cardiac output is heart rate-dependent. Atropine (0.02-0.05 mg/kg) can be administered to block the cardiac vagal nerves and prevent excessive salivation. Inhibition of salivary and respiratory tract secretions is the primary rationale for using glycopyrrolate (0.01 mg/kg) as a premedication. Inclusion of an anticholinergic as part of preoperative medication is not indicated in cardiac surgery, because of potential for ventricular tachycardia and bigeminal patterns.

991

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA Table XII Nonhuman Primates: Drug Doses a Drug

Dosage

Route

Old World nonhuman primates (Macaca and Papio species) Dissociative agents and combinations Ketamine Ketamine + xylazine Ketamine + diazepam Ketamine + medetomidine Tiletamine-zolazepam (Telazol) a2-antagonist antipamezole Barbiturates Thiopentol Pentobarbital Other injectable anesthetics Alphaxolone-alphadolone (Saffan) Propofol Etomidate Fentanyl Fentanyl Anticholinergics Atropine Glycopyrrolate Muscle relaxants Pancuronium Vecuronium Inhalational anesthesia Halothane Isoflurane Analgesics Opioids Morphine Morphine Oxymorphone Buprenorphine Opioid antagonist: naloxone Local anesthetic: bupivicaine NSAIDs Ketorolac Aspirin Miscellaneous Antiarrhythmic: lidocaine Vasoactive drugs and inotropes Dopamine Norepinephrine Dobutamine Phenylephrine

5.0-20 mg/kg 7-10 mg/kg 0.6-2 mg/kg 5-10 mg/kg 0.35 mg/kg 5 mg/kg 60-150 gg/kg 4 - 6 mg/kg 0.3-0.75 mg/kg

IM IM IM IM IM IM IM IM

5-7 mg/kg 15-17 mg/kg/hr 25-30 mg/kg

IV (induction) IV infusion (maintenance) IV

18 mg/kg 6-12 mg/kg 2.5-5.0 mg/kg 0.5 mg/kg 0.2 mg/kg boluses every 6-12 min 5-10 gg/kg 70-100 gg/kg/hr

IM (induction) IV (maintenance boluses) IV IV IV (repeated boluses) IV in combination with low MAC of isoflurane Continuous IV infusion

0.02-0.05 mg/kg 0.005-0.01 mg/kg

IM IM

0.1 mg/kg 0.04-0.06 mg/kg

IV IV

1 MAC = 0.89-1.15% 1 MAC = 1.28%

1-2 mg/kg 0.1 mg/kg 0.15 mg/kg 0.015 mg/kg 0.1-0.2 mg 1.5 mg/kg intercostal nerve block

IM, SC Epidurally IM IM, SC IV as needed

5-15 mg 125 mg/5 kg

IM Suppositories

1-2 mg/kg IV, 20-50 gg/kg/hr

Continuous IV infusion

3-5 gg/kg/min 0.05-0.2 gg/kg/min 2-10 gg/kg/min 1-2 gg/kg 0.5-1.0 gg/kg/min

posttransplant IV infusion Continuous IV infusion Continuous IV infusion IV bolus Continuous IV infusion

(continues)

992

M. MICHAEL SWINDLE, ETAL.

Table XII (Continued) Nonhuman Primates: Drug Doses a Drug

Dosage

Route

New World nonhuman primates (Saimiri and CaUithrix species) Dissociative agents and combinations Ketamine Ketamine + xylazine Ketamine + diazepam Tiletamine-zolazepam (Telazol) Other injectable anesthetics Alphaxolone-alphadolone (Saffan) Propofol Medetomidine Atipamezole Fentanyl-fluanisone (Hypnorm) Anticholinergics Atropine Analgesics Opioids Morphine Oxymorphone Buprenorphine Butorphanol Opioid antagonist: naloxone a

10-30 mg/kg 10-30 mg/kg 1.5-3.0 mg/kg 15-20 mg/kg 1.0 mg/kg 5-10 mg/kg

IM IM IM IM IM IM

11-19 mg/kg 2.0-6.0 mg/kg 100 ~tg/kg 200 ~tg 0.3 ml/kg

IM IV IM, SC IV IM, SC

0.04 mg/kg

IM

1-2 mg/kg 0.075 mg/kg 0.015 mg/kg 0.02 mg/kg 0.01-0.02 mg/kg

IM, SC IM IM, SC SQ IV as needed

See text for references.

c.

Intraoperative Anesthesia

gender, higher values for lactate dehydrogenase (LDH) and creatine kinase (CK) are noted for males. In contrast, females tend 1. Injectable Anesthetics to have higher values for amylase and cholesterol (Fernie et al., 1994). Ketamine does not appear to alter the magnitude of ena. Dissociatives docrine responses even after multiple injections, thereby makKetamine has a wide margin of safety in many species of non- ing it a suitable anesthetic for studies on hormonal change (Cashuman primates, and it has been used as an agent for restraint tro et al., 1981; Malaivijitnond et al., 1998). and induction for subsequent administration of other injectable Combining xylazine, an a2-agonist, with ketamine provides or gaseous anesthetics. At the dose of 5-20 mg/kg IM, induc- muscular relaxation and analgesia sufficient for minor surgical tion is rapid and chemical restraint is provided for 15-30 min procedures. Ketamine (7 mg/kg) and xylazine (0.6 mg/kg) IM that is sufficient for minor procedures. Complete recovery oc- given to M a c a c a mulatta provides adequate anesthesia for digit curs within 1 hr, depending on the dosage used. Because non- amputation, tattooing, and urinary and spinal catheterization human primates retain pharyngeal and laryngeal reflexes after (Banknieder et al., 1978). However, combining xylazine with ketamine administration, laryngospasm is not uncommon ketamine produces bradycardia, second-degree heart block, during intubation. Poor muscle relaxation and, in some in- and significant hypotension. The undesirable cardiovascular stances, tonic-clonic movements commonly accompany keta- changes preclude the use of this combination in young primates mine anesthesia. For this reason, if a procedure requires com- and animals with cardiovascular deficits. Atropine is useful in plete immobilization and adequate muscle relaxation, other preventing some of these complications. Yohimbine may be agents are administered in combination with ketamine. The used for xylazine reversal. Addition of medetomidine (60influence of ketamine on hematologic, biochemical, and hor- 150 ~tg/kg) IM to ketamine ( 2 - 6 mg/kg) IM produces rapid inmonal values should be considered in interpreting experimental duction, stable immobilization with good myorelaxation, and data. Ketamine consistently reduces total leukocyte and ab- calm recovery in a variety of nonhuman primates (Lewis, 1993; solute numbers of lymphocyte and monocytes (Bennett et al., Vie, 1998). An advantage of inclusion of this a2-agonist with 1992; Fernie et al., 1994). The red blood cell count, hemoglo- ketamine is that medetomidine can be reversed with atipamebin, and hematocrit are also significantly lower when ketamine zole (300-750 ~tg/kg) with significantly shortened recovery is administered. When comparing the effects of ketamine on times.

22.

PREANESTHESIA,ANESTHESIA,ANALGESIA,AND EUTHANASIA

A combination of ketamine (5-10 mg/kg) and diazepam (0.2-0.4 mg/kg) is used to induce reliable sedation and avoid perioperative excitement in Old World primates. In Saimiri sciureus and Callithrix jacchus the dose of diazepam is higher, 1 mg/kg IM. Although sedation is short, this combination is effective for minor procedures requiring muscle relaxation (Woolfson, 1980). In contrast to diazepam, midazolam is better absorbed after IM injection, making it less painful, with more predictable anxiolytic sedation after this route of administration. Shorter elimination half-life makes it suitable to be used as an infusion (0.05-0.15 mg/kg) in combination with ketamine (15 mg/kg IM) for various imaging techniques in Macaca mulatta and Cercopithecus aethiops (Jacobs et al., 1993). b.

Tiletamine-Zolazepam (Telazol)

Telazol at 4.0-6.0 mg/kg IM has been reported to be useful as an anesthetic for minor procedures for about 4 5 - 6 0 min in various species of macaques (Cohen and Bree, 1978). New World primates require a higher dose of Telazol to produce immobilization; in Saimiri sciureus the dose is 10 mg/kg, and in Alouatta seniculus a dose of 22-30 mg/kg IM produces light to moderate anesthesia sufficient for restraint in wildlife conditions (Agoramoorthy and Rudran, 1994). c.

Miscellaneous Anesthetics

Although not available in the United States, alphaxolonealphadolone (Saffan) has been reported to produce effective anesthesia in New World primates. A single IM injection of Saffan at 11.5-15.5 mg/kg provides up to 1 hr of surgical anesthesia with good muscle relaxation in Saimiri sciureus. Rapid induction and uneventful recovery accompany Saffan anesthesia. However, respiratory depression and hypothermia are also noted (Logdberg, 1988). In Old World primates the dose of Saffan required to produce anesthesia is much higher. In macaques, effective surgical anesthesia will be achieved at a dosage of 120 mg/kg IM given as a single bolus or, alternatively, an IM injection of Saffan at 18 mg/kg, followed by 6-12 mg/kg IV as needed (Box and Ellis, 1973). d.

Propofol

Propofol provides a smooth induction with adequate muscle relaxation sufficient for procedures of short duration. Because rapid clearance of propofol contributes to relatively fast awakening, repeated boluses of 2 - 5 mg/kg IV can be administered to extend the duration of anesthesia without delaying recovery. At 2.5 mg/kg IV, propofol provides good muscle relaxation sufficient for laparoscopy, with minimal effects on cardiovascular and respiratory functions in Macacafascicularis (Sainsbury et al., 1991). However, at the dosages of propofol exceeding 2.5 mg/kg IV, it is not uncommon to produce apnea. A slow induc-

993

tion will minimize the respiratory effect of this agent. Supplemental oxygen via endotracheal tube or face masks may also be required. The other potential problem associated with the infusion of propofol is a moderate hypotension. The hypotension is dose-dependent and can be partially corrected by adjusting the propofol infusion. Maintaining adequate hydration is important to prevent propofol-induced hypotension. e.

Etomidate

Etomidate is an IV short-acting hypnotic agent that has been used in neuroanesthesia to monitor motor-evoked potentials after transcranial stimulation (Ghaly et al., 1990). Because it is very short-acting, an initial dose of 0.5 mg/kg IV, followed by repeated boluses of etomidate 0.2 mg/kg every 6-12 min, is needed to maintain anesthesia in Macacafascicularis. f

Barbiturates

Thiopental is used for induction to facilitate intubation prior to maintenance of anesthesia with inhalational agents. One-half of the calculated dosage of 5-10 mg/kg IV is given as a bolus, followed by additional drug to effect. The dosage is reduced if the animal has received ketamine. Slow infusion of thiopental at 15-17 mg/kg/hr provides satisfactory chemical restraint with stable physiological values for 90 min in Papio ursinus. Animals recover uneventfully within 20 min after discontinuation of the infusion (Goosen et al., 1984). Despite its long history of use in research animal anesthesia, pentobarbital is now less frequently used as an anesthetic for survival procedures. Pronounced respiratory depression at the dosages required to produce adequate analgesia, and inability to modulate the depth of anesthesia with a long period of recovery, make it a less popular general anesthetic. The dose of pentobarbital in nonhuman primates is 25-30 mg/kg IV; however, in animals that have been chemically restrained with ketamine, the dose is reduced by about one-third to one-half. The duration of surgical anesthesia will vary between 30 and 60 min. In some instances, there are applications for which pentobarbital may be useful. Because pentobarbital induces minimal changes in cerebrospinal fluid and decreases cerebral blood flow and metabolic rate, it may be beneficial for some neurosurgical procedures. g.

Opioids

Opioids have been used in nonhuman primates to reduce the requirement for inhalational anesthetics and to enhance intraoperative analgesia during balanced anesthesia. Fentanyl is a valuable supplement to isoflurane anesthesia for cardiovascular procedures in Papio species and macaques. It can be administered as a bolus (5-10 ~tg/kg) or as a continuous infusion (1050 ~tg/kg/hr) in combination with low inspired concentration of isoflurane (0.4-1.25%), with minimal effects on heart rate and

M. MICHAEL SWINDLE,ET AL.

994

blood pressure. Respiratory depression, however, is detectable in nonhuman primates even after a relatively small dosage of narcotic analgesic. In M a c a c a mulatta, 2 ~tg/kg of fentanyl produces a noticeable decrease in respiratory rate; a significant increase in Pco2 is seen after 4 ~tg/kg of fentanyl (Nussmeier et al., 1991). Reversal of opioid-induced respiratory depression is achieved with naloxone at 0.01 mg/kg IV.

2. InhalationalAnesthesia a.

Nitrous Oxide

Unlike the potent volatile anesthetics, nitrous oxide has high minimum alveolar concentration (MAC = 200%), preventing its use as a complete surgical anesthetic. Nitrous oxide has been used in combination with inhalational anesthetics such as halothane or isoflurane because it allows for a lower concentration of these agents. This in turn results in a less pronounced circulatory depression, which may accompany the sole administration of isoflurane or halothane. Tinker et al. (1977) demonstrated that the MAC of halothane was reduced from 1.15% to 0.75% when it was supplemented with 30% of nitrous oxide. b.

Halothane

There seems to be some variance of halothane among closely related species of nonhuman primates. For example, in M a c a c a arctoides, the MAC of halothane is 0.89% (Steffey et al., 1974), but in M a c a c a fascicularis it is 1.15% (Tinker et al., 1977). Cardiovascular effects of halothane have been well documented in nonhuman primates. The decline in blood pressure and cardiac output is proportional to the depth of anesthesia, with significant effects usually seen above 1% of halothane. Increasing halothane from 1.0 to 1.5% MAC results in a 30-50% decline in blood pressure and heart rate in M a c a c a mulatta (Ritzman et al., 1976). Renal perfusion and glomerular filtration are also decreased secondary to a decline in cardiac output and blood pressure. This may influence radiorenography studies because of the delay in excretion of various isotopes and contrast material (Dormehl et al., 1992). Cerebral vasodilation and increased cerebral blood flow are more pronounced with halothane than with other volatile anesthetics. This effect is dosedependent in macaques, with a maximum increase in cerebral blood flow observed at 2% of halothane (Morita et al., 1977). Because this may also have a potentially adverse effect on intracranial pressure, halothane should be used with caution in neurosurgical procedures.

c.

pression of cardiac output. However, isoflurane produces a dose-dependent decrease in blood pressure because of its reduction in systemic vascular resistance. The hypotension is especially pronounced during mask induction or when animals are maintained at or above 2%. Inclusion of fentanyl attenuates the decrease in blood pressure associated with isoflurane. In young animals, isoflurane-induced hypotension most likely reflects unrecognized hypovolemia. Isoflurane produces direct cerebral vasodilatory effects that are dose-related. Although isoflurane-induced hypotension reduces cerebral blood flow, it has been shown to be a safe anesthetic from the cerebral oxygenation point of view in M a c a c a mulatta (Enlund et al., 1997). Isoflurane anesthesia also does not influence the binding of dopamine receptor ligands, making it suitable for various positron emission tomography studies (Nader et al., 1999). The effect of isoflurane on calcium metabolism in cynomolgus monkeys has been studied. Unexpectedly, isoflurane has been shown to lower ionized calcium, with secondary increases in parathyroid hormone and osteocalcin concentrations in cynomolgus monkeys (Hotchkiss et al., 1998). A cautious approach may be needed with respect to the use of isoflurane for studies of osteoporosis and the effects of various antiosteoporotic agents.

Isoflurane

Isoflurane has a MAC of 1.28% in M a c a c a fascicularis (Tinker et al., 1977). Isoflurane, unlike halothane, does not sensitize the myocardium to the arrhythmogenic properties of circulating catecholamines. Heart rhythm is stable, and there is minimal de-

d.

Sevoflurane

The new inhalational anesthetic sevoflurane is characterized by a low blood/gas partition coefficient, making it useful for rapid induction and fast recovery from anesthesia. Because of its pleasant smell and lack of respiratory irritant properties, mask induction is a feasible alternative to other inhalant agents. However, it is degraded by carbon dioxide absorbents into a haloalkele known commonly as compound A. Compound A at high doses has been shown to be nephrotoxic in nonhuman primates, causing proximal tubular necrosis (Kharasch, 1998). In common with other inhalational anesthetics, sevoflurane causes a dose-dependent cardiovascular depression. During sevoflurane anesthesia, cerebral blood flow is generally preserved; however, at 3.0% sevoflurane there is a clear inhibition of autoregulation of cerebral blood flow in rhesus monkeys (Yoshikawa et al., 1997). On the other hand, even at high concentrations of sevoflurane, the ratio of oxygen consumption and delivery is well maintained throughout the brain regions.

D.

Intraoperative Monitoring and Support

Intraoperative monitoring of nonhuman primates provides valuable information on physiological function during anesthesia. Routine monitoring includes electrocardiogram, oximetry, and body temperature assessment. Indirect blood pressure monitoring, with the pediatric cuff of appropriate size placed around the area of the radial or tibial artery, should be reserved for procedures on pregnant nonhuman primates. For complex surgical

22. PREANESTHESIA,ANESTHESIA,ANALGESIA,AND EUTHANASIA

procedures, cannulation of the femoral artery will provide for direct arterial pressure monitoring and allow for frequent blood sampling to determine arterial blood gases. Noninvasive evaluation of pulmonary function can be achieved with monitors such as pulse oximetry and end-tidal CO2 monitors. A pediatric digit oxygen transducer may be placed on the tongue or ear of nonhuman primates to provide continuous measurement of arterial oxygen saturation. Smaller nonhuman primates are susceptible to significant hypothermia. The use of warm intravenous fluids and convective warming devices, such as Bair Hugger and thermal blankets, will minimize the severity of hypothermia.

E.

Special Anesthesia Considerations ~

1.

Cardiac Transplant Anesthesia

Cardiac anesthesia for cardiac transplantation can be separated into distinct periods. The preincision period is one of minimal stimulation. The goal is to maintain primates on an isoflurane concentration (usually 1.0 - 1.25 %) sufficient to produce loss of consciousness and avoid hemodynamic instability. Maintaining primates on high concentrations of isoflurane in the absence of surgical stimuli often leads to reduction in blood pressure. The period from incision to initiation of cardiopulmonary bypass (CPB) is characterized by periods of intense surgical stimulation. Addition of fentanyl 10-20 ~tg/kg, followed by fentanyl infusion (10-25 ~tg/kg/hr) while nonhuman primates are maintained on 0.7-1.0% isoflurane, seems to produce minimal cardiac depression while maintaining the ability to adjust to changing intraoperative conditions. Perioperative anesthesia in conjunction with surgery may produce a complex and unstable physiologic status. The hypotension that occurs during maintenance of anesthesia is treated with IV infusion of fluids and the use of vasopressors. A bolus of 1-2 ~tg/kg phenylephrine followed by phenylephrine infusion is an excellent choice for intraoperative hypotension. Institution of CPB is often associated with declines in mean arterial pressures, presumably reflecting hemodilution that results from the infusion of priming solutions. Any reduction in perfusion pressure below 40 mm Hg can be effectively treated with phenylephrine. Although anesthetic requirements are usually reduced during hypothermic CPB, blood pressure often begins to rise, reflecting activation of the sympathetic nervous system. Repeated doses of 1-2 ~tg/kg of fentanyl and 0.2-0.4 mg/kg of midazolam are needed to prevent the increase in MAP and maintain it within 5 0 - 6 0 mm Hg. After completion of surgical anastomosis and rewarming, defibrillation followed by lidocaine (12 mg/kg) is directed toward restoring electrical activity to the heart. Ventricular volume and contractility can be evaluated visually, and cardiac indices and preload and volume status can be obtained with the aid of a Swan-Ganz catheter. A dopamine infusion is administered (3-5 ~tg/kg/min) to augment cardiac

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contractility and improve renal blood flow. Continuous IV infusions of norepinephrine (0.05-0.2 ~tg/kg/min) and dobutamine (2-10 ~tg/kg/min) may be necessary to maintain cardiac output and blood pressure. During the postbypass period the bleeding is controlled with a dose of protamine (at the ratio of 1 mg to 80-100 U of heparin) to reverse heparin activity. The protamine must be given slowly to avoid pronounced hypotension. Transplanted nonhuman primates do not tolerate surgical levels of isoflurane well; following bypass, animals are maintained on low inspired concentration of isoflurane supplemented with a fentanyl infusion (5-7 ~tg/kg/hr). Before closure of the sternotomy incision, intercostal injections of 0.25% bupivicaine are given to produce prolonged sensory analgesia. Postsurgically, maintenance of an arterial catheter until the nonhuman primate regains sufficient consciousness allows one to determine arterial blood gases in the animal as it spontaneously breathes air. Extubation is considered only if the nonhuman primate is hemodynamically stable and can maintain arterial oxygenation above 60 mm Hg on room air. Before extubation, the oropharynx and trachea are thoroughly suctioned to prevent obstruction of the respiratory passages with secretions. 2.

Obstetric Anesthesia

Physiologic changes occuring during pregnancy (such as increased cardiac output and decreased requirements for inhaled anesthetics) can directly influence anesthesia management for obstetric surgery. Maintenance of anesthesia for obstetric procedures is usually achieved with volatile anesthetics, such as isoflurane or halothane. Low inspired concentrations of either agent, supplemented with at least 5 0 - 6 0 % of inspired nitrous oxide, provide an advantage of producing a less pronounced circulatory depression, which may be seen with a sole administration of isoflurane or halothane. It has been shown that maintaining pregnant macaques at around 1.5% halothane reduces maternal blood pressure and cardiac output with a consequential reduction in uterine blood flow (Eng et al., 1975; Sanders et al., 1991). This in turn will decrease fetal perfusion, resulting in fetal acidosis and hypoxia. Placing pregnant animals in a supine position may contribute to the hypotension because of compression of the caudal vena cava and abdominal aorta by the gravid uterus. Therefore the animal should be tilted to the left by placing a wedge under the right hip. Adequate fluid therapy is also indicated to prevent hypovolemia and hypotension. Placement of a urinary catheter allows one to monitor the effectiveness of fluid therapy. Because hypotension is the most common complication encountered during anesthesia, monitoring of blood pressure should be instituted during experimental procedures in pregnant nonhuman primate. If maternal hypotension persists despite hydration and positioning, consideration should be given to vasopressor therapy. Ephedrine (2.5-5.0 mg) is the safest of vasopressors to be used during maternal hypotension. Because of its predominant [3-adrenergic activity, ephedrine will maintain arterial perfusion by increasing maternal

M. MICHAELSWINDLE,ETAL.

996

cardiac output and restoring uterine blood flow without uterine arterial vasoconstriction. Maternal normocapnia should be maintained during the intraoperative period because the effects of hypoventilation may be associated with premature ventricular contraction in macaques (Sanders et al., 1991). Accordingly, controlled ventilation is the most efficient way of maintaining normal Pco2. 3. Pediatric Anesthesia

There are important physiologic differences between adults and neonates. Neonatal cardiac output is heart rate-dependent. Therefore, one of the main goals of preanesthesia and induction is avoidance of heart rate reduction. Administration of ketamine and atropine provides protection against possible reduction in heart rate during the initial stages of anesthesia in pediatric primates. Although mask induction is also an accepted technique in pediatric animals, it must be kept in mind that isoflurane is pungent and produces airway irritation, which could possibly result in laryngospasm during induction. Smooth induction can be easily achieved with the IV dose of propofol at 2 - 4 mg/kg. Because neonatal primates are usually allowed to nurse until anesthesia time, assisted ventilation during mask induction may lead to insufflation of the stomach with gases and put neonates at risk of aspiration. Cuffed or uncuffed tracheal tubes may be used in intubation of pediatric primates. Postintubation laryngeal edema is unlikely if the size of the tube in the trachea is such that slightly audible air leaks occur around it. Small tidal volume in pediatric primates requires the use of nonrebreathing systems such as modified Jackson-Rees and Bain. The fresh gas flow of 1.5-2.0 liter/min is adequate to prevent rebreathing during spontanous ventilation. Because isoflurane depresses spontaneous ventilation, controlled or assisted ventilation will prevent CO2 accumulation. Isoflurane appears to be the anesthetic of choice for anesthesia maintenance in pediatric primates. It may increase heart rate slightly but maintains a stable rhythm. The hypotension that accompanies administration of isoflurane is not uncommon in pediatric primates. Isoflurane-induced hypotension can be treated with phenylephrine or in some cases with a slow infusion of norepinephrine. Because vasoconstrictive responses of neonates to hemorrhage are less than those of adults, even moderate blood loss will result in precipitous decline in blood pressure. Any blood losses should be immediately replaced with 3 - 4 times the volume of crystalloid solution. 4. Anesthesia for Magnetic Resonance Imaging (MRI)

MRI has become the imaging modality of choice because it is noninvasive and provides multiplanar, high-contrast images that are sensitive to myelin saturation and blood flow. However, MRI also introduces unique problems for the provision of general

anesthesia. Magnetic and radiofrequency interactions between the imager and anesthetic equipment may result in image degradation and interference with anesthetic monitoring devices. Typically, IV anesthetics are used to provide sedation for MRI. Propofol has many characteristics of the ideal IV anesthetic for MRI. It is commonly administered by IV bolus followed by infusion. Some potential problems associated with the infusion of propofol are moderate hypotension and respiratory depression. The hypotension is dose-dependent and can be partially corrected by adjusting the propofol infusion. It is also important to emphasize the need for adequate hydration to prevent propofol-induced hypotension. Propofol-induced depression of respiration results in hypoxemia; supplemental oxygen is recommended. 5. Neurosurgical Anesthesia

Selection of a specific anesthetic depends on the effect of the agents on intracranial pressure, cerebral blood flow, cerebral perfusion pressure and metabolic rate, promptness of return of consciousness, and drug-related protection from cerebral ischemia. Thiopental is one of the safest induction agents, owing to its ability to decrease cerebral blood flow and metabolic rate. Although the use of muscle relaxants is somewhat controversial, a muscle relaxant such as vecuronium (0.05 mg/kg IV) is a useful adjunct to general anesthesia by virtue of its ability to allow establishment of effective controlled ventilation. Maintenance of anesthesia is usually achieved with the use of nitrous oxide, isoflurane, and fentanyl in various combinations. The present authors routinely administer a high dose of fentanyl (70-100 lxg/kg/hr) in combination with a low inspired concentration of isoflurane (0.3-0.5%). Establishing hypocarbia to Pco2 levels of 28-32 mm Hg blunts the tendency of inhalant anesthetics to increase intracranial pressure. Because glucosecontaining solutions have been shown to worsen cerebral injury in macaques, judicious use of glucose-free isotonic electrolyte solutions provides the most appropriate fluid therapy (Lanier et aL, 1987).

F.

Postoperative Recovery

Unlike monitoring of postoperative procedures in other species, monitoring recovery in nonhuman primates is usually limited to the immediate postoperative period. Although extubation is performed after the nonhuman primate regains the swallowing reflex, it may lead to gagging or vomiting. If vomiting occurs, the animal should be placed in a prone position with its head lowered to avoid aspiration of the vomitus, and the oropharynx should be suctioned. Gentle suctioning of the trachea is important in smaller primates because respiratory passages can be easily obstructed with bronchial secretions. Monitoring of cardiopulmonary functions in the immediate

997

22. PREANESTHF_~IA, ANESTHESIA, ANALGESIA, AND EUTHANASIA

postoperative period will depend on the extent and type of procedure. Monitoring of arterial pressure is often needed until the nonhuman primate regains sufficient consciousness after cardiac or neurosurgical procedures. Maintaining a femoral arterial catheter helps to determine the status of arterial blood gases and avoid hypoxemia in the immediate postoperative period. Postoperative hypothermia is frequently encountered during surgical procedures more often in young and small nonhuman primates. Hypothermia and associated shivering can be treated effectively with convective warming devices such as Bair Hugger. Administering warm fluids will help avoid pronounced hypothermia. 1. Analgesic Therapy

Postoperatively, nonhuman primates often show little reaction to surgical procedures, and it should be recognized that the signs of pain may not be evident until the animal is in severe pain. Therefore, treating postoperative pain in a nonhuman primate in an effective and timely matter is one of the most important tasks of the immediate postoperative period.

Tuohy epidural needle, which is introduced into epidural space. Entry into epidural space is confirmed by the ease of depression of the plunger in the syringe. Epidural morphine, at 0.1 mg/kg, has been used for postoperative pain relief after major abdominal surgeries (Popilskis et al., 1994). Administration of epidural morphine before surgery may also provide preemptive analgesia and potentially reduce the need for additional analgesia in the immediate postoperative period. 4. Local, Regional, and Epidural Anesthesia

Local and regional anesthesia is usually administered as an adjunct for postoperative pain relief. Intercostal nerve blocks with bupivicaine (0.25-0.5%) provide better pain relief after thoracotomy in nonhuman primates. Generally, a combination of local anesthetics (lidocaine or bupivicaine at 1.5 mg/kg) with opioids (morphine epidurally at 0.05-0.1 mg/kg) provides more complete postoperative analgesia in nonhuman primates by altering more than one pathway in the nociceptive process.

G. 2. NSAIDs

The use of NSAIDs in postoperative analgesia has been gaining popularity since the introduction of potent injectable and oral drugs such as ketorolac and carprofen. Ketorolac (515 mg) produces moderate analgesia and, unlike opioids, has not been associated with respiratory depression or sedation. As with other NSAIDs, it inhibits platelet function for 2 4 - 4 8 hr and therefore should be used with caution in animals with acquired or natural coagulopathies. Aspirin (125 mg in suppository form) provides mild pain relief after minor lacerations or procedures involving catheter placements. 3.

Opioids

Traditionally, management of pain consisted of a standard dose of opioid given IV or IM. Effective postoperative analgesia can be attained by using morphine IM or SC or oxymorphone IM every 4 hr (Table XII). Buprenorphine has been widely used for postoperative analgesia in a variety of nonhuman primates. Buprenorphine IM or SC provides mild to moderate pain relief without excessive sedation in Saimiri sciureus, Galago spp., and Macaca mulatta. The disadvantages of using parenteral analgesics include a relatively short effective analgesia time and unpredictable absorption in hypotensive and hypothermic animals. Epidural administration of opioids provides the benefit of prolonged analgesia with minimal sympathetic blockade and no sedation or respiratory depression. Placement of an epidural needle is most commonly done at a lower lumbar interspace using the "loss of resistance" technique. With this technique, a glass syringe with a freely movable plunger is attached to

Euthanasia

An overdose of pentobarbital (> 100 mg/kg) administered IV is the preferred form of euthanasia in nonhuman primates. Prior to pentobarbital injection, animals are usually restrained with a dose of ketamine at 5-10 mg/kg IM. KC1 injection administered under anesthesia is also an acceptable method of euthanasia.

REFERENCES

Aeschbacher, G., and Webb, A. I. (1993a). Propofol in rabbits. 1. Determination of an induction dose. Lab. Anim. Sci. 43, 324-327. Aeschbacher, G., and Webb, A. I. (1993b). Propofol in rabbits. 2. Long-term anesthesia. Lab. Anim. Sci. 43, 328-335. Agoramoorthy, G., and Rudran, R. (1994). Field application of Telazol (tiletamine + zolazepam) immobilaze wild howler monkeys (Alouatta seniculus) in Venezuela. J. Wildl. Dis. 30(3), 417-420 Alibhai, H. I. K., Clarke, K. W., Lee, Y. H., and Thompson, J. (1996). Cardiopulmonary effects of combinations of medetomidine hydrochloride and atropine sulphate in dogs. Vet. Rec. 138, 11-13 Allen, M. J., and Borkowski, G. L. (1999). "The Laboratory Small Ruminant." CRC Press, Boca Raton, Florida. Andersen, H. E., Fosse, R. T., Kuiper, K., Nordrehaug, J. E., and Pettersen, R. J. (1998). Ketorolac (Toradol) as an analgesic in swine following angioplastic and angiographic procedures. Lab. Anim. 32(3), 307-315. Andrews, E. J., Bennet, B. T., Clark J. D., et al. (1993). Report of the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc. 202, 229-249. Banknieder, A. R., Phillips, J. M., Jackson, K. T., and Vinal, S. I. (1978). Comparison of ketamine with the combination of ketamine and xylazine for effective analgesia in the rhesus monkey (Macaca mulatta). Lab. Anim. Sci. 28, 742-745.

998

Barquet, I. S., Soriano, E. S., Green, W. R., and O'Brien, T. E (1999). Provision of anesthesia with single application of lidocaine 2% gel. J. Cataract Refract. Surg. 25, 626-631. Bennett, J. S., Gossett, K. A., McCarthy, M. P., and Simpson, E. D. (1992). Effects of ketamine hydrochloride on serum biochemical and hematologic variables in rhesus monkeys (Macaca mulatta). Vet. Clin. Pathol. 21, 1518. Benson, G. J. (1992). "Defining Pain in Laboratory Animals." Office for Protection from Research Risks, Vanderbilt Univ. Medical Center, and Maharry Medical College Workshop on Minimizing Pain and Distress in Laboratory Animals, Nashville. Benson, G. J., and Thurmon, J. C. (1986). Regional analgesia of food animals. In "Current Veterinary Therapy 2: Food Animal Practice" (J. L. Howard, ed.), pp. 71-83. Saunders, Philadelphia. Berg, R. J., and Orton, E. C. (1986). Pulmonary function in dogs after intercostal thoracotomy: comparison of morphine, oxymorphone, and selective intercostal nerve block. Am. J. Vet. Res. 47, 471-474. Blake, D. W., Jover, B., and McGrath, B. P. (1988). Haemodynamic and heart rate reflex responses to propofol. Br. J. Anaesth. 61, 194-199. Blake, D. W., Way, D., Trigg, L., Langton, D., and McGrath, B. P. (1991). Cardiovascular effects of volatile anesthesia in rabbits: influence of chronic heart failure and enapril treatment. Anesth. Analg. 73, 441-448. Bonath, K., Nolte, I., Schniewind, A., Sandmann, H., and Failing, K. (1982). Food deprivation as precaution and aftercare measure for anaesthesia--the influence of fasting on the acid base status and glucose concentration in blood of rabbits. Berl. Munch. Tierarztl. Wochenschr. 95, 126-130. Borkowski, G. L., Danneman, P. J., Russell, G. B., and Lang, C. M. (1990). Evaluation of three intravenous anesthetic regimens in New Zealand rabbits. Lab. Anim. Sci. 4t), 270-276. Box, P. G., and Ellis, K. R. (1973). Use of CT 1341 anaesthetic (Saffan) in monkeys. Lab. Anim. 7, 161-170 Brammer, A., West, C. D., and Allen, S. L. (1993). A comparison of propofol with other injectable anesthetics in a rat model for measuring cardiovascular parameters. Lab. Anim. 27, 250-257. Bree, M. M.,. and Cohen, B. J. (1965). Effects of urethane anesthesia on blood and blood vessels in rabbits. Lab. Anim. Care 15, 254-259. Buelke-Sam, J., Hoson, J. E, Bazare, J. J., and Young, J. E (1978). Comparative stability of physiological parameters during sustained anesthesia in rats. Lab. Anim. Sci. 28(2), 157-162. Burton, S., Lemke, K. A., Ihle, S. L., and Mackenzie, A. L. (1998). Effects of medetomidine on serum osmolality; urine volume, osmolality, and pH; free water clearance; and fractional clearance of sodium, chloride, potassium, and glucose in dogs. Am. J. Vet. Res. 59(6), 756-761. Cambron, H., Latulippe, J. E, Nguyen, T., and Cartier, R. (1995). Orotracheal intubation in rats by transillumination. Lab. Anim. Sci. 45(3), 303-304. Castro, M. I., Rose, J., Green, W., Lehner, N., Peterson, D., and Taub, D. (1981). Ketamine-HCL as a suitable anesthetic for endocrine, metabolic, and cardiovascular studies in Macaca fascicularis monkeys. Proc. Soc. Exp. Biol. Med. 168, 389-394. Chakrabarty, S., Thomas, P., and Sheridan, D. J. (1991). Arrhythmias, haemodynamic changes, and extent of myocardial damage during coronary ligation in rabbits anaesthetized with halothane, alpha chloralose, or pentobarbitone. Int. J. Cardiol. 31, 9-14. Clark, J. A., Myers, P. H., Goelz, M. E, Thigpen, J. E., and Forsythe, D. B. (1997). Pica behavior associated with buprenorphine in the rat. Lab. Anim. Sci. 47(3), 300-303. Cohen, B. J., and Bree, M. M. (1978). Chemical and physical restraint of nonhuman primates. J. Med. PrimatoL 7, 193-201. Cooper, D. M., DeLong, D. E., and Gillett, C. S. (1997). Analgesic efficacy of acetaminophen and buprenorphine administered in the drinking water of rats. Lab. Anim. Sci. 36(3), 58-62. Cope, D. K., Impastato, W. K., Cohen, M. V., and Downey, J. M. (1997). Volatile anesthetics protect the ischemic rabbit myocardium from infarction. Anesthesiology 86, 699-709.

M. MICHAEL SWINDLE, E T A L . Danneman, E J. (1997). Monitoring of analgesia. In "Anesthesia and Analgesia in Laboratory Animals" (D. H. Kohn, S. K. Wixon, W. J. White, and G. J. Benson, eds.) pp. 83-103. Academic Press, New York. Danneman, P. J., and Man&ell, T. D. (1997). Evaluation of five agents/methods for anesthesia of neonatal rats. Lab. Anim. Sci. 47(4), 386-395. Danneman, P. J., White, W. J., Marshall, W. K., and Lang, C. M. (1988). Evaluation of analgesia associated with the immobility response in laboratory rabbits. Lab. Anim. Sci. 38, 51-56. Danneman, P. J., Stein, S., and Walshaw, S. O. (1997). Humane and practical implications of using carbon dioxide mixed with oxygen for anesthesia or euthanasia of rats. Lab. Anim. Sci. 47(2), 376-385. DeCastro, G., and Viars, P. (1968). Anesthetie analgeoique sequentielle, ou A.A.S. Arch. Med. 23, 170-176. Delaporte-Cerceau, S., Samama, C. M., Riou, B., Bonnin, P., Guillosson, J. J., and Coriat, P. (1998). Ketorolac and enoxaparin affect arterial thrombosis and bleeding in the rabbit. Anesthesiology 88, 1310 - 1317. De Mulder, P, A., Van Kerckhoven, R. J., Adriaensen, H. E, Gillebert, T. C., and De Hert, S. G. (1997). Continuous total intravenous anesthesia using propofol and fentanyl in an open-thorax rabbit model: evaluation of cardiac contractile function and biochemical assessment. Lab. Anita. Sci. 47, 367375. Dodam, J. R., Kruse-Elliot, K. T., Aucion, D. E, and Swanson, C. R. (1990). Duration of etomidate-induced adrenocortical suppression during surgery in dogs. Am. J. Vet. Res. 51, 786-788. Doerning, B. J., Brammer, D. W., Chrisp, C. E., and Rush, H. G. (1992). Nephrotoxicity of tiletamine in New Zealand White rabbits. Lab. Anita. Sci. 42, 267-269. Dormehl, I. C., Redelinghuys, E, Hugo, N., Oliver, D., and Pilloy,.W. (1992). The baboon model under anesthesia for in vivo cerebral blood flow studies using single photon emission computed tomographic (SPECT) techniques. J. Med. Primatol. 21, 270-274. Dorr, W., and Weber-Frisch, M. (1998). Short-term immobilization of mice by methohexitone. Lab. Anita. 33, 35-40. Drummond, J. C. (1985). MAC for halothane, enflurane, and isoflurane in the New Zealand White rabbit: and a test for the validity of MAC determinations. Anesthesiology 62, 336-338. Dunlop, C. I., and Hoyt, R. E (1997). Anesthesia and analgesia in ruminants. In "Anesthesia and Analgesia in Laboratory Animals" (D. E Kohn, S. K. Wixon, W. J. White, and G. J. Benson, eds.), pp. 281-331. Academic Press, New York. Dyson, D., and Pettifer G. (1997). Evaluation of the arrhythmogenicity of a low dose of acepromazine: comparison with xylazine. Can. J. Vet. Res. 61(4), 241-254. Ecobichon, D. J., and Comeau, A. M. (1974). Genetic polymorphism of plasma carboxylesterases in the rabbit: correlation with pharmacologic and toxicologic effects. Toxicol. Appl. Pharmacol. 27, 28-40. Eisele, E H., Talken, L., and Eisele, J. H. (1985). Potency of isoflurane and nitrous oxide in conventional swine. Lab. Anita. Sci. 35, 76-78. Eng, M., Bonica, J. J., Alamatsu, T. J., Berges, E U., Yuen, D., and Ueland, K. (1975). Maternal and fetal responses to halothane in pregnant monkey. Acta Anaesthesiol. Scand. 19, 154-158. Enlund, M., Andersson, J., Hartvig, E, Valtysson, J., and Wiklund, L. (1997). Cerebral normoxia in the rhesus monkey during isoflurane or propofol induced hypotension and hypocapnia, despite disparate blood-flow patterns. Acta Anaesthesiol. Scand. 41(8), 1002-1010. Erhardt, W., Hebestedt, A., and Aschenbrenner, G. A. (1984). Comparative study with various anesthetics in mice (pentobarbitone, ketamine-xylazine, carfentanyl-etomidate). Res. Exp. Med. 184, 159-169. Ewing, K. K. (1990). Anesthesia techniques in sheep and goats. Vet. Clin. North Am. Food Anita. Pract. 6(3), 759-778. Fernie, S., Wrenshall, E., Malcolm, S., Bryce, E, and Arnold, D. L. (1994). Normative hematological and serum biochemical values for adult and infant rhesus monkeys (Macaca mulatta) in a controlled laboratory environment. J. Toxicol. Environ. Health 42, 53-72.

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA Field, K. J., White, W. J., and Lang, C. M. (1993). Anaesthetic effects of chloral hydrate, pentobarbitone, and urethane in adult male rats. Lab. Anim. 27, 258-269. Flecknell, P. A. (1987). "Laboratory Animal Anesthesia." Academic Press, London. Flecknell, P. A. (1989). Rodent and rabbit anesthesia lecture. Paper presented at 40th Annual American Association for Laboratory Animal Science Meeting, Little Rock, Arkansas. Flecknell, P. A. (1996a). "Laboratory Animal Anaethesia," 2nd ed. Academic Press, London. Flecknell, P. A. (1996b). Anesthesia and analgesia for rodents and rabbits. In "Handbook of Rodent and Rabbit Medicine" K. Laber, M. M. Swindle, and P. Flecknell, eds.), pp. 219-238. Pergamon (Elsevier Science), Oxford. Flecknell, P. A. (1996c). Anaesthesia. In "Laboratory Animal Anaethesia," 2nd ed, p. 68. Academic Press, London. Flecknell, P. A. (1997). Medetomidine and atipamezole: potential uses in laboratory animals. Lab. Anim. 26, 21-25. Flecknell, P. A., and Liles, J. H. (1990). Assessment of the analgesic action of opioid agonist-antagonists in the rabbit. J. Assoc. Vet. Anaesth. 17, 24-29. Flecknell, P. A., and Mitchell, M. (1984). Midazolam and fentanyl-fluanisone: assessment of anaesthetic effects in laboratory rodents and rabbits. Lab. Anim. 18, 143-146. Flecknell, P. A., and Waterman-Pearson, A. (2000). "Pain Management in Animals." W. B. Saunders, London. Flecknell, P. A., John, M., Mitchell, M., Shurey, C., and Simpkin, S. (1983). Neuroleptanalgesia in the rabbit. Lab. Anim. 17, 104-109. Flecknell, P. A., Liles, J. H., and Wooton, R. (1989). Reversible fentanyl/ fluanisone neuroleptanalgesia in the rabbit using mixed agonist/antagonist opioids. Lab. Anim. 23, 147-155. Flecknell, P. A., Liles, J. H., and Williamson, H. A. (1990). The use of lignocaine-prilocaine local anaesthetic cream for pain-free venepuncture in laboratory animals. Lab. Anim. 24, 142-146. Flecknell, P. A., Roughan, J. V., and Stewart, R. (1999a). Use of oral buprenorphine ("buprenorphine jello") for postoperative analgesia in rats--a clinical trial. Lab. Anim. 32(2), 169-174. Flecknell, P. A., Orr, H. E., Roughan, J. V., and Stewart, R. (1999b). Comparison of the effects of oral or subcutaneous carprofen or ketoprofen in rats undergoing laparotomy. Vet. Rec. 144, 65-67. Forsythe, D. B., Payton, A. J., Dixon, D., Meyers, P. H., Clark, J. A., and Snipe, J. R. (1992). Evaluation of Telazol-xylazine as an anesthetic combination for use in Syrian hamsters. Lab. Anim. Sci. 42(5), 497-502. Foster, P. S., Hopkinson, K. C., and Denborough, M. A. (1992). Propofol anaesthesia in malignant hyperpyrexia susceptible swine. Clin. Exp. Pharmacol. Physiol. 19, 183-186. Fox, S. M., and Johnston, S. A. (1997). Use of carprofen for the treatment of pain and inflammation in dogs. J. Am. Vet. Med. Assoc. 210(10), 14931498. Fraser, A. (1995). Sheep. In "The Experimental Animal in Biomedical Research" (B. Rollin and M. L. Kessel, eds.), Vol. 2, pp. 87-118. CRC Press, Boca Raton, Florida. Fujimoto, J. L. (1992). Dosage tables for opiate agonists and agonist-antagonists in laboratory animals. In "Animal Pain" (C. E. Short and A. V. Poznak, eds.), pp. 539-542. Churchill Livingstone, New York. Fulton, L. K., M. S. Clarke, and H. E. Farris. (1994). The goat as a model for biomedical research and teaching. ILAR News 36(2), 21-29. Gardiner, T. W., and Toth, L. A. (1999). Stereotactic surgery and long-term maintenance of cranial implants in research animals. Contemp. Top. Lab. Anita. Sci. 38(1), 56-63. Ghaly, R. E, Stone, J. M., Levy, W. J., Roccaforte, E, and Brunner, E. B. (1990). The effects of etomidate on motor evoked potentials induced by transcranial magnetic stimulation in the monkey. Neurosurgery 27(6), 936-942. Gleed, R. D. (1987). Tranquilizers and sedatives. In "Principles and Practice of Veterinary Anesthesia" (C. E. Short, ed.), pp. 20-21,384-386. Williams and Wilkins, Baltimore.

999 Glen, J. B., and Hunter, S. C. (1984). Pharmacology of an emulsion formulation of IC135868. Br. J. Anaesth. 52, 617-626. Goosen, D. J., Davies, J. H., Maree, M., and Dormehl, I. C. (1984). The influence of physical and chemical restraint on the physiology of the chacma baboon (Papio ursinus). J. Med. Primatol. 13, 339-351. Green, C. J. (1975). Neuroleptanalgesic drug combinations in the anaesthetic management of small laboratory animals. Lab. Anim. 9, 161-178. Green, C. J. (1979). Order Carnivora. In "Animal Anaesthesia," pp.199-215. Laboratory Animals, London. Green, C. J., Halsey, M. J., and Precious, S. (1978). Alphaxolone-alphadolone anaesthesia in laboratory animals. Lab. Anim. 12, 85-90. Green, C. J., Knight, J., Precious, S., and Simpkin, S. (1981). Ketamine alone and combined with diazepam or xylazine in laboratory animals: a 10 year experience. Lab. Anim. 15, 163-170. Grisneaux, E. Pibarot, P., Dupuis, J., and Bias, D. (1999). Comparison of ketoprofen and carprofen administered prior to orthopedic surgery for control of postoperative pain in dogs. J. Am. Vet. Med. Assoc. 215, 1105-1110. Hackbarth, H., Kiippers, N., and Bohnet, W. (1999). Euthanasia of rats with carbon dioxidelanimal welfare aspects. Lab. Anim. 34, 91-96. Hall, L. W., and Clarke, K. W. (1991). "Veterinary Anaesthesia." Bailliere Tindall, London. Harvey, R. C., Paddleford, R. R., Popilskis, S., and Wixson, S. K. (1997). In "Anesthesia and Analgesia in Laboratory Animals" (D. E Kohn, S. K. Wixson, W. J., White, and G. J. Benson, eds.), pp. 257-280. Academic Press, New York. Hedenqvist, P., Roughan, J. V., and Flecknell, P. A. (2000). Effects of repeated anaesthesia with ketamine/medetomidine and of pre-anaesthetic administration of buprenorphine in rats. Lab. Anim. 34, 207-211. Hellebrekers, L. J., de Boer, E.-J. W., van Zuylen, M. A., and Vosmeer, H. (1996). A comparison between medetomidine-ketamine and medetomidine-propofol anaesthesia in rabbits. Lab. Anim. 31, 58-69. Hellebrekers, L. J., van Herpen, H., Hird, J. E, Rosenhagen, C. U., Sap, R., and Vainio, O. (1998). Clinical efficacy and safety of propofol or ketamine anaesthesia in dogs premedicated with medetomidine. Vet. Rec. 142(23), 631-634. Hellyer, P. (1991). Anesthesia in small ruminants. Lecture presented at Clemson Univ., Clemson, South Carolina. Heniff, M. S., Moore, G. P., Trout, A., Cordell, W. H., and Nelson, D. R. (1997). Comparison of routes of flumazenil administration to reverse midazolaminduced respiratory depression in a canine model. Acad. Emerg. Med. 4(12), 1115-1118. Hildebrand, S. V. (1997). Paralytic agents. In "Anesthesia and Analgesia in Laboratory Animals" (D. H. Kohn, S. K. Wixon, W. J. White, and G. J. Benson, eds.), pp. 57-72. Academic Press, New York. Hindman, B. J., Funatsu, N., Cheng, D. C. H., Bolles, R., Todd, M. M., and Tinker, J. H. (1990). Differential effect of oncotic pressure on cerebral and extracerebral water content during cardiopulmonary bypass in rabbits. Anesthesiology 73, 951-957. Hotchkiss, C. E., Bromage, R., Du, M., and Jerome, C. E (1998). The anesthetic isoflurane decreases ionized calcium and increases parathyroid hormone and osteocalcin in cynomolgus monkeys. Bone 23(5), 479-484. Hrapkiewicz, Stein, S., and Smiler, K. L. (1989). A new anesthetic agent for use in the gerbil. Lab. Anita. Sci. 39(4), 338-341. Hsu, W. H., Bellin, S. I., and Dellmon, H. D. (1986). Xylazine-ketamine induced anesthesia in rats and its antagonism by yohimbine. J. Am. Vet. Med. Assoc. 189(9), 1040-1043. Hughes, E J., Doherty, M. M., and Charman, W. N. (1993). A rabbit model for the evaluation of epidurally administered local anaesthetic agents. Anaesth. Intensive Care 21, 298-303. Ilkiw, J. E. (1992). Other potentially useful new injectable anesthetics agents. Vet. Clin. North Am. Small Anita. Pract. Jacobs, B., Harris, G. C., Allada, V., Chugani, H. T., Pollack, D. B., and Raleigh, M. J. (1993). Midazolam as an effective intravenous adjuvant to prolonged ketamine sedation in young rhesus (Macaca mulatta) and vervet

1000 (Cercopithecus aethiops sabaeus) monkeys: a preliminary report. Am. J. Primatol. 29, 291-298. Jacobsen, C. (2000). Adverse effects on growth rates caused by buprenorphine administration. Lab. Anim. 34, 202-206. Jensen, E M., Dahl, J. B., and Frigast, C. (1992). Direct spinal effect of intrathecal acetaminophen on visceral noxious stimulation in rabbits. Acta Anaesthesiol. Scand. 36, 837-841. Kero, P., Thomasson, B., and Soppi, A. M. (1981). Spinal anaesthesia in the rabbit. Lab. Anim. 15, 347-348. Kharasch, E. D. (1998). Compound A: toxicology and clinical relevance. Anaesthesist 47(Suppl. 1), $7-S 10. Ko, J. C. H., Thurmon, J. C., Benson, G. J., Gard, J., Tranquilli, W. J., and Olson, W. A. (1992a). Evaluation of analgesia induced by epidural injection of detomidine or xylazine in swine. J. Vet. Anesth. 19, 56-60. Ko, J. C. H., Thurmon, J. C., Tranquili, W. J., Benson, G. J. and Olson, W. A. (1992b). Comparison of medetomidine-propofol and medetomidinemidazolam-propofol anesthesia in rabbits. Lab. Anim. Sci. 42, 503-507. Ko, J. C. H., Williams, B. L., McGrath, C. J., Short, C. E., and Rogers, E. R. (1997). Comparison of anesthetic effects of Telazol-xylazine-xylazine, Telazol-xylazine-butorphanol, and Telazol-xylazine-azaperone combinations in swine. Lab. Anita. Sci. 35, 71-74. Ko, J. C., Nicklin, C. E, Melendaz, M., Hamilton, E, Kuonen, C. D. (1998). Effects of a microdose of medetomidine on diazepam-ketamine induced anesthesia in dogs. J. Am. Vet. Med. Assoc. 213(2), 215-219. Kohn, D. E, Wixson, S. K., White, W. J., Benson, G. J., eds. (1997). "Anesthesia and Analgesia in Laboratory Animals." Academic Press, New York. Korner, E I., Uther, J. B., and White, S. W. (1968). Circulatory effects of chloralose-urethane and sodium pentobarbitone anaesthesia in the rabbit. J. Physiol. 199, 253-265. Kushner, L. I., Trim, C. M., Madhusudhan, S., and Boyle, C. R. (1995). Evaluation of the hemodynamic effects of interpleural bupivicaine in dogs. Vet. Surg. 24, 180-187. Kyles, A. E., Papich, M., and Hardie, E. M. (1996). Disposition of transdermally administered fentanyl in dogs. Am. J. Vet. Res. 57(5), 715-719. Lagutchik, M. S., Mundie, T. G., and Martin, D. G. (1992). Methemoglobinemia induced by a benzocaine-based topically administered anesthetic in eight sheep. J. Am. Vet. Med. Assoc. 201, 1407-1410. Landi, M. S., Kreider, J. W., and Lang, C. M. (1982). Effects of shipping on the immune function of mice. Am. J. Vet. Res. 43(9), 1654-1657. Langerman, L., Chaimsky, G., Golomb, E., Tverskoy, M., and Kook, A. (1990). Rabbit model for evaluation of spinal anesthesia: chronic cannulation of the subarachnoid space. Anesth. Analg. 71, 529-535. Lanier, W. L, Strangland, K. J., Scheithauer, B. W., Milde, J. H., and Michenfelder, J. D. (1987). The effects of dextrose infusion and head position on neurologic outcome after complete cerebral ischemia in primates: examination of a model. Anesthesiology 66(1), 39-48. Lascelles, B. D., Cripps, P. J., Jones, A., and Waterman-Pearson, A. E. (1998). Efficacy and kinetics of carprofen, administered preoperatively or postoperatively, for the prevention of pain in dogs undergoing ovariohysterectomy. Vet. Surg. 27(6), 568-582. Latt, R. H., and Ecobichon, D. J. (1984). Self-mutilation in guinea pigs following the intramuscular administration of ketamine-acepromazine. Lab. Anim. Sci. 34(5), 516. Leash, A. M., Beyer, R. D., and Wilber, R. G. (1973). Self-mutilation following Innovar-Vet injection in the guinea pig. Lab. Anim. Sci. 23(5), 720-721. Lewis, J. C. (1993). Medetomidine-ketamine anaesthesia in the chimpanzee (Pan troglodytes). J. Vet. Anaesthesiol. 20, 18-20. Liles, J. H., and Flecknell, P. A. (1992). The use of non-steroidal antiinflammatory drugs for the relief of pain in laboratory rodents and rabbits. Lab. Anim. 26, 241-255. Lindquist, P. A. (1972). Induction of methoxyflurane anesthesia in the rabbit after ketamine hydrochloride and endotracheal intubation. Lab. Anita. Sci. 22, 898-899.

M. MICHAEL SWINDLE, E T A L . Lipman, N. S., Marini, R. E, and Erdman, S. E. (1990). Comparison of ketamine/xylazine and ketamine/xylazine/acepromazine anesthesia in the rabbit. Lab. Anita. Sci. 40, 395-398. Lipman, N. S., Marini, R. E, and Flecknell, E A. (1997). Anesthesia and analgesia in rabbits. In "Anesthesia and Analgesia in Laboratory Animals" (D. E Kohn, S. K. Wixson, W. J. White, and G. J. Benson, eds.), pp. 205232. Academic Press, New York. Logdberg, B. (1988). Alphaxolone-alphadolone for anesthesia in squirrel monkeys of different ages. J. Med. Primatol. 17, 163-167. Lovell, D. E (1986a). Variation in pentobarbitone sleeping time in mice. 1. Strain and sex differences. Lab. Anita. 20, 85-90. Lovell, D. E (1986b). Variation in pentobarbitone sleeping time in mice. 2. Variables affecting test results. Lab. Anita. 20, 91-96. Lovell, D. P. (1986c). Variation in pentobarbitone sleeping time in mice. 3. Strain X environment interactions. Lab. Anim. 20, 307-312. Madsen, J., Jensen, E, Faber, T., and Bill-Hansen, V. (1993). Chronic catheterization of the epidural space in rabbits: a model for behavioural and histopathologial studies--examination of meptazinol neurotoxicity. Acta Anaesthesiol. Scand. 37, 307-313. Maggi, C. A., Manzini, S., Parlani, M., and Meli, A. (1984). Analysis of the effects of urethane on cardiovascular responsiveness to catecholamines in terms of its interference with Ca ++ mobilization from both intra and extracellular pools. Experientia 40, 52-59. Malaivijitnond, S., Takenaka, O. Sankai, T., Yoshida, T., Cho, F., and Yoshikawa, Y. (1998). Effects of single and multiple injections of ketamine hydrochloride on serum hormone concentrations in male cynomolgus monkeys. Lab. Anim. Sci. 48(3), 270-274. Malinosky, J. M., Bernard, J. M., Baudrimont, M., Dumand, J. B., and Lepage, J. Y. (1997). A chronic model for experimental investigation of epidural anesthesia in the rabbit. Reg. Anaesth. 22, 80-85. Marano, G., Formigari, R., Grigioni, M., and Vergari, A. (1997). Effects of isoflurane versus halothane on myocardial contractility in rabbits: assessment with transthoracic two-dimensional echocardiography. Lab. Anim. 31, 144-150. Marini, R. P., Avison, D. L., Corning, B. E, and Lipman, N. S. (1992). Ketamine/xylazine/butorphanol: a new anesthetic combination for rabbits. Lab. Anita. Sci. 42, 57-62. Marini, R. P., Hurley, R. J., Avison, D. L., and Lipman, N. S. (1993). Evaluation of three neuroleptanalgesic combinations in the rabbit. Lab. Anim. Sci. 43, 338-345. Mills, P., Sessler, D. I., Moseley, M., Chew, W., Pereira, B., James, T. L., and Litt, L. (1987). In vivo 19F nuclear magnetic resonance study of isoflurane elimination from the rabbit brain. Anesthesiology 67, 169-173. Miyamoto K., Wakita K., Okuda T., Fuji K., Suekane K. (1992). Suppressant effects of midazolam on responses of spinal dorsal horn neurones in rabbits. Neuropharmacology 31(1), 49-53. More, R. C., .Kody, M. H., Kabo, J. M., Dorey, E J., and Meals, R. A. (1989). The effects of two non-steroidal anti-inflammatory drugs on limb swelling, joint stiffness, and bone torsional strength following fracture in a rabbit model. Clin. Orthop. 247, 306-312. Morita, H., Nemoto, E. M., Bleyaert, A. L., and Stezoski, S. W. (1977). Brain flow autoregulation and metabolism during halothane anesthesia in monkeys. Am. J. Physiol. 233, H670-H676. Morita, H., Nashida, Y., Uemura, N., and Hosomi, H. (1987). Effect of pentobarbital anesthesia on renal sympathetic nerve activity in the rabbit. J. Auton. Pharmacol. 20, 57-64. Muir, W. W., and Gadawski, J. (1998). Cardiorespiratory effects of low-flow and closed circuit inhalational anesthesia, using sevoflurane delivered with an in-circuit vaporizer and concentrations of compound A. Am. J. Vet. Res. 59(5), 603-608. Muir, W. W., Hubbell, J. A. E., and Skarda, R. (1989). "Handbook of Veterinary Anesthesia," pp. 220-227. Mosby, St. Louis. Mutoh, T., Nishimura, R., and Kim, H. Y. (1997). Cardiopulmonary effects of

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA sevoflurane compared with halothane, enflurane, and isoflurane in dogs. Am. J. Vet. Res. 58(6), 885-890.

Nader, M. A., Grant, K. A., Gage, H. D., Ehrenkraufer, R. L., Kaplan, J. R., and Mach, R. H. (1999). PET imaging of dopamine D2 receptors with [18] fluoroclebopride in monkeys: effects of isoflurane- and ketamine-induced anesthesia. Neuropsychopharmacology 21(4), 589-596. National Institutes of Health (NIH). (1991). "Preparation and Maintenance of Higher Mammals during Neuroscience Experiments." NIH Publication 913207, Washington, D.C. Nevlainen, T., Pyhala, L. Voipio, H. M., and Virtanen, R. (1989). Evaluation of anaesthetic potency of medetomidine-ketamine combination in rats, guinea-pigs, and rabbits. Acta Vet. Scand. 85, 139-143. Newell, S. M., Ko, J. C., Ginn, P. E., Heaton-Jones, T. G., Hyatt, D. A., Cardwell, A. L., Mauragis, D. F., and Harrison, J. M. (1997). Effects of three sedative protocols on glomerular filtration rate in clinically normal dogs. Am. J. Vet. Res. 58(5), 446-450. Nussmeier, N. A., Buenthuysen, J. L., Steffey, E. P., Anderson, J. H., Carstens, E. E., Eisele, J. H., and Stanley, T. H. (1991). Cardiovascular, respiratory, and analgesic effects of fentanyl in unanesthetized rhesus monkeys. Anesth. Analg. 72, 221-226. Olson, M. E., Vizzutti, D., Morck, D. W., and Cox, A. K. (1994). The parasympatholytic effects of atropine sulfate and glycopyrrolate in rats and rabbits. Can. J. Vet. Res. 57, 254-258. Paddleford, R. R. (1992). "Post-operative pain control and chronic pain management in primates, small ruminants, and swine." Office for Protection from Research Risks, Vanderbilt Univ. Medical Center, and Meharry Medical College Workshop on Minimizing Pain and Distress in Laboratory Animals, Nashville. Papaioannou, V. E., and Fox, J. G. (1993). Use and efficacy of tribromoethanol anesthesia in the mouse. Lab. Anim. Sci. 43(2), 189-192. Parks, C. M., Clegg, K. E., Harvey-Clark, C. J., and Hollenberg, M. J. Improved techniques for successful neonatal rat surgery. Lab. Anim. Sci. 42(5): 508-513. Peeters, M. E., Gil, D., Teske, E., Eyzenbach, V., van der Brom, W. E., Lumeij, J. T., and de Vries, H. W. (1988). Four methods for general anesthesia in the rabbit: a comparative study. Lab. Anim. 22, 355-360. Popilskis, S. J., and Kohn, D. K. (1997). Anesthesia and analgesia in nonhuman primates. In "Anesthesia and Analgesia in Laboratory Animals" (D. K. Kohn, S. K. Wixson, W. J. White, and G. J. Benson, eds.), pp. 233255. Academic Press, New York. Popilskis, S. J., Kohn, D. E, Sanchez, J. A., and Gorman, P. (1991). Epidural versus intramuscular oxymorphone analgesia after thoracotomy in dogs. Vet. Surg. 20, 462-467. Popilskis, S. J., Danilo, P., Acosta, H., and Kohn, D. K. (1992). Is preoperative fasting necessary? J. Med. Primatol. 21, 349-352. Popilskis, S. J., Kohn, D. E, Laurent, L., and Danilo, P. (1993). Efficacy of epidural morphine versus intravenous morphine in alleviation of postthoracotomy pain in dogs. J. Vet. Anaesthesiol. 20, 22-26. Popilskis, S., Daniel, S., and Smiley, R. (1994). Effects of epidural versus intravenous morphine analgesia on postoperative catecholamine response in baboons. In "Proceedings of the Fifth International Congress of Veterinary Anaesthesia," August 21-25, Guelph, Canada. Popilskis, S. J., Cancel, D., Danilo, P, Kohn, D. E (2000). Analgesic effect of epidural fentanyl infusion after thoracic surgery in dogs: a clinical report. Presented at the Seventh World Congress of Veterinary anaesthesia, September 20-23, Bern, Switzerland. Portier, D. B., and Slusser, C. A. (1985). Azaperone: a review of a new neuroleptic agent for swine. Vet. Med. 80, 88-92. Radde, G. R., Hinson, A., Crenshaw, D., and Toth, L. A. (1996). Evaluation of anaesthetic regimens in guineapigs. Lab. Anim. 30, 220-227. Ramsey, D. E., Aldred, N., and Power, J. M. (1993). A simplified approach to the anesthesia of porcine laparoscopic surgical subjects. Lab. Anim. Sci. 43, 336-337.

1001 Report of the AVMA Panel on Euthanasia (1993). J. Am. Vet. Med. Assoc. 202 (2), 229-249. Rich, G. E, Sullivan, M. P., and Adams, J. M. (1990). Is distal sampling of end tidal CO2 necessary in small subjects? Anesthesiology 73, 265-268. Riebold, T. W. (1994). Ruminants: anesthesia and analgesia. In "Research Animal Anesthesia, Analgesia, and Surgery" (A. C. Smith and M. M. Swindle, eds.), pp. 111-127. Scientists Center for Animal Welfare, Greenbelt, Maryland. Riebold, Thomas W. (1996). Ruminants. In "Lumb and Jones' Veterinary Anesthesia" (J. C. Thurmon, W. J. Tranquilli, and G. J. Benson, eds.), pp. 610626. Williams and Wilkins, Baltimore. Riebold, T. W., Goble, D. O., and Geiser, D. R. (1995). "Large Animal Anesthesia: Principles and Techniques in Swine." Iowa State Univ. Press, Ames. Ritzman, J. R., Erickson, H. H., and Miller, E. D. (1976). Cardiovascular effects of enflurane and halothane on the rhesus monkey. Anesth. Analg. 55, 85-90. Robson, W. L., Bayliss, C. E., Feldman, R., Goldstein, M. B., Chen, C. B., Richardson, R. M. A., Stinebaugh, B. K., Tam, S. C. and Halperin, M. L. (1981). Evaluation of the effect of pentobarbital anaesthesia on the plasma potassium concentration in the rabbit and the dog. Can. Anaesth. Soc. J. 28, 210-216. Rooks, W. H., II, Maloney, E J., Shott, L. D., Schuler, M. E., Sevelius, H., Strosberg, A. M., Tanenbaum, L., Tomolonis, A. J., Wallach, M. B., Waterbury, D., et al. (1985). The analgesic and anti-inflammatory profile of ketorolac and its tromethamine salt. Drugs Exp. Clin. Res. 11, 479-492. Sainsbury, A. W., Eaton, B. D., and Cooper, J. E. (1991). An investigation into the use of propofol (Rapinovet) in long-tailed macaques (Macaca fascicularis). J. Vet. Anaesthesiol. 18, 38-41. St. Jean, G., and Anderson, D. E. (1999). Anesthesia and analgesia in swine. In "Diseases of Swine" (B. E. Straw, S. D'Allaire, W. L. Mengeling, and D. J. Taylor, eds.), pp. 1133-1154. Iowa State Univ. Press, Ames. Sanders, E. A., Gleed, R. D., and Nathanielsz, R. W. (1991). Anesthetic management for instrumentation of the pregnant rhesus monkeys. J. Med. PrimatoL 20, 223-228. Santus, G., Rivolta, R., Bottoni, G., Testa, B., Canali, S., and Peano, S. (1993). Nasal formulations of ketorolac tromethamine: technological evaluationw bioavailability and tolerability in rabbits. Farmaco 48, 1709-1723. Scrivani, P. V., Bednarski, R. M., and Myer, C. W. (1998). Effects of acepromazine and butorphanol on positive-contrast upper gastrointestinal tract examination in dogs. Am. J. Vet. Res. 59 (10), 1227-1233. Sebel, P. S., and Lowdon, J. D. (1989). Propofol: a new intravenous anesthetic. Anesthesiology 71, 250-277. Sedgwick, C. J. (1986). Anesthesia for rabbits. Vet. Clin. North Am. Food Anim. Pract. 2, 731-736. Short, C. E. (1987). Pain, analgesics, and related medications. In "Principles and Practice of Veterinary Anesthesia" (C. E. Short, ed.). Williams and Wilkins, Baltimore. Shutttebarger, J. V., Doyle, J., Roth, T., Maguire, K., and Rothkopf, D. M. (1996). The effect of ketorolac on microvascular thrombosis in an experimental rabbit model. Plast. Reconstr. Surg. 98, 140-145. Silverman, J., and Muir, W. W. (1993). A review of laboratory animal anesthesia with chloral hydrate and chloralose. Lab. Anim. Sci. 43(3), 210-216. Silverman, J., Huhndorf, M., and Balk, M. (1983). Evaluation of a combination of tiletamine and zolazepam as an anesthetic for laboratory rodents. Lab. Anim. Sci. 33(5), 457-460. Skarda, R. T. (1989). Local anesthesia in dogs and cats. In "Handbook of Veterinary Anesthesia" (W. W. Muir, J. A. E. Hubbell, and R. T. Skarda eds.). Mosby, Washington, D.C. Smith, A. C., and Swindle, M. M. (1994). "Research Animal Anesthesia and Surgery" (A. C. Smith and M. M. Swindle, eds.), pp. 107-110. Scientists Center for Animal Welfare, Bethesda, Maryland. Smith, A. C., Zellner, J. L., Spinale, E G., and Swindle, M. M. (1991). Sedative and cardiovascular effects of midazolam in swine. Lab. Anim. Sci. 41, 157-161.

1002 Smith, A. C., Ehler, W., and Swindle, M. M. (1997). Anesthesia and analgesia in swine. In "Anesthesia and Analgesia in Laboratory Animals" (D. E Kohn, S. K. Wixson, W. J. White, and G. J. Benson, eds.), pp. 313-336. Academic Press, New York. Smith, M. C., and D. Sherman. (1994). "Goat Medicine," pp. 509-517. Lea and Febiger, Philadelphia. Steffey, E. P., Gillespie, J. R., Berry, J. D., Eger, E. I. II, and Munson, E. S. (1974a). Anesthetic potency (MAC) of nitrous oxide in the dog, cat, and stump-tailed macaque. J. Appl. Physiol. 36, 530-532. Stepien, R. L., Bonagura, J. D., Bednarski, R. M., and Muir, W. W. (1995). Cardiorespiratory effects of acepromazine maleate and buprenorphine hydrochloride in clinically normal dogs. Am. J. Vet. Res. 56(1), 78-84. Swift, J. Q., Roszkowski, M. T., Alton, T., and Hargreaves, K. M. (1998). Effect of intra-articular versus systemic anti-inflammatory drugs in a rabbit model of temporomandibular joint inflammation. Int. J. Oral Maxillofac. Surg. 56, 1288-1295. Swindle, M. M. (1998). "Surgery, Anesthesia, and Experimental Techniques in Swine." Iowa State Univ. Press, Ames. Swindle, M. M., Thompson, R. P., Carabello, B. A., Smith, A. C., Green, C., and Gillette, P. C. (1992). Congenital cardiovascular diseases. In "Swine as Models in Biomedical Research" (M. M. Swindle, ed.), pp. 176-184. Iowa State Univ. Press, Ames. Swindle, M. M., Wiest, D. B., Smith, A. C., Garner, S. S., Case, C. C., Thompson, R. P., Fyfe, D. A. & Gillette, P. C. (1996). Fetal surgical protocols in Yucatan miniature swine. Lab. Anim. Sci. 46, 90-95. Taguchi, H., Murao, K., Nakamura, K., Uchida, M., and Shingu, K. (1996). Percutaneous chronic epidural catheterization in the rabbit. Acta Anaesthesiol. Scand. 40, 232-236. Taylor, P. (1991). Anesthesia in sheep and goats. Practice 91(January), 31-36. Thompson, S. E., and Johnson, J. M. (1991). Analgesia in dogs after intercostal thoracotomy: a comparison of morphine selective intercostal nerve block, and interpleural regional analgesia with bupivicaine. Vet. Surg. 20, 73-77. Thurmon, J. C., and G. J. Benson. (1986). Anesthesia in ruminants and swine. In "Current Veterinary Therapy 2: Food Animal Practice" (J. L. Howard, ed.), pp. 51-71. Saunders, Philadelphia. Thurmon, J. C., and Benson, G. J. (1996). "Lumb and Jones' Veterinary Anesthesia," 3rd ed. Williams and Wilkins, Baltimore. Thurmon, J. C., Tranquilli, W. J., and Benson, G. J. (1986). Cardiopulmonary responses of swine to intravenous infusion of guaifenesin, ketamine, and xylazine. Am. J. Vet. Res. 47, 2138-2140. Thurmon, J. C., Benson, G. J., Tranquilli, W. J., and Olson, W. A. (1988). The anesthetic and analgesic effects of Telazol and xylazine in pigs: evaluating clinical trials. Vet. Med. 83, 841- 845. Tinker, J. H., Sharbrough, E H., and Michenfelder, J. D. (1977). Anterior shift of the dominant EEC rhythm during anesthesia in the Java monkey: correlation with anesthetic potency. Anesthesiology 46, 252-259. Tran, D. Q, and Lawson, D. (1986). Endotracheal intubation and manual ventilation of the rat. Lab. Anim. Sci. 36(5), 540-541. Tranquilli, W. J., Thurmon, J. C., Benson, G. J., and Steffey, E. P. (1983). Halothane potency in pigsmSus scrofa. Am. J. Vet. Res. 44, 1106-1107. Vachon, P., Faubert, S., Blais, D., Comtois, A., and Bienvenue, J. G. (1999). A pathophysiological study of abdominal organs following intraperitoneal injections of chloral hydrate in rats: comparison between two anaesthesia protocols. Lab. Anim. 34(2), 84-90. Vainio, O., and Ojala, M. (1994). Medetomidine, an alpha 2-agonist, alleviates post-thoracotomy pain in dogs. Lab. Anim. 28, 369-375. Vainio, O. M., Bloor, B. C., and Kim, C. (1992). Cardiovascular effects of a ketamine-medetomidine combination that produces deep sedation in Yucatan mini swine. Lab. Anim. Sci. 42, 582-588. Vesal, N., Cribb, P. H., and Frketic, M. (1996). Postoperative analgesic and cardiopulmonary effects in dogs of oxymorphone administered epidurally and intramuscularly, and medetomidine administered epidurally: a comparative clinical study. Vet. Surg. 25, 361-369.

M. MICHAEL SWINDLE, E T AL. Vie, J. C., DeThoisy, B., Fournier, E, Fournier-Chambrillon, C., Genty, C., and Keravec, J. (1998). Anesthesia of wild red howler monkeys (Alouatta seniculus) with medetomidine/ketamine and reversal by atipamezole. Am. J. PrimatoL 45(4), 399-410. Vogler, G. A. (1997). Anesthesia equipment: types and uses. In "Anesthesia and Analgesia in Laboratory Animals" (D. H. Kohn, S. K. Wixon, W. J. White, and G. J. Benson, eds.), pp. 105-147. Academic Press, New York. Vogler, G. A., Morgenthaler, W. A., Frank, P. A., and Baudendistoe, L. J. (1992). Control of tympany and salivation during anesthesia of small ruminants. Contemp. Top. 31(2), 24-25. Voipio, H. M., Navalainen, T. O., and Virtanen, R. (1990). Evaluation of anaesthetic potency of medetomidine-ketamine combination in mice. In "ICLAS (International Council on Laboratory Animal Science) Symposium Proceedings," pp. 298-299. Warren, D. J., and Ledingham, J. G. G. (1978). Renal vascular response to haemorrhage in the rabbit after pentobarbitone, chloralose-urethane, and ether anaesthesia. Clin. Sci. Mol. Med. 54, 489-494. Weiskopf, R. B., Holmes, M. A., Eger, E. I. II, Yasuda, N., Rampil, I. J., Johnson, B. H., Targ, A. G., Reid, I. A., and Keil, L. C. (1992). Use of swine in the study of anesthetics. In "Swine as Models in Biomedical Research" (M. M. Swindle, ed.), pp. 96-117. Iowa State Univ. Press, Ames. Whelan, G., and Flecknell, P. A. (1992). The assessment of depth of anaesthesia in animals and man. Lab. Anim. 26, 153-162. Williams, C. S. E (1990). Routine sheep and goat procedures. Vet. Clin. North Am. Food Anim. Pract. 6(3), 737-758. Williams, C. E S. (1995). Goats. In "The Experimental Animal in Biomedical Research" (B. Rollin and M. L. Kessel, eds.), pp. 119-143. CRC Press, Boca Raton, Florida. Wilson, R. P., Zagon, I. S., and Larach, D. R. (1992). Antinociceptive properties of tiletamine-zolazepam improved by addition of xylazine or butorphanol. Pharmacol. Biochem. Behav. 43, 1129-1133. Wilson, R. P., Zagon, I. S., and Larach, D. R. (1993). Cardiovascular and respiratory effects of tiletamine-zolazepam. Pharmacol. Biochem. Behav. 44, 1-8. Wixson, S. K. (1994). Anesthesia and analgesia for rabbits. In "The Biology of the Laboratory Rabbit" (P. J. Manning, ed.), pp. 87-109. Academic Press, San Diego. Wixson, S. K., and Smiler, K. L. (1997a). Anesthesia and analgesia of rodents. In "Anesthesia and Analgesia in Laboratory Animals" (D. H. Kohn, S. K. Wixon, W. J. White, and G. J. Benson, eds.), pp. 165-203. Academic Press, New York. Wixson, S. K., and Smiler, K. L. (1997b). Anesthesia and analgesia in rodents. In "Anesthesia and Analgesia in Laboratory Animals" (D. H. Kohn, S. K. Wixon, W. J. White, and G. J. Benson, eds.), p. 173. Academic Press, New York. Wixson, S. K., and Smiler, K. L. (1997c). Anesthesia and analgesia in rodents. In "Anesthesia and Analgesia in Laboratory Animals" (D. H. Kohn, S. K. Wixon, W. J. White, and G. J. Benson, eds.), p. 179. Academic Press, New York. Wixson, S. K., White, W. J., and Hughes, H. C. (1987a). The effects of pentobarbital, fentanyl-droperidol, ketamine-xylazine, and ketamine-diazepam on core and surface body temperature regulation in adult male rats. Lab. Anim. Sci. 37(6), 743-749. Wixson, S. K., White, W. J., and Hughes, H. C. (1987b). The effects of pentobarbital, fentanyl-droperidol, ketamine-xylazine, and ketamine-diazepam on noxious stimulus perception in adult male rats. Lab. Anim. Sci. 37(6), 731-735. Wong, P. L. (1997). Anesthesia and monitoring during MRI. ASLAP Newsl. 30(2), 15-18. Worek, E S., Blumel, G., Zeravik, J., Zimmerman, G. J., and Pfeiffer, U. J. (1988). Comparison of ketamine and pentobarbital anesthesia with the conscious state in a porcine model of Pseudomonas aeruginosa septicemia. Acta Anaesthiol. Scand. 32, 509-515.

22. PREANESTHESIA, ANESTHESIA, ANALGESIA, AND EUTHANASIA Woolfson, M. W., Foran, J. A., Freedman, H. M., Moore, E A., Shulman, L. B., and Schnitman, P. A. (1980). Immobilization of baboons (Papio anubus) using ketamine and diazepam. Lab. Anim. Sci. 30, 902-904. Wyatt, J. D., Scott, R. A. W., and Richardson, M. E. (1989). Effects of prolonged ketamine-xylazine intravenous infusion on arterial blood pH, blood gases, mean arterial blood pressure, heart and respiratory rates, rectal temperature, and reflexes in the rabbit. Lab. Anim. Sci. 39, 411-416.

1003 Yoshikawa, T., Ochiaia, R., Kaneko, T., Takeda, J., Fukushima, K., Tsudaka, H., Seki, C., and Kakiuchi, T. (1997). The effect of sevoflurane on regional cerebral metabolism and cerebral blood flow in rhesus monkeys. Masui 46(2), 237-243. Zemel, E., Loewenstein, A., Lazar, M., and Perlman, I. (1995). The effects of lidocaine and bupivacaine on the rabbit retina. Doc. Ophthalmol. 90, 189-199.

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Chapter 2 3 Techniques of Experimentation Robert J. Adams

I. II. III.

IV. V. VI. VII.

Introduction .................................................

1006

Identification M e t h o d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1006

B l o o d C o l l e c t i o n and I n t r a v e n o u s Injection . . . . . . . . . . . . . . . . . . . . . . . .

1008

A.

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1008

B.

Rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1011

C.

Ferrets and M i n k . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1011

D.

D o g s and Cats

E.

Nonhuman Primates

1011 1012

IX.

X.

LABORATORY ANIMAL MEDICINE, 2nd edition

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E

Other Mammals ..........................................

G.

Birds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1012

H.

U n u s u a l Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

S u b c u t a n e o u s and I n t r a m u s c u l a r Injection . . . . . . . . . . . . . . . . . . . . . . . . .

1013 1013 1015 1015

Digestive S y s t e m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1015

Vascular C a n n u l a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intraperitoneal Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1012

A.

Oral E x a m i n a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1015

B.

Oral A d m i n i s t r a t i o n of S u b s t a n c e s . . . . . . . . . . . . . . . . . . . . . . . . . . .

1015 1016

C.

Tooth E x t r a c t i o n

D.

P u l p e c t o m y and P u l p o t o m y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1016

E. E

Bile D u c t M a n i p u l a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Partial H e p a t e c t o m y and Liver Biopsy . . . . . . . . . . . . . . . . . . . . . . . .

1017 1018

G. H.

P a n c r e a t i c E x o c r i n e and E n d o c r i n e Studies . . . . . . . . . . . . . . . . . . . . Intestinal C a n n u l a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1019 1019 1019

I. VIII.

Rodents

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Intestinal L o o p Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Urinary System Techniques

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Respiratory S y s t e m T e c h n i q u e s

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1019 1021

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Collection of P h a r y n g e a l S a m p l e s

B.

Endotracheal Intubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1021

C.

T r a c h e o b r o n c h i a l W a s h i n g and I n o c u l a t i o n

1022

D.

Tracheal Pouch Formation ..................................

1022

E.

Tracheostomy

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1023

F.

Bronchoscopy

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G.

Bronchoalveolar Lavage

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1021

1023 1023

Reproductive S y s t e m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1023

A.

Laparoscopy .............................................

1024

B.

Testicular Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1024

C.

Castration ...............................................

1025

Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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ROBERT J. ADAMS

XI.

XII.

XIII. XIV.

XV. XVI. XVII.

I.

D. SemenCollection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Artificial Insemination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E PregnancyDiagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. EmbryoTransfer and Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . H. Intrauterine Fetal Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CardiovascularTechniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. BloodPressure Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Carotid-Jugular Shunt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Microangiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Experimental Heart Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endocrine System Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hypophysectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Pinealectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Adrenalectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Thyroidectomy and Parathyroidectomy . . . . . . . . . . . . . . . . . . . . . . . . . Orthopedic Procedures for Laboratory Animals . . . . . . . . . . . . . . . . . . . . . Neurosurgical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. SurgicalProcedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. SpinalCatheters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. CerebrospinalFluid Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor Transplantation . . . . . . . . . . . . . . . . ........................ Imaging Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Radiotelemetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION

Since 1984, when the first edition of this book was published, there has been a dramatic change in the use of laboratory animals, attributable almost entirely to the development of genetically manipulated rodents, particularly mice. The creation of transgenic and knockout mice with a wide variety of genotypes has revolutionized the study of many disease entities, some of which were previously available only in larger animal species. Although the study of these animals is still largely descriptive, the next phase will be the applied use of these animals in studies that will require various experimental manipulations. The challenge for the future will be to adapt many of the techniques described in the following sections to the mouse. Developments in miniaturization, telemetry, catheter implantation, and noninvasive imaging systems will greatly aid in these studies. Readers are encouraged to utilize the Internet-based mailing lists dealing with laboratory animals. In the author's experience one of the most useful is C O M P M E D , which is available through the American Association for Laboratory Animal Science (AALAS). The information presented in the literature covers a wide selection of animal species and a voluminous number of the experimental techniques. The reader should be aware of general reference texts that are available (Gay, 1965-1986; Melby and Altman, 1974-1976; UFAW Staff, 1976; Waynforth and Flecknell, 1992; Swindle and Adams, 1988; American College of Laboratory Animal Medicine Series; Hillyer & Queensberry, 1997; Laboratory Animal Pocket Reference Series). The pur-

1025 1025 1025 1026 1027 1027 1027 1027 1028 1028 1028 1028 1028 1028 1029 1029 1029 1029 1031 1031 1032 1033 1034 1034

pose of this chapter is to select and summarize the available information in an attempt to emphasize two major concepts: (1) the technique employed in animal experimentation is often the critical factor in determining the success or failure of a research protocol, and (2) a mastery of selected techniques is extremely useful to the veterinary clinician in performing diagnostic and therapeutic procedures. This chapter discusses or outlines by organ system one or more of the following: (1) procedures for administration of drugs and collection of biological specimens; (2) collection of physiological data; (3) surgical procedures, postoperative care, and advantages and disadvantages of alternative ways to perform the same procedure; and (4) references for access to detailed descriptions for a described technique. References are cited for complex techniques not described in this text. Since anesthesia techniques are covered in depth in another chapter, anesthesia will not be discussed here as it relates to specific surgical procedures. Reference will be made to the type of anesthesia and instrumentation required when they are critical in the performance of the described technique.

II.

IDENTIFICATION METHODS

Cage cards may be used to identify groups of rodents or individually housed animals. The information on the card should include the name and contact information of the responsible investigator, the approved protocol number under which the

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23. TECHNIQUES OF EXPERIMENTATION

Table I Identification Markers Animals

Marker a

Mouse, rat, hamster Guinea pig Rabbit Dog Cat Nonhuman primate

Ear punch, toe clip, dye, tail tattoo, ear tags, SC chip Ear tags, dye, ear punch, ear tattoo, SC chip Ear tattoo, ear tags, dye, SC chip Collar with tags, tattoo, ear tag, SC chip Collar with tags, tattoo, SC chip Tattoo, collar with tags, ear tag, SC chip

aSC, subcutaneous.

animals are being used, the source of the animal, and the strain or stock ("Guide for the Care and Use of Laboratory Animals," 1996). Natural characteristics and coat coloration could be recorded and used to identify individual animals, but this process is cumbersome for large numbers of animals and impossible to use for animals identical in appearance. Table I lists some of the common markers for laboratory animal species. Cage cards may also incorporate barcodes that can be used to provide additional information regarding the animals, and they may also greatly facilitate routine colony management functions like census taking. Animals may be easily marked by the application of dyes or ink to the fur or tail. Felt tip pens work well, but the mark is not permanent. Holes and notches may be placed in the ear with commercially available forceps (Michi-Crown, Bay City, Michigan). An established code should be followed (Fig. 1) (Dickie, 1975).

lf~L~

This method produces a permanent mark if the ear is not selfmutilated or chewed on by cagemates. Holes and notches may potentially close with time. Amputation of toes with scissors according to an established code will produce a permanent mark (Kumar, 1979). The technique is more traumatic than ear notching, and infection may develop. Anesthesia should be used with toe amputation performed after 2 weeks of age. This method of identification is prohibited by the U.S. Food and Drug Administration in Good Laboratory Practice studies. Tattoos are applied with pliers (Stone Manufacturing and Supply Co., Kansas City, Missouri) or electrovibrators. Tattoos may include various letter/number combinations, common with larger species such as rabbits and nonhuman primates, or colored dot patterns (Iwaki et al., 1989). The tail and feet are the best sites for tattooing rats and mice; specialized tattoo equipment and training are available for tattooing rodents (Avery and Spyker, 1977; Schoenborne et aL, 1977; Greenham, 1978). The inner surface of the ear can be tattooed in guinea pigs, rabbits, dogs, and cats. Nonhuman primates are usually tattooed on the abdomen, chest, or thigh. The technique should be performed aseptically, and instruments must be disinfected after use on each animal. A variety of ear tags is commercially available for all species of animals. Small aluminum ear studs are used in rats and guinea pigs, and larger Ketchum tags are used in rabbits and dogs. All tags should be placed in the ear according to the manufacturers' directions. It is essential that the tag is not too tight; otherwise, pressure necrosis and infection will occur. If the tag is too loose, it may be easily torn out.

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0 Fig. 1. Ear notch code. (Adapted from Harkness and Wagner, 1983.)

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Implantable transponders may be used in many species for rapid identification (Ball et al., 1991; Mrozek et al., 1995). These devices are miniature radio transponders capable of transmitting a unique identification number when queried using a low-power radio-frequency signal transmitter and reader. These small devices are individually packaged within a sterile large-gauge needle and are implanted subcutaneously. Some transponders also are capable of providing additional physiologic information such as body temperature (BioMedic Data Systems, Inc., Maywood, New Jersey).

IIl. BLOOD COLLECTION AND INTRAVENOUS INJECTION

The techniques for obtaining blood from a variety of animal species are described in the following sections. Many studies now describe the use of indwelling catheters, swivels, and protective jackets and devices like subcutaneous access ports for repeated, long-term sampling of blood and the administration of drugs and other experimental substances (Table II). Two useful references regarding blood sampling from laboratory animals are McGuill and Rowan (1989) and UFAW Joint Working Group on Refinement (1993).

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Rodents

Cardiac puncture is used for the collection of small serial samples as well as for the collection of a large quantity of blood within a short period of time. However, the technique is difficult, and an inexperienced operator may be frustrated by missing the heart entirely or by causing cardiac tamponade and death when serial samples are needed. From a humane perspective, cardiac puncture should be performed only on anesthetized animals as a terminal procedure. Cardiac puncture may be accomplished by inserting the needle through the ventral abdominal wall just lateral to the xiphoid process or through the lateral thoracic wall. The needle (23-gauge, 1 inch) is inserted at a 10~ ~ angle above the plane of the abdomen and directed cephalad toward the heart (Ambrus et al., 1951; Falabella, 1967; Krause, 1980; Simmons and Brick, 1970; Waynforth, 1980). Frankenberg (1979) describes insertion of the needle through the thoracic inlet. For animals larger than mice, the needle may be inserted through the lateral thoracic wall in the region of maximum palpitation of the heart (Fig. 2) (Burhoe, 1940; Moreland, 1965). The technique of collecting blood from the orbital sinus or plexus is easily learned, requires minimal equipment, and reliably produces small blood samples. The eye and health of the animal seem to be unaffected when the procedure is properly

Table II Needle Sizes and Recommended Injection Volumes a Species Mouse Rat Hamster Guinea pig Rabbit

Cat Dog Primate (marmoset) Primate (baboon)

Intravenous

Intraperitoneal

Lateral tail vein, 0.2 ml, < 25 gauge Lateral tail vein, 0.5 ml, < 23 gauge Femoral or jugular vein (cut down), 0.3 ml, < 25 gauge Ear vein, saphenous vein, 0.5 ml, < 23 gauge Marginal ear vein, 1-5 ml (slowly), < 21 gauge

2 - 3 ml, < 21 gauge

Cephalic vein, 2 - 5 ml (slowly), < 21 guage Cephalic vein, 10-15 ml (slowly), < 20 gauge Lateral tail vein, 0.5-1 ml (slowly), < 21 gauge Cephalic vein, recurrent tarsal vein, jugular vein, 10-20 ml (slowly), < 20 gauge

50-100 ml, < 20 gauge

aModified from Flecknell (1987); AALAS (1990).

5 - 1 0 ml, < 21 gauge 3 - 4 ml, < 21 gauge 10-15 ml, < 21 gauge 50-100 ml, < 20 gauge

200-500 ml, < 20 gauge 10-15 ml, < 21 gauge 50-100 ml, < 20 gauge

Intramuscular

Subcutaneous

Quadriceps/posterior thigh, 0.05 ml, < 23 gauge Quadriceps/posterior thigh, 0.3 ml, < 21 gauge Quadriceps/posterior thigh, 0.1 ml, < 21 gauge Quadriceps/posterior thigh, 0.3 ml, < 21 gauge Quadriceps/posterior thigh, lumbar muscles, 0.5-1 ml, < 20 gauge Quadriceps/posterior thigh, 1 ml, < 20 gauge Quadriceps/posterior thigh, 2 - 5 ml, < 20 gauge Quadriceps/posterior thigh, 0.3-0.5 ml, < 21 gauge Quadriceps/posterior thigh, triceps, 1-3 ml, < 20 gauge

Scruff, 2 - 3 ml, < 20 gauge Scruff, back, 5 - 1 0 ml, < 20 gauge Scruff, 3 - 4 ml, < 20 gauge Scruff, back, 5 - 1 0 ml, < 20 gauge Scruff, flank, 3 0 - 5 0 ml, < 20 gauge Scruff, back, 50-100 ml, < 20 gauge Scruff, back, 100-200 ml, < 20 gauge Scruff, 5 - 1 0 ml, < 20 gauge Scruff, 10-30 ml, < 20 gauge

23.

TECHNIQUESOF EXPERIMENTATION

Fig. 2. Cardiacpuncture in a rat. A 20-gaugeneedle is inserted throughthe right thoracic wall at the point of maximumheart palpitation.

performed on an anesthetized animal (Cate, 1969; Grice, 1964; Pansky et al., 1961; Simmons and Brick, 1970; Sorg and Buckner, 1964; Stone, 1954). In one study, hemorrhage was found in the puncture track, eye muscles, and periosteum immediately following blood sampling, but these lesions usually healed without scar formation following a single puncture. Four weeks following the procedure no lesions were found (Van Herck et al., 1992). The animal is held on a flat surface, and the operator's thumb is used to apply pressure to the external jugular vein immediately caudal to the mandible and thus occlude venous return from the orbital sinus. The forefinger of the same hand is used to pull the dorsal eyelid back and produce slight exophthalmos (Fig. 3). Usually a glass capillary tube or Pasteur pipette is used to penetrate the orbital conjunctiva and rupture

Fig. 3. Collectionof bloodfromthe orbital sinus of a gerbil with a capillary tube. Note how traction appliedby the forefingerproducesexophthalmos.

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the orbital sinus; however, Cate (1969) describes the use of small-bore polyethylene tubing cut with a beveled tip. Sorg and Buckner (1964) state that introduction of the tube into the lateral rather than the medial canthus reduces the incidence of epistaxis and eye trauma associated with the technique. Further anatomical studies by Timm (1989) have shown that this lateral approach should be used in the mouse, hamster, and gerbil. However, in an earlier publication, Timm (1979) describes the orbital venous anatomy of the rat and recommends directing the tube in a caudal and medial direction through the dorsal conjunctiva. This is necessary because the rat has an orbital plexus rather than a venous sinus, and the largest vein of this plexus is located deep within the orbit. Once the sinus or plexus has been ruptured, blood will flow through and around the tube into a collection vessel. Blood flow will cease when the tube is released and pressure is removed from the external jugular vein. Bleeding from this location may result in damage to the orbital structures. Retro-orbital hematoma, abscess, ocular injury, and phthisis bulbi may occur but are uncommon. Damage may be correlated with the experience and technique of the individual performing the procedure and the frequency of repetition. Collection of blood from the tail is easily accomplished, and minimal equipment is required. Serial samples may be collected, bleeding can be controlled, and it is safe for the animal. Tail bleeding usually involves amputation of the tip of the tail or laceration of the blood vessel within the tail. If a vacuum apparatus is used, large samples can be obtained (Levine et al., 1973; Nerenberg and Zedler, 1975; Stuhlman et al., 1972). The primary disadvantages of tail bleeding techniques, which involve laceration or amputation of a portion of the tail, are that blood may not flow freely from the wound, and a clot may form before a sample of adequate volume is obtained. Heparin or citrate solution may be applied directly to the wound to slow clot formation (Ambrus et al., 1951; Lewis et al., 1976). Another technique is to partially lacerate the ventral artery of the tail with a sharp razor blade. This technique prevents constriction of the vessel and improves the yield of blood (Fields and Cunningham, 1976). Rats and mice can be easily bled from the lateral caudal tail vein by using a 21-gauge butterfly set, after the animal is warmed in a 40~ chamber (Conybeare et al., 1988). Proper restraint is essential for tail bleeding or injection, and effective restraint chambers are commercially available or may be constructed from a plastic syringe (Fumer and Mellett, 1975). Transillumination of the tail with a light source will improve visualization of the veins (Kaplan and Wolf, 1972; Keighley, 1966; Mylrea and Abbrecht, 1967), and occlusion of the veins at the base of the tail will also facilitate injection (Barrow, 1968). Omaye et al. (1987) described the use of a simple tourniquet on the tail to facilitate venous engorgement and blood sampiing of rats. Compression of the lateral tail vein without compressing the middle coccygeal artery may be accomplished with a pair of forceps designed for wound clips (Bergstrom, 1971).

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A 27-gauge or smaller needle is used for venipuncture in the mouse, and a 19-gauge or smaller for the rat. Warming the tail (Ambrus et al., 1951; Fields and Cunningham, 1976; Levine et al., 1973) or warming the entire animal (Lewis et al., 1976; Stuhlman et al., 1972) seems to increase blood flow. Topical vasodilators like limonene and oil of wintergreen may facilitate blood flow. It is essential that the tail be cleaned to remove all chemicals on completion. Other disadvantages of the tail bleeding techniques include stimulation of the sympathetic nervous system, with resulting vasoconstriction (Carvalho et al., 1975). Significant differences were shown between samples obtained from the orbital sinus and tail of the same mouse; there was sample-to-sample variation of blood samples taken from the tail vessels in the same animal (Sakaki et al., 1961); and the mixing of venous and arterial blood plus extravascular tissue fluids in samples may occur. Percutaneous puncture and bleeding from the jugular vein of rats and other rodents with a needle and syringe have been described (Kassel and Leviton, 1953; Phillips et al., 1973). Because the technique is relatively safe, it can be used to collect serial samples. Success with the procedure is largely dependent on proper restraint and positioning of the animal. The neck should be held in hyperextension by fastening a strip of gauze behind the upper incisors and pulling the head back or to the side. Removal of hair from the ventral neck region by shaving or by use of a depilatory will make identification of landmarks easier. The site for venipuncture is just cephalad to the point where the external jugular vein passes between the pectoral muscle and the clavicle. If the needle is inserted through the pectoral muscle, it is stabilized better within the vein. Reliability of jugular vein techniques for blood collection can be improved by anesthetizing the animal and surgically exposing the vessel through a skin incision. Once the vessel is exposed, blood can be collected with a needle and syringe, by cannulation, or by severing the vessel and allowing blood to flow directly into a collection vial. Usually one jugular vein is occluded while blood is collected from the opposite vein. The dorsal metatarsal vein in the rat (Nobonaga et al., 1966) is an excellent site for simple intravenous injection with a needle and syringe. When the animal's limb is grasped at the stifle joint, the vein is compressed and the leg is immobilized in extension. It is necessary to clip hair from the venipuncture site, and a 27-gauge or smaller needle is used. Other sites well suited for simple intravenous injection in rodents are the sublingual vein (Greene and Wade, 1967; Waynforth and Parkin, 1969) and the penile vein (Grice, 1964; Karlson, 1959). The animals must be anesthetized, and venipuncture performed with a 26-gauge needle. Snitily et al. ( 1991) have described a technique in which the interdigital space of the hindfoot of an anesthetized rat is punctured with a 20-gauge needle, yielding between 0.5 and 1.0 ml of blood. Oil of wintergreen is used to stimulate blood flow to the foot beforehand. In the hamster, the cephalic vein may be used for intravenous injections (Ransom, 1984).

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Decapitation may be used to collect blood from smaller rodents, and the procedure may be accomplished with a commercially available guillotine or autopsy shears (Krause, 1980). Following decapitation, blood flowing from the severed neck is collected in a funnel. The technique is esthetically offensive and potentially dangerous for the operator, and electroencephalographic activity persists for a short period of time following decapitation (Mikeska and Klemm, 1975). Blood collected in this manner will also be contaminated with tracheal and salivary secretions. The American Veterinary Medical Association (AVMA) Panel on Euthanasia (2001) describes this technique as provisionally acceptable if required by the experimental design, approved by the Institutional Animal Care and Use Committee, and performed correctly by trained personnel. Large quantities of blood may be obtained in terminal experiments by severing large vessels and exsanguinating the animal. The inherent disadvantage of such a technique is that only one sample can be collected. Exsanguination techniques should be performed only on anesthetized animals. One to 1.5 ml of whole blood may be collected from mice by incising the brachial vessels (Young and Chambers, 1973). Large amounts of blood can be collected from the abdominal aorta by severing the vessel (Lushbough and Moline, 1961) or by aspirating with a needle and syringe (Grice, 1964). Terminal sampling from the caudal vena cava of mice can yield up to 2.5 ml of blood free from hemolysis and contamination (Adeghe and Cohen, 1986). In the rat, coagulation times in blood obtained at euthanasia from the orbital venous plexus were abnormally prolonged, and serum magnesium and phosphorus levels were markedly lower than in blood obtained from the posterior vena cava (Dameron et al., 1992). Blood may be collected from fetal and newborn rodents by severing the jugular and carotid vessels (Smith and McMahon, 1977), by decapitation or amputation of an extremity (Grazer, 1958), or by cardiac puncture through the thoracic inlet (Gupta, 1973). Animals should be anesthetized, preferably by inhalant, prior to this procedure. Blood collection and intravenous injection in the guinea pig are difficult because of the relatively small peripheral veins. Small amounts of blood can be collected by cutting a toenail close to the nail bed (Vallejo-Freire, 1951). Warming the animal in an incubator (40 ~C) tends to increase blood flow. The veins of the ear may be punctured with a 25-gauge needle or lacerated with a scalpel blade, and small amounts of blood may be collected with a capillary tube (Bullock, 1983; Enta et al., 1968; Grice, 1964). The auricular vein is also suitable for intravenous injection (Decad and B irnbaum, 1981). Blood can easily be aspirated from the medial saphenous vein of the anesthetized guinea pig (Carraway and Gray, 1989). A vacuum-assisted bleeding apparatus may be used to collect blood from the lateral or medial metatarsal veins. A small incision is made just distal to the malleolus, and a vacuum of 5 mm Hg is applied (Dolence and Jones, 1975; Lopez and Navia, 1977; Rosenhaft et al., 1971).

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The lateral metatarsal or lateral saphenous vein is also suitable for intravenous injection or blood sampling (Nau and Schunck, 1993). The dorsolateral vein of the penis may also be used for both blood sampling and intravenous injection (Reuter, 1987). As noted previously, cardiac puncture by introduction of a needle (20- to 23-gauge, 1 inch) through the lateral thoracic wall in the region of maximum heart palpitation may also be used to collect blood from anesthetized guinea pigs. Although not without some degree of postprocedural hemorrhage, the anterior vena cava may be accessed in a manner similar to that used to bleed domestic swine (Reuter, 1987). Unanesthetized, restrained guinea pigs may be bled from the left jugular vein by inserting the needle in the hollow of the right shoulder above the clavicle and directing it toward the left hip (Shomer et al., 1999). Blood samples may be obtained from a number of sites in chinchillas, including the orbital sinus and a variety of peripheral veins, such as the auricular, femoral, cephalic, dorsal penile, saphenous, lateral abdominal, and tail veins (Tappa et al., 1989). A technique utilizing the transverse sinus medial to the auditory bulla in anesthetized animals has been described. Samples of 0.5 ml were obtained at 3-day intervals (Boettcher et al., 1990). By using a modification of the technique described by Boettcher, up to 10 ml of blood can be obtained from the transverse venous sinus (Paolini et al., 1993).

B.

Rabbits

Intravenous injection and blood collection techniques commonly utilize the auricular artery or marginal ear veins of the rabbit. Blood collection from these sites may be facilitated by the topical application of a lignocaine-prilocaine local anesthetic cream 1 hr prior to sampling (Flecknell et al., 1990). Small amounts of blood may be collected from a puncture wound in the vessels produced by a 23-gauge needle. Collection J0f large amounts of blood and intravenous injection are facilitated if the blood vessels are dilated beforehand. This can be accomplished by the application of heat using a low-wattage bulb, the application of agents such as depilatories over the site of blood sampling, or topical application of 40% d-limonene to the posterior margin of the ear (Lacy et aL, 1987). These agents will dilate the vessels, and a 20-gauge needle may then be used. Vasodilatation may also be produced by the general administration of acepromazine or of a combination of acepromazine and butorphanol (Thulin, 1994) or by multiple local injections of lidocaine along the marginal vein or medial artery (Paulsen and Valentine, 1984). A vacuum apparatus (Hoppe et al., 1969) or a miniperistaltic pump (Stickrod et al., 1981) may be used to collect even larger quantities (30-50 ml) of blood. Cardiac puncture in the anesthetized rabbit is done with an 18-gauge, 1.5inch needle (Kaplan and Timmons, 1979). The needle is inserted through the lateral chest wall at the site of maximal car-

diac impulse or is inserted just caudal to the xiphoid cartilage, held at a 30 ~ angle above the plane of the abdomen, and directed cranially (Bivin and Timmons, 1974). Once the needle is within the heart, blood may be collected by aspiration into a syringe by use of a miniperistaltic pump (Stickrod et al., 1981) or by tubing directly into evacuated blood collection tubes or into a centrifuge tube (Kaplan and Timmons, 1979). Blood samples may also be collected by penetration of the orbital sinus in anesthetized animals with nonglazed microhematocrit capillary tubes. To facilitate rupture of this sinus, the tubes may be broken to form a sharp edge. The best site for inserting the tube is within the dorsal conjunctival sac midway between the medial and lateral canthi (Lumsden et al., 1974). The ease of other methods of blood collection has made this technique uncommon. Cranney and Zajac (1993) have described a technique for obtaining blood via jugular puncture in awake, restrained rabbits. The technique requires two people to perform it, and animals are restrained in dorsal recumbency.

C.

Ferrets and M i n k

Small amounts of blood may be collected from the toenail of a ferret. The nail is clipped close to the nail bed, and blood is collected with a capillary tube. Larger amounts (3-5 ml) may be safely collected from the caudal artery of the tail (Bleakley, 1980). A 20- or 21-gauge needle is inserted into a groove in the ventral surface of the tail. The artery is superficially located, and care must be taken to prevent going through the artery. Blood may also be collected by cardiac puncture, using a syringe and a 20-gauge, 1.5-inch needle (Baker and Gorham, 1951). The needle is inserted on the midline just caudal to the xiphoid cartilage. Cardiac puncture should be done only in anesthetized animals as a terminal procedure. Jugular and cephalic venipuncture in mink and ferret has been described (Fletch and Wabeser, 1970; Otto et aL, 1993), as well as jugular vein cannulation (Bergman et al., 1972; Mesina et al., 1988). The jugular vein may be seen by occluding the vessel by pressing a finger in the area between the sternum and the shoulder. Ferrets may be conditioned to allow jugular venipuncture while awake. Towel and scruff restraint of the animal while it is distracted with the nutritive paste Nutrical is used by an assistant. A 20- to 25-gauge needle is required, depending on gender and size of the ferret. A cranial vena cava and caudal tail artery technique may also be used (Quesenberry, 1997; Marini and Fox, 1998).

D.

Dogs and Cats

Common sites for collection of blood from the dog and cat include the cephalic, recurrent metatarsal, jugular, and femoral veins. Collection of blood from the cephalic vein is usually

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accomplished while the animal is restrained in sternal recumbency on a table. The vein is stabilized and occluded by grasping the foreleg just behind the elbow. The vein may then be seen or palpated on the dorsal surface of the forelimb. As with the ear vein of the rabbit, the use of a topical anesthetic cream prior to sampling simplifies the process (Flecknell et al., 1990). The recurrent metatarsal vein is located on the lateral surface of the hock joint. Collection of blood from the recurrent metatarsal vein is accomplished with the dog restrained in lateral recumbency. The vein is occluded by grasping the stifle joint and extending the limb. The vein is easily seen as it crosses the lateral surface of the hock joint, but venipuncture may be difficult because of a tendency for the vein to roll away from the needle. The jugular vein is best for collection of blood from dogs with small peripheral veins and when large volumes are required. The animal is restrained with its neck extended and held slightly to one side. Pressure applied at the base of the neck will occlude the vein, which may then be visualized. Clipping the hair from the neck will aid in visualization of the vein. Two people are typically required for jugular venipuncture. Frisk and Richardson (1979), however, describe a technique that requires only one person. Femoral venipuncture is performed with the animal in lateral recumbency and the hindlimb extended. The vein is located just medial to the femoral pulse.

E.

Nonhuman Primates

Intravenous injection and blood collection techniques utilize the cephalic, saphenous, coccygeal, and femoral veins of nonhuman primates (Bowen et al., 1976; Hall, 1966; Whitney et al., 1973). The femoral vein is most commonly used for blood collection; it lies within the femoral triangle just medial to the

Fig. 4. Femoralvenipuncturein a rhesus monkey.The needle is inserted in the femoraltrianglejust medial to the femoralpulse.

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femoral artery and can be quite superficial in some species of primates (Fig. 4). If the femoral artery is inadvertently punctured, direct pressure must be applied to the site for a period of about 5 min in order to prevent excessive hemorrhage and hematoma formation. This may be especially critical in the owl monkey (Aotus trivirgatus) (Loeb et al., 1976). Blood may be collected from the jugular vein of a nonhuman primate if the animal is anesthetized. Small amounts of blood may be collected from neonatal primates by lancet puncture of a finger or toe or by superficial incision of the ear; however, the cellular indices may differ from those of a sample obtained from the femoral vein (Berchelman et al., 1973).

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Other Mammals

The laboratory opossum (gray short-tailed opossum, Monodelphis domestica) may be repeatedly bled via cardiac puncture under anesthesia (Robinson and VandeBerg, 1994). The common opossum may be bled from a number of sites, including the heart, lateral and ventral tail veins, femoral vessels, and pouch veins. Moore (1984) has described a method for obtaining blood from the brachiocephalic veins in a fashion similar to that used to bleed swine from the anterior vena cava. Daily samples ranging from 0.5 to 3.0 ml have been obtained from unanesthetized nine-banded armadillos (Dasypus novemcinctus) via puncture of the caudal tail vein between the second and third, or third and fourth, bony tail segments. A modified piece of polyvinyl chloride (PVC) pipe is used for restraint (Herbst and Webb, 1988).

G.

Birds

Common sites for blood collection and intravenous injection in avian species are the brachial veins of the wing, the jugular veins, and the heart. The brachial vein can be easily seen on the medial surface of the wing if the feathers are plucked or separated at the region of the elbow joint. Venipuncture is easily accomplished in the brachial vein if the bird is not too small; however, hematoma formation is a common sequela (Fredrickson et al., 1958). The right jugular vein, usually largest in birds, is superficially located on the dorsolateral surface of the neck between the dorsal and ventral cervical feather tracts (Law, 1960; Stevens and Ridgeway, 1966). Occlusion of the vein may be accomplished by applying pressure to the base of the neck. Jugular venipuncture is safer than cardiac puncture, and hematoma formation rarely occurs. Repeated collections may be done if venipuncture is done first at the base of the neck, and subsequent collections are made from sites nearer to the head. Jugular venipuncture is the blood collection method of choice in Japanese quail (Coturnix coturnixjaponica). Cardiac puncture may also be used to collect blood from birds, and it has the advan-

23. TECHNIQUES OF EXPERIMENTATION

tage of yielding large amounts in a short period of time. The needle is inserted 1 inch lateral and 1 inch caudal to the point of the keel in chickens. The syringe and needle are held at a 45 ~ angle above the body and directed toward the opposite shoulder. If the procedure is properly done, mortality rates are low (Hofstad, 1950). Garren (1959) constructed a vacuum apparatus that maintained constant negative pressure for bleeding from the wing vein or heart of chickens. Another vacuum-assisted technique of cardiac puncture has more recently been described (Foytik et al., 1989). Cannulation of both the jugular and brachial veins may also be used for chronic blood sampling and intravenous infusion in the chicken (Cravener and VasilatosYounken, 1989; Zhou and Brown, 1988).

H.

Unusual Species

Cooper (1993) has described a method for obtaining hemolymph from African land snails (Achatina spp.) that avoids perforation of the shell or incision of soft tissues by insertion of the needle at a variable distance below the pneumostome, depending on the size of the snail. Earthworms may constitute 6 0 - 8 0 % of the animal biomass in some soils, and have been proposed as a surrogate species for vertebrates in toxicity studies related to environmental pollution. Such studies may require the collection of earthworm leukocytes (coelomocytes). Coelomic puncture using a sharpened Pasteur pipette is an invasive way to obtain such cells (Eyambe et al., 1991). Such an invasive technique may injure the worm and frequently collects other cells in addition to the leukocytes. Two noninvasive methods have been described, which minimize contamination of the sample or injury to the worm (Eyambe et al., 1991; Diogene et al., 1997).

IV.

VASCULAR CANNULATION

For short-term experiments, the animal may be anesthetized or physically restrained during infusion using one of the external vessels described above. However, when the study requires repeated sampling or administration over an extended period of time, a more permanent infusion system can be used. Such a system may involve the implantation and exteriorization of chronic catheters, or the use of a subcutaneous access port (Vascular Access Port, Access Technologies, Skokie, Illinois; Soloport, Instech-Solomon, Plymouth Meeting, Pennsylvania) (Fitzgerald et al., 1996; Wojnicki et al., 1994). If catheters are to be used for a long period of time, the technique of cannulation must provide for protection of the catheter and allow freedom of movement for the animal. To accomplish these objectives, many ingenious methods and apparatuses are described in

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the literature and will be briefly summarized here (Table III). While catheters and access ports make blood sampling and vascular injections easier and less stressful for both animal and human, appropriate aseptic catheter maintenance techniques must be followed to prevent infection. Care must be taken to properly prepare the catheter or skin site prior to injection or sampling. In conjunction with proper catheter handling, the instillation of antibiotic and enzyme solutions into the catheter has been shown to be an effective way to prevent catheter-related sepsis (Palm et al., 1991) Following tail vein cannulation in rats and mice, a glass, metal, or plastic tube may be placed over the tail and attached to the top of the cage (Born and Moiler, 1974; Conner et aL, 1980; Plager, 1972; Rhodes and Patterson, 1979; Saarni and Viikari, 1976). This effectively protects the cannulation site and allows freedom of movement for the animal. Conti et al. (1979) describes the use of a fiberglass cast applied to the limb of a rhesus monkey to protect a cannula in the saphenous vein. Access to the vena cava from the lateral tail vein, and to the aorta from the ventral tail artery, have been described in the rat (Fejes-Toth et al., 1984). Venous and arterial cannulation with catheter protection, using a spring device, has been described in the rat (Kurowski et al., 1991). Using this system, direct blood pressure recordings were obtained from conscious rats for a period of 5 weeks. The most common method of protecting the cannula is by creating a subcutaneous tunnel from the site of vessel cannulation to the dorsum of the neck. The tunnel may be formed by blunt dissection with scissors or with a modified intramedullary pin (Wingfield et al., 1974). Exiting the catheters from the dorsal surface of the neck minimizes the possibility of damage by the animal to the setup. Installation of pouches or boxes over the neck is an additional measure taken to protect the infusion setup (Born and Moiler, 1974; Wingfield et al., 1974). A pouch may also be formed from skin folds (Hall et al., 1974). Goetz and Hermreck (1972) and Zambraski and DiBona (1976) describe exteriorization devices for chronically implanted catheters that further protect the catheter and facilitate infusion. Leather or canvas vests that protect implanted catheters are commercially available for a variety of species. Such protective devices have been used in the ferret (Jackson et al., 1988); dog (Foss and Barnard, 1969); rabbit (Knize and Weatherby-White, 1974); chicken (Hamilton, 1978); and different species of nonhuman primates, including macaques and African green monkeys (Bryant, 1980; McNamee et al., 1984), baboons (Lukas et al., 1982), and marmosets (O'Byrne, 1988). Subcutaneous access devices consist of a length of catheter attached to a reservoir buried beneath the skin so that it may be repeatedly punctured by a needle. Such devices give access to vascular or other structures to allow injections or withdrawal of blood without the risk of animal-induced damage or infections associated with catheters that perforate the skin. 'Such access ports are available in different sizes to accommodate most

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Table l l I

Blood Vessel Cannulation Species Mouse Rat

Ferret Guinea pig Rabbit

Dog Nonhuman primate

Swine

Sheep Cattle Chicken Pigeon Atlantic salmon

Vessel Tail vein Tail vein Jugular vein Dorsal aorta Carotid artery Cranial mesenteric vein Jugular vein Jugular vein Carotid artery Jugular vein Auricular vein Carotid artery Renal, mesenteric, iliac, hepatic arteries; portal vein Jugular vein Jugular vein Coccygeal vein Saphenous artery Saphenous vein Aorta and vena cava Internal jugular vein (marmoset) Jugular vein Femoral artery and vein Portal vein Anterior vena cava Inferior vena cava Hepatic vein and artery Cervical AV fistula Jugular vein Brachial vein Jugular vein Carotid artery Dorsal aorta

species, including rodents, rabbits (Perry-Clark and Meunier, 1991), cats (Webb e t al., 1995), and swine (Bailie e t al., 1986). Subcutaneous access devices have also been used for repeated blood sampling of woodchucks (Woolf e t al., 1989), desert tortoises (Wimsatt e t al., 1998), and nonhuman primates (Fitzgerald e t al., 1996; Wojnicki e t al., 1994). In dogs and ferrets, vascular access ports have been used to directly measure blood pressure (Mann e t al., 1987; Yao e t al., 1992). However, for studies in which blood pressure measurements may be required, there may be more variability when vascular access ports are used than when conventional catheters are used (Tartarini e t al., 1996). Osmotic minipumps (Alzet osmotic pump, ALZA Corporation, Palo Alto, California) are implantable devices that deliver a specified volume of fluid over a defined time period. These

Reference(s) Conner et al., 1980; Plager, 1972 Born and Moller, 1974; Rhodes and Patterson, 1979; Saarni and Viikari, 1976 Terkel and Urbach, 1974; Waynforth, 1980 Still and Whitcomb, 1956 Wixson et al., 1987 Zammit et al., 1979 Mesina et al., 1988 Christison and Curtin, 1969 Shrader and Everson, 1968 Hall et al., 1974 Knize and Weatherby-White, 1974; Melich, 1990 Conn and Langer, 1978 Sils et al., 1994 Branham, 1976; Dudrick et al., 1970; Foss and Barnard, 1969; Goetz and Hermreck, 1972; Platts et al., 1972; Engelhardt et al., 1993 Craig et al., 1969 Stickrod and Pruett, 1979 Munson, 1974 Conti et al., 1979 Scalese et al., 1990 O'Byrne, 1988 Ford and Maurer, 1978; Wingfield et al., 1974; Zanella and Mendl, 1992 Jackson et al., 1972 Knipfel et al., 1975 Moritz et aL, 1989 Smith et al., 1992 Drougas et al., 1996 Dennis et al., 1984 Ladewig and Stribrny, 1988 Hamilton, 1978; Zhou and Brown, 1988 Cravener and Vasilatos-Younken, 1989 Wendt et al., 1982 Pye-MacSwain et al., 1994

pumps have a flexible, impermeable reservoir chamber surrounded by a sealed layer containing an osmotic agent, all surrounded by a semipermeable membrane. When put into an aqueous environment (including subcutaneous or intraperitoneal implantation), the osmotic agent imbibes water at a rate determined by the semipermeable membrane. The imbibed water generates hydrostatic pressure, which compresses the flexible reservoir chamber to produce a constant flow of the contained material. Numerous papers describe the use of these pumps to deliver drugs intravenously, intra-arterially, intrathecacally, intraperitoneally, and subcutaneously. A limiting factor in the use of such pumps is the size of the pump relative to that of the animal to be used. Small animals can be implanted only with small pumps, which limits the volume, infusion rate, and duration of infusion. One method to eliminate this difficulty has

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been described in marmosets. Whereas most applications require that the pump be implanted within the animal, a system has been described in marmosets in which the pump was contained in an aqueous chamber in an externally worn backpack. This device allowed continuous delivery of gonadotropinreleasing hormone over a period of 3 months by simply replacing the osmotic pump every 2 weeks (Ruiz de Elvira and Abbott, 1986) If the infusion is to be done in a freely moving animal, the tubing, which connects the cannula with the infusion pump, must be shielded with some type of durable flexible sleeve. In addition, swivels and pulleys with counterweights are necessary to prevent the infusion tube from becoming twisted or kinked. Use of tether systems of this type is described by Conn and Langer (1978), Hamilton (1978), and Desjardins (1986).

V.

INTRAPERITONEAL INJECTION

Intraperitoneal injection is a common method of administering drugs to rodents. The injection site should be in the lower left quadrant of the abdomen because vital organs are absent from this area. Only the tip of the needle should penetrate the abdominal wall, to prevent injection into the intestine (Waynforth, 1980). Many operators recommend restraining the animal in a "head down," or Trendelenburg, position, assuring that the viscera will be displaced craniad in this maneuver. Injection into the right caudal abdominal quadrant of rodents restrained with the head lowered minimizes the risk of inadvertent injection into a viscus. As with injection in other sites, the operator should draw back the plunger of the syringe to determine that bowel contents, blood, or urine does not appear.

VI. SUBCUTANEOUS AND INTRAMUSCULAR I N J E C T I O N

The preferred site for subcutaneous injection in most laboratory animals is the back or neck region. A fold of skin is held with one hand, and the needle is inserted just under skin at the base of the fold. Lifting the needle after its insertion in the skin helps assure that it is properly placed. The thigh muscles are most commonly used for intramuscular injection. When large volumes of irritating substances are to be injected, the quadriceps group rather than the posterior thigh muscles should be used. The sciatic nerve lies posterior to the femur, and any substance injected into a fascial plane of the posterior thigh muscles may be carried directly to the nerve (Leash et al., 1973). Proper restraint is essential, and an assistant may be required for larger rodents. Needles should be advanced perpendicular to the skin

and should not be driven so deep as to strike bone. Alternative sites for injection in nonrodent species are the epaxial musculature of the rabbit, the triceps in most larger animal species, and the lateral neck of swine.

Vll.

DIGESTIVE SYSTEM

The following techniques will be described in this section: oral examination, oral administration, tooth extraction, pulpectomy, pulpotomy, cannulation of the common bile-pancreatic duct, biopsies of the liver and spleen, placement of intestinal cannulas, and formation of isolated intestinal loops. Complex surgical procedures, including gastroscopy, gastric fistulas, pyloric cuff techniques, a denervated Heidenhain pouch, a Pavlov's pouch, repeated intestinal biopsies, esophagogastroscopy, surgical removal of the pyloric antrum, and cecectomy, have been described in the literature (Markowitz et al., 1964; Pare et al., 1979; Pazin et al., 1978; Cook and Williams, 1978; Bruckner-Kardoss and Worstmann, 1967; Harris and Decker, 1969; Houghton and Jones, 1977) but will not be discussed in this text. Additional, more recent surgical models are reviewed by Swindle and Adams (1988). A.

Oral Examination

Many types of specialized restraining devices for oral examination are described in the literature (Evans et al., 1968; Davies and Grice, 1962; Redfern, 1971). A tubular device described by Macedo-Sobrinho et al. (1978) overcomes the difficulties created by the large diastema and small oral cavity of rodents. This device has proved to be reliable for intraoral examination of mice, rats, hamsters, and guinea pigs, even in the hands of inexperienced technical personnel. Certain species have unique anatomical adaptations that are utilized in research investigations. One example is the hamster cheek pouch. In order to expose this pouch, the hamster is held around the body with a thumb across its cheek. The fifth finger of the opposite hand is placed near the caudal end of the cheek pouch. By pushing cranially, while pulling gently at the corner of the hamster's mouth with the thumb of the free hand, the cheek pouch can be completely everted. This technique may be performed on an unanesthetized animal, the hamster does not bite its own everted cheek pouch, and traumatic instruments are not required (Haisley, 1980). B.

Oral Administration of Substances

Per os administration of solids and liquids to laboratory animals is an essential technique for a variety of experimental

ROBERT J. ADAMS

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protocols. Gastric intubation ensures that all the material is administered. Flexible catheters may be used for gavage in small rodents, but durable, stainless steel, ball-tipped needles are used more frequently (animal feeding needles, Popper and Sons, New Hyde Park, New York). These needles minimize trauma to oropharyngeal tissues and make inadvertent endotracheal passage less likely (Fig. 5). Common complications associated with gastric intubation are damage to the esophagus and administration of substances into the trachea. Careful and gentle passage of the gavage tube will greatly reduce these possibilities. In addition, introduction of the tube from the pharynx into the esophagus is best accomplished when the animal is in the act of swallowing. Usually one can determine that the tube is not within the trachea by observing the tube's profile as it moves within the esophagus. A method for administering gelatin capsules to awake guinea pigs using an intravenous catheter modified to contain a cup at one end has been described. The cup is sized to hold the gelatin capsule, such that, once passed into the stomach, the gelatin capsule can be pushed out of the cup by use of a wire through the catheter (DeBrant and Remon, 1991). Some species have anatomic variations that may cause difficulty in gastric intubation. Guinea pigs, chinchillas, and other hystricomorph rodents have a communication between the oropharynx and the pharynx on the dorsal midline of the soft palate termed the palatal ostium. Care must be taken to pass the needle or tube through this ostium to prevent damage to the adjacent velopharyngeal recess (Timm et al., 1987). Swine have a median cul-de-sac dorsal to the entrance to the esophagus termed the pharyngeal diverticulum that may also interfere with successful gastric or endotracheal intubation (Sisson and Grossman, 1966). Oral specula such as those constructed from syringe cases or tongue depressors may be used to prevent the animal from chewing on gavage tubes when introduced into the oral cavity.

Nasogastric intubation prevents tube damage, and it is easily accomplished in nonhuman primates or cats when the animals are anesthetized or severely depressed. For nonhuman primates a mouth speculum has been described that minimizes the risk to the handlers. The device consists of a central stainless steel speculum tube with two stainless steel arms welded on either side (Halliday et al., 1998). Surgically implanted gastric cannulas may also be used to deliver drugs to the stomach. As described in larger domestic animals like cats and dogs, pharyngostomy tubes may also be placed in rabbits (Rogers et al., 1988). In cats and other larger species, gastric cannulas may be placed endoscopically from the stomach through the body wall (Keshavarzian et al., 1989). Gastric cannulas may be placed during an abdominal laparotomy; the catheter may be tunneled subcutaneously to exit at the back, and the catheter may be maintained by using a protective sheath and jacket system, as previously described for vascular cannulas (Muller et al., 1992).

C.

Because of their anatomical configuration, the teeth of some animals, such as the primate, are very difficult to remove. The technique of tooth extraction using the dental elevator involves (1) inserting the dental elevator between the alveolar bone and the tooth; (2) applying pressure on the elevator, using a rotating wrist action while directing it toward the apex of the root on all sides; and finally, (3) attempting to pull the tooth only after all periodontal ligaments have been severed. Dental procedures may be adapted from those described from dogs and cats. One method of avoiding the risks of trauma, bleeding, and fracture of the mandible in nonhuman primates is to use a nonsurgical technique for tooth extraction. Serial injections of the peridontal tissues with a 20% solution of potassium hydroxide, which is made into a gel with Carbopol 934 (a water-soluble resin, B. E Goodrich Chemical Company, Cleveland), causes a loosening of mandibular and maxillary teeth. Following 1-3 injections, the tooth is easily removed from the alveolus. Using this same technique for hydrolyzing the membranes, it is possible to loosen malpositioned teeth, reposition them in the alveoli, splint, and allow alveolar reattachment (Patrick et al., 1968).

D.

Fig. 5. Stainlesssteel, ball-tipped needles used for a gavagein rodents.

Tooth Extraction

Pulpectomy and Pulpotomy

More recently, the surgical techniques of pulpectomy and pulpotomy have been popularized as alternatives to canine tooth extraction. These procedures have been used in veterinary medicine for treating tooth fractures and pulp infections and for disarming dangerous animals. Pulpotomies refer to the opening of the root canal and the partial removal of the pulpal tissue. In-

23. TECHNIQUESOF EXPERIMENTATION creased sensitivity may result from pulpotomy because of the potential reaction of the remaining pulpal tissue. Pulpectomies, on the other hand, refer to the total removal of the pulpal tissue. Although variations in techniques and chemicals used are described, the pulpectomy procedure, as outlined by Tomson et al. (1979) is excellent. This procedure involves anesthetizing the animal, in this case a nonhuman primate, and placing it on a surgery table in dorsal recumbency. Rubber dams are used on all teeth to prevent contamination of the root canal with microorganisms from the oral cavity. Each canine tooth is cut off level with adjacent teeth, using tapering crosscut bars or Damascus separating disks (William Dixon, Inc., Carlstadt, New Jersey) attached to a dental drill. Broaches are used to remove all pulpal tissue from the root canal. The root canal is then irrigated with sterile saline. Paper points are used to dry the canal and check for the presence of blood (Tomson et al., 1979). If bleeding does occur, the canal must be explored, so that all pulpal tissue is removed. Other techniques have employed epinephrine flushes or electrocautery to stop the bleeding and dry the root canal. Root canal paste formulas include an RC-2W mixture consisting of 3% BaSO 4, 6% Titamicin oxide, 8% bismuth subnitrate, 18% bismuth subcarbonate, 6.5% paraformaldehyde, and 62.5% zinc oxide (Tomson et al., 1979), or the commercial product Hypo-Cal (Hellmann Dental Manufacturing, Hewlett, New York), which is calcium hydroxide in a calibrated disposable syringe with a special applicator needle. After the canal has been filled, excess paste is removed, and an amalgam plugger is used to firmly pack the hardening paste into the filled canal. A silver alloy amalgam (Caulk Company, Milford, Delaware, or White Dental Products International, Philadelphia) filling is used to permanently seal the filled root canal. Excess alloy is removed, and the tooth is sculptured smooth (Tomson et al., 1979) (Fig. 6). Some have advocated a more permanent technique involving the placement of a permanent pulp cap (Reynolds and Hall, 1979). Although this technique is more costly and time-consuming, it does give the investigator an opportunity to easily convert troublesome temporary caps to more durable crowns at a later date. Pulpotomy using formocresol and electrosurgery has also been described (Shulman et al., 1987). In baboons and macaques, a technique termed submucosal vital root retention, involving the amputation of the crown of the canine tooth below the margin of the gum, has been described (Schofield et al., 1991). In this technique, the gum flap is repositioned over the remaining tooth, with retention of nerve function and minimization of subsequent problems such as tooth root abscesses and devitalization of the tooth.

E.

Bile Duct Manipulations

The common bile duct may be manipulated in a number of ways to accomplish various research goals. Complete biliary re-

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Fig. 6. Stepsin pulpectomy. Diagram illustrates the level at which the canine tooth shouldbe cut in relationto adjacentteeth. Followingextractionof the pulpal tissue, the root canal is filledwith a root canal paste. (Reprintedwithpermission fromTomsonet al., 1979.)

tention can be accomplished easily by ligation of the common bile duct. Although effective, this technique results in obstructive jaundice. Partial obstruction, to mimic disease conditions such as bile duct stricture, biliary tumors or extrinsic compression of the bile duct can be produced in the rat by ligation of the bile duct over a 0.5 mm rod, followed by removal of. the rod (Sekas, 1990). Complete biliary retention without obstruction can be accomplished by implanting a catheter between the common bile duct and the caudal vena cava via the right lumbar vein (Clements et al., 1985). This technique allows for studies of complete bile recirculation without the pathologic effects of complete obstruction. Animal models in which bile can be sampled have many applications, especially in pharmacokinetic studies. Some techniques, such as that described by Spalton and Clifford (1979), involve the oral passage of a tube into the duodenum. After application of a vacuum for several minutes, the animal is given an intravenous injection of pancreozymic enzymes and cholecystokinin, which causes contraction of the gallbladder. Aspiration of the bile sample is carried out via the duodenal tube. A simple, short-term method for collecting bile is direct surgical cannulation of the gallbladder and ligation of the common bile duct (Talbot and Hynd, 1985). In larger species, such as the nonhuman primate, bile can be aspirated by cholecystocentesis under ultrasound guidance (Pekow et al., 1994). Attempts to cannulate the common bile duct are described by several authors in a variety of species (Boegli and Hall, 1969; Barringer et al., 1982; Soli and Birkeland, 1977; Knapp et al., 1971). Most authors reflect on the difficulty of maintaining an experimental preparation of this type. It is also well known that some anesthetics interfere with biliary secretion (Berthelot et al., 1970).

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To overcome such artifacts, a double recurrent choledocholedocal biliary fistula has been described for rabbits. Through a midline laparotomy, the bile duct is exposed and dissected for a distance of 10 mm from the duodenal wall. Two silastic medical catheters (external diameter 3.16 mm, internal diameter 1.97 mm) are inserted into the bile duct. One is directed toward the liver, making sure that its end does not pass the cystic and secondary hepatic ducts. The other catheter is directed toward the duodeneum but is stopped short of the sphincter of Oddi. The catheters are then brought to the body surface through stab wounds in the lateral abdominal wall and connected to taps that allow the bile samples to be taken and solutions infused. The catheters are protected with a specially constructed bandage in which small polyethylene bags are placed for collection of samples (Jimenez et al., 1982). In smaller species, such as mice and rats, a stereoscopic microscope may be used to identify the common bile-pancreatic duct. Once the junction between the hepatic ducts and cystic duct is individualized, the bile duct is closed immediately below their junction by a nylon ligature. The common bile-pancreatic duct ends in an ampulla that connects it to the duodenal lumen. A small cut is made on the ampulla wall with Bellucci scissors, and a polyethylene cannula (internal diameter 150 ~tm) is inserted approximately 1 mm into the common bile-pancreatic duct lumen. This cannula is secured by a 7-0 nylon ligature. A calibrated capillary tube is placed in the free end of the cannula to collect the sample (Maillie et al., 1981). Chronic indwelling catheters for continuous collection of bile can be exteriorized through the skin at the base of the skull and protected by a metal coil covering (Balabaud et aL, 1981). Where maintenance of the enterohepatic recirculation of bile must be maintained during bile collection, a number of surgical techniques for catheter placement have been described in various species (Raggi et al., 1985; Rath and Hutchison, 1989; Rolf et al., 1991; Wang and Reuning, 1994; Clegg, 1997). Such techniques usually involve the insertion of two cannulas, one going proximally and the second distally in the bile duct or the duodenum, with both distal catheter ends being connected after exiting the body. Bile may be collected by opening the connection between the catheters, aspirating the sample and then reinserting the connection. A modification of this technique in young swine used an external Y connection between the two catheters, allowing aspiration without opening the system (Faidley et al., 1991). A method described in rats utilized an intra-abdominally implanted reservoir with three cathetersmtwo.implanted proximally and distally in the bile duct, and a third exiting the body, through which samples could be collected (Heitmeyer and Powers, 1992). A procedure in dogs utilized a T piece with a central occluding diaphragm. Each arm of the T and the occluding chamber were connected to separate catheters. Both arms of the T piece were implanted into the bile duct, with the three catheters exiting the body. With the central occluding diaphragm unexpanded, bile freely flowed through the T piece, as it

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would through the normal bile duct. Expansion of the central diaphragm with saline occluded the cannula and allowed bile to flow through the collection catheter attached to the proximal arm of the T piece for collection. Additionally, various solutions, uncontaminated with bile, could be injected through the infusion catheter attached to the distal arm of the T piece (Kissinger et al., 1998).

F.

Partial Hepatectomy and Liver Biopsy

In rodents, partial hepatectomy is performed to get sufficient tissue for certain types of studies or to remove a sufficient amount to induce regeneration with associated gene activation. Entire lobes may be removed relatively quickly with little hemorrhage by using hemoclips to ligate blood vessels (Schaeffer et al., 1994). The most straightforward methods for performing liver biopsies involve a transcutaneous skin puncture; large tissue samples may require abdominal surgery. Voss (1970) describes a simple, rapid, closed biopsy technique for primates utilizing a 16-gauge Klatskin needle and a subcostal approach. Smallergauge biopsy needles are used in a similar manner on rodent species and ducks (Varagona et al., 1991). Bacher et al. (1989) has described the use of a 6-mm Keyes skin punch for serial liver biopsies in Pekin ducks. When larger tissue samples are needed in larger species, an open liver biopsy procedure may be required. Following anesthesia and surgical preparation of the abdominal area, a cranial midline abdominal incision is made to expose the liver. After identification of the area to be excised, a scalpel or scissors are used to remove the wedge of tissue. The incised area is then packed with a folded piece of absorbable gelatin sponge (Gelfoam, U.S.E, Upjohn Company, Kalamazoo, Michigan) (Voss, 1970). In Pekin ducks an atraumatic cardiovascular clamp has been used to delineate the tissue to be removed (Carp et al., 1991). Large pieces of liver can also be removed by using the finger or ligature fracture technique. Using the fingers, the liver parenchyma is crushed along the line of tissue to be removed. The portal triads remain connected between the two segments and are easily double-ligated and separated. Similarly, an absorbable suture can be loosely tied around a segment of liver to be removed. When slowly tightened, the suture cuts through the liver parenchyma, isolating the portal triads, which are then ligated with the suture (Talcott and Dysko, 1991). Some authors have recommended the use of a TA-90 stapling gun (U.S. Surgical Corporation, Stamford, Connecticut) or similar instrument. The advantage of this technique is that larger samples of biopsy material are provided and hemostasis of the severed liver lobe is excellent (Nolan and Conti, 1980). Laparoscopic biopsies of liver and spleen have been described in the S c h i s t o s o m a infected baboon. Under halothane anesthesia, baboons had up to three biopsies obtained using an Olympus (Olympus, Tuttlin-

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gen, Germany: 0 ~ 10 mm 29 cm telescope) laparoscope xenon light source in a three-trocar technique (Rawlings et al., 2000).

G.

Pancreatic Exocrine and Endocrine Studies

Studies of pancreatic exocrine function may be performed in a variety of species by utilizing a number of techniques, which are well described by Sarr (1988) and Niebergall-Roth et al. (1997). Many of these older techniques involve the surgical creation of pancreatic fistulas, duodenal fistulas, or duodenal pouches. More recent studies usually involve catheterization of the pancreatic duct (Niebergall-Roth et al., 1997; Naranjo et al., 1986). A study employing catheterization of a surgically created pancreatic fistula allowed studies to be done in conscious, freely moveable rats (Toriumi et al., 1994). Studies of pancreatic endocrine function usually involve the chemical ablation of the pancreatic islets, using alloxan or streptozotocin, or surgical removal of the pancreas. In laboratory rodents, surgical removal of the pancreas may be virtually impossible because of its diffuse anatomy, and chemical ablation is the method of choice (Hatchell et aL, 1986; Sarr, 1988; Miller, 1990). Chemical ablation is also effective in rabbits and nonhuman primates (Sarr, 1988; Litwak et al., 1998). Cats are resistant to the diabetogenic effects of both alloxan and strep.tozotocin, and surgical removal, or a combination of surgical and chemical techniques, is necessary (Hatchell et al., 1986; Reiser et aL, 1987). Total pancreatectomy in dogs and swine is readily accomplished (Sarr, 1988; Stump et al., 1988).

H.

Intestinal Cannulation

There are many designs and materials used for insertion of rigid or flexible cannulas into alimentary tract fistulas of experimental animals. The choice of cannula depends on several factors, such as the size of the lumen of the organ to be fistulated, the particle size and viscosity of the material to be sampled or infused, the site of exteriorization of the cannula, and, in the case of reentrant fistulas, the volume and consistency of ingesta expected to flow through the cannulas to avoid producing excessive resistance to flow. Because of these variables, flexible plastic cannulas (polyvinyl chloride [PVC], Tygon, polyethylene) may be preferable to rigid cannulas in some cases (Buttle et aL, 1982; Banks et al., 1989; Rodhouse et al., 1988). Many more recent papers, however, still describe the use of rigid cannulas, often composed of stainless steel and plastic components (Carman and Waynforth, 1994; Maragos et al., 1990; Kloots et al., 1995; Hill et al., 1996; Swindle et al., 1998). Vascular access ports may be used for delivering drugs and solutions to various segments of the gastrointestinal tract without the problems associated with externalized catheters or cannulas (Meunier et al., 1993).

Intestinal Loop Isolation

Numerous research protocols for the study of gastrointestinal physiology and pathology require segments of the digestive tract that have no continuity with the fecal stream. These segments are provided by preparation of isolated loops at different levels of the gut. The crucial stage in preparing such a segment is the method of anastomosis used on the intestinal wall. For most species, the preferred technique is the use of a simple, interrupted, approximating suture pattern that limits intestinal tissue trauma and minimizes luminal narrowing (Toofanian and Targowski, 1982). This is particularly true in the guinea pig, because its small bowel is highly friable. This species requires using interrupted 6-0 silk sutures meticulously placed 1 mm apart and 1 mm deep (Bett et al., 1980). Suture patterns in other rodent species would require a similar technique. A technique called the RITARD model (reversible intestinal tie-adult rabbit diarrhea) has been developed to allow temporary occlusion of a section of bowel for bacterial colonization studies in the rabbit. This technique utilizes sterile umbilical tape tied in such a way that the ligature can be loosened and removed by gentle traction on the ends that are exteriorized through the incision site (Spira et al., 1981; Kesel and Ellis, 1988). Although Variations exist, all models are characterized by temporary small intestinal ligation and permanent cecal ligation at a site just distal to the sacculus rotundus. Occlusion of the cecum prevents absorption of fluids secreted from the small intestine.

VIII.

URINARY SYSTEM TECHNIQUES

The techniques described in this section include methods of urine collection, exteriorization of the ureters, and implantation of the ureters into the intestine. Because meaningful descriptive techniques require extensive detail, the following procedures are referenced only: reconstructive surgery of the ureters, fistula of the urinary bladder, transposition of the kidney into the iliac fossa, and denervation and decapsulation of the kidney (Lopukhin, 1976). Renal allotransplantation and xenotransplantation procedures may be reviewed in Bernsteen et al. (2000) and Platt (1999), respectively. Urine is usually removed from the bladder by one of four methodsm(a) collection during spontaneous micturition, (b) manual compression of the urinary bladder, (c) catheterization, (d) cystocentesismand use of metabolic cages (Khosho et aL, 1985). With spontaneous collection and following manual expression, the first part of the urine stream should not be used for urinalysis or bacterial culture, because it may contain debris, bacteria, or exudate flushed from the urethra or genital tract. A collecting device to collect urine uncontaminated with

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feces during micturition in turkeys and chickens has been described by St. Cyr et al. (1987). In male rats, which are difficult to catheterize, urine may be collected by retraction of the prepuce and attachment of silicone tubing over the glans penis (White, 1971: Rahlmann et al., 1976). The urethra of male baboons is also difficult to catheterize. Gavellas et al. (1987) used an external condom catheter in restraint chain-trained male baboons to collect urine over a 24 hr period. Garvey and Alseth (1971) capitalized on the fact that abdominal muscle control is poorly developed in the newborn. Many females stimulate their newborn animals to urinate by stroking the lower abdomen. Although the technique is described for the rabbit, the investigator or clinician can collect urine samples from the newborn of several species by applying gentle strokes and pressure on the lower abdomen. Stroking and pressure are continued until the muscles relax and a drop of urine is observed; then pressure is applied over the bladder until the flow stops. Although there is individual variation, up to 5 ml of urine has been collected from newborn rabbits using this method (Garvey and Alseth, 1971). Success in obtaining urine by this method is often greater in females because of reduced urethral resistance. Other methods of urine collection have required the attachment of a modified pediatric urine bag to fully cover the vulva in swine and thus prevent fecal contamination (Galitzer et al., 1979) and the use of specialized metabolic cages, screens, and baffles to separate the urine and feces (Black and Claxter, 1979; Smith et al., 1981). For some species, urethral and/or ureteral catheterization is the method of choice. Diagnostic catheterization is indicated for (1) collecting bladder urine for urinalysis or bacterial culture, (2) studying renal function, (3) instilling contrast media for radiography, (4) evaluating the urethral lumen for strictures and/ or obstruction, and (5) surgically repairing the urethra and surrounding structures. These methods are routinely used for dogs and have also been described for calves (Allen, 1974), domestic fowl (Wideman and Braun, 1982), ferrets (Marini et al., 1994), and rats (Cohen and Oliver, 1964). In the female dog, visualization of the external urethral meatus aids in the placement of the catheter. A variety of devices have been used for this purpose. Regardless of the specific procedure employed, meticulous aseptic and gentle atraumatic technique should be used for catheterization of all species. Conscious patients should be restrained by an assistant in order to minimize contamination of the catheter as well as trauma to the urethra. The smallestdiameter catheter that will permit catheterization should be used. If a stylet is used in the catheter, it should be lubricated before it is inserted into the lumen of the catheter. If it is not lubricated, difficulty may be encountered in removing the stylet after the catheter has been placed in the patient. Regardless of species or sex, the approximate distance from the external urethral orifice to the neck of the bladder should be determined and mentally transposed to the catheter. This step will minimize the

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likelihood of traumatizing the bladder wall due to insertion of an excessive length. Proper lubrication of the catheter with a liberal quantity of sterilized aqueous lubricant will minimize discomfort to the patient and catheter-induced trauma to the urethra. Although usually unnecessary, local anesthesia of the urethra may be provided with a topical anesthetic. Aseptic technique must be used in advancing the catheter into the urethra. If difficulty is encountered in inserting the catheter, the catheter should be withdrawn a short distance and inserted again while rotating. With unsuccessful catheterization, a smaller-diameter catheter should be used. The tip of the catheter should be positioned so that it is located just beyond the junction of the neck of the bladder within the urethra. Verification of this position may be accomplished by injection of a known quantity of air through the catheter; inability to remove all of the air indicates improper positioning of the catheter. Proper positioning of the catheter facilitates removal of all of the urine from the bladder. Urine may be aspirated from the bladder with the aid of a syringe and two-way valve. Aspiration must be gentle to prevent trauma to the bladder mucosa as a result of sucking it into the eye of the catheter. Catheters that are to remain in the bladder for some time but that are not designed to permit self-retention may be sutured to the skin. Potential complications associated with catheterization in all species include hematuria and infection. Although hematuria is usually seif-limiting, it may interfere with interpretation of the urinalysis. Because of the risk of inducing infection of the bladder as a result of catheterization, this technique should be reserved for diagnostic or therapeutic purposes (Painter et al., 1971; Goodpasture et al., 1982). Procedures that may be used to reduce the incidence of infection following catheterization include (1) strict adherence to principles of asepsis, (2) administration of oral or parenteral antibiotics, (3) use of catheters impregnated with antibacterial agents, and (4) irrigation of the bladder with antibacterial solutions, such as neomycin or furacin (Osborne et al., 1972). Urine collection in rodents may be accomplished by surgical cystotomy (Moreland, 1965) or one of the methods previously described. The small size of the animals is one complicating factor; another is the unique reproductive system of some species. For example, catheterization of the male guinea pig can be accomplished rather easily; however, in almost all cases the male will ejaculate as the catheter is passed, and the coagulum will quickly plug the catheter. Some success has been attained with urethral catheterization of the female rat using a No. 4 Coude ureteral catheter that has a bend adjacent to the tip. This angulation allows the catheter to atraumatically slide by most obstructive areas in the urethra (Cohen and Oliver, 1964). Operators must recognize that female rats have distinct urethral and vaginal meatus. In order to avoid lower urinary tract infections and to facilitate renal function studies, techniques have been developed for catheterizing the ureter, urinary bladder (Black et al., 1996),

23. TECHNIQUES OF EXPERIMENTATION or urethra. Many studies describe the use of such catheters in the tethered, freely moving animal (Oz et al., 1989; Mandavilli et al., 1991). In some species, such as the rabbit, the ureters are very friable and subject to intraluminal bleeding. Reduced urine output during anesthesia, combined with bleeding tendencies, will often result in occlusion of the catheter with blood clots. In order to compensate for this problem, Harris and Best (1979) designed a double-lumen catheter. Irrigation of blood from the catheter is accomplished by perfusion of heparinized saline through a small inner catheter. Other, still more invasive techniques have involved relocating or exteriorizing the ureters (Abernathy and Anderson, 1974). The exteriorized ureter can then be easily cannulated, or it can be temporarily obstructed for research purposes. At times, resection of the ureters is required in order to facilitate a surgical procedure. If this is necessary, an elastic catheter with a diameter equal to or a little smaller than the ureter is inserted into both ends of the transected ureter, and the ends are then approximated. Interrupted catgut or silk sutures are placed in the periureteral cellular tissue and muscular coat, with care taken not to enter the lumen. The catheter is removed through a lateral longitudinal incision in the ureter below the anastomosis. This incision is closed by using transverse interrupted sutures, taking care not to narrow the ureteral lumen (Lopukhin, 1976). In swine, the upper urinary tract can be easily accessed via a retroperitoneal approach (Parlett et al., 1993; Bowen et al., 1994). Ureteral transplantation into the intestines or Heidenhain pouch has also been described (Lopukhin, 1976).

IX.

RESPIRATORY SYSTEM TECHNIQUES

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tamination by bacteria has been described (Snyder and Soave, 1970). A straight tube formed from a 1 ml tuberculin syringe is used as a speculum for the passage of sterile swabs into the pharynx. The transparent syringe barrel allows for easy visualization of the larynx. Commercial sources for sheathed applicator swabs are also available; however, the described technique can be performed in several species with materials readily available in the laboratory and at a much lower unit cost.

B.

Endotracheal Intubation

A patent airway is essential in reducing mortality during and after surgical procedures. A number of papers describe methods of intubating rodents with a variety of instruments. The simplest method described for anesthetized rats is a transillumination technique performed with the animals in dorsal recumbency on an inclined restraint board. With the superior incisors held by a rubber band, a 50-watt lighted bulb is placed 5 cm from the skin of the neck. A small curved spatula is then used like a laryngoscope to elevate the tongue, allowing easy visualization of the larynx because of the transillumination produced by the light bulb. A 16-gauge flexible intravenous catheter is then passed into the trachea (Cambron et al., 1995). Other techniques described for mice, hamsters, and guinea pigs use modified laryngoscope or otoscope specula to allow visualization of the larynx (Costa et al., 1986; Tran and Lawson, 1986; Blouin and Cormier, 1987; Turner et al., 1992). Another method of endotracheal intubation in rodents in dorsal recumbency is accomplished by retracting the upper jaw downward with a rubber band passed over the upper incisor teeth, retracting the tongue to one side, and observing the laryngeal region through a surgical microscope under x 6 and x 10 magnification. After the mucus is cleared with a cotton-tipped applicator to allow visualization of the epiglottis, glottis, and paired arytenoid cartilages, the endotracheal tube is inserted into the trachea. As the end of the tube is advanced past the larynx, water vapor is seen passing from the tube during expiration, indicating proper placement (Pena and Cabrera, 1980). Oral endotracheal intubation in the rabbit has been described by numerous authors, including Davis and Malinin (1974), Berthelot et al. (1970), Alexander and Clark (1980), Macrae and Guerreiro (1989), and Bechtold and Abrutyn (1991). The present author believes that the technique by Alexander and Clark (1980) is the simplest and most frequently used. The rabbit is anesthetized with gas anesthesia and placed on a flat surface in an outstretched prone position. Anesthesia is continued until all laryngeal reflexes are abolished. The rabbit's head is tipped and extended into an upright position at right angles to the rest of the body. This position provides a straight passage from the lips to the larynx. A sterile, uncuffed, nylon-reinforced, latex endotracheal tube (4.0-4.5 mm internal diameter) lubricated with a water-soluble sterile lubricant is passed into the ,

This section will discuss the following techniques as useful tools in the diagnosis and treatment of respiratory diseases: collection of pharyngeal fluids, tracheobronchial washings, endotracheal inoculation and intubation, tracheal pouch formation, tracheostomy, ventriculocordectomy, bronchoscopy, and bronchopulmonary lavage. The references should be consulted for other techniques involving the lower respiratory system, including lobectomies (Bernstein and Agee, 1964; Markowitz et al., 1964), bioinstrumentation of the thorax (Harvey and Jones, 1982), and the development of chronic lung-lymph fistulas (Brown et al., 1982). Because these techniques involve extensive protocols, a description could not be included in this chapter.

A.

Collection of Pharyngeal Samples

A simple, effective, and inexpensive method for the collection of pharyngeal cultures from nonhuman primates without con-

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diastema over the tongue and positioned over, but not touching, the larynx. Correct positioning is ascertained by listening to the respiration through the tube and adjusting to obtain maximum ventilation. At inspiration, when the vocal cords are maximally opened, the endotracheal tube is inserted with a straight push to the desired depth (Alexander and Clark, 1980). Conlon et al. (1990) describe a technique in rabbits that utilizes an esophageal stethoscope attached to an uncuffed endotracheal tube. A small elliptical hole is cut in the end of the stethoscope to allow air to escape when the animal coughs. Under halothane anesthesia the rabbit is placed in a supine position and the neck is extended. The endotracheal tube is inserted into the mouth to the left of the incisor teeth, and by using the stethoscope to identify the inspiratory and expiratory phases of respiration, the tip of the tube can be placed precisely over the glottis. On the next inspiratory phase the endotracheal tube is advanced into the trachea. Correct placement of the endotracheal tube in the trachea causes the rabbit to cough. Additional blind techniques include use of an endoesophageal tube (Kim and Han, 2000) and use of capnography during tube placement (Han et al., 2000). Visual techniques require the use of laryngoscopes and laryngoscope blades of specific size (0-1 Wisconsin or Miller 1). Animals are restrained in sternal recumbancy, dorsal cervical flexion, and lingual traction. The laryngoscope blade is inserted, and the tongue is depressed by the blade until the epiglottis is visualized. The tube is then advanced through the aditus laryngis (Lipman et al., 1997).

C.

Tracheobronchial Washing and Inoculation

Tracheobronchial washing techniques have been described for the anesthetized primate. With the head tilted slightly off the edge of the table, the tongue is grasped with a gauze sponge and traction is applied. A fiberoptic light source aids in visualization of the orifice, and a sterile straight Kelly forceps is used to grasp the central body of the epiglottis. A pediatric laryngoscope with individual sterile blades permits easy visualization of the larynx and passage of the tubes. Tracheobronchial washings are obtained using a 3 French feeding tube 40.6 cm long. To permit sterile placement of the tube into the trachea, the scabbard or outer tube is cut with a blunt taper on one end. This outer tube is lubricated with a sterile lubricant and introduced directly into the trachea on expiration to a position about 5 - 8 cm posterior to the larynx. The 3 French rubber feeding tube is subsequently passed through the outer tube and introduced to about the level of the carina. A 5 ml syringe containing sterile saline is then attached to the feeding tube and used to infuse saline into the bronchi. Saline is cleared from the tube using 2 - 3 ml of air. At this point the animal is rotated from side to side several times and light pressure is applied to the chest, while simulatenously aspirating the 5 ml syringe. Generally, 1-2 ml of foamy tracheobronchial fluid is obtained (Ilievski and Fleischman, 1981). A second method is to use the transtracheal aspiration tech-

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nique that involves passing a needle between the tracheal rings or cricothyroid membrane and into the tracheal lumen. A catheter is then directed through the needle and advanced to a bronchus. Sterile saline (10-15 ml for a macaque) is infused and aspirated through the catheter (Stills et al., 1979). This technique, as with the previous technique, circumvents the potential problems of pharyngeal and laryngeal contamination. An advantage of this technique is that it does not require chemical restraint in the acutely ill animal. Some experimental protocols require that chemical substances be administered intratracheally. If the animal already has an endotracheal tube in place, this procedure is quite simple. The material is injected directly through the tube. Other techniques for endotracheal inoculation in rodents have been described by Nicholson and Kinkead (1982), Yap (1982), and Pena and Cabrera (1980). The equipment required for intratracheal inoculation in small rodents includes two pairs of small curved forceps; a 100 ~tl microsyringe; a 38 x 1 mm diameter needle with a blunt tip bent at a 30 ~ angle 10 mm from the end, and a laryngoscope made from a disposable polypropylene micropipette tip (Yap, 1982). The anesthetized rodent is placed on its back, and a pair of forceps is used to grasp the tongue and hold it to one side. The specially designed laryngoscope (Yap, 1982) is inserted, narrow end first, into the mouth. The inoculation needle, curved end uppermost, is then inserted through the laryngoscope. On entry, the syringe is held so that the portion of the inoculation needle up to the bend is parallel to the throat. With further insertion, the needle is gradually tilted upward, and when resistance is felt, the needle is withdrawn very slightly and tilted up further. On reinsertion, a give is felt when the epiglottis is passed (Yap, 1982). Another specialized speculum has been designed by Nicholson and Kinkead (1982) to facilitate intratracheal inoculations. This speculum is inserted into the rat's mouth, keeping the tongue fiat beneath the blade and thus facilitating inoculation. Additional reports describe methods of acute intratracheal instillation in mice (Starcher and Williams, 1989), rats (Smith, 1991; Ruzinski et al., 1995; Wheeldon et aL, 1992), and newborn rabbits (Venkatesh et al., 1988). Chronic, repeated intratracheal administration of materials may be done through implanted catheters. Two such techniques have been described in laboratory rodents (Blouin et al., 1994) and ferrets (Chimes, 1993) and can likely be adapted to any species under study.

D.

Tracheal Pouch Formation

The development of a model for studying tracheal secretions by using a surgically isolated segment of trachea with an intact nerve and blood supply, the trachael pouch, was first described in the dog (Wardell et al., 1970). The technique involves resecting a segment of cervical trachea, with blood and nerve supply intact, and relocating this closed segment subcutaneously for ease in sampling. A more simplified technique for creating a

23. TECHNIQUESOF EXPERIMENTATION tracheal pouch has been described in the ferret. Following anesthesia, an 8-10 cm ventral midline skin incision is made three cartilage rings caudal to the larynx to expose the trachea. The trachea is transected at two points between cartilage rings, while exercising care not to disturb the dorsolateral recurrent laryngeal nerves and dorsal vascular supply. Pouches of eight cartilage rings in length can be made without causing discomfort. A stay suture is used to hold the tracheal segment away from the surgical field so that the ferret can easily breathe. Tracheal continuity is reestablished by anastomosis, using four 3-0 gut sutures. The cranial end of the tracheal segment is then sutured to the subcutis with four 3-0 silk sutures. The caudal end is closed with three 3-0 chromic gut sutures, thus forming a pouch (O1son, 1974; Barber and Small, 1977). More recently, the tracheal pouch technique has been used to study nonadrenergic, noncholinergic inhibitory (NANCI) neurotransmission in guinea pigs (Venugopalan et al., 1998), evaluation of mucolytic drops in miniature swine (Livingstone et al., 1990), and vasoactive intestinal peptide as a neurotransmitter of nonadrenergic inhibition of guinea pigs (Venugopalan et al., 1984).

E.

Tracheostomy

Permanent tracheostomies have been described in a number of species. As with humans, some procedures create a permanent stoma into which a removable tracheostomy tube is inserted (Vogler et al., 1992). Although effective, this technique does necessitate daily management of the stoma and tube. A permanent tracheostomy has been developed for dogs that eliminates the need for the tracheostomy tube. The procedure consists of dissecting portions of the cartilagenous rings free from the underlying tracheal mucosa, cutting through the mucosa, and suturing the mucosa to the skin. This procedure results in a permanent, maintenance-free, mucocutaneous stoma (Dalgard et al., 1979; Ritter, 1984). A similar technique has also been described in sheep (Dueck et al., 1985).

F.

Bronchoscopy

Flexible fiberoptic bronchoscopy has been well established as a diagnostic and therapeutic tool in human medicine. Because the anatomy of the rhesus monkey is quite similar to that of the human, this technique is now utilized as a diagnostic and therapeutic tool in primate medicine. The monkey is anesthetized with ketamine, and the vocal cords, larynx, and trachea are desensitized with supplemental topical anesthesia. Either a pediatric fiberoptic bronchoscope (outer diameter 4.5 mm; length 605 mm) or an adult bronchoscope (outer diameter 5.8 mm; length 605 mm) is used for inspection and photography (Strumpf et al., 1979). Rha and Mahoney (1999) have provided a review of indications, instrumentation, and techniques of bronchoscopy in small-animal medicine. Bronchoscopy of swine has

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been recommended for instruction of physicians in otolaryngology (Ram et al., 1999)

G.

Bronchoalveolar Lavage

Bronchoalveolar lavage is used as a therapeutic procedure in humans and as a means of recovering cells, surfactant, and inhaled particulates from the lungs of animals (Muggenberg and Maudefly, 1975; Brain and Frank, 1968; Myrvik et al., 1961; Maxwell et al., 1964; Medin et aL, 1976). For a more complete description of the mechanical equipment required for this procedure, the reader is referred to the works of Mauderly (1977). Highlights of the technique described by Mauderly for rabbits, guinea pigs, and small rodents are as follows. The subject is deeply anesthetized and placed in a prone position on a lavage platform. A mouth speculum is placed behind the incisor teeth and a high-intensity lamp is used to visualize the epiglottis and trachea. A specially designed tracheal catheter and stylet are moistened and passed into the trachea. The volume of saline to be used for each wash is determined by individual pressurevolume measurements prior to lavage. The subject is hyperventilated with a syringe until either apnea is induced or breathing frequency is significantly reduced. A syringe-manometer system is then connected to the luer fitting, and the lungs are inflated until transthoracic pressure reaches 20 cm H20. After multiple determinations, the average syringe volume required to reach that pressure is corrected for manometer displacement and gas compression to calculate the actual volume change of the lung. Lavage is accomplished by hyperventilating the subject, instilling the calculated volume of warmed normal saline, and immediately withdrawing the saline until a slight resistance is felt on the plunger. Another syringe and the tracheal aspiration tube are used to clear residual fluid from the catheter, and the animal is then ventilated until effective spontaneous breathing is reestablished. A total of four wash sequences is completed, taking care to drain as much fluid as possible from the lungs between washes. After the last wash, the anesthetic vaporizer is turned off, and the animal is extubated at the first sign of spontaneous movement (Mauderly, 1977). Additional methods for performing bronchoalveolar lavage in small laboratory rodents (mice, rats, guinea pigs) have been described (Moores et al., 1989; Van Soolingen et al., 1990; Tremblay et al., 1990).

X.

REPRODUCTIVE SYSTEM

Many animal models have contributed to the voluminous literature on reproductive biology. In this section, the following techniques will be described or referenced: laparoscopy as an

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aid for ovarian biopsy, aspiration of follicle contents, ovarian injections, and artificial insemination; testicular biopsy; castration; semen collection; artificial insemination; pregnancy diagnosis; embryo transfer; and intrauterine fetal surgery.

A.

Laparoscopy

The earliest known report of endoscopy occurred in 1806 and described the projection of candlelight through a double-lumen urethral cannula. Traditionally, the basic research scientists have made discoveries and developed techniques that were then applied to clinical problems. Laparoscopy evolved in a reverse manner, with the extensive development of practical, clinically important techniques relating to ovarian biopsy, cyst removal, pregnancy diagnosis, implantation site quantification, recovery of uterine fluid, and sterilization by ligation of the oviducts in many laboratory animal species (Dukelow et al., 1971; Dukelow and Ariga, 1976; Dukelow, 1978; Wildt et al., 1975; Morcom and Dukelow, 1980). Laparoscopic ovariectomy in swine has been described by Boulton et al. (1995). The use of the laparoscope for examining the reproductive tracts of woodchucks and guinea pigs has also been described (Woolf and Curl, 1987; Porter et al., 1997). The basic technique involves anesthetizing the animal and preparing the lower abdominal region for surgery. A Verres cannula is attached to an insufflator, and the abdomen is inflated with either 5% carbon dioxide or 5% nitrogen. Sufficient abdominal insufflation is required to prevent collapse of the abdominal wall when the laparoscopic trocar is inserted. Several companies are currently marketing this type of laparoscopy equipment (Richard Wolf Medical Instrument Corp., Rosemont, Illinois). The diameter of the endoscope sleeve will vary with the species under investigation. Also there is a marked difference in maneuverability and clarity of vision on the part of the investigator, depending on the instrument. Once the abdominal wall has been penetrated, a high-intensity light source and the prewarmed endoscope are inserted through the cannula. For ease in observation, the animal is placed in a steep Trendelenburg position. The technique of manipulation and observation of the abdominal organs, in this case the ovary, requires repeated examinations in the species selected before a person can produce reliable interpretation. In many cases this has required the insertion of two or more trocars to displace organs or mesentery that is obstructing a clear view with the endoscope. However, perfection of this technique has permitted visualization and color photography of the ovaries, ovarian biopsy, injections into the ovary, aspiration of follicle contents, and artificial insemination (Graham, 1976). The technique can be repeated at short intervals with no apparent ill effects. Following laparoscopy, the skin incision is closed with a simple interrupted or mattress suture, and a systemic broad-spectrum antibiotic is administered (Dukelow et al., 1971). Laparoscopy of

experimental animals as a generalized technique has been used extensively for training of physicians (West et al., 1999; Ravizzini et aI., 1999; Olinger et al., 1999; Quintero et al., 1994; Gutt et al., 1998). In veterinary practice, laparoscopy may be used for examination of abdominal contents and peritoneal surfaces and for a range of procedures (Jones, 1990).

B.

Testicular Biopsy

Existing methods of sampling testes include castration and biopsy. The most commonly used method of testis biopsy involves incision of the scrotum and tunica albuginea with subsequent removal of a small wedge of testis tissue (McFee and Kenelly, 1964; Simmons, 1952). Although this method is preferred over castration, it presents technical difficulties with small testes (Simmons, 1952) and has limited value in repeated sampling from the same individual. The most promising procedure for obtaining multiple samples is described by Martin and Richmond (1972). The instrument for biopsy is constructed from a 3/4-inch, 16-gauge needle and a 2-inch, 25-gauge needle. A small hook is made on the tip of the 25-gauge needle, which is then passed through the lumen of a 16-gauge needle. To effect this procedure, the animal is anesthetized, the scrotum is surgically scrubbed, and a sterile 16-gauge needle is introduced through the testis parenchyma. The sterile 25-gauge needle probe is then introduced through the lumen of the 16-gauge needle, and the hub is used to rotate the hook in the testis. As the 25-gauge needle is gently withdrawn, the tissue sample is extracted on the hook of the needle. Usually one or two samples of seminiferous tubules provide an adequate sample for paraffin embedding (Martin and Richmond, 1972). The risk of damage by needle biopsy appears to be less than that from surgical excision. Semen quality and libido are not impaired by this technique. Liang et al. (1997) has described the use of a modified rotating cutting needle for obtaining testicular biopsy samples from cynomolgus monkeys (Macaca fasicularis). Tissue may be obtained from other portions of the reproductive tracts of male and female animals by postmortem harvest, laparotomy, and biopsy or with less invasive techniques. Olson and Sternfeld (1987) describe a percutaneous method of obtaining uterine biopsy samples from female rhesus monkeys (Macaca mulatta). Ultrasound-guided biopsy of the canine prostate is a relatively easy, minimally invasive procedure (Chang et al., 1996). Vaginal biopsy in the female canine and other species can be performed by using a vaginoscope and biopsy forceps (Schmitt, 1988). For smaller species, equipment modifications are necessary to make a vaginoscope of a size small enough to enter the vagina. An indwelling catheter system for obtaining repeated samples of uterine fluid in the cat has been described by Verhage et al. (1986) and could also be adapted to many other species.

23. TECHNIQUES OF EXPERIMENTATION C.

Castration

Castration has been well described for most domesticated species, but descriptions are unavailable for many laboratory animal species. Two techniques that may be of value to the surgeon for rabbits and guinea pigs are recorded by Hodesson and Miller (1964) and McGlinn et al. (1976). McGlinn's technique is unique in that it is designed to remove seminiferous tubules and Leydig's cells from the tunica albuginea, leaving the epididymis and its nerve and vascular supply intact. This technique is valuable in studies concerned with reproductive physiology of spermatozoa, the epididymis, and the vas deferens of many other mammals.

D.

Semen Collection

Rectal probe ejaculation (RPE) and electrical stimulation of the penis have been utilized since the 1930s as means of obtaining semen from domestic agricultural animals, exotic animals, and some nonhuman primates (Seier et al., 1989). More recently, these techniques have been extended to common laboratory animals. It is also possible to use electroejaculation to obtain semen samples from woodchucks (Concannon et al., 1996). In some species, such as bulls, dogs, and rabbits, it is often more convenient to train individuals to serve an artificial vagina. This method eliminates the need to restrain or tranquilize the animal and also eliminates the risk of contamination of the ejaculate with urine (Fussell et al., 1973; Seager and Fletcher, 1972; Fayrer-Hosken et al., 1987; DeBoer and Krueger, 1991). In these species, RPE is more appropriately used for incidental collections from untrained animals. For a more complete description of the instrumentation and techniques required for RPE, the reader is referred to the works of Gould et al. (1978), Van Pelt and Keyser (1970), Lang (1967), and Fussell et al. (1967). Although the majority of the literature supports RPE as the preferred method of electroejaculation, some authors have indicated that direct stimulation of the penis is superior to RPE (Valerio et al., 1969). Collection of spermatozoa from male mice has usually been done as an antemortem or postmortem procedure. With the increasing use of genetically manipulated mice, it may be useful to be able to obtain serial samples of spermatozoa from an individual male. Two techniques have been described, which involve the flushing of spermatozoa, postcoitus, from the uterine horns of a female mouse after euthanasia or the flushing of the uterus per vagina in the anesthetized female (King et al., 1994; Foxworth et al., 1996). Collection of rete testis fluid is reported in monkeys (Waites and Einer-Jensen, 1974), rats (Cooper and Waites, 1974), and bulls and rams (Voglmayr et al., 1970). For a detailed description of catheter implantation of the rete testis in the ovine, the reader is referred to the works of Ellery and Kinnen (1981). Using this technique, up to 38 ml of rete testis fluid may be collected per testes for up to 3 weeks.

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E.

Artificial Insemination

Artificial insemination has long been recognized as a valuable technique in breeding domesticated mammals and poultry (Jones, 1971). Fresh semen, diluted fresh semen, or diluted stored semen can be used for artificial insemination. The osrnotic tension, pH, buffer capacity, and electrolyte balance of the diluents must be compatible with the ejaculate. Many types of diluent have been tried, including tomato juice, coconut milk, glycerol, egg yolk, lactose, and skim milk solutions. Other diluent extenders have also included DMSO (dimethyl sulfoxide), commercial bovine extender, reconstituted dried skim milk, Locke's solution, and sodium chloride (Seager and Fletcher, 1972). Prior to insemination, the female is determined to be in a receptive stage for implantation by examination of vaginal smears. The presence of well-defined cornified epithelial cells on the vaginal smear is indicative of ovulation in primates and most other species (Davis et al., 1975; Blakely et al., 1981). Those females that are not naturally receptive may be induced to ovulate by injection of pregnant mare serum gonadotropin. The actual dosages for each species should be carefully selected, as low doses produce unreliable ovulation and high dosages may lead to fragmentation of ova (Wolfe, 1967). Once the female has been determined to be in a receptive state, the insemination pipette is introduced into the cranial portion of the vagina or through the cervical canal. To ensure fertilization of most ova, the number of viable sperm inseminated should equal or exceed 106 (Stavy et al., 1978; Wolfe, 1967; Sojka et al., 1970). In some species, such as swine, one can introduce the semen directly into the oviductal lumen by using laparoscopy. This procedure may have application for basic studies of sperm and egg physiology and may be adapted to other species (Morcom and Dukelow, 1980).

F.

Pregnancy Diagnosis

Pregnancy diagnosis varies from observation of external appearance to digital palpation, radiographs, ultrasonography, and chemical tests. For the most part, rodent species are very receptive to mating, and if copulation does occur, a high percentage of the females will become pregnant. Therefore, it is a common practice either to observe copulation or to look for the vaginal plug that forms immediately following copulation. To assure accuracy, vaginal smears may be obtained, and if sperm are found, the animal is designated as being in day 0 of pregnancy (Moler et al., 1979). A second method, developed primarily for use in primates, is digital palpation. Rectal bimanual palpation of the uterus in the rhesus macaque was described by Hartman (1932), with few improvements added to the technique in subsequent years. The palpator inserts a gloved lubricated index

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finger into the rectum (the "pinky" finger is inserted into the smaller cynomolgus macaque (Macacafascicularis). The cervix is encountered upon initial entry, but the finger must be inserted until the anterior free edge of the uterus is palpated (Van Pelt, 1974). The free hand is cupped on the caudoventral abdomen, and the uterus is immobilized between the thumb and first two fingers of the free hand ventrally or dorsally by the rectally positioned finger. The normal nonpregnant uterus may vary in width from 7 to 21 mm or more (Catchpole and van Wagenen, 1975). The first palpable sign of pregnancy is a dorsoventral rounding of the anterior aspect of the body of the uterus (Fig. 7) (Mahoney, 1975a,b). Individual animal records containing approximate uterine sizes on previous palpations, as well as menstrual cycle and breeding records, should be reviewed prior to palpating the animal (Moore, 1983). Gloves should be changed and relubricated before palpating additional animals. Early embryonic abortions may occur if palpation is performed prior to implantation or if excess manipulation of the uterus occurs soon after implantation. Skilled, cautious palpators can determine pregnancy at 30 days from the onset of the last cycle, with a recheck performed 2 weeks after the initial positive palpation. Females should be palpated prior to assignment to breeding males to determine if they are in fact pregnant, thus freeing the male for use with another cycling female. Rectal palpation in concert with daily cycle records aids in the evaluation of females as potential breeding stock. A long, 7 mm wide "worm" uterus often indicates a poor breeding prognosis. Palpable adhesions of the uterus to abdominal structures may indicate complications in future pregnancies. If spotting of blood is neither observed nor recorded in cycle records, either by technician error or "personal hygiene" practice by the mon-

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key, the female may be erroneously labeled as having irregular cycles. Review of cycle records should reveal the inconsistency with subsequent cycles occurring at the appropriate cycle interval for that female (Moore, 1983). Radiographs have also been used to confirm pregnancy in its latter stages. This technique is most useful following calcification of the skeleton. Due to the variation in gestation periods among species, the effective date at which a diagnostic radiograph may be taken will vary. Therefore the investigator or clinician needs to be knowledgeable of the calcification dates for the species under investigation. Laboratory tests may also be used for pregnancy determination. The nonhuman primate pregnancy test has been used for diagnosis in macaques (Hodgen and Ross, 1974), baboons (Hobson, 1976; Hodgen and Niemann, 1975), marmosets (Hodgen et al., 1976a,b, 1978), chimpanzees, and orangutans (Hodgen et al., 1977). This hemagglutination inhibition test for urinary chorionic gonadotropin uses an antiserum (H-26) that cross-reacts with the chorionic gonadotropin of a variety of primates and provides results within 2 hr. Conventional bioassays and radioimmune assay systems are also useful procedures for detection of chorionic gonadotropin. The nonhuman primate pregnancy test, employed in conjunction with uterine palpation, is a useful method in most nonhuman primates (Hodgen et al., 1978; Lequin et al., 1981; Hall and Hodgen, 1979). In the domestic cat, abdominal palpation can determine pregnancy by 21-26 days of gestation, while serum progesterone levels are elevated as early as 6 days after breeding (Hammer and Howland, 1991). Ultrasound pregnancy diagnosis is a simple, commonly used technique for determining pregnancy in humans and a variety of animal species.

G. Embryo Transfer and Cryopreservation

Fig. 7. Rectalbimanual pregnancypalpation. This figure illustrates the positioning of the fingersfor digital palpation of the femalenonhumanprimatereproductive tract. (Reprintedwith permission from Van Pelt, 1974.)

Embryo transfer, used extensively in research with laboratory animals, involves the removal of the developing embryo from the reproductive tract of one female and transferal to another. A technique commonly used for the production of transgenic and knockout mice, embryo transfer may also be used for the production of pathogen-free colonies (Rouleau et al., 1993). Experiments involving separation of maternal and fetal genetic effects are possible with this technique. The transfer of preimplantation mouse embryos may be accomplished either surgically, by making an incision through the abdominal wall and exposing the uterus, or nonsurgically, by gaining access to the uterine lumen through the vagina. Embryo transfer has also been successful in rabbits (Prins and Fox, 1984), cats (Swanson and Godke, 1994), and woodchucks (Concannon et al., 1997). The creation and maintenance of archives of cryopreserved oocytes, ovaries, embryos, and spermatozoa are the necessary consequence of the study of mouse genomics (Glenister and

23. TECHNIQUESOF EXPERIMENTATION Thornton, 2000). Mouse embryos may be collected and stored at - 196 ~C. Successful cryoprotectants used for long-term storage of embryos include dimethyl sulfoxide, ethylene glycol, glycerol, and erythritol. Each cryoprotectant has its optimum freezing and thawing rate that must be followed in order to achieve the highest survivability of embryos (Leibo et al., 1974; Kasai et al., 1981). Cryopreservation of a number of tissues has recently been reviewed (Sztein et al., 1999; Pinkert, 1998; Nakagata, 2000).

H.

Intrauterine Fetal Surgery

Surgical procedures performed on developing fetuses in a number of animal species have been excellent models for similar procedures in human fetuses and the study of maternal fetal physiology. At present, life-threatening congenital malformations, such as diaphragmatic hernia, obstructive uropathy, and some cardiac anomalies, have been corrected on human fetuses. Sheep have been the classic model for such studies because the developing lamb is close in size to the human fetus, and sheep usually have only 1 or 2 fetuses at a time. Ultrasound may be used to confirm pregnancy and to determine the number of fetuses present in the uterus (Hoffman et al., 1996; Keller-Wood et al., 1998). Rabbits, swine, and nonhuman primates can also be used and may serve as excellent models for some disease conditions (Moise et al., 1992; Kizilcan et al., 1994; Swindle et al., 1996; Stark et al., 1989).

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successful. Tail cuff techniques generally are satisfactory in rats (Zatz, 1990; Ikeda et al., 1991) and have been used for many years. A tail cuff containing a photoelectric sensor has proven to be effective in miniature swine (Cimini and Zambraski, 1985). Using a commercial oscillometric pressure monitor in the baboon, Hartford et al. (1996) found no advantage in using the arm over the tail and concluded that such a device showed good correlation with, but not good agreement with, invasive measurements. In anesthetized dogs, Sawyer et al. (1991) showed that indirect blood pressure measurements were most accurate when the ratio of cuff width to limb circumference was between 0.4 and 0.6 and when systolic pressure was between 80 and 100 mm Hg. Studies comparing indirect and direct blood pressure in ferrets have also be.en performed (Ko et al., 1997; Olin et al., 1997). Both studies found divergence in measured values. Direct arterial pressure measurements in sheep, cows, and horses may be obtained by using the carotid artery loop procedure, which externalizes the carotid artery (Lagutchik et al., 1992; Orsini and Roby, 1997). In cardiopulmonary pathophysiology, the measurement of pulmonary arterial (PA) pressure is of considerable importance. This measurement is useful in studying the effects of drugs on pulmonary vasculature and in the diagnosis and treatment of lung and heart diseases. Using a bent-tip 23-gauge light-wall Teflon catheter (PE-50) inserted into the right jugular vein, Carrillo and Aviado (1969) and Hayes and Will (1978) successfully catheterized the pulmonary artery of the rat. The Teflon catheter was inserted until the "shepherd's crook" tip reached the right atrium and was then manipulated until its bent tip entered the pulmonary artery. Stinger et al. (1981) describe a similar method.

CARDIOVASCULAR TECHNIQUES

B. Animal models have been used to provide major insights into techniques of cardiovascular manipulation. Techniques have included developing surgical methods for organ transplants, vessel transplants and prostheses, arteriovenous shunts, blood pressure studies, electromagnetic flow probes, and microangiography (Bishop, 1980; Chung et al., 1999; Zhong, 1999). Additional models are described in Simon and Rogers (2001).

A.

Blood Pressure Techniques

Systolic and diastolic blood pressures are difficult to monitor in animals because of the problems associated with physical restraint. Garner et al. (1988) have described the use of the implanted vascular access port for direct blood pressure measurement in conscious rats. Attempts to use noninvasive methods to determine blood pressure in various species have been variably

Carotid-Jugular Shunt

The carotid-jugular shunt technique has been used for attachment of an experimental animal to an extracorporeal circuit or for obtaining repeated blood samples. Following anesthesia, the carotid artery and jugular vein are surgically exposed. The jugular vein is tied off cranially with two strands of No. 2 surgical silk. An incision is made into the vessel, and a vascular thumb forceps is used to slip the vessel onto the plastic connector. The vessel is then secured to the connector, and the anterior jugular vein is secured to the shunt. In a similar manner, the carotid artery is attached to the connector, and the shunt is secured to both vessels. The shunt is then attached to the skin with a nonabsorbable suture passed through an autoclaved clothing button (Belding et al., 1976; Corbitt et al., 1981; Payne et al., 1974). Larger species, including goats and dogs, have been used for carotid-jugular shunts. Arteriovenous fistulas have been described in cats, rats (Korber and Flye, 1987), rabbits, and larger species (Desjardins, 1986).

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C. Microangiography Microangiography is a technique in which blood vessels are visualized by the use of roentgen contrast medium and microradiographic techniques. It is a technique that is applicable to a variety of experimental animals, including pigs, dogs, rabbits, and rats, as well as many anatomical regions. The animal is placed under general anesthesia, the artery is exposed and cannulated with a corresponding cannula, and 1 mg of heparin per kilogram is administered. The level of cannulation and corresponding transection of vessels for the efflux of blood and the perfusion of finely ground barium sulfate for each species are well described by Erol et al. (1980). Following injection of the contrast medium, rapid film microradiographic techniques are employed.

D. Experimental Heart Surgery Surgical techniques involving the heart include correction of mitral, pulmonary, and aortic valve insufficiencies; stenosis of the pulmonary trunk; aortic banding; septal defects; infarction; aneurysm; revascularization; electrical stimulation; heart-lung preparations; heart transplants; and implantation of artificial hearts.

the auditory canal until it reaches the medial wall, and advancing the needle until the outer steel sheath contacts the capsule (a distance of 2.0-4.5 mm), utilizing the correct needle size. The tip of the needle is now located directly beneath the hypophysis. The needle is then connected to a suction device, and gentle suction is used to withdraw the hypophysis in 4 - 7 sec. A 100 ml, two-way flask is interposed for collection of aspirated hypophyses. With proficiency, this technique can be completed in 25-35 sec per rodent (Falconi and Rossi, 1964). A modification of this approach is described by Sato and Yoneda (1966). Postoperatively, survival rates are increased if 5% glucose and tetracycline are administered intraperitoneally for 3 days. Also, cortisone may be used for the first 2 days to reduce inflammation and swelling. A parapharyngeal method has also been described in the rat (Waynforth and Flecknell, 1992). Hypophysectomies have been performed in the dog by an extracranial route through the oral cavity (transsphenoidal), by an intracranial route through the anterior or middle cranial fossa, and by the retropharyngeal access. These techniques are described by Lopukhin (1976) and Markowitz et al. (1964). Other techniques described include a paraoccular approach for the rabbit (Lopukhin, 1976) and a combination of the transtemporal and parapharyngeal approaches for hypophysectomizing calves (Whipp et al., 1970).

B. Pinealectomy XII.

ENDOCRINE SYSTEM TECHNIQUES

Studies involving mechanisms of the endocrine system often require the sacrifice of large numbers of experimental animals in the experimental protocol. Therefore, the rodent species have assumed a major role of importance in these studies. Endocrine techniques discussed in this section include hypophysectomy, pinealectomy, adrenalectomy, thyroidectomy, and parathyroidectomy.

A. Hypophysectomy Rodents possess an almost flat sella turcica that positions the hypophysis for rapid and easy removal (Sato and Yoneda, 1966). The position of the hypophysis under the midbrain is directly on the midline at a point perpendicular to a line joining openings of the auditory canals. As the rat grows older, the hypophysis becomes displaced rostrally. The transauricular technique of hypophysectomy in the rodent is the most popular and involves anesthetizing the animal, placing the animal in a prone position with the head held toward the surgeon, introducing a modified hypodermic needle sheathed in a steel tube into the left auditory canal, guiding the needle with the right hand along

The rodent pineal body is a median epithalamic structure originating from the dorsal diencephalon and extending dorsally to the superior saggital sinus (Peterborg et al., 1980). The first technique for pinealectomy was presented in 1910 (Baker et al., 1980). Newer techniques were described by Hoffman and Reiter (1965), Bliss and Bates (1973), and, most recently, Kuszak and Robin (1977) and Jurek (1977). The newer technique involves the following: the anesthetized animal is placed in a stereotaxic instrument; a 1.75 cm longitudinal skin incision is made over the cranium; a dental drill is used to remove a rectangular section of the skull over the saggital and lamboid sutures; the dura is incised; the superior saggital vein is doubly ligated and resected to expose the gland; the pineal gland is freed with a fine pair of curved forceps; and, finally, the gland is removed by grasping the base of the gland with the forceps and retracting it. This technique appears to have the advantage of being more direct, produces a minimal amount of hemorrhage, and allows for a perfect sham operation because no sympathectomy is required.

C. Adrenalectomy Adrenalectomy is the surgical removal of one or both adrenal glands. Removal of both glands causes severe physiologic

23. TECHNIQUESOF EXPERIMENTATION changes that are difficult to correct with replacement therapy. Therefore, in clinical practice it is a rarely used technique, with the exception of use in the ferret, in which adrenal gland tumors are prevalent and in which staged unilateral adrenalectomies and subtotal bilateral adrenalectomies are established techniques. However, from a research standpoint this technique has proved to be very valuable (Lang, 1976). The technique of adrenalectomy for the dog is well described by Lang (1976) and Lopukhin (1976). Hoar (1966) cites several references indicating the difficulties of performing this procedure in guinea pigs. He also describes a technique for the guinea pig that is shorter, simpler, and less traumatic than other similar procedures. A brief description of his procedure is as follows: surgical preparation of the thoracoabdominal region, incision of the skin over the penultimate intercostal space; incision of the intercostal muscles (avoiding cutting the peritoneum); incision of the peritoneum using a blunt-nosed scissors; and use of saline-soaked gauze to pack off the intestines and liver for exposure of the kidney and adrenal. Mobilization of the adrenal is a four-step procedure: (1) the cranial pole of the kidrtey is freed; (2) the loosened fascia attached to the adrenal is freed from the diaphragm; (3) the adrenal is now freed by blunt dissection; and (4) the fascial connections between the liver and posterior vena cava are dissected free. When the gland is adequately freed, the adrenal vein is clamped off, and the gland removed. A Gelfoam sponge is used to control hemorrhage over the severed stump of the adrenal vein (Hoar, 1966). If required, removal of the opposite adrenal is usually accomplished at a later date. An adrenalectomy procedure using a paralumbar approach has been described in the rat (Waynforth and Flecknell, 1992). Adrenalectomy techniques in the ferret utilize both ventral abdominal and paralumbar approaches (Marini and Fox, 1998).

D.

Thyroidectomy and Parathyroidectomy

The role of the thyroid in reproduction, metabolism, myocardial enzyme activity, and tissue catalase activity has been investigated in several species (Kromka and Hoar, 1975; Lopukhin, 1976; Lang, 1976). The parathyroid glands are critical for the body to maintain the proper calcium and phosphorus metabolism. Although references to thyroparathyroidectomy procedures are scattered throughout the literature, they are difficult to find and generally are lacking in details necessary for surgical proficiency. The following technique is described for guinea pigs by Kromka and Hoar (1975). The animal is anesthetized, and the ventral neck area is surgically prepared. A 2.5 cm skin incision is made just caudal to the larynx. With the aid of a dissecting microscope, the infrahyoid musculature is separated on a midline exposing the trachea. Retractors are used to visualize the thyroids and parathyroids on either side of the trachea. The anatomy of the thyroid differs from species to species and should be reviewed prior to surgery. The thyroids and/or para-

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thyroids are dissected free, being careful not to damage the recurrent laryngeal nerve as it passes deep and medial to the thyroids. Cautery is helpful in freeing the gland to expose the vascular bed, which lies on the medial aspect of the cranial pole. This vasculature is then ligated and/or cauterized to free the thyroid and parathyroid glands. Surgical thyroidectomy is enough to produce a temporary hypothyroid state, but long-term experimentation requires the additional use of 1311to destroy developing thryoid rests (Kromka and Hoar, 1975). The parathyroids are not always attached to the thyroids in guinea pigs, and so care should be taken to identify these organs before excising (Peterson et al., 1952). The number and the location of the parathyroids vary in each species. This technique is also described for the dog by Lang (1976) and Lopukhin (1976) and for the rat by Waynforth and Flecknell (1992).

XIII. ORTHOPEDIC PROCEDURES FOR LABORATORY ANIMALS

The need for surgical correction of fractures in laboratory animals is usually determined through a thorough physical examination and/or diagnostic radiography. The advisability of fracture repair should be determined by suitability of the animal for further study, the severity of the injury, and other considerations. Fracture repair and stabilization techniques should be adapted from procedures described for companion animals (Richards et al., 1972; Anson, 1993; DeYoung et al., 1993; Egger, 1993; DeCamp, 1993). Current orthopedic research using animals tends to be directed toward issues such as fracture healing, prosthesis design and implantation, and bone transplantation. Most recently studies have focused on the use of growthenhancing materials (bone morphogenic protein; low-level electromagnetic stimulation) to speed the healing of bone fractures; the continuing search for safe and effective prosthetic materials; and methods to speed healing in the presence of antineoplastic drugs. Many of the techniques used in these studies are well described by Adams (1988).

XIV.

NEUROSURGICAL TECHNIQUES

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Surgical Procedures

1. Lumbar Sympathectomy Lumbar sympathectomy has been used for conditions of humans such as Raynaud's disease, arteriosclerosis, and thromboangiitis obliterans. In experimental animals the technique

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involves anesthesia; surgical preparation of the abdomen; a midline incision; use of a self-retaining retractor; packing off the small intestine with warmed, saline-soaked sterile towels; and exposure of the lumbar sympathetic chain with subsequent sympathectomy or excision of the hypogastric nerve. Renal sympathectomy,is theoretically a sound operation for chronic glomerulonephritis, especially before there is too much destruction of the renal parenchyma. It should be noted that there is regeneration of the excised sympathetic chain in most species. Total extirpation of the sympathetic chains in cats, dogs, and monkeys does not seem to affect the life of the animal (Markowitz et al., 1964). Sympathectomy has been used in swine to investigate the effect of vasospasm on restenosis following angioplasty (Lamawansa et al., 1999). Bilateral lumbar sympathectomy was performed in swine subjected to femoral balloon endarterectomy. Although sympathectomy did not inhibit intimal thickening, it did result in an increase in luminal area. Lumbar sympathectomy has been used recently in a rat model of chronic limb ischemia (van Dielen et al., 1998) and in the rat model of neuropathic pain induced by ligation of the L5 spinal nerve (Ringkamp et al., 1999). 2.

Exposure of the Cerebellomedullar Region and the Skull Base

Techniques have been established in smaller laboratory animals to approach areas of the brain and skull base without damage to associated structures. Rennella and Hussein (1986) have described a microsurgical approach to the cerebellomedullary region of the rat, which provides access to the medulla and basal cerebellum. The spheno-occipital synchondrosis located in the posterior basicranium is important for normal craniofacial growth. Experimental studies in laboratory animals have helped define the importance of this area. Surgical exposure in the neonatal rat (Reidenberg and Laitman, 1990b) and the rabbit (Haworth et al., 1992) has been described. 3.

Spinal Laminectomy

Spinal laminectomy is a relatively common procedure to expose the spinal cord in larger laboratory animals. With the increase in the availability of genetically manipulated mice, it may be necessary to access the spinal cord surgically for various studies. Ellegalla et al. (1996) have described the approach to the thoracolumbar spinal cord in mice by using fine-toothed forceps rather than rongeurs for removing the dorsal spinal lamina. 4.

Intracerebral Implantation

A number of experiments have been designed to demonstrate the effects of the direct action of neonatal hormones on a mammal's developing brain (Hayashi and Gorski, 1974). The instru-

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ments required for this technique in the rat include a converted 5 ml syringe that can be attached to a stereotaxic instrument. The syringe has a central threaded metal rod that is fitted with a clip to hold the steel wire of the implanting stylet. The stylet (27-gauge hypodermic needle) tubing and wire are attached to the syringe, and the wire is firmly held by the clip. A hormone implant is fused to the end of the wire, and as the wire is pulled through the tubing, the implant is left unattached to the apparatus. This technique of implantation in the gerbil involves anesthetizing the animal; making a scalp incision; placing the head in a stereotaxic apparatus; and, using coordinates, drilling a hole into the skull. The stylet is now lowered to the proper depth, and the implant is detached. The stylet is then removed, and the wound is closed with collodion (Holman, 1980). Other methods for neurological examination in the gerbil include the implantation of platinum-wire recording electrodes and the implantation of a chronic ventricular infusion cannula (Herndon and Ringle, 1969). 5.

Stereotaxic Electrode Implantation and Headplate Attachment

Stereotaxic brain electrode implantation involves the use of an instrument for immobilizing the animal's head and calibrated to identify coordinates in the brain for placement of electrodes in premapped structures. The coordinates are read from horizontal, coronal, and sagittal planes. Stereotaxic atlases are available for most common laboratory animals and may be developed for others (Cain and Dekergommeaux, 1979; Harris and Walker, 1980). The zero point for the coordinate system may be alternately the intersection of the coronal and sagittal skull suturesmbregma--or the middle of an interaural line. Atlases have been developed based on these two zero systems. Bregma zero points have the advantage of being grossly visible and introducing less error with different-sized heads. Stereotaxic devices come with varying frame sizes for different-sized animals but have interchangeable coordinate bars so that one frame might be used on mouse- through dog-size heads and another on goats. Parts include a cranial-caudal adjustment for coronal planes, lateral adjustment for sagittal planes, depth adjustment for horizontal planes, and electrode holder, ear bars, incisor bars, nose clamp, and infraorbital bars for dogs, cats, and monkeys. For stereotaxic electrode implantation in the rat brain, the rat is anesthetized, and atropine is given at 1 mg/kg to reduce airway secretions and help keep airways open. The shaved head is steadied in a stereotaxic device by the two ear bars placed in the auditory meatus, and the incisor bar is fitted behind the front incisors with a nose clamp over the snout. Placement must be precise for symmetric alignment of the brain between these points. Mineral oil or petrolatum is placed in both eyes to keep them moist and protect them from alcohol or dental acrylic spills. The scalp is appropriately prepped, and a midline scalp incision is made from between the eyes to between the ears and to the end of the external occipital crest. The incision is ex-

23. TECHNIQUESOF EXPERIMENTATION tended to the skull, and the periosteum is scraped back with a bone curette or periosteal elevator. The periosteum is held back with hemostats on each side of both ends of the incision. Bleeding is controlled with cotton swabs and pressure. An electrode drill is attached to the electrode carrier. The skull is leveled visually or by making horizontal readings at bregma (intersection of sagittal and coronal sutures) and lambda (intersection of sagittal and lambdoidal sutures). Different atlases may specify various horizontal readings above or below zero. If bregma is not used as the zero, this point will have to have been established prior to positioning the ear bars on the rat to get the right interaural depth reading for zero. The coordinates for electrode placement are read off an atlas for the structure being examined as millimeters from zero in each plane. If bregma is zero in the atlas, the coordinates of bregma are read off the stereotaxic device on the rat to an accuracy of 0.1 mm, using a vernier scale. The structure site for implantation is identified in the serial sections of an atlas at its largest size, and the coordinates of the atlas are added or subtracted from the coordinates attributed to the zero reading in the rat being implanted. Only the bony skull is drilled, with the dura left intact. Additional smaller holes are drilled in four corners of the skull within the incision line for placing jeweler's screws to help anchor the dental acrylic cap. The holes should not be placed in the relatively weak suture lines or too close to the electrode hole. The dura is slit with a sterile needle, and the jeweler's screws are placed 1 mm into the cortex, avoiding the dura and brain surface. The electrode is placed, using either the surface of the brain or the surface of the skull as a reference point for depth. Atlas readings are generally from the brain surface. One millimeter is added for skull thickness if this is used for reference (Cooley and Vanderwolf, 1978). A small piece of Gelfoam dipped in saline and pressed nearly dry is placed around the electrode to keep toxic dental acrylic off the brain (Skinner, 1971). The electrode is fixed in dental cement. Powder and solvent are mixed to syrupy consistency and allowed to harden around the electrode and jeweler's screws. The electrode end(s) are fitted with contacts that attach to a connector base, and they are incorporated in additional dental acrylic layers applied over the skull to create a secure cap. Acrylics and bone cements generate heat during curing; these materials should be applied in thin laminae to avoid thermal injury to subjacent tissue. The connector base eases connection of the electrodes to electrical recording instruments. The acrylic cap should be free of sharp edges that could hinder incision healing. The bottom of the connector base should be as close as possible to the skull, or else the implant cap may be dislodged. Skin closure is made with simple interrupted sutures, bringing the skin tight against the cement cap. The rat is removed from the stereotaxic device and allowed to recover (Skinner, 1971). Headplates are commonly attached to the skull of laboratory animals to protect implanted electrodes; to provide a point of restraint to prevent head movement; and to hold and protect cephalic cylinders used to provide repeated access to various

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structures of the brain. Gardiner and Toth (1999) provide extensive description of methods to place and maintain cranial implants in rats. These techniques are readily adaptable to other laboratory species. Headplates often consist of a combination of a stainless steel implant with dental acrylic, such that the device is affixed to the skull with screws and then partially covered with dental acrylic. Rabbits have a well-developed sagittal crest that prevents the attachment of some types of headplates. Rickards and Mitchell (1992) have described a headplate that has been modified to eliminate this problem. Headplates and access cylinders require meticulous care to minimize infection (Lee et al., 1998).

B.

Spinal Catheters

Catheterization of the spinal canal is done for both therapeutic and experimental purposes. Epidural administration of analgesics is a commonly performed practice in humans for relief of pain associated with a variety of surgical procedures and medical conditions and is becoming more common for pain control in surgically manipulated large laboratory animal species such as dogs. In laboratory animals such catheters are more likely used for the delivery of drugs for experimental reasons. Such studies may be directed at investigating the effects of a drug on the spinal cord or at studying the effects of such drugs on the fetus. Epidural catheters may be inserted surgically or percutaneously. In the guinea pig, epidural catheters can be inserted surgically by performing a dorsal laminectomy in the L 3 - L 4 space to expose the dura. The catheter is then carefully inserted in the epidural space and implanted subcutaneously for future access (Eisele et al., 1994). In the rabbit, Arkan et al. (1996) describe the placement of an epidural catheter by amputating the tail and inserting the catheter through the exposed epidural space. Rosenquist et al. (1996) describe the placement of an epidural catheter in the rabbit via percutaneous puncture of the L 5 - L 6 space. Remedios and Duke (1993) described the surgical placement of an epidural catheter attached to a vascular access port in the L 4 - L 7 region of the cat. The dorsal lamina of the selected spinal segment was exposed by dissection of the overlying musculature. By use of a handheld drill chuck, an intramedullary pin was used to create a hole through the dorsal lamina into the epidural space though which the catheter was inserted. Percutaneous insertion of an epidural catheter attached to a vascular access port through the L7-S 1 site has been described in the rhesus macaque (DeWeert et al., 1995). Proper placement of the catheter is determined by a loss of resistance during insertion and is confirmed by contrast radiography.

C.

Cerebrospinal Fluid Sampling

The collection of ventricular and cisternal cerebrospinal fluid has been described for cattle (Cox and Littledike, 1978), swine

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(Boogerd and Peters, 1986), goats (Peregrine and Mamman, 1993), rhesus monkeys (Snead and LaCroix, 1977), marmosets (Geretschlager et al., 1987), horses (Spinelli et al., 1968), rats (Brakkee et al., 1979; Waynforth and Flecknell, 1992), guinea pigs (Suckling and Reiber, 1984), and rabbits (Kusumi and Plouffe, 1979). For obtaining rabbit cerebrospinal fluid, the rabbit is anesthetized, and the dorsal cervical area and occipital area of the skull are shaved. The rabbit is positioned in lateral recumbency, and with the ears firmly secured, the neck is flexed to expose the base of the skull. This area is aseptically prepared, and a 22-gauge, 3.81 cm (1V2-inch) needle (Stoelting, Chicago) is inserted with the free hand approximately 2 mm caudal to the external occipital protuberance. The needle is kept parallel to the table and is advanced slowly toward the animal's mouth. At times a slight rotation of the head is needed to raise the anterior skull segments to allow this alignment. The needle is advanced through the caudal spinous muscle until a slight decrease in resistance is felt upon entering the fourth ventricle. The stylet is then removed, and 1.5-2 ml of cerebrospinal fluid can be collected (Fig. 8). This procedure may be accomplished in less than 5 min following anesthesia (Kusumi and Plouffe, 1979). Cerebrospinal fluid may be obtained from mice and rats following surgical exposure of the atlanto-occipital region of the spine (Vogelweid and Kier, 1988; Hudson et al., 1994). Catheters may be percutaneously inserted into the subdural space in the L4-L7 location and pushed cranially into the cisterna magna for cerebrospinal fluid sampling in the rabbit (Haslberger and Gaab, 1986). The chronic catheterization of the cisterna magna,

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or third ventricle of the brain, has been used to obtain cerebrospinal fluid in rabbits (Glue et al., 1988; Vistelle et al., 1994); swine, sheep, and goats (Prelusky and Hartin, 1991; Forte et al., 1989; Eisenhauer et al., 1994); and rhesus monkeys (McCully et al., 1990; Bacher et al., 1994).

XV.

TUMOR TRANSPLANTATION

Since 1965, stable lines of transplantable tumors have been developed that are well characterized in terms of speed of growth, size, morphology, histology, local invasiveness, tendency to metastasize or regress, and response to chemotherapeutic agents. Serially transplantable tumors are serving several important functions in current research (Roberts, 1969). The most notable service is the use of human tumors transplanted into rodents for the purpose of testing potential anticancer chemotherapeutic agents. Human xenograft tumors are preferred over rodent-origin tumors because the former retain their human morphology, functional behavior, and chemotherapeutic responsiveness (Shorthouse et al., 1980; Kyriazis et al., 1982). Active research is being conducted, using diabetic rats, in attempts to prolong survival of transplanted pancreatic tissue (Akimaru et al., 1981; Janney et al., 1982). A basic understanding of transplantation terminology is necessary when discussing methods of transplantation.

Fig. 8. Landmarksfor cerebrospinal fluid samplingin the rabbit. (Reprintedwithpermissionfrom Kusumiand Plouffe, 1979.)

23.

TECHNIQUES OF EXPERIMENTATION Autograft: A graft of tissue from one site to another on the same individual. Rejection is rare. Allograft: A graft between genetically dissimilar animals of the same species. This graft may undergo rejection. Xenograft: A graft between animals of different species. Rejection may be quite violent.

The outcome of transplantation is dependent on many factors (Sugiura, 1965). Following the initial transplantation of a tumor, there is a slight tendency toward dedifferentiation. In general, tumors will grow best in the same strain of animal in which they spontaneously appear or can be induced. After many transplantations, the host requirements are less specific. Tumor growth is better in young, vigorous, well-fed rats and mice than in older, food-restricted animals. Sex of the recipient animal will not affect the number of tumor takes but may influence subsequent growth. Pregnancy has an unpredictable effect on tumor growth. Site of transplantation may also influence percentage of tumor takes, subsequent growth, and therapeutic response (Double and Ball, 1975). The histologic classification of tumors also has a profound effect on transplantability. Malignant melanoma and colon, lung, kidney, and bone neoplasias have been among the more successful donors (Fogh et al., 1980; Shimosato et al., 1976). The difficulty of transplanting human mammary tumors is well documented (Bailey et al., 1980; Outzen and Custer, 1975; Boesen and Cobb, 1974). Endocrine, hormone-dependent, and lymphohematopoietic malignancies are not readily transplantable (Gazdar et al., 1981). Higher take rates for recurrent and metastatic neoplasms than for primary tumors have also been observed (Fogh et al., 1980). One of the most important aspects of assuring a long-lasting transplant take is proper selection and/or preparation of the host to prevent a host-versus-graft reaction (Festing, 1980). The use of immunodeficient rodents in tumor transplantation is reviewed in "Immunodeficient Rodents" (NRC, 1989). The physical techniques involved with tumor transplantation are fairly simple. Careful selection of tumor and host combination is necessary. Healthy host animals must be chosen, and the transplant should be an actively dividing, nonulcerated tumor if possible. High standards of sterile technique are mandatory. The donor is anesthetized or euthanized, and the tumor is removed to a sterile petri dish. Chilled sterile saline or refrigerated culture medium is used to moisten the tissue, and nonhemorrhagic, nonnecrotic areas are aseptically minced into 2 - 3 mm fragments. If tumor cells are being grown by tissue culture, the cells are dispersed by using 0.02% EDTA in phosphate-buffered saline. Cell viability is determined using trypan blue staining. The amount of media necessary to adjust the viable cells to 106/0.2 ml is added (Kyriazis et al., 1982). For optimal results, transplantation should occur quickly following removal from the donor.

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Subcutaneous inoculation is the oldest and most commonly used site for transplantation. If tumor fragments are being utilized, a 3 - 5 mm fragment is inoculated subcutaneously, using a trocar. Alternatively 106-107 tumor cells may be injected. Growth is measured using calipers. The tumor should not be palpated, because of the danger of fatal hemorrhage from highly vascular tissues. Intracranial inoculation is useful, especially because the minimum number of tumor cells necessary for a take is relatively small. The small-cell carcinoma invariably grows in the meninges and is very locally invasive, compared with subcutaneous inoculation (Gazdar et al., 1981). For intracranial injection, a 26-gauge needle and an insulin syringe are used to inject 0.05 ml of cells. The needle is inserted one-half to two-thirds of the way in at a site just above the midpoint of a line connecting the lateral canthus of the eye and the external auditory meatus. Injected mice seem to go into a state of shock for several seconds and then recover, but some of the mice may convulse and die within hours. Due to the rich vascular supply of renal tissue, implantation of tumors under the renal capsule may be desirable for enhanced tumor growth. Fragments of 1 mm 3 are implanted (Bogdon et al., 1979). The technique of induction of cystitis and infusion of tumor cells into the bladder is used for induction of bladder tumors (Edwards et al., 1978). The bladder is infused with Cetavlon, which is left in for 30 min. The bladder is then emptied, irrigated with 0.9% saline, and filled with tumor suspension. This method is very effective for induction of bladder tumors in rabbits and rats. Rats will also show renal tumor growth, because of a species-characteristic vesicoureteral reflux. Many obstacles must be overcome in the search for the perfect animal models for human cancer. Differences in growth rate, invasiveness, metastatic tendencies, metabolism, pharmacokinetics, and the use of a stroma provided by the host species must be considered. These problems may be minimized by the further development of immunodeficient animals, as well as improved immunosuppression techniques.

XVI.

IMAGING TECHNIQUES

Diagnostic and experimental imaging techniques include plain and contrast radiography; fluoroscopy; computerized axial tomography (CAT); ultrasonography; nuclear scintigraphy; magnetic resonance imaging (MRI); positron emission tomography (PET); and single-photon emission computed tomography (SPECT). All of these techniques share the attribute of being noninvasive or relatively noninvasive. Radiography, CAT and MRI scans, and ultrasonography are widely and commonly used in human medicine for diagnostic purposes. Laboratory animals have been used to develop all of these techniques, and

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they continue to be used to improve and expand the capability of existing techniques and to develop new ones. The rapid availability of mice genetically altered to model a variety of human disease conditions will push the capability of these techniques to be of use in the smallest animals. Radiography is a standard diagnostic tool that uses X-rays passing through a body structure to produce an image on film. Human dentistry utilizes high-speed film to obtain diagnostic images of the teeth and jaw. Such techniques may also be used in laboratory animals for the same purpose (Petursson et al., 1998). Human mammography utilizes specialized X-ray equipment, film, and film cassettes to obtain high-definition images of soft tissue. This same type of system can be used in mice and rats for research and diagnostic purposes (Vogelweid and Dreesen, 1995; Zeligman et al., 1992). Fluoroscopy is a modification of X-ray examination that is used to visualize the continuous movement of internal structures. In many instances a contrast medium, such as barium or an iodinated compound, is administered to provide more definition of the soft tissue in the area of interest (e.g., gastrointestinal system, central nervous system, or cardiovascular system). In rats, Reidenberg and Laitman (1990a) have described the use of a paste of barium sulfatetantalum that adheres to the soft tissue of the upper respiratory and digestive tracts for increased radiographic visualization. Ultrasonography uses the echo produced by high-frequency sound to create visual images of internal structures. Ultrasonography is commonly used for diagnostic purposes in both humans and animals to discriminate cystic versus solid masses; to interrogate body cavities filled with fluids that prohibit radiographic techniques; to discriminate the texture of suspected solid masses; to locate nonradiopaque calculi within organs; to evaluate cardiac (echocardiography), muscle, and tendon tissues; to diagnose pregnancy; and to provide biopsy guidance for internal masses and cystocentesis. Ultrasonography is also of value in examinations of the eye and in confirmation of tissue densities visualized by radiography (Cartee et aL, 1993). Ultrasonography has been used to diagnose pregnancy in many domestic and laboratory animal species, including ferrets (Peter et al., 1990) and nonhuman primates (Herring et al., 1991). Echocardiogaphy has been used to detect myocardial infarction in the mouse (Manning et al., 1993). MRI is based on the fact that tissues placed in a magnetic field can be excited when irradiated with radiowaves at the resonant frequency determined by the magnetic field strength. The excited tissue emits a radio signal that can be detected by a coil placed about the tissue, and these may be used to create an image. Previously called nuclear magnetic resonance (NMR) imaging, the name was changed to magnetic resonance imaging because of the general public's fear of the term nuclear. Magnetic resonance imaging is a common diagnostic tool in human medicine and is becoming more commonly used in domestic and laboratory animal diagnosis and experimental techniques. Although MRI was initially used most frequently for evaluation

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of the head and pelvic structures, MRI is also commonly used for bone and joint evaluations. MRI studies have most commonly been performed in larger animals, such as rabbits, cats, dogs, and nonhuman primates (Price et al., 1997; Waterton et al., 1992). However, using small-bore and strong-magnet machines, MRI has been used to produce images of entire mouse and rat bodies (Munasinghe et al., 1995; Fiel and Button, 1990; Wolf et al., 1992).

XVII.

RADIOTELEMETRY

Radiotelemetry combines a physiologic sensor with a radiotransmitter to allow the acquisition of data from a freely moving animal without the need for any type of restraint (such as tether, jacket, etc.) (DataSciences International, St. Paul, Minnesota; Mini-Mitter Company, Inc., Sunriver, Oregon). Telemetry devices have generally been time-limited because the power source is a nonrechargeable implanted battery. Current devices now have batteries that can be turned on and off remotely to extend their life. Currently such devices may be used to transmit data on body temperature, electrocardiogram (ECG), blood pressure, movement, electromyogram (EMG), and electroencephalogram (EEG), although not all parameters can currently be transmitted with one device. A surgical procedure is needed to implant the device and to connect the catheters or wires to the structures to be monitored. Telemetry to obtain physiologic data has been described in most laboratory animal species, including rabbits (Varosi et al., 1990; Brackee et al., 1995), marmosets (Schnell and Wood, 1995), rhesus monkeys (Sadoff et al., 1992), and baboons (Pearce et al., 1989).

REFERENCES

AALAS (American Association for Laboratory Animal Science) (1990). "Laboratory Animal Technician." Training manual series, Vol. 2. AALAS, Cordova. Abernathy, R. E., and Anderson, C. B. (1974). Exteriorized ureters in the dog. Lab. Anim. Sci. 24, 946-947. Adams, R. J. (1988). Musculoskeletal system. In "Experimental Surgery and Physiology: Induced Animal Models of Human Disease" (M. M. Swindle and R. J. Adams, eds.), pp. 10-41. Williams and Wilkins, Baltimore. Adeghe, A. J.-H., and Cohen, J. (1986). A better method for terminal bleeding of mice. Lab. Anim. 20, 70-72. Akimaru, K., Stuhmiller, G. M., and Sugler, H. E (1981). Allotransplantation of insulinoma into the testis of diabetic rats. Transplantation 32, 227-232. Alexander, D. J., and Clark, G. C. (1980). A simple method of oral endotracheal intubation in rabbits ( Oryctolagus cuniculus). Lab. Anim. Sci. 30, 871-873. Allen, S. A. (1974). A method for bladder catheterization of the male calf. Lab. Anim. Sci. 24, 96-98. Ambrus, J. L., Ambrus, C. M., Harrison, E W. E., Leonard, C. A., Moser, C. E.,

23. TECHNIQUES OF EXPERIMENTATION and Cravitz, H. (1951). Comparison of methods for obtaining blood from mice. Am. J. Pharm. 123, 100-104. American College of Laboratory Animal Medicine Series. Academic Press, Orlando, Florida. Andrews, E. J., Ward, B. C., and Altman, N. H., eds. (1979-1980). "Spontaneous Animal Models of Human Disease," Vol. 1 (1979); Vol. 2, "Research Applications" (1980). Baker, H. J., Lindsey, J. R., and Weisbroth, S. H., eds. (1979-1980). "The Laboratory Rat," Vol. 1, "Biology and Diseases" (1979); Vol. 2, "Research Applications" (1980). Bennett, B. T., Abee, C. R., and Henrickson, R., eds. (1995). "Nonhuman Primates in Biomedical Research: Biology and Management." Foster, H. L., Small, J. D., and Fox, J. G., eds. (1981-1983). "The Mouse in Biomedical Research," Vol. 1, "History, Genetics, and Wild Mice" (1981); Vol. 2, "Diseases" (1982); Vol. 3, "Normative Biology, Immunology, and Husbandry" (1983); Vol. 4, "Experimental Biology and Oncology" (1982). Fox, J. G., Cohen, B. J., and Loew, F. M., eds. (1984). "Laboratory Animal Medicine." Kohn, D. E, Wixson, S. K., White, W. J., and Benson, G. J., eds. (1997). "Anesthesia and Analgesia in Laboratory Animals." Manning, P. J., Ringler, D. H., and Newcomer, C. E., eds. (1994). "The Biology of the Laboratory Rabbit," 2nd ed. VanHoosier, G. L., Jr., and McPherson, C. W., eds. (1987). "Laboratory Hamsters." Wagner, J. E., and Manning, E J., eds. (1976). "The Biology of the Guinea Pig." Weisbroth, S. H., Flatt, R. E., and Kraus, A. L., eds. (1974). "The Biology of the Laboratory Rabbit." Anson, L. W. (1993). Emergency management of fractures. In "Textbook of Small Animal Surgery" (D. Slatter, ed.), 2nd ed., Vol. 2, pp. 1603-1610. Saunders, Philadelphia. Arkan, A., Kucukguclu, S., Kupelioglu, A., Maltepe, E, and Gokel, E. (1996). New technique for catheterization of the sacral canal in rabbits. Contemp. Top. 35, 96-98. Avery, D. L., and Spyker, J. M. (1977). Foot tattoo of neonatal mice. Lab. Anita. Sci. 27, 110-112. Bacher, J., Kassianides, C., Moskal, T. J., Mathews, D. M., and Hoofnagle, J. H. (1989). A technique for liver biopsy in Pekin ducks. Lab. Anita. Sci. 39, 6768. Bacher, J. D., Balis, E M., McCully, C. L., and Godwin, K. S. (1994). Cerebral subarachnoid sampling of cerebrospinal fluid in the rhesus monkey. Lab. Anita. Sci. 44, 148-152. Bailie, M. B., Wixson, S. K., and Landi, M. S. (1986). Vascular-access-port implantation for serial blood sampling in conscious swine. Lab. Anim. Sci. 36, 431-433. Bailey, M. J., Gazet, J. C., and Peckham, M. J. (1980). Human breast cancer xenografts in immunosuppressed mice. Br. J. Cancer 42, 524-529. Baker, G. A., and Gorham, J. R. (1951). A technique for bleeding ferrets and mink. Cornell Vet. 41, 235-236. Baker, H. J., Lindsey, J. R., and Weisbroth, S. H., eds. (1980). "The Laboratory Rat," Vol. 2. Academic Press, New York. Balabaud, C., Saric, J., Gonzales, E, and Deiphy, C. (1981). Bile collection in free moving rats. Lab. Anita. Sci. 31, 273-275. Ball, D. J., Argentieri, G., Krause, R., Lipinski, M., Robison, R. L., Stoll, R. E., and Visscher, G. E. (1991). Evaluation of a microchip implant system used for animal identification in rats. Lab. Anita. Sci. 41, 185-186. Banks, R. E., Roy, M., Hellems-Lewis, G., and Hadick, C. (1989). Antigen delivery to gut associated lymphoid tissue of rabbits. Lab. Anita. Sci. 39, 582586. Barber, H. W., and Small, E A., Jr. (1977). Construction of an improved tracheal pouch in the ferret. Am. Rev. Respir. Dis. 115, 165-169. Barringer, M., Sterchi, J. M., Jackson, D., and Meredith, J. (1982). Chronic bil-

1035 iary sampling via a subcutaneous system in dogs. Lab. Anim. Sci. 32, 283285. Barrow, M. V. (1968). Modified intravenous injection technique in rats. Lab. Anim. Care 18, 570-571. Bechtold, S. V., and Abrutyn, D. (1991). An improved method of endotracheal intubation in rabbits. Lab. Anim. Sci. 41, 630-631. Belding, R. C., Quarles, J. M., Beaman, T. C., and Gerhardt, P. (1976). Exteriorized carotid-jugular-shunt for hemodialysis of the goat. Lab. Anim. Sci. 26, 951-954. Berchelman, M. L., Kolter, S. S., and Britton, H. A. (1973). Comparison of hematologic values from peripheral blood of the ear and venous blood of infant baboons (Papio cynocephalus). Lab. Anim. Sci. 23, 48-52. Bergman, R. K., Lodmel, D. L., and Hadlow, W. J. (1972). A technique for multiple bleedings or intravenous inoculations of mink at prescribed intervals. Lab. Anim. Sci. 22, 93-95. Bergstrom, A. (1971). A simple device for intravenous injection in the mouse. Lab. Anim. Sci. 21, 600-601. Bernsteen, L., Gregory, C. R., Kyles, A. E., Wooldridge, J. D., and Valverde, C. R. (2000). Renal transplantation in cats. Clin. Tech. Small Anim. Pract. 15, 40-45. Bernstein, L. I., and Agee, J. (1964). Successful lobectomy in the guinea pig. Lab. Anim. Care 14, 519-523. Berthelot, R. D., and Hughes, C. (1980). Endotracheal intubation: an easy way to establish a patent airway in rabbits. Lab. Anim. Sci. 30, 227-230. Berthelot, P., Erlinger, S., Dhumeaux, D., and Pavaux, A. M. (1970). Mechanism of phenobarbital induced hypercholeresis in the rat. Am. J. Physiol. 219, 809-813. Bett, N. J., Hynd, J. W., and Green, C. J. (1980). Successful anesthesia and small-bowel anastomosis in the guinea pig. Lab. Anim. Care 14, 225-228. Bishop, S. P. (1980). Cardiovascular research. In "The Laboratory Rat" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth. eds.), Vol. 2, pp. 168-173. Academic Press, New York. Bivin, W. S., and Timmons, E H. (1974). Basic biomethodology. In "The Biology of the Laboratory Rabbit" (S. H. Weisbroth, R. E Flatt, and A. L. Kraus, eds.), pp. 73-90. Academic Press, New York. Black, D., and Claxter, M. (1979). A simple reliable and inexpensive method for the collection of rat urine. Lab. Anim. Sci. 29, 253-254. Black, K. W., Roberts, P. R., and Zaloga, G. P. (1996). New urine collection technique for monitoring acute changes in renal function of the rat. Contemp. Top. 35, 69-70. Blakely, G. B., Beamer, T. W., and Dukelow, W. R. (1981). Characteristics of the menstrual cycle in nonhuman primates. IV. Timed mating in Macaca nemestrina. Lab. Anim. 15, 351-353. Bleakley, S. P. (1980). Simple technique for bleeding ferrets (Mustela putorius furo). Lab. Anim. 14, 59-60. Bliss, D. K., and Bates, P. L. (1973). A rapid and reliable technique for pinealectomizing rats. Physiol. Behav. 11, 111-112. Blouin, A., and Cormier, Y. (1987). Endotracheal intubation in guinea pigs by direct laryngoscopy. Lab. Anim. Sci. 37, 244-245. Blouin, A., Kingma, I., and Boutet, M. (1994). An innovative technical approach for repetitive intratracheal instillation without anesthesia in small animals. Lab. Anim. Sci. 44, 274-279. Boegli, R. G., and Hall, I. H. (1969). A surgical external biliary fistula for the total collection of bile from rabbits. Lab. Anim. Care 19, 657-658. Boesen, E. A. M., and Cobb, L. M. (1974). Benign and malignant human breast tumors as an intramuscular xenograft in the hamster. J. Natl. Cancer Inst. 52, 281-293. Boettcher, E A., Bancroft, B. R., and Salvi, R. J. (1990). Blood collection from the transverse sinus in the chinchilla. Lab. Anim. Sci. 40, 223-224. Bogdon, A. E., Haskell, P. M., Le Page, D. L., Kelton, D. E., Cobb, W. R., and Esber, H. J. (1979). Growth of human tumor xenographs implanted under the renal capsule of normal immunocompetent mice. Exp. Cell Biol. 47, 281-293.

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0

3

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R

O

Boogeerd, W., and Peters, A. C. B. (1986). A simple method for obtaining cerebrospinal fluid from a pig model of herpes encephalitis. Lab. Anim. Sci. 36, 386-388. Born, C. T., and Moiler, M. L. (1974). A simple procedure for long-term intravenous infusion in the rat. Lab. Anim. Sci. 29, 78. Boulton, E. P., Gould, A. V., and Shwaery, G. T. (1995). Laparoscopic ovariectomy of miniature swine. Contemp. Topics 34, 78-82. Bowen, J., Cranley, J., and Glough, D. (1994). The flank approach to the porcine upper urinary tract: safe and reliable. Lab. Anim. 29, 204-206. Bowen, W. H., Coid, C. R., T-W-Fiennes, R. N., and Mahoney, C. J. (1976). Primates. In "The UFAW Handbook on the Care and Management of Laboratory Animals" (UFAW Staff, eds.), 5th ed., pp. 377-427. Churchill Livingstone, Edinburgh. Brackee, G., Young, E., Coffey, T., Perry, R., and Bankneider, R. (1995). Technique for telemetric determination of blood pressure and heart rate in the conscious, pregnant rabbit. Contemp. Top. 34, 90-92. Brain, J. D., and Frank, N. R. (1968). Recovery of free cells from rat lungs by repeated washings. J. Appl. Physiol. 25, 63-69. Brakkee, J. H., Wiegant, V. M., and Gispen, W. H. (1979). A simple technique for rapid implantation of a permanent cannula into the rat brain ventricular system. Lab. Anim. Sci. 29, 78-81. Branham, G. W. (1976). A device for continuous intravenous fluid injections in dogs. Lab. Anim. Sci. 26, 75-77. Brown, M. J., Erichsen, D. E, Helgerson, R., and Will, J. A. (1982). A modification for preparing the chronic lung-lymph fistula in sheep. J. Appl. Physiol. 52(6), 1664-1666. Bruckner-Kardoss, E, and Worstmann, B. S. (1967). Cecectomy in germfree rats. Lab. Anim. Care 17, 542-546. Bryant, J. M. (1980). Vest and tethering system to accommodate catheters and temperature monitors for nonhuman primates. Lab. Anim. Sci. 30, 706 -708. Bullock, L. P. (1983). Repetitive blood sampling from guinea pigs. Lab. Anim. Sci. 33, 70-71. Burhoe, S. O. (1940). Methods of securing blood from rats. J. Hered. 31, 445-448. Burke, J. G. (1977). Blood collecting by the ear artery method. Lab. Anim. 11, 49. Buttle, H. L., Clapham, C., and Oldham, J. D. (1982). A design for flexible intestinal cannulas. Lab. Anim. 16, 307-309. Cain, D. P., and Dekergommeaux, S. E. (1979). Electrode implantation in small rodents for kindling and long-term brain recording. Physiol. Behav. 22, 799-801. Cambron, H., Latulippe, J.-F., Nguyen, T., and Cartier, R. (1995). Orotracheal intubation of rats by transillumination. Lab. Anim. Sci. 45, 303-304. Carman, R. J., and Waynforth, H. B. (1994). Chronic fistulation and cannulation of the rabbit cecum. Lab. Anita. 18, 258-260. Carp, N. Z., Saputelli, J., Halbherr, T. C., Mason, W. S., and Jilbert, A. R. (1991). A technique for liver biopsy performed in Pekin ducks using anesthesia with Telazol. Lab. Anita. Sci. 41, 474-475. Carraway, J. H., and Gray, L. D. (1989). Blood collection and intravenous injection in the guinea pig via the medial saphenous vein. Lab. Anita. Sci. 39, 623 -624. Carrillo, L., and Aviado, D. M. (1969). Monocrotaline induced pulmonary hypertension and p-chlorophenylalanine (PCPA). Lab. Invest. 20, 243-248. Cartee, R. E., Hudson, J. A., and Finn-Bodner, S. (1993). Ultrasonography. Vet. Clin. North Am. Small Anita. Pract. 23, 345-377. Carvalho, J. S., Shapiro, R., Hopper, E, and Page, L. B. (1975). Methods for serial study of renin-angiotensin system in the unanesthetized rat. Am. J. Physiol. 228, 369-375. Catchpole, H. R., and van Wagenen, G. (1975). Reproduction in the rhesus monkey, Macaca mulatta. In "The Rhesus Monkey" (G. H. Bourne, ed.), Vol. 2, pp. 118-140. Academic Press, New York. Cate, C. C. (1969). A successful method for exsanguinating unanesthetized mice. Lab. Anita. Care 19, 256-258. Chang, S. S. C., Anthony, S., Koder, E C., and Brown, S. G. (1996). Transrec-

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tal ultrasound guided manipulation of the canine prostate with minimum intervention. Lab. Anim. 31, 219-224. Chimes, M. J. (1993). A technique for catheterization of ferrets for chronic intratracheal material administration. Lab. Anim. Sci. 43, 346-349. Christison, G. E., and Curtin, R. M. (1969). A simple venous catheter for sequential blood sampling from unrestrained pigs. Lab. Anim. Care 19, 259262. Chung, W. S., Cho, C., Kim, S., Wang, Y., Lee, S., Tarin, T., Chung, R., Housman, L., and Jamieson, S. W. (1999). Review of significant microvascular surgical breakthroughs involving the heart and lungs in rats. Microsurgery 19, 71-77. Cimini, C. M., and Zambraski, E. J. (1985). Non-invasive blood pressure measurement in Yucatan miniature swine using tail cuff sphygmomanometry. Lab. Anim. Sci. 35, 412-416. Clegg, J. M. (1997): A practical in-house method for bile duct cannulation of rats. Contemp. Top. 36, 49-50. Clements, D., Iqbal, S., Mills, C., and Elias, E. (1985). A new method of complete biliary retention. Lab. Anim. 19, 277-278. Cohen, E., and Oliver, M. (1964). Urethral catheterization of the rat. Lab. Anim. Sci. 29, 781-784. Concannon, P., Roberts, P., Parks, J., Bellezza, C., and Tennant, B. (1996). Collection of seasonally spermatozoa-rich semen by electroejaculation of laboratory woodchucks (Marmota monax), with and without removal of bulbourethral glands. Lab. Anim. Sci. 46, 667-675. Concannon, E, Roberts, E, Ball, B., Schlafer, D., Yang, X., Baldwin, B., and Tennant, B. (1997). Estrus, fertility, early embryo development, and autologous embryo transfer in laboratory woodchucks (Marmota monax). Lab. Anim. Sci. 47, 63-74. Conlon, K. C., Corbally, M. T., Bading, J. R., and Brennan, M. E (1990). Atraumatic endotracheal intubation in small rabbits. Lab. Anita. Sci. 40, 221-222. Conn, H., and Langer, R. (1978). Continuous long-term intraarterial infusion in the unrestrained rabbit. Lab. Anita. Sci. 28, 598-602. Conner, M. K., Dombroske, R., and Cheng, M. (1980). A simple device for continuous intravenous infusion of mice. Lab. Anim. Sci. 30, 212-214. Conti, E A., Nolan, T. E., and Gehret, J. (1979). Immobilization of a chronic intravenous catheter in the saphenous vein of African green and rhesus monkeys. Lab. Anita. Sci. 29, 234-236. Conybeare, G., Leslie, G. B., Angles, K., Barrett, R. J., Luke, J. S. H., and Gask, D. R. (1988). An improved simple technique for the collection of blood samples from rats and mice. Lab. Anita. 22, 177-182. Cook, R. W., and Williams, J. E (1978). Surgical removal of the pyloric antrum in weanling rats. Lab. Anita. Sci. 28, 437-439. Cooley, R. K., and Vanderwolf, C. H. (1978). "Stereotaxic Surgery in the Rat: A Photographic Series," 2nd ed. A. J. Kirby Co., Ontario. Cooper, J. E. (1993). Bleeding of pulmonate snails. Lab. Anita. 28, 277-278. Cooper, T. G., and Waites, G. M. H. (1974). Testosterone in rete testis fluid and blood of rams and rats. J. Endocrinol. 62, 619-629. Corbitt, R. H., McCormick, K. A. A., and Bowles, C. A. (1981). A modification of the canine carotid-jugular shunt. Lab. Anita. Sci. 31, 516-518. Costa, D. L., Lehmann, J. R., Harold, W. M., and Drew, R. T. (1986). Transoral tracheal intubation of rodents using a fiberoptic laryngoscope. Lab. Anita. Sci. 36, 256-261. Cox, E, and Littledike, E. T. (1978). Techniques for sampling ventricular and cisternal cerebrospinal fluid from unanesthetized cattle. Lab. Anita. Sci. 28, 465 -469. Craig, D. J., Trost, J. G., and Talley, W. (1969). A surgical procedure for implantation of a chronic, indwelling jugular catheter in a monkey. Lab. Anita. Care 19, 237-239. Cranney, J., and Zajac, A. (1993). A method for jugular blood collection in rabbits. Contemp. Top. 32, 6. Cravener, T. L., and Vasilatos-Younken, R. (1989). A method for catheterization, harnessing, and chronic infusion of undisturbed chickens. Lab. Anita. 23, 270-275.

23. TECHNIQUES OF EXPERIMENTATION Dalgard, D. W., Marshall, E, Fitzgerald, G. H., and Rendon, E (1979). Surgical technique for a permanent tracheostomy in beagle dogs. Lab. Anita. Sci. 29, 367-370. Dameron, G. W., Weingand, K. W., Duderstadt, J. M., Odioso, L. W., Dierckman, T. A., Schwecke, W., and Baran, K. (1992). Effect of bleeding site on clinical laboratory testing of rats: orbital venous plexus versus posterior vena cava. Lab. Anim. Sci. 42, 299-301. Davies, L., and Grice, H. C. (1962). A device for restricting the movements of rats suitable for a variety of procedures. Can. J. Comp. Med. Vet. Sci. 26, 62. Davis, L., and Malinin, T. I. (1974). Rabbit intubation and halothane anesthesia. Lab. Anim. Sci. 24, 617-621. Davis, R. H., Kramer, D. L., Sackman, J. W., and Kyriazis, G. (1975). A simple staining method for vaginal smears using red ink. Lab. Anim. Sci. 25, 319320. DeBoer, K. E, and Krueger, D. (1991). A simplified artificial vagina for use in rabbits and other species. Lab. Anim. Sci. 41, 187-188. DeBrant, V., and Remon, J. P. (1991). A simple method for the intragastric administration of drugs to fully conscious guinea pigs. Lab. Anim. 25, 308309. Decad, G. M., and Birnbaum, L. S. (1981). Noninvasive techniques for intravenous injection of guinea pigs. Lab. Anim. Sci. 31, 85-86. DeCamp, C. E. (1993). External coaptation, In "Textbook of Small Animal Surgery" (D. Slatter, ed.), 2nd ed., Vol. 2, pp. 1661-1676. Saunders, Philadelphia. Dennis, M. B., Cole, J. J., Jensen, W. M., and Scribner, B. H. (1984). Long-term blood access by catheters implanted into arteriovenous fistulas of sheep. Lab. Anim. Sci. 34, 388-392. Desjardins, C. (1986). Indwelling vascular cannulas for remote blood sampling, infusion, and long term instrumentation of small laboratory animals. In "Methods of Animal Experimentation" (W. I. Gay and J. E. Heavner, eds.), Vol. 7, pp. 143-194. Academic Press, Orlando. DeWeert, T. M., Golub, M. S., and Kaaekuahiwi, M. A. (1995). Long-term epidural catheterization of rhesus macaques: loss of resistance technique. Lab. Anim. Sci. 45, 94-97. DeYoung, D. J., Probst, C. W., and Pardo, A. D. (1993). Methods of internal fracture fixation. Chap. 122, 1610-1640. Slatter, D. (Ed.) In "Textbook of Small Animal Surgery" (D. Slatter, ed.), 2nd ed., Vol. 2, pp. 1610-1640. Saunders, Philadelphia. Dickie, M. M. (1975). Keeping records. In "Biology of the Laboratory Mouse" (E. L. Green, ed.), 3rd ed., pp. 23-27. Dover, New York. Diogene, J., Dufour, M., Poirier, G. G., and Nadeau, D. (1997). Extrusion of earthworm coelomocytes: comparison of the cell populations recovered from the species Lumbricus terrestris, Eisenia fetida, and Octolasion tyrtaeum. Lab. Anita. 31, 326-336. Dolence, D., and Jones, H. E (1975). Pericutaneous phlebotomy and intravenous injection in the guinea pig. Lab. Anita. Sci. 25, 106-107. Double, J. A., and Ball, C. R. (1975). Influence of transplant site on the establishment of human tumor xenografts in immunosuppressed mice. Br. J. Cancer 54, 233-234. Drougas, J. G., Barnard, S. E., Wright, J. K., Sika, M., Lopez, R. R., Stokes, K. A., Williams, P. E., and Pinson, C. W. (1996). A model for the extended studies of hepatic hemodynamics and metabolism in swine. Lab. Anim. Sci. 46, 648-655. Dudrick, S. J., Steiger, E., Wilmore, D. W., and Vars, H. M. (1970). Continuous long-term intravenous infusion in unrestrained animals. Lab. Anim. Care 20, 521-529. Dueck, R., Davidson, T. M., and Rathbun, M. (1985). Intermittent tracheostomy in sheep. Lab. Anim. Sci. 35, 509-512. Dukelow, W. R. (1978). Laparoscopic research techniques in mammalian embryology. In "Methods in Mammalian Reproduction" (J. C. Daniel, Jr., ed.), pp. 437-460. Academic Press, New York. Dukelow, W. R., and Ariga, S. (1976). Laparoscopic techniques for biomedical research. J. Med. Primatol. 5, 82-99.

1037 Dukelow, W. R., Jarosz, S. J., Jewett, D. A., and Harrison, R. M. (1971). Laparoscopic examination of the ovaries in goats and primates. Lab. Anim. Sci. 21, 594-597. Edwards, L., Rosin, D., Leaper M., Swedan, M., Trott, P., and Vertido, R. (1978). The induction of cystitis and the implantation of tumours in rats and rabbit bladders. Br. J. Urol. 50, 502-504. Egger, E. L. (1993). External skeletal fixation. In '~Fextbook of Small Animal Surgery" (D. Slatter, ed.), 2nd ed., Vol. 2, pp. 1641-1661. Saunders, Philadelphia. Eisele, P. H., Kaaekuahiwi, M. A., Canfield, D. R., Golub, M. S., and Eisele, J. H. (1994). Epidural catheter placement for testing of obstetrical analgesics in female guinea pigs. Lab. Anim. Sci. 44, 486-490. Eisenhauer, C. L., McCullen, A. H., Ichimura, W. M., and Claybaugh, J. R. (1994). Technique for placement of chronic third cerebroventricular cannula in female goats (Capra hircus). Lab. Anim. Sci. 44, 55-59. Ellegalla, D. B., Tassone, J. C., Avellino, A. M., Pekow, C. A., Cunningham, M. L., and Kliot, M. (1996). Dorsal laminectomy in the adult mouse: a model for nervous system research. Lab. Anim. Sci. 46, 86-89. Ellery, A., and Kinnen, L. (1981). Operative procedures for the collection of rete testis fluid from conscious sheep. Lab. Anim. 15, 187-188. Engelhardt, J. A., Zeilinga, M. J., Phelps, J. O., and Turner, D. A. (1993). Chronic jugular vein cannulation in beagles. Contemp. Top. 32, 7-9. Enta, T., Lockey, S. D., Jr., and Reed, C. F. (1968). A rapid safe technique for repeated blond collection from small laboratory animals. The farmer's wife methods. Proc. Soc. Exp. Biol. Med. 127, 136-137. Erol, O. O., Spira, M., and Levy, B. (1980). Microangiography: a detailed technique of perfusion. J. Surg. Res. 29, 406-413. Evans, C. S., Smart, J. L., and Stoddart, R. C. (1968). Handling methods for wild house mice and wild rats. Lab. Anim. 2, 29. Eyambe, G. S., Goven, A. J., Fitzpatrick, L. C., Venables, B. J., and Cooper, E. L. (1991). A non-invasive technique for sequential collection of earthworm (Lumbricus terrestris) leukocytes during subchronic immunotoxicity studies. Lab. Anim. 25, 61-67. Faidley, T. D., Galloway, S. T., Luhman, C. M., Foley, M. K., and Beitz, D. C. (1991). A surgical model for studying biliary bile acid and cholesterol metabolism in swine. Lab. Anim. Sci. 41, 477-450. Falabella, E (1967). Bleeding mice: a successful technique of cardiac puncture. J. Lab. Clin. Med. 70, 981-982. Falconi, G., and Rossi, G. L. (1964). Transauricular hypophysectomy in rats and mice. Endocrinology 74, 301-303. Fayrer-Hoskin, R. A., Reynolds, D. C., and Brackett, B. G. (1987). An efficient rabbit artificial vagina and its use in assessing sperm fertilizing ability in vitro. Lab. Anita. Sci. 37, 359-361. Fejes-Toth, G., Naray-Fejes-Toth, A., Ratge, D., Frolich, J. C. (1984). Chronic arterial and venous catheterization of conscious, unrestrained rats. Hypertension 6, 926-930. Festing, M. W. F. (1980). Inherited immunological defects in laboratory animals. In "Immunodeficient Animals for Cancer Research" (J. E. Castro, ed.). Oxford Univ. Press, London. Fiel, R. J., and Button, T. M. (1990). Magnetic resonance imaging of small laboratory animals. Lab. Anim. Sci. 40, 215-216. Fields, B. T., Jr., and Cunningham, D. R. (1976). A tail artery technique for collecting one-half milliliter of blood from a mouse. Lab. Anim. Sci. 26, 505506. Fitzgerald, A. L., Dillon, L. M., Altrogge, D. M., Bleavins, M. R., and Breider, M. A. (1996). Use of subcutaneous vascular access ports in common marmosets (Callithrix jacchus). Contemp. Top. 35, 57-59. Flecknell, P. A. (1987). "Laboratory Animal Anesthesia: An Introduction for Research Workers and Technicians." Academic Press, Orlando, Florida. Flecknell, P. A., Liles, J. H., and Williamson, H. A. (1990). The use of lignocaine-prilocaine anaesthetic cream for pain-free venipuncture in laboratory animals. Lab. Anim. 24, 142-146. Fletch, S. M., and Wabeser, G. (1970). A technique for safe multiple bleedings, or intravenous injections in mink. Can. Vet. J. 11, 33.

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Fogh, J., Thomas, O., Tiso, J., Sharkey, E E., Fogh, J. M., and Daniels, J. W. (1980). Twenty-three new human tumor lines established in nude mice. Exp. Cell BioL 48, 229-239. Ford, J. J., and Maurer, R. R. (1978). Simple technique for chronic venous catheterization of swine. Lab. Anim. Sci. 28, 615-618. Forte, V. A., Jr., Devine, J. A., and Cymerman, A. (1989). A reusable adapter for collection of cerebrospinal fluid in chronically cannulated goats. Lab. Anim. Sci. 39, 433-436. Foss, M. L., and Barnard, R. J. (1969). A vest to protect chronic implants in dogs. Lab. Anim. Care 19, 113 - 114. Foxworth, W. B., Carpenter, E., Kraemer, D. C., and Kier, A. B. (1996). Nonsurgical and nonlethal retrieval of mouse spermatozoa. Lab. Anim. Sci. 46, 352-354. Foytik, J. E., Satterfield, W. C., Bailey, J. W., and Keeling, M. E. (1989). Vacuum-assisted cardiac puncture in chickens. Lab. Anim. Sci. 39, 626-628. Frankenberg, L. (1979). Cardiac puncture in the mouse through the anterior thoracic aperture. Lab. Anim. 13, 311-312. Fredrickson, T. N., Chute, H. L., and O'Meara, D. C. (1958). Simple improved method for obtaining blood from chickens. J. Am. Vet. Med. Assoc. 132, 390-391. Frisk, C. S., and Richardson, M. R. (1979). Rapid methods for jugular bleeding of dogs requiring one technician. Lab. Anim. Sci. 29, 371-373. Fumer, R. L., and Mellett, L. B. (1975). Mouse restraining chamber for tail-vein injection. Lab. Anim. Sci. 25, 648-649. Fussell, E. N., Roussel, J. D., and Austin, C. R. (1967). Use of the rectal probe method for electrical ejaculation of apes, monkeys, and a prosimian. Lab. Anim. Care 17, 528-530. Fussell, E. N., Franklin, L. E., and Frantz, R. C. (1973). Collection of chimpanzee semen with an artificial vagina. Lab. Anim. Sci. 23, 252-255. Galitzer, J., Hayes, R. H., and Oehme, E W. (1979). A simplified urine collection method for female swine. Lab. Anim. Sci. 29, 404-405. Gardiner, T. W., and Toth, L. A. (1999). Stereotaxic surgery and long-term maintenance of cranial implants in research animals. Contemp. Top. 38, 56-63. Garner, D., McGivern, R., Jagels, G., and Laks, M. M. (1988). A new method for direct measurement of systolic and diastolic pressures in conscious rats using vascular-access-ports. Lab. Anim. Sci. 38, 205-207. Garren, H. W. (1959). An improved method for obtaining blood from chickens. Pouit. Sci. 38, 916-918. Garvey, J. S., and Alseth, B. L. (1971). Urine collection from newborn rabbits. Lab. Anim. Sci. 21, 739. Gavellas, G., Disbrow, M. R., Hwang, K. H., Hinkle, D. K., and Bourgoignie, J. J. (1987). Glomerular filtration rate and blood pressure monitoring in awake baboons. Lab. Anim. Sci. 37, 657-662. Gay, W. I., ed. (1965-1986). "Methods of Animal Experimentation," Vols. 17. Academic Press, New York. Gazdar, A. F., Carney, D. M., Sims, H. L., and Simmons, A. (1981). Heterotransplantation of small-cell carcinoma of the lung into nude mice: comparison of intracranial and subcutaneous routes. Int. J. Cancer 36, 777783. Geretschlager, E., Russ, H., Mihatsch, W., and Przuntek, H. (1987). Suboccipital puncture for cerebrospinal fluid in the common marmoset (Callithrix jacchus). Lab. Anim. 21, 91-94. Glenister P. H., and Thornton, C. E. (2000). Cryoconservation--archiving for the future. Mamm. Genome 11, 565-571. Glue, P., Bacher, J. D., and Nutt, D. J. (1988). A technique for chronic catheterization of the cisterna magna in rabbits. Lab. Anim. Sci. 38, 740-742. Goetz, K. L., and Hermreck, A. S. (1972). A simple, inexpensive, diaphragmtype skin connector for implanted catheters. Lab. Anim. Sci. 22, 538-540. Goodpasture, J. C., Cianci, J., and Zanefeld, L. J. D. (1982). Long-term evaluation of the effect of catheter materials on urethral tissue in dogs. Lab. Anim. Sci. 32, 180-182. Gould, K. G., Warner, H., and Martin, D. E (1978). Rectal probe ejaculation in primates. J. Med. PrimatoL 7, 213-222.

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Graham, C. E. (1976). Technique of laparoscopy in the chimpanzee. J. Med. Primatol. 5, 111-123. Grazer, F. M., (1958). Technique for intravascular injection and bleeding of newborn rats and mice. Proc. Soc. Exp. Biol. Med. 99, 407-409. Greene, F. E., and Wade, A. E (1967). A technique to facilitate sublingual vein injection in the rat. Lab. Anim. Care 17, 604-606. Greenham, L. W. (1978). Tattooing newborn albino mice in life-span experiments. Lab. Anim. Sci. 28, 346. Grice, H. C. (1964). Methods for obtaining blood and for intravenous injections in laboratory animals. Lab. Anim. Care 14, 483-493. Gupta, B. N. (1973). Technique for collecting blood from neonatal rats. Lab. Anim. Sci. 23, 559. Gutt, C. N., Riemer, V., Brier, C., Berguer, R., and Paolucci, V. (1998). Standardized technique of laparoscopic surgery in the rat. Dig. Surg. 15, 135-139. Haisley, A. D. (1980). A technique for cheek pouch examination of Syrian hamsters. Lab. Anim. Sci. 30, 107-109. Hall, A. S. (1966). Methods and techniques of manipulating laboratory primates. Lab. Anim. Dig. 2, 3-5. Hall, L. L., DeLopez, O. H., Roberts, A., and Smith, E A. (1974). A procedure for chronic, intravenous catheterization in the rabbit. Lab. Anim. Sci. 24, 79-83. Hall, R. D., and Hodgen, R. D. (1979). Pregnancy diagnosis of owl monkeys (Aotus trivirgatus): evaluation of the hemagglutination inhibition test for urinary chorionic gonadotropin. Lab. Anim. Sci. 29, 345-348. Halliday, L. C., Fortman, J. D., and Bennett, B. T. (1998). A mouth speculum for orogastric administration of compounds to nonhuman primates. Contemp. Top. 37, 76-77. Hamilton, R. M. G. (1978). Intravenous cannulation of hens for long-term infusion. Lab. Anim. Sci. 28, 746-750. Hammer, J. G., and Howland, D. R. (1991). Use of serum progesterone levels as an early, indirect evaluation of pregnancy in the timed pregnant domestic cat. Lab. Anim. Sci. 41, 42-45. Han, J. S., Kim, J. S., and Lee, J. J. (2000). Intubation in rabbits using capnography. Contemp. Top. Lab. Anim. Med. 39, 80. Harkness, J. E., and Wagner, J. F. (1983). "The Biology and Medicine of Rabbits and Rodents," p. 3. Lea and Febiger, Philadelphia. Harris, D. L., and Decker, W. J. (1969). A method for obtaining repeated serial intestinal biopsies from the dog. Lab. Anim. Care 19, 849-852. Harris, D. V., and Walker, J. M. (1980). A semi-chronic electrode implant for very small animals. Brain Res. Bull. 5, 479-480. Hartford, C. G., Marcos, E. E, and Rogers, G. G. (1996). Noninvasive versus invasive blood pressure measurement in normotensive and hypotensive baboons. Lab. Anim. Sci. 46, 231-233. Hartman, C. G. (1932). Studies in the reproduction of the monkey Macacus (pithecus) rhesus with special reference to menstruation and pregnancy. Contrib. Embryol. Carnegie Inst. 23, 1-161. Harvey, R. G., and Jones, E. E (1982). A technique for bioinstrumentation of the thorax of miniature swine. Lab. Anim. Sci. 32, 94-96. Haslberger, A. G., and Gaab, M. R. (1986) A technique for repeated sampling of pure cerebrospinal fluid from the conscious rabbit. Lab. Anim. Sci. 36, 181-182. Hatchell, D. L., Reiser, H. J., Bresnahan, J. E, and Whitworth, U. G., Jr. (1986). Resistance of cats to the diabetogenic effect of alloxan. Lab. Anim. Sci. 36, 37-40. Haworth, R. D., Rosenberg, P. H., Hoffman, L. A., and Latrenta, G. (1992) Anterior cervical microsurgical approach to the cranial base in the rabbit: technical note. Lab. Anim. 26, 196-199. Hayashi, S.. and Gorski, R. A. (1974). Critical exposure time for androgenization by intracranial crystals of testosterone proprionate in neonatal female rats. Endocrinology 94, 1161-1167. Hayes, B. E., and Will, J. A. (1978). Pulmonary artery cathetenzation in the rat. Am. J. PhysioL 235, H452-H455. Heitmeyer, S. A., and Powers, J. E (1992). Improved method for bile collection in unrestrained conscious rats. Lab. Anim. Sci. 42, 312-315.

23. TECHNIQUES OF EXPERIMENTATION Herbst, L. H., and Webb, A. I. (1988). A simple technique for sampling blood from fully conscious nine-banded armadillos. Lab. Anim. Sci. 38, 335336. Herring, J. M., Fortman, J. D., Anderson, R. J., and Bennett, B. T. (1991). U1trasonographic determination of fetal parameters in baboons (Papio anubis). Lab. Anim. Sci. 41, 602-605. Hill, R. C., Ellison, G. W., Burrows, C. E, Bauer, J. E., and Carbia, B. (1996). Ileal cannulation and associated complications in dogs. Lab. Anim. Sci. 46, 77-80. Hillyer, E. V., and Quesenberry, K. E., eds. (1977). "Ferrets, Rabbits and Rodents: Clinical Medicine and Surgery." Saunders, Philadelphia. Hoar, R. M. (1966). A technique for bilateral adrenalectomy in guinea pigs. Lab. Anim. Care 16, 4 1 0 - 416.

Hobson, B. M. (1976). Evaluation of the subhuman primate tube test for pregnancy of primates. Lab. Anim. 10, 87-89. Hodesson, S., and Miller, J. N. (1964). Testis and popliteal lymph node removal from the living rabbit using aseptic techniques and other anesthesia. Lab. Anim. Care 14, 494-498. Hodgen, G. D., and Niemann, W. H. (1975). Application of the subhuman primate pregnancy test kit to pregnancy diagnosis in baboons. Lab. Anim. Sci. 25, 757-759. Hodgen, G. D., and Ross, G. T. (1974). Pregnancy diagnosis by a hemagglutination inhibition test for urinary macaque chorionic gonadotropin (mCG). J. Clin. Endocrinol. Metab. 3, 927-930. Hodgen, G. D., Wolfe, L. G., Ogden, J. D., Adams, M. R., Descalzi, C. C., and Hildebrand, D. F. (1976a). Diagnosis of pregnancy in marmosets: hemagglutination inhibition test and radio immunoassay for urinary chorionic gonadotropin. Lab. Anim. Sci. 26, 224-229. Hodgen, G. D., Niemann, W. H., Turner, C. K., and Chen, H. C. (1976b). Diagnosis pregnancy in chimpanzees using the nonhuman primate pregnancy test kit. J. Med. Primatol. 5, 247-252. Hodgen, G. D., Turner, C. K., Smith, E. E., and Bush, R. M. (1977). Pregnancy diagnosis in the orangutan (Pongo pygmaeus) using the subhuman primate pregnancy test. Lab. Anim. Sci. 27, 99-101. Hodgen, G. D., Stolzenberg, S. J., Jones, D. C. L., Hildebrand, D. E, and Turner, C. K. (1978). Pregnancy diagnosis in squirrel monkeys: hemagglutination test, radio immunoassay, and bioassay of chorionic gonadotropin. J. Med. Primatol. 7, 59-64. Hoffman, K. M., Timmel, G. B., Meuli-Simmen, C., Meuli, M., Yingling, C. D., and Adzick, N. S. (1996). Experimental fetal neurosurgery: the normal neurology of neonatal lambs and abnormal findings after in utero manipulation. Contemp. Top. 35, 53-56. Hoffman, R. A., and Reiter, R. J. (1965). Rapid pinealectomy in hamsters and other small rodents. Anat. Rec. 153, 19-21. Hofstad, M. S. (1950). A method of bleeding chickens from the heart. J. Am. Vet. Med. Assoc. 66, 353-354. Holman, D. (1980). A method for intracerebrally implanting crystalline hormones into neonatal rodents. Lab. Anim. 14, 263-266. Hoppe, P. C., Laird, C. W., and Fox, R. R. (1969). A simple technique for bleeding the rabbit ear vein. Lab. Anim. Care 19, 524-525. Houghton, P. W., and Jones, C. L. (1977). A chronic gastrostomy and test system for evaluation of gastric secretion in rhesus monkeys. Gastroenterology 73, 252-254. Hudson, L. C., Hughes, C. S., Bold-Fletcher, N. O., and Vaden, S. L. (1994). Cerebrospinal fluid collection in rats: modification of a previous technique. Lab. Anim. Sci. 44, 358-361. Ikeda, K., Nara, Y., and Yamori, Y. (1991). Indirect systolic and mean blood pressure determination by a new tail cuff method in spontaneously hypertensive rats. Lab. Anim. 25, 26-29. Ilievski, V., and Fleischman, R. W. (1981). A technique for obtaining tracheobronchial washings from rhesus monkeys (Macaca mulatta). Lab. Anim. Sci. 31, 524-525. Iwaki, S., Matsuo, A., and Kast, A. (1989). Identification of newborn rats by tattooing. Lab. Anim. 23, 361-364.

1039 Jackson, I., Cook, D. B., and Gill, G. (1972). Simultaneous intravenous infusion and arterial sampling in piglets. Lab. Anim. Sci. 22, 552-555. Jackson, R. K., Kieffer, V. A., Sauber, J. J., and King, G. L. (1988). A tetheredrestraint system for blood collection from ferrets. Lab. Anim. Sci. 38, 625628. Janney, C. G., Lacy, P. E., Finke, E. H., and Davis, J. M. (1982). Prolongation of intrasplenic islet xenograft survival. Am. J. Pathol. 107, 1-5. Jimenez, R., Esteller, A., and Lopez, M. A. (1982). Biliary secretions in conscious rabbits: surgical technique. Lab. Anim. 16, 182-185. Jones, B. D. (1990). Laparoscopy. Vet. Clin. North Am. Small Anim. Pract. 20, 1243-1263. Jones, R. C. (1971). Uses of artificial insemination. Nature (Lond.) 229, 534537. Jurek, E W. (1977). Antigonadal effect of melatonin in pinealectomized and intact male hamsters. Proc. Soc. Exp. Biol. Med. 155, 31-34. Kaplan, A., and Wolf, I. (1972). A device for restraining and intravenous iniection of mice. Lab. Anim. Sci. 22, 223-224. Kaplan, H. M., and Timmons, E H. (1979). "The Rabbit: A Model for the Principles of Mammalian Physiology and Surgery." Academic Press, New York. Karlson, A. G. (1959). Intravenous injections in guinea pigs via veins of the penis. Lab. Invest. 8, 987-989. Kasai, M., Neva, K., and Tritani, A. (1981). Effects of various cryoprotective agents on the survival of unfrozen and frozen mouse embryos. J. Reprod. Fertil. 63, 175-180. Kassel, R., and Leviton, S. (1953). A jugular technique for the repeated bleeding of small animals. Science 118, 563-564. Keighley, G. (1966). A device for intravenous injection of mice and rats. Lab. Anim. Care 16, 185-187. Keller-Wood, M., Cudd, T. A., Norman, W., Caldwell, S. M., and Wood, C. E. (1998). Sheep model for study of maternal adrenal gland function during pregnancy. Lab. Anim. Sci. 48, 507-512. Kesel, M. L., and Ellis, R. P. (1988). A reversible-tie technique of the rabbit gut for bacterial colonization and toxin production. Lab. Anim. Sci. 38, 621623. Keshavarzian, A., Schoenau, G., and Isaac, R. (1989). Percutaneous endoscopic gastrostomy tube placement in cats. Lab. Anim. Sci. 39, 459-461. Khosho, E K., Kaufmann, R. C., and Amankwah, K. S. (1985). A simple and efficient method for obtaining urine samples from rats. Lab. Anim. Sci. 35, 513-514. Kim, J. S., and Han, J. S. (2000). Endoesophageal intubation for controlled ventilation in rabbits: a rapid and easy technique to establish a patent airway. Contemp. Top. Lab. Anim. Med. 39, 80. King, W. W., St. Amant, L. G., and Lee, W. R. (1994). A technique for serial spermatozoa collection in mice. Lab. Anim. Sci. 44, 295-296. Kissinger, J. T., Garver, E. M., Schnell, M. A., Schantz, J. D., Coatney, R. W., and Meunier, L. D. (1998). A new method to collect bile and access the duodenum in conscious dogs. Contemp. Top. 37, 89-93. Kizilcan, E, Tanyel, E C., Buyukpamukcu, N., and Hicsonmez, A. (1994). Fetal survival after in-utero experimentation in the rabbit. Lab. Anim. Sci. 44, 144-147. Kloots, W. J., van Amelsvoort, J. M. M., Brink, E. J., Ritskes, J., and Remie, R. (1995). Technique for creating a permanent cecal fistula in the rat. Lab. Anim. Sci. 45, 588-591. Knapp, W. C., Leeson, G. A., and Wright, B. J., (1971). An improved technique for the collection of bile in the unanesthetized rat. Lab. Anim. Sci. 21,403405. Knipfel, J. E., Peace, R. W., and Evans, J. A. (1975). Multiple vascular and gastric cannulations of swine for studies of gastrointestinal, liver, and peripheral tissue metabolism. Lab. Anim. Sci. 25, 74-78. Knize, D. M., and Weatherby-White, R. C. A. (1974). Restraint of rabbits during prolonged administration of intravenous fluids. Lab. Anim. Sci. 19, 394-397. Ko, J. C. H., Harrison, J. M., and Maldelach, C. H. (1997). Comparison of

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0

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B

invasive and non-invasive cardiopulmonary monitoring techniques in ferrets. Vet. Anesth. 26, 160. (Abstract). Korber, K. E., and Flye, M. W. (1987). Arteriovenous shunt construction for vascular access in the rat. Microsurgery 8, 245-246. Krause. A. L. (1980). Research methodology. In 'q'he Laboratory Rat" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), Vol. 2, pp. 1-42. Academic Press, New York. Kromka, M. C., and Hoar, R. M. (1975). An improved technique for thyroidectomy in guinea pigs. Lab. Anim. Sci. 25 82-84. Kumar, R. K. (1979). Toe clipping procedure for individual identification of rodents. Lab. Anim. Sci. 29, 679-680. Kurowski, S. Z., Slavik, K. J., and Szilagy, J. E. (1991). A method for maintaining and protecting chronic arterial and venous catheters in conscious rats. J. Pharmacol. Methods 26, 249-256. Kusumi, R. K., and Plouffe, J. F. (1979). A safe and simple technique for obtaining cerebrospinal fluid from rabbits. Lab. Anim. Sci. 29, 681-682. Kuszak, J., and Rodin, M. (1977). A new technique for the pinealectomy of adult rats. Experientia 33, 283-284. Kyriazis, A. P., Kyriazis, A. A., Scarpelli, D. G., Fogh, J., Rao, S., and Lepers, L. (1982). Human pancreatic adenocarcinoma line Capan-1 in tissue culture and the nude mouse. Am. J. Pathol. 106, 250-260. Laboratory Animal Pocket Reference Series. CRC Press, Boca Raton, Florida. Allen, M. J., and Borkowski, G. L. (1999). "The Laboratory Small Ruminant." Field, K., and Sibold, A. L. (1998). "The Laboratory Hamster and Gerbil." Martin, B. J. (1997). "The Laboratory Cat." Sharp, P., LaRegina, M. (1998). "The Laboratory Rat." Suckow, M. A. (1997). "The Laboratory Rabbit." Suckow, M. A., Danneman, P., and Brayton, C. (2000). "The Laboratory Mouse." Terril-Robb, L. (1997). "The Laboratory Guinea Pig." Lacy, M. J., Kent, C. R., and Voss, E. W. (1987). D-Limonene: an effective vasodilator for use in collecting rabbit blood. Lab. Anim. Sci. 37, 485486. Ladewig, J. and Stribrny, K. (1988). A simplified method for stress free continuous blood collection in large animals. Lab. Anim. Sci. 38, 333-335. Lagutchik, M. S., Sturgis, J. W., Martin, D. G., and Bley, J. A. (1992). J. Invest. Surg. 5, 79-89. Lamawansa, M. D., Wysocki, S. J., House, A. K., and Norman, P. E. (1999). The changes seen in balloon-injured porcine femoral arteries following sympathectomy. Cardiovasc. Surg. 7, 526-531. Lang, C. M. (1967). A technique for the collection of semen from squirrel monkeys (Saimiri sciureus) by electroejaculation. Lab. Anim. Care 17, 218221. Lang, C. M. (1976). "Animal Physiologic Surgery," pp. 107-120. Springer-Verlag, Berlin. Law, G. R. J. (1960). Blood samples from jugular vein of turkeys. Poult. Sci. 39, 1450-1452. Leash, A. M., Beyer, R. D., and Wilber, R. G. (1973). Self-mutilation following Innovar-Vet injections in the guinea pig. Lab. Anim. Sci. 23, 720-721. Lee, G. E., Danneman, P. J., Rufo, R. D., Kalesnykas, R. P., and Eng, V. M. (1998). Use of chlorine dioxide for antimicrobial prophylactic maintenance of cephalic recording devices in rhesus macaques (Macaca mulatta). Contemp. Top. 37, 59-63. Leibo, S. P., Mazur, P., and Jackowski, S. C. (1974). Factors affecting survival of mouse embryos during freezing and thawing. Exp. Cell Res. 89, 79-88. Lequin, R. M., Elvers, L. H., and Bertens, A. P. M. G. (1981). Early detection of pregnancy in rhesus and stumptailed macaques (Macaca mulatta and Macaca arctoides). J. Med. Primatol. 10, 189-198. Levine, G., Lewis, L., and Cember, H. (1973). A vacuum-assisted technic for repetitive blood sampling in the rat. Lab. Anim. Sci. 26, 211-213. Lewis, V. J., Thacker, W. L., Mitchell, S. H., and Baer, G. M. (1976). A new technique for obtaining blood from mice. Lab. Anim. Sci. 26, 211-213.

E

R

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J. ADAMS

Liang, J-H., Sankai, T., Yoshida, T., and Yoshikawa, Y. (1997). Use of a modified rotating cutting needle for testicular biopsy in cynomolgus monkeys (Macaca fascicularis). Contemp. Top. 36, 77-79. Litwak, K. N., Cefalu, W. T., and Wagner, J. D. (1998). Streptozotocin-induced diabetes mellitus in cynomologus monkeys: changes in carbohydrate metabolism, skin glycation, and pancreatic islets. Lab. Anim. Sci. 48, 172178. Livingstone, C. R., Andrews, M. A., Jenkins, S. M., and Marriott, C. (1990). Model systems for the evaluation of mucolytic drugs: acetylcysteine and Scarboxymethylcysteine. J. Pharm. PharmacoL 42, 73-78. Loeb, W. E, Ciemanec, J. L., and Wickum, M. (1976). A coagulopathy of the owl monkey (Aotus trivirgatus) associated with high antithrombin III activity. Lab. Anim. Sci. 26, 1084-1086. Lopez, H., and Navia, J. M. (1977). A technique for repeated collection of blood from the guinea pig. Lab. Anim. Sci. 27, 522-523. Lopukhin, Y. M. (1976). "Experimental Surgery," pp. 191-236, 271-308, 350-366. M.I.R. Publishers, Moscow. Lukas, S. E., Griffiths, R. R., Bradford, D., Brady, J. V., and Daley, L. (1982). A tethering system for intravenous and intragastric drug administration in the baboon. Pharm. Biochem. Behav. 17, 823-829. Lumsden, J. H., Presidenta, P. J. A., and Quinn, P. J. (1974). Modification of the orbital sinus bleeding technique for rabbits. Lab. Anim. Sci. 24, 345348. Lushbough, C. H., and Moline, S. W. (1961). Improved terminal bleeding method. Proc. Anim. Care Panel 11, 305-308. Macedo-Sobrinho, B., Roth, G., and Grellner, T. (1978). Tubular device for intraoral examination of rodents. Lab. Anim. 12, 137-139. Macrae, D. J., and Guerreiro, D. (1989). A simple laryngoscopic technique for the endotracheal intubation of rabbits. Lab. Anim. 23, 59-61. Mahoney, C. J. (1975a). The accuracy of bimanual rectal palpation for determining the time of ovulation and conception in the rhesus monkey (Macaca mulatta). In "Breeding Simians for Developmental Biology" (E T. Perkins and P. N. O'Donoghue, eds.), Laboratory Animals Handbook 6, pp. 127138. Laboratory Animals, London. Mahoney, C. J. (1975b). Practical aspects of determining early pregnancy, stage of fetal development, and imminent parturition in the monkey (Macaca fascicularis). In "Breeding Simians for Developmental Biology" (F. T. Perkins and P. N. O'Donoghue, eds.), Laboratory Animal Handbook 6, pp. 261274. Laboratory Animals, London. Maillie, A. J., Calvo, E L., Vaccaro, M. I., Caboteau, L. I., and Pivetta, O. H. (1981). An experimental model to study bile and exocrine pancreatic secretion from mice. Lab. Anim. Sci. 31, 707-709. Mandavilli, U., Schmidt, J., Rattner, D. W., Watson, W. T., and Warshaw, A. L. (1991). Continuous complete collection of uncontaminated urine in conscious rodents. Lab. Anim. Sci. 41, 258-261. Mann, W. A., Landi, M. S., Horner, E., Woodward, P., Campbell, S., and Kinter, L. B. (1987). A simple procedure for direct blood pressure measurements in conscious dogs. Lab. Anim. Sci. 37, 105-108. Manning, W. J., Wei, J. Y., Katz, S. E., Douglas, P. S., and Gwathmey, J. K. (1993). Echocardiographically detected myocardial infarction in the mouse. Lab. Anim. Sci. 43, 583-585. Maragos, C. M., Fubini, S. L., and Hotchkiss, J. H. (1990). A two stage cannula for gastic fistulation of swine. Lab. Anim. Sci. 40, 217-219. Marini, R. P., and Fox, J. G. (1988). Anesthesia, surgery, and biomethodology. In "Biology and Diseases of the Ferret" (J. G. Fox, ed.), pp. 449-484. Williams and Wilkins, Baltimore. Marini, R. P., Esteves, M. I., and Fox, J. G. (1994). A technique for catheterization of the urinary bladder in the ferret. Lab. Anim. 28, 155-157. Markowitz, J., Archibald, J., and Downie, H. G. (1964). "Experimental Surgery," 5th ed., pp. 326-352, 332-381,581-598, 630-643. Williams and Wilkins, Baltimore. Martin, K. H., and Richmond, M. E. (1972). A method for repeated sampling of testis tissue from small mammals. Lab. Anim. Sci. 22, 541-545.

23. TECHNIQUES OF EXPERIMENTATION Maudedy, J. L. (1977). Bronchopulmonary lavage of small laboratory animals. Lab. Anim. Sci. 27, 255-261. Maxwell, K. W., Dietz, T., and Marcus, S. (1964). An in situ method for harvesting guinea pig alveolar macrophages. Am. J. Vet. Med. Res. 37, 237-238. McCully, C. L., Balis, E M., Bacher, J., Phillips, J., and Poplack, D. G. (1990) A rhesus monkey model for continuousinfusion of drugs into cerebrospinal fluid. Lab. Anita. Sci. 40, 520-525. McFee, A. E, and Kenelly, J. J. (1964). Evaluation of a testicular biopsy technique in the rabbit. J. Reprod. FertiL 8, 141-144. McGlinn, S. M., Shepherd, B. A., and Martan, J. (1976). A new castration technique in the guinea pig. Lab. Anim. Sci. 26, 203-205. McGuill, M. W., and Rowan, A. N. (1989). Biological effects of blood loss: implications for sampling volumes and techniques. ILAR News 31, 5-20. McNamee, G. A., Wannemacher, R. W., Dinterman, R. E., Rozmiarek, H., and Montrey, R. (1984). A surgical procedure and tethering system for chronic blood sampling, infusion, and temperature monitoring in caged nonhuman primates. Lab. Anim. Sci. 34, 303-307. Medin, N. I., Osebold, J. W., and Zee, Y. C. (1976). A procedure for pulmonary lavage in mice. Am. J. Vet. Res. 37, 273-238. Melby, E. C., Jr., and Altman, N. H., eds. (1974-1976). "Handbook of Laboratory Animal Science," Vols. 1-3. CRC Press, Cleveland. Melich, D. (1990). A method for chronic intravenous infusion of the rabbit via the marginal ear vein. Lab. Anim. Sci. 40, 327-328. Mesina, J. E., Sylvina, T. S., Hotaling, L. C., Pecquet Goad, M. E., and Fox, J. G. (1988). A simple technique for chronic jugular catheterization in ferrets. Lab. Anim. Sci. 38, 89-90. Meunier, L. D., Kissinger, J. T., Marcello, J., Nichols, A. J., and Smith, E L. (1993). A chronic access port model for direct delivery of drugs into the intestine of conscious dogs. Lab. Anim. Sci. 43, 466-470. Mikeska, J. A., and Klemm, W. R. (1975). EEG evaluation of humaneness of asphyxia and decapitation euthanasia in the laboratory rat. Lab. Anim. Sci. 25, 175-179. Miller, D. L. (1990). Experimental diabetes: effect of streptozotocin on the golden Syrian hamster. Lab. Anim. Sci. 40, 539-540. Moise, K. J., Jr., Hesketh, D. E., Belfort, M. M., Saade, G., Van den Veyver, I. B., Hudson, K. M., and Rodkey, L. S. (1992). Ultrasound-guided blood sampling of rabbit fetuses. Lab. Anim. Sci. 42, 398-401. Moler, T. L., Donahue, S. E., and Anderson, G. B. (1979). A simple technique for nonsurgical embryo transfer in mice. Lab. Anim. Sci. 29, 353-356. Moore, D. M. (1983). Dept. of Comparative Medicine, UTHSC Medical School at Houston, Texas. Unpublished paper. Moore, D. M. (1984). A simple technique for blood collection in the opossum (Didelphis virginiana). Lab. Anim. 18, 52-54. Moores, H. K., Janigan, D. T., Hajela, R. P., and Luner, S. J. (1989). Use of a fluid dispensing apparatus for bronchoalveolar lavage in mice. Lab. Anim. Sci. 39, 149-152. Morcom, C. B., and Dukelow, W. R. (1980). A research technique for the oviductal insemination of pigs using laparoscopy. Lab. Anim. Sci. 30, 1030-1031. Moreland, A. E (1965). Collection and withdrawal of body fluids and infusion techniques. In "Methods of Animal Experimentation" (W. I. Gay, ed.), Vol. 1, pp. 18-19. Academic Press, New York. Moritz, M. M., Dawe, E. J., Holliday, J. E, Elliott, S., Mattei, J. A., and Thomas A. L. (1989). Chronic central vein catheterization for intraoperative and long-term venous access in swine. Lab. Anim. Sci. 39, 153-155. Mrozek, M., Fischer, R., Trendelenburg, M., and Zillmann, U. (1995). Microchip system used for animal identification in laboratory rabbits, guinea pigs, woodchucks, and in amphibians. Lab. Anim. 29, 339-433. Muggenberg, B. A., and Maudedy, J. L. (1975). Lung lavage using a single lumen endotracheal tube. J. AppL Physiol. 38, 922-926. Muller, G., Schaarschmidt, K., and Stratmann, U. (1992). A new device for long-term continuous enteral nutrition of rats by elementary diet via gastrostomy, following extensive oesophageal or lower gastrointestinal surgery. Lab. Anim. 26, 9-14.

1041 Munasinghe, J. P., Gresham, G. A., Carpenter, T. A., and Hall, L. D. (1995). Magnetic resonance imaging of the normal mouse brain: comparison with histologic sections. Lab. Anim. $ci. 45, 674-679. Munson, E. S. (1974). Arterial cannulation in awake restrained monkeys. Lab. Anim. Sci. 24, 793-795. Mylrea, K. C., and Abbrecht, P. H. (1967). An apparatus for tail vein injection in mice. Lab. Anita. Care 17, 602-603. Myrvik, Q. N., Leake, E. S., and Fariss, B. (1961). Studies on pulmonary alveolar macrophages from the normal rabbit: a technique to produce them in a high state of purity. J. Immunol. 86, 128-132. Nakagata, N. (2000). Cryopreservation. Mature. Genome 11, 572-576. Naranjo, J. A., Valverde, A., Martinez de Victoria, E., Manas, M., and Moreno, M. (1986). Surgical preparation for the study of pancreatic exocrine secretion in the conscious preruminant goat. Lab. Anim. 20, 231-233. Nau, R., and Schunck, O. (1993). Cannulation of the lateral saphenous vein-a rapid method to gain access to the venous circulation in anaesthetized guinea pigs. Lab. Anim. 27, 23-25. Nerenberg, S. T., and Zedler, E (1975). Sequential blood samples from the tail vein of rats and mice obtained with modified Liebig condenser jackets and vacuum. J. Lab. Clin. Med. 85, 523-526. Nicholson, J. W., and Kinkead, E. R. (1982). A simple device for intratracheal injections in rats. Lab. Anim. Sci. 32, 509-510. Niebergall-Roth, E., Teyssen, S., and Singer, M. V. (1997). Pancreatic exocrine studies in intact animals: historic and current methods. Lab. Anim. Sci. 47, 606 -614. Nobonaga, T., Nabamura, K., and Imamichi, T. (1996). A method for intravenous injection and collection of blood from rats and mice without restraint and anesthesia. Lab. Anim. Care 16, 40-49. Nolan, T. E., and Conti, E A. (1980). Liver wedge biopsy in chimpanzees (Pan troglodytes) using an automatic stapling device. Lab. Anim. Sci. 30, 578580. O'Byrne, K. T. (1988). Long-term blood sampling technique in the marmoset. Lab. Anita. 22, 151-153. Olin, J. M., Smith, T. J., and Talcott, M. R. (1997). Evaluation of noninvasive monitoring techniques in domestic ferrets. Am. J. Vet. Res. 58, 1065. Olinger, A., Pistorius, G., Lindemann, W., Vollmar, B., Hlidebrandt, U., and Menger, M. D. (1999). Effectiveness of a hands-on training course for laparoscopic spine surgery in a porcine model. Surg. Endosc. 13, 118122. Olson, G. A. (1974). A method for surgical construction of a tracheal pouch in the ferret. Lab. Anim. Dig. 9, 47-49. Olson, L. C., and Sternfeld, M. D. (1987). A percutaneous method for obtaining uterine biopsies. Lab. Anita. Sci. 37, 663-664. Omaye, S. T., Skala, J. H., Gretz, M. D., Schaus, E. E., and Wade, C. E. (1987). Simple method for bleeding the unanesthetized rat by tail venipuncture. Lab. Anim. 21, 261-264. Orsini, J. A., and Roby, K. A. (1997). Modified carotid artery transposition for repetitive arterial blood gas sampling in large animals. J. Invest. Surg. 10, 125-128. Osborne, C. A., Low, D. G., and Finco, D. R. (1972). "Canine and Feline Urology," pp. 28-30. Saunders, Philadelphia. Otto, G., Rosenblad, W. D., and Fox, J. G. (1993). Practical venipuncture techniques for the ferret. Lab. Anim. 27, 26-29. Outzen, H. D., and Custer, R. E (1975). Growth of human normal and neoplastic mammary tissues in cleared mammary fat pad of nude mouse. J. Natl. Cancer Inst. 55, 1461-1463. Oz, M. C., Popilskis, S. J., Morales, A., and Nowygrod, R. (1989). Long term catheterization of the rat ureter. Lab. Anim. Sci. 39, 349-350. Painter. N. W., Borski, A. A., and Trevino, G. S. (1971). Urethral reaction to foreign objects. J. Urol. 106, 227-230. Palm, U., Boemke, W., Bayed, D., Schnoy, N., Juhr, N.-C., and Renihardt, H. W. (1991). Prevention of catheter-related infections by a new, catheterrestricted antibiotic filling technique. Lab. Anim. 25, 142-152.

1042 Pansky, B., Jacobs, M., and House, E. L. (1961). The orbital region as a source of blood samples in the golden hamster. Anat. Rec. 39, 409. Paolini, R. V., Rossman, J. E., Patel, V., and Stanievich, J. E (1993). A reliable method for large volume blood collection in the chinchilla. Lab. Anim. Sci. 43, 524-525. Pare, W. P., Vincent, G. P., and Isom, K. E. (1979). Comparison of pyloric ligation and pyloric cuff techniques for collecting gastric secretion in the rat. Lab. Anim. Sci. 29, 218-220. Parlett, W. R., George, T. E, III, and Bley, J. A. (1993). A paramedian retroperitoneal approach to the kidney and ureter in pigs. Lab. Anim. Sci. 43, 520523. Patrick, R. L., Kaplan, C. M., Margetis, P. M., and Pani, K. C. (1968). Nonsurgical extraction of teeth in small primates. Lab. Anim. Care 18, 442-449. Paulsen, R., and Valentine, J. L. (1984). A procedure for intravenous administration of drugs and repeated arterial blood sampling in the rabbit. Lab. Anim. 13, 34-36. Payne, J. E., Bischel, M. D., and Berne, T. W. (1974). Carotid-jugular arteriovenous fistulas for chronic hemodialysis in dogs. Lab. Anim. Sci. 24, 367369. Pazin, G. J., Wu, B. A., and Van Theil, D. H. (1978). Fiberoptic esophagogastroscopy, brushings, and biopsy in rabbits. Lab. Anim. Sci. 28, 733-736. Pearce, P. C., Halsey, M. J., Ross, J. A. S., Luff, N. P., Bevilacqua, R. A., and Maclean, C. J. (1989). A method of remote physiological monitoring of a fully mobile primate in a single animal cage. Lab. Anim. 23, 180-187. Pekow, C. A., WeUer, R. E., Kimsey, B. B., and Allen, M. K. (1994). Ultrasound-guided cholecystocentesis in the owl monkey. Lab. Anim. Sci. 44, 365-369. Pena, H., and Cabrera, C. (1980). Improved endotracheal intubation technique in the rat. Lab. Anim. Sci. 30, 712-713. Peregrine, A. S., and Mammar, M. (1993). Pharmacology of diminazene: A review. Acta. Trop. 54, 185-203. Perry-Clark, L. M., and Meunier, L. D. (1991). Vascular access ports for chronic serial infusion and blood sampling in New Zealand White rabbits. Lab. Anim. Sci. 41, 495-497. Peter, A. J., Bell, J. A., Manning, D. D., and Bosu, W. T. K. (1990). Real-time ultrasonographic determination of pregnancy and gestational age in ferrets. Lab. Anim. Sci. 40, 91- 92. Peterborg, L. J., Philo, R. C., and Reiter, R. J. (1980). The pineal body and pinealectomy in the cotton rat (Sigmodon hispidus). Acta Anat. 107, 108113. Peterson, R. R., Webster, R. C., and Rayner, B. (1952). The thyroid and reproductive performance in the adult male guinea pig. Endocrinology 5, 504518. Petursson, C. M., Brammer, D. W., Peter, G. K., and Hofing, G. L. (1998). Determination of optimal angles for intraoral occlusal views for the radiographic imaging of dental structures in the cynomolgus monkey (Macaca fascicularis). Contemp. Top. 37, 52-54. Phillips, W. A., Stafford, W. W., and Stunt, J. (1973). Jugular vein technique for serial blood sampling and intravenous injection in the rat. Proc. Soc. Exp. Biol. Med. 143, 733-735. Pinkert, C. A. (1998). Mouse sperm cryopreservation: a legacy in the making. Lab. Anim. Sci. 48, 224. Plager, J. E. (1972). Intravenous, long-term infusion in the unrestrained mousemethod. J. Lab Clin. Med. 79, 669-672. Platt, J. L. (1999). Xenotransplantation of the kidney: a pediatric perspective. Pediatr. Nephrol. 13, 966-973. Platts, R. G. S., Wilson, P., and Shaw, K. M. (1972). Design for a chronic right ventricular catheter for dogs. Lab. Anim. Sci. 22, 900-903. Porter, K. B., Tsibris, J. C. M., Porter, G. W., O'Brien, W. E, and Spellacy, W. N. (1997). Use of endoscopic and ultrasound techniques in the guinea pig leiomyoma model. Lab. Anim. Sci. 47, 537-539. Prelusky, D. B., and Hartin, K. E. (1991). A technique for serial sampling of cerebrospinal fluid from conscious swine and sheep. Lab. Anim. Sci. 41, 481-485.

ROBERT J. ADAMS

Price, R. E., Leeds, N. E., Hazle, J. D., Jackson, E. F., Stephens, L. C., and Ang, K. K. (1997). Magnetic resonance imaging of the central nervous system of the rhesus monkey. Lab. Anim. Sci. 47, 304-311. Prins, J.-B., and Fox, R. R. (1984). A successful technique for the preservation of rabbit embryos. Lab. Anim. Sci. 34, 484-487. Pye-MacSwain, J. K., Cawthorn, E. G., Rainnie, D. J., and Johnson, G. R. (1994). Modification of the cannulation for the dorsal aorta of Atlantic salmon (Salmo salar). Lab. Anim. Sci. 44, 540-541. Quesenberry, K. E. (1997). Basic approach to veterinary care. In "Ferrets, Rabbits and Rodents: Clinical Medicine and Surgery," p. 1425. WB Saunders, Philadelphia. Quintero, R. A., Puder, K. S., Bardicef, M., Rossman, K., Acosta, L., Esclapes, M., and Cotton, D. B. (1994). Hydrolaparoscopy in the rabbit: a fine model for the development of operative fetoscopy. Am. J. Obstet. Gynecol. 171, 1139-1142. Raggi, L. A., Manas, M., Martinez de Victoria, E., Lupiani, M. J., and Mataix, F. J. (1985). Biliary secretion in the conscious preruminant goat: use of a reentrant cannula. Lab. Anim. 19, 35-38. Rahlmann, D. F., Mains, R. C., and Kodama, A. M. (1976). A urine collection device for use with the male pigtail macaque (Macaca nemestrina). Lab. Anim. Sci. 26, 829-831. Ram, B., Oluwole, M., Blair, R. L., Mountain, R., Dunkley, P., and White, P. S. (1999). Surgical simulation: an animal tissue model for training in therapeutic and diagnostic bronchoscopy. J. Laryngol. Otol. 113, 149-151. Ransom, J. H. (1984). Intravenous injection of unanesthetized hamsters (Mesocricetus auratus). Lab. Anim. Sci. 34, 200-201. Rath, L., and Hutchison, M. (1989). A new method of bile duct cannulation allowing bile collection and re-infusion in the conscious rat. Lab. Anim. 23, 163-168. Ravizzini, P. I., Shulsinger, D., Guarnizo, E., Pavlovich, C. P., Marion, D., and Sosa, R. E. (1999). Hand-assisted laparoscopic donor nephrectomy versus standard laparoscopic donor nephrectomy: a comparison study in the canine model. Tech. Urol. 5, 174-178. Rawlings, C. A., Van Lue, S., King, C., Freeman, L., Damian, R. T., Greenacre, C., Chernosky, A., Mohamed, F. M., and Chou, T. M. (2000). Serial laparoscopic biopsies of liver and spleen from schistosoma-infected baboons (Papio spp.). Comp. Med. 50(5), 551-555. Redfern, R. (1971). Techniques for oral intubation of wild rats. Lab. Anim. 5, 169-172. Reidenberg, J. S., and Laitman, J. T. (1990a). A new method for radiographically locating upper respiratory and upper digestive tract structures in rats. Lab. Anim. Sci. 40, 72-76. Reidenberg, J. S., and Laitman, J. T. (1990b). A new surgical approach to the skull base in rats. Lab. Anim. Sci. 40, 312-315. Reiser, H. J., Whitworth, U. G., Jr., Hatchell, D. L., Suthefland, F. S., Nanda, S., McAdoo, T., and Hardin, J. R. (1987). Experimental diabetes in cats induced by partial pancreatectomy alone or combined with local injection of alloxan. Lab. Anim. Sci. 37, 449-452. Remedios, A. M., and Duke, T. (1993). Chronic epidural implantation of vascular access catheters in the cat lumbosacrum. Lab. Anim. Sci. 43, 262-264. Renella, R. R., and Hussein, S. (1986). Microsurgical approach to the cerebellomedullar region in rat. Lab. Anim. 20, 210-212. Report of the AVMA Panel on Euthanasia. (2001). J. Am. Vet. Med. Assoc. 218, 669-696. Reuter, R. E. (1987). Venipuncture in the guinea pig. Lab. Anim. Sci. 37, 245246. Reynolds, J. A., and Hall, A. S. (1979). A rapid procedure for shortening canine teeth of nonhuman primates. Lab. Anim. Sci. 29, 521-524. Rha, J. Y., and Mahony, O. (1999). Bronchoscopy in small animal medicine: indications, instrumentation, and techniques. Clin. Tech. Small Anim. Pract. 14, 207-212. Rhodes, M. L., and Patterson, C. E. (1979). Chronic intravenous infusion in the rat: a nonsurgical approach. Lab. Anim. Sci. 29, 82-84. Richards, D. A., Hinko, P. J., and Morse, E. M. (1972). Orthopaedic procedures

23. TECHNIQUES OF EXPERIMENTATION

for laboratory animals and exotic pets. J. Am. Vet. Med. Assoc. 161, 729732. Rickards, E. S., and Mitchell, G. (1992). Improved stereotaxic headplates for rabbits. Lab. Anim. Sci. 42, 81-82. Ringkamp, M., Eschenfelder, S., Grethel, E. J., Habler, H. J., Meyer, R. A., Janig., W., and Raja, S. N. (1999). Lumbar sympathectomy failed to reverse mechanical allodynia- and hyperalgesia-like behavior in rats with L5 spinal nerve injury. Pain. 79, 143-153. Ritter, J. W. (1984). Permanent tracheostomy for expired air collection in conscious dogs. Lab. Anim. Sci. 34, 79-81. Roberts, D. C. (1969). "Transplanted Tumors of Rats and Mice: An Index of Tumors and Host Strains." Laboratory Animals, London. Robinson, E. S., and V.andeBerg, J. L. (1994). Blood collection and surgical procedures for the laboratory opossum (Monodelphis domestica). Lab. Anim. Sci. 44, 63-68. Rodhouse, S. L., Herkelman, K. L., Veum, T. L., and Zinn, G. M. (1988). A flexible T-cannula for ileal cannulation of 10 to 20 kg pigs. Lab. Anim. Sci. 38, 92-94. Rogers, G., Taylor, C., Austin, J. C., and Rosen C. (1988). A pharyngostomy technique for chronic oral dosing of rabbits. Lab. Anim. Sci. 38, 619-620. Rolf, L. L., Jr., Bartels, K. E., Nelson, E. C., and Berlin K. D. (1991). Chronic bile duct cannulation in laboratory rats. Lab. Anim. Sci. 41,486-492. Rosenhaft, M. E., Bing, D. H., and Knudson, K. C. (1971). A vacuum-assisted method for repetitive blood sampling in guinea pigs. Lab. Anim. Sci. 21, 598-599. Rosenquist, R. W., Bunte, R. M., and Artwohl, J. E. (1996). Percutaneous spinal catheterization in the rabbit. Contemp. Top. 35, 76-78. Rouleau, A. M. J., Kovacs, P. R., Kunz, H. W., and Armstrong, D. T. (1993). Decontamination of rat embryos and transfer to specific pathogen-free recipients for the production of a breeding colony. Lab. Anim. Sci. 43, 611-615. Ruiz de Elvira, M.-C., and Abbott, D. H. (1986). A backpack system for longterm osmotic minipump infusions into unrestrained marmoset monkeys. Lab. Anim. 20, 329-334. Ruzinski, J. T., Skerrett, S. J., Chi, E. Y., and Martin, T. R. (1995). Deposition of particles in the lungs of infant and adult rats after direct intratracheal administration. Lab. Anim. Sci. 45, 205-210. Saarni, H., and Viikari, J. (1976). A simple technique for continuous intravenous infusion under neuroleptic tranquillization. Lab. Anim. 10, 69-72. Sadoff, D. A., Fischel, R. J., Carroll, M. E., and Brockway, B. (1992). Chronic blood pressure radiotelemetry in rhesus macaques. Lab. Anim. Sci. 42, 78-80. St. Cyr, J., Bianco, R., Judd, D., Pierpont, M. E., Staley, N., Noren, G., Foker, J. E., and Einzig, S. (1987). A new method of urine collection in turkeys and chickens. Lab. Anim. Sci. 37, 366-367. Sakaki, K., Tanaka, K., and Hirasawa, K. (1961). Hematological comparison of the mouse blood taken from the eye and the tail. Exp. Anim. 10, 14-19. Sarr, M.G. (1988). Pancreas. In "Experimental Surgery and Physiology: Induced Animal Models of Human Disease" (M. M. Swindle and R. J. Adams, eds.), pp. 204-216. Williams and Wilkins, Baltimore. Sato, M., and Yoneda, S. (1966). An efficient method for transauricular hypophysectomy in rats. Acta Endocrinol. (Copenhagen) 51, 43-48. Sawyer, D. C., Brown, M., Striler, E. L., Durham, R. A., Langham, M. A., and Rech, R. H. (1991). Comparison of direct and indirect blood pressure measurement in anesthetized dogs. Lab. Anim. Sci. 41, 134-138. Scalese, R. J., DeForrest, J. M., Hammerstone, S., Parente, E., and Burkett, D. E. (1990). Long term vascular catheterization of the cynomolgus monkey. Lab. Anim. Sci. 40, 530-532. Schaeffer, D. O., Hosgood, G., Oakes, M. G., St. Amant, L. G., and Koon, C. E. (1994). An alternative techniclue for partial hepatectomy in mice. Lab. Anim. Sci. 44, 189-190. Schmitt, P.-J. (1988). Vaginal biopsy in long-term toxicity studies of beagle dogs. Lab. Anim. Sci. 38, 86-88. Schnell, C. R., and Wood, J. M. (1995). Measurement of blood pressure and

1043 heart rate by telemetry in conscious unrestrained marmosets. Lab. Anim. 29, 258-261. Schoenborne, B. M., Schrader, R. E., and Canolty, N. L. (1977). Tattooing newborn mice and rats for identification. Lab. Anim. Sci. 27, 110. Schofield, J. C., Alves, M. E. A. F., Hughes, K. W., and Bennett, B. T. (1991). Disarming canine teeth of nonhuman primates using the submucosal vital root retention technique. Lab. Anim. Sci. 41, 128-133. Seager, S. W. J., and Fletcher, W. S. (1972). Collection, storage, and insemination of canine semen. Lab. Anim. Sci. 22, 177-182. Seier, J. V., Fincham, J. E., Menkveld, R., and Venter, F. S. (1989). Semen characteristics of vervet monkeys. Lab. Anim. 23, 43-47. Sekas, G. (1990). A technique for creating partial obstruction of the common bile duct in the rat. Lab. Anim. 24, 284-287. Shimosato, Y., Kameya, T., Nagai, S., Hirohashi, T., Koide, T., Hayashi, H., and Momura, T. (1976). Transplantation of human tumors in nude mice. J. Natl. Cancer Inst. 56, 1251-1255. Shomer, N. H., Astrofsky, K. M., Dangler, C. A., and Fox, J. G. (1999). Biomethod for obtaining gastric juice and serum from unanesthetized guinea pigs (Cavia porcellus). Contemp. Top. 38, 32-35. Shorthouse, A. J., Smyth, J. F., Steel, G. G., Ellison, M., Mills, J., and Peckham, M. J. (1980). The human tumor xenograftma valid model in experimental chemotherapy? Br. J. Surg. 67, 715-722. Shrader, R. E., and Everson, G. J. (1968). Intravenous injection and blood sampiing using cannulated guinea pigs. Lab. Anim. Care 18, 214-219. Shulman, E. R., Mclver, F. T., and Burkes, E. J., Jr. (1987). Comparison of electrosurgery and formocresol as pulpotomy techniques in monkey primary teeth. Pediatr. Dent. 9, 189-194. Sils, I. V., Szlyk-Modrow, P. C., Tartarini, K. A., Hubbard, L. J., Glass, E., Caretti, D. M., and Darrigrand, A. A. (1994). Chronic implantation of nonocclusive catheters and flow probes in the splanchnic and hindlimb vasculature of the rabbit. Lab. Anim. Sci. 44, 319-323. Simmons, F. A. (1952). Correlation of testicular biopsy material with semen analysis in male infertility. Ann. N.Y. Acad. Sci. 5, 643-656. Simmons, M. L., and Brick, J. O. (1970). "The Laboratory Mouse-Selection and Management." Prentice-Hall, Englewood Cliffs, New Jersey. Simon, D. I., and Rogers, C. (2001). "Vascular Disease and Injury: Preclinical Research." Humana Press, Totowa, New Jersey. Sisson, S., and Grossman, J. D. (1966). "The Anatomy of the Domestic Animals," p. 489. Saunders, Philadelphia. Skinner, J. E. (1971). "Neuroscience: A Laboratory Animal." Saunders, Philadelphia. Smith, C. J., and McMahon, J. B. (1977). Method for collecting blood from fetal and neonatal rats. Lab. Anim. Sci. 27, 112-113. Smith, C. R., Felton, J. S., and Taylor, R. T. (1981). Description of a disposable individual mouse collection apparatus. Lab. Anim. Sci. 31, 80-82. Smith, D. M., Lieberman, R. P., Stribley, J. A., and Sharp, J. G. (1992). Chronic catheterization of the inferior vena cava in Yucatan miniature swine. Lab. Anim. Sci. 42, 602-606. Smith, G. (1991). A simple non-surgical method of intrabronchial instillation for the establishment of respiratory infections in the rat. Lab. Anim. 25, 4 6 49. Snead, O. C., III, and LaCroix, J. T. (1977). Lumbar puncture obtaining cerebrospinal fluid in the rhesus monkey (Macaca mulatta). Lab. Anim. Sci. 27, 1039-1040. Snitily, M. U., Gentry, M. J., Mellencamp, M. A., and Preheim, L. C. (1991). A simple method for collection of blood from the rat foot. Lab. Anim. Sci. 41, 285 -287. Snyder, S. B., and Soave, O. A. (1970). A method for the collection of throat specimens for laboratory primates for the rapid identification of respiratory infection. Lab. Anim. Care 20, 518-520. Sojka, N. J., Jennings, L. L., and Hamner, C. E. (1970). Artificial insemination in the cat (Felis cams). Lab. Anim. Care 20, 198-204. Soli, N. E., and Birkeland, R. (1977). A method for collection of bile in conscious sheep. Acta Vet. Scand. 18, 221-226.

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Sorg, D. A., and Buckner, B. (1964). A simple method for obtaining venous blood from small laboratory animals. Proc. Soc. Exp. Biol. Med. 115, 1131-1132. Spalton, P. N., and Clifford, J. M. (1979). Nasogastric intubation technique for bile sampling in the baboon, Papio ursinus. Lab. Anim. Sci. 29, 237-239. Spinelli, J., Holliday, T., and Homer, J. (1968). Technical notes: collection of large samples of cerebrospinal fluid from horses. Lab. Anim. Care 18, 565567. Spira, W. M., Sack, R. B., and Froehlich, J. L. (1981). Simple adult rabbit model for Vibrio cholerae and enterotoxigenic Escherichia coli diarrhea. Infect. Immun. 32, 739-747. Starcher, B., and Williams, I. (1989). A method for intratracheal instillation of endotoxin into the lungs of mice. Lab. Anim. 23, 234-240. Stark, R. I., Daniel, S. S., James, L. S., MacCarter, G., Morishima, H. O., Niemann, W. H., Rey, H., Tropper, P. J., and Yeh, M.-N. (1989). Chronic instrumentation and long term investigation in the fetal and maternal baboon: tether system, conditioning procedures, and surgical techniques. Lab. Anim. Sci. 39, 25-32. Stavy, M., Terkel, J., and Marder, U. (1978). Artificial insemination in the European hare (Lepus europaeus syriacus). Lab. Anim. Care 28(2), 163-166. Stevens, R. W. C., and Ridgeway, G. J. (1966). A technique for bleeding chickens from the jugular vein. Poult. Sci. 45, 204-205. Stickrod, G., and Pruett, D. K. (1979). Multiple cannulation of the primate superficial lateral coccygeal vein. Lab. Anim. Sci. 29, 398-399. Stickrod, G., Ebaugh, T., and Garnett, C. (1981). Use of a miniperistaltic pump for collection of blood from rabbits. Lab. Anim. Sci. 31, 87-88. Still, J. W., and Whitcomb, E. A. (1956). Techniques for long-term intubation of rat aorta. J. Lab. Clin. Med. 48, 152. Stills, H. E, Jr., Balady, M. A., and Liebenberg, S. P. (1979). A comparison of bacterial flora isolated by transtracheal aspiration and pharyngeal swabs in Macaca fasicularis. Lab. Anim. Sci. 29, 229-233. Stinger, R. B., Iacopino, V. J., Alter, I., Fitzpatrick, T. M., Rose, J. C., and Kot, P. A. (1981). Catheterization of the pulmonary artery in the closed-chest rat. J. Appl. Physiol. 51, 1047-1050. Stone, S. H. (1954). Method for obtaining venous blood from the orbital sinus of the rat or mouse. Science 1tl9, 100. Strumpf, I. R., Bacher, J. D., Gadek, J. E., Morin, M. L., and Crystal, R. G. (1979). Flexible fiberoptic bronchoscopy of the rhesus monkey (Macaca mulatta)., Lab. Anim. Sci. 29, 785-788. Stuhlman, R. A., Packers, J. T., and Rose, S. D. (1972). Repeated blood sampiing of Mystromys albicaudatus. Lab. Anim. Sci. 22, 268-270. Stump, K. C., Swindle, M. M., Saudek, C. D., and Strandberg, J. D. (1988). Pancreatectomized swine as a model of diabetes mellitus. Lab. Anim. Sci. 38, 439-443. Suckling, A. J., and Reiber, H. (1984). Cerebrospinal fluid sampling from guinea pigs: sample volume-related changes in protein concentration in control animals and animals in the relapsing phase of chronic relapsing experimental allergic encephalomyelitis. Lab. Anim. 18, 36-39. Sugiura, K. (1965). Tumor transplantation. In "Methods of Animal Experimentation" (W. I. Gay, ed.), Vol. 2, pp. 171-222. Academic Press, New York. Swanson, W. E, and Godke, R. A. (1994). Transcervical embryo transfer in the domestic cat. Lab. Anim. Sci. 44, 288-291. Swindle, M. M., and Adams, R. J., eds. (1988). "Experimental Surgery and Physiology: Induced Animal Models of Human Disease." Williams and Wilkins, Baltimore. Swindle, M. M., Wiest, D. B., Smith, A. C., Garner, S. S., Case, C. C., Thompson, R. P., Fyfe, D. A., and Gillette, P. C. (1996). Fetal surgical protocols in Yucatan miniature swine. Lab. Anim. Sci. 46, 90-95. Swindle, M. M., Harvey, R. B., Kasari, E., and Buckley, S. A. (1998). Chronic cecal cannulation in Yucatan miniature swine. Contemp. Top. 37, 68-69. Sztein, J. M., McGregor, T. E., Bedigian, H. J., and Mobraaten, L. E. (1999). Transgenic mouse strain rescue by frozen ovaries. Lab. Anim. Sci. 49, 99100.

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Talbot, R. W., and Hynd, J. W. (1985). Intermittent sampling of portal venous blood and bile from guinea pigs. Lab. Anim. 19, 173-176. Talcott, M. R., and Dysko, R. C. (1991). Partial lobectomy via a ligature fracture technique: a method for multiple hepatic biopsies in nonhuman primates. Lab. Anim. Sci. 41, 476-480. Tappa, B., Amao, K. W., and Takahashi, K. W. (1989). A simple method for intravenous injection and blood collection in the chinchilla (Chinchilla laniger). Lab. Anim. 23, 73-75. Tartarini, K. A., Sils, I. V., and Szlyk-Modrow, P. C. (1996). Comparison of blood pressure measurements using vascular access ports and conventional catheters. Contemp. Top. 35, 57-61. Terkel, L., and Urbach, L. (1974). A chronic intravenous cannulation technique adapted for behavioral studies. Horm. Behav. 5, 141-148. Thulin, J. (1994). Re: Vasodilator for rabbit blood collection. COMPMED, AALAS. Timm, K. I. (1979). Orbital venous anatomy of the rat. Lab. Anim. Sci. 29, 636638. Timm, K. I. (1989). Orbital venous anatomy of the Mongolian gerbil with comparison to the mouse, hamster, and rat. Lab. Anim. Sci. 39, 262-264. Timm, K. I., Jahn, S. E., and Sedgwick, C. J. (1987). The palatial ostium of the guinea pig. Lab. Anim. Sci. 37, 801-802. Tomson, E N., Schulte, J. M., and Bertsch, M. L. (1979). Root canal procedure for disarming nonhuman primates. Lab. Anim. Sci. 29, 382-386. Toofanian, F., and Targowski, S. (1982). Small intestinal anastomosis and preparation of intestinal loops in the rabbit. Lab. Anim. Sci. 32, 80-82. Toriumi, Y., Samuel, I., Wilcockson, D. P., and Joehl, R. J. (1994). A new model for study of pancreatic exocrine secretion: the tethered pancreatic fistula rat. Lab. Anim. Sci. 44, 270-273. Tran, D. Q., and Lawson, D. (1986). Endotracheal intubation and manual ventilation of the rat. Lab. Anim. Sci. 36, 540-541. Tremblay, G., Gagnon, L., and Cormier, Y. (1990). Sequential bronchoalveolar lavages by endotracheal intubation in guinea pigs. Lab. Anim. 24, 63-67. Turner, M. A., Thomas, P., and Sheridan, D. J. (1992). An improved method for direct laryngeal intubation in the guinea pig. Lab. Anim. 26, 25-28. UFAW (Universities Fund for Animal Welfare) Joint Working Group on Refinement (1993). Removal of blood from laboratory mammals and birds. Lab. Anim. 27, 1-22. UFAW Staff, eds. (1976). "The UFAW Handbook on the Care and Management of Laboratory Animals," 5th ed. Churchill-Livingstone, Edinburgh. Valerio, D. A., Ellis, E. B., Clark, M. L., and Thompson, G. E. (1969). Collection of semen from macaques by electroejaculation. Lab. Anim. Care 19, 250-252. Vallejo-Freire, A. (1951). A simple technic for repeated collection of blood samples from guinea pigs. Science 114, 524-525. Van Dielen, E M., Kurvers, H. A., Dammers, R., Oude Egbrink, M. G., Slaaf, D. W., Tordoir, J. H., and Kitslaar, P. J. (1998). Effects of surgical sympathectomy on skin blood flow in a rat model of chronic limb ischemia. World J. Surg. 22, 807-811. Van Herck, H., Baumans, V., Van der Craats, N. R., Hesp, A. P. M., Meijer, G. W., Van Tintelen, G., Walvoort, H. C., and Beynen, A. C. (1992). Histological changes in the orbital region of rats after orbital puncture. Lab. Anim. 26, 53-58. Van Pelt, L. F. (1974). Clinical assessment of reproductive function in female rhesus monkeys. Lab. Anim. 8, 199-212. Van Pelt, L. F., and Keyser, P. E. (1970). Observations on semen collection and quality in macaques. Lab. Anim. Care 20, 726-733. Van Soolingen, D., Moolenbeek, C., and Van Loveren, H. (1990). An improved method of bronchoalveolar lavage of lungs of small laboratory animals: short report. Lab. Anim. 24, 197-199. Varagona, G., Ellis, L. A., Moore, D., Penney, D., and Dusheiko, G. M. (1991). A percutaneous liver biopsy technique in ducks (Anas platyrhynchos) experimentally infected with duck hepatitis B virus. Lab. Anim. 25, 254-257. Varosi, S. M., Brigmon, R. L., and Besch, E. L. (1990). A simplified telemetry

23. TECHNIQUES OF EXPERIMENTATION system for monitoring body temperature in small animals. Lab. Anim. Sci. 40, 299-302. Venkatesh, V. C., Whyte, H. E. A., and Read, S. E. (1988). A simple technique for repeated intratracheal instillation of foreign material in newborn rabbits. Lab. Anim. Sci. 38, 618-619. Venugopalan, C. S., Said, S. I., and Drazen, J. M. (1984). Effect of vasoactive intestinal peptide on vagally mediated tracheal pouch relaxation. Respir. Physiol. 56, 205-216. Venugopalan, C. S., Krautmann, M. J., Holmes, E. P., and Maher, T. J. (1998). Involvement of nitric oxide in the mediation of NANC inhibitory neurotransmission of guinea-pig trachea. J. Auton. Pharmacol. 5, 281-286. Verhage, H. G., Murray, M. K., Brown, M. J., and Bennett, B. T. (1986). An indwelling catheter for the collection of cat uterine fluids. Lab. Anim. Sci. 36, 71-73. Vistelle, R., Jaussaud, R., Trenque, T., and Wiczewski, M. (1994). Rapid and simple cannulation technique for repeated sampling of cerebrospinal fluid in the conscious rabbit. Lab. Anim. Sci. 44, 362-364. Vogelweid, C. M., and Dreesen, J. (1995). Radiographic imaging of rodents using a portable mammographic system. Conte'mp. Top. 34, 86-88. Vogelweid, C. M., and Kier, A. B. (1988). A technique for the collection of cerebrospinal fluid from mice. Lab. Anim. Sci. 38, 91-92. Vogler, G. A., Yagi, K., Frank, P. A., and Baudendistel, L. J. (1992). Permanent tracheostomy in goats. Contemp. Top. 31, 15-17. Voglmayr, J. K., Larsen, L. H., and White, I. G. (1970). Metabolism of spermatozoa and composition of fluid collected from the rete testis of living bulls. J. Reprod. Fertil. 21, 449-460. Voss, W. R. (1970). Primate liver and spleen biopsy procedures. Lab. Anim. Care 20, 995-997. Waites, G. M. H., and Einer-Jensen, N. (1974). Collection and analysis of rete testis fluid from macaque monkeys. J. Reprod. FertiL 41, 505-508. Wang, Y.-M. C., and Reuning, R. H. (1994). A comparison of two surgical techniques for preparation of rats with chronic bile duct cannulae for the investigation of enterohepatic circulation. Lab. Anim. Sci. 44, 479-485. Wardell, J. R., Jr., Chakrin, L. W., and Payne, B. J. (1970). The canine tracheal pouch: a model for use in respiratory mucus research. Am. Rev. Respir. Dis. 101, 741-754. Waterton, J. C., Miller, D., Morrell, J. S. W., Dukes, M., West, C. D., and Wadsworth, P. E (1992). A case of endometriosis in the macaque diagnosed by nuclear magnetic resonance imaging. Lab. Anim. 26, 59-64. Waynforth, H. B. (1980). "Experimental and Surgical Technique in the Rat." Academic Press, New York. Waynforth, H. B., and Flecknell, P. A. (1992). "Experimental and Surgical Technique in the Rat," 2nd ed. Academic Press, New York. Waynforth, H. B., and Parkin, R. (1969). Sublingual vein injection in rodents. Lab. Anim. 3, 35-37. Webb, A. I., Bliss, J. M., and Herbst, L. H. (1995). Use of vascular access ports in the cat. Lab. Anim. Sci. 45, 110-113. Wendt, D. J., Normile, H. J., Dawe, E. J., Trumpeter, T., and Barraco, R. A. (1982). Chronic intracarotid cannulation of pigeons for administration 6f behaviorally active peptides. Lab. Anim. 16, 335-338. West, D. A., Rallo, M. C., Moore, R. G., Vogler, G. A., Niehoff, M., Parra, R. O., Varma, C., and Smith, G. S. (1999). Laparoscopic v. laparoscopy-assisted donor nephrectomy in the porcine model. J. Endourol. 13, 513-515. Wheeldon, E. B., Walker, M. E., Murphy, D. J., and Turner, C. R. (1992). Intratracheal aerosolization of endotoxin in the rat: a model of the adult respiratory distress syndroms (ARDS). Lab. Anim. 26, 29-37.

1045 Whipp, S. C., Littledike, E. T., and Wangsness, S. (1970). A technique of hypophysectomy of calves. Lab. Anim. Care 20, 533-538. White, W. A. (1971). A technique for urine collection from anesthetized male rats. Lab. Anita. Sci. 2.1, 401-402. Whitney, R. A., Jr., Johnson, D. J., and Cole, W. C. (1973). "Laboratory Primate Handbook." Academic Press, New York. Wideman, R. E, and Braun, E. J. (1982). Urethral urine collection from anesthetized domestic fowl. Lab. Anim. Sci. 32, 298-301. Wildt, D. E., Morcom, C. B., and Dukelow, W. R. (1975). Laparoscopic pregnancy diagnosis and uterine fluid recovery in swine. J. Reprod. Fertil. 44, 301-304. Wimsatt, J., Johnson, J. D., and Mangone, B. A. (1998). Use of a cardiac access port for repeated collection of blood samples form desert tortoises (Gopherus agassizii). Contemp. Top. 37, 81-83. Wingfield, W. E., Tumbleson, M. E., Hicklin, K. W., and Mather, E. C. (1974). An exteriorized cranial vena caval catheter for serial blood sample collection from miniature swine. Lab. Anim. Sci. 24, 359-361. Wixson, S. K., Murray, K. A., and Hughes, H. C. (1987). A technique for chronic arterial catheterization in the rat. Lab. Anim. Sci. 37, 108-111. Wojnicki, E H. E., Bacher, J. D., and Glowa, J. R. (1994). Use of subcutaneous vascular access ports in rhesus monkeys. Lab. Anim. Sci. 44, 491-494. Wolf, R. E E., Lam, K. H., Mooyaart, E. L., Bleichrodt, R..E, Nieuwenhuis, E, and Schakenraad, J. M. (1992). Magnetic resonance imaging using a clinical whole body system: an introduction to a useful technique in small animal experiments. Lab. Anim. 26, 222-227. Wolfe, H. G. (1967). Artificial insemination of the laboratory mouse (Mus musculus). Lab. Anim. Care 17, 426-432. Woolf, A., and Curl, J. L. (1987). A technique for laparoscopic examination of woodchuck ovaries. Lab. Anim. Sci. 37, 664-665. Woolf, A., Curl, J., and Gremillion-Smith, C. (1989). The use of subcutaneous ports in the woodchuck (Marmota monax). Lab. Anim. Sci. 39, 620-622. Yao, Z., Adler, A., Kovar, P., and Kleinert, H. (1992). A restraint device for blood sampling and direct blood pressure measurement in conscious ferrets. Contemp. Top. 31, 19-21. Yap, K. L. (1982). A method for intratracheal innoculation of mice. Lab. Anim. 16, 143-145. Young, L., and Chambers, T. (1973). A mouse bleeding technique yielding consistent volume with minimal hemolysis. Lab. Anita. Sci. 23, 428-430. Zambraski, E. J., and DiBona, G. E (1976). A device for the cutaneous exteriorization of chronic intravascular catheters. Lab. Anim. Sci. 26, 939-941. Zammit, M., Toledo-Pereya, L. H., Malcom, S., and Konde, W. N. (1979). Longterm cranial mesenteric vein cannulation in the rat. Lab. Anim. Sci. 29, 364-366. Zanella, A. J., and Mendl, M. T. (1992). A fast and simple technique for jugular catheterization in adult sows. Lab. Anita. 26, 211-213. Zatz, R. (1990). A low cost tail-cuff method for the estimation of mean arterial pressure in conscious rats. Lab. Anim. Sci. 40, 198-201. Zeligman, B. E., Howard, R. B., Marcell, T., Chu, H., Rossi, R. P., Mulvin, D., and Johnston, M. R. (1992). Chest roentgenographic techniques for demonstrating human lung tumor xenografts in nude rats. Lab. Anim. 26, 100106. Zhong, R. (1999). Organ transplantation in mice: current status and future prospects. Microsurgery 19, 52-55. Zhou, C., and Brown, L. A. (1988). Intravenous cannulation of chickens for blood sampling. Lab. Anita. Sci. 38, 631-632.

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Chapter 24 Control of Biohazards Associated with the Use of Experimental Animals Thomas E. Hamm, Jr.

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Risk A s s e s s m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M a n a g i n g Facilities in W h i c h Biohazards Are U s e d . . . . . . . . . . . . . . . . . A. Biohazard C o n t a i n m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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INTRODUCTION

In every laboratory animal facility there is an inherent risk of exposure to biological, physical, and chemical hazards. Biological hazards include pathogenic microorganisms and allergens, physical hazards include bites and falls, and chemical hazards include cleaning and disinfecting agents and pest-control agents. To ensure a safe working environment, a comprehensive occupational health and safety program is a critical component of all animal care and use programs. The law also mandates it. The LABORATORYANIMALMEDICINE, 2nd edition

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Occupational Safety and Health Act of 1970 (29 USC 651) requires employers to provide safe working conditions and empowers the Occupational Safety and Health Administration (OSHA) to promulgate standards (29 CFR 1910). This chapter focuses on biological hazards; however, all exposures must be considered in designing a health and safety program. The National Research Council publication "Occupational Health and Safety in the Care and Use of Research Animals" (NRC, 1997) is an excellent guide for establishing or reviewing and upgrading a health and safety program for an animal care and use program. Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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The first stage in any biohazard control program is to conduct a risk assessment to determine current and anticipated biohazards and the personnel, facilities, and programs needed to support the proposed experiments. Valuable information on performing a risk assessment can be found in "Biosafety in Microbiological and Biomedical Laboratories" (CDC-NIH, 1999) and "Occupational Health and Safety in the Care and Use of Research Animals" (NRC, 1997). Risk assessment must include a number of factors so that a level of containment that will protect the workers can be determined. One such factor is the availability of quantitative information. When quantitative information is lacking, a conservative approach should be taken. Data from animal studies might be useful, but caution should be exercised in extrapolating that data to humans. Knudsen (1998) has published a good discussion of performing a qualitative risk assessment for an infectious agent. Another important risk factor is the route of transmission. A good source of information on disease transmission is the "Control of Communicable Diseases Manual" (APHA, 1995). The most common routes of disease transmission associated with experimental animals are inhalation of aerosols; direct inoculation (e.g., a stick from a contaminated needle); contact of mucous membranes with contaminated materials, hands, or surfaces; and ingestion (Barkley and Richardson, 1984). Of these routes, agents that can be transmitted by the aerosol route have caused most laboratory infections (CDC-NIH, 1999). Infectious aerosols are produced by the activity of infected animals, animal care and husbandry procedures, and experimental manipulations. Size determines the length of time a particle remains airborne. Smaller particles remain airborne longer, are more likely to move with air currents, and are more likely to be respirable (Barkley and Richardson, 1984). Pathogenicity (disease incidence and severity) also affects risk. The more severe the disease, the higher the risk. However, pathogenicity alone does not determine the level of containment needed. Infectious agents that are transmitted by the aerosol route or for which there is no effective prophylaxis require more containment than those transmitted by some other route or for which there is a vaccine or treatment. For example, human immunodeficiency virus (HIV) and hepatitis B virus do not require the same level of containment as work with Ebola, Marburg, or Lassa fever viruses, because HIV is not transmitted by the aerosol route, and there is an effective vaccine for hepatitis B (CDC-NIH, 1999). Other factors that affect the level of containment are stability (the time an organism can survive outside the host), infectious dose (how many organisms are necessary to cause disease), concentration (the number of infectious organisms per unit of volume), and origin of the infectious organism.

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MANAGING FACILITIES IN W H I C H BIOHAZARDS ARE USED

The principal investigator and facility veterinary staff share responsibility for the safe use of biohazards by the people under their supervision. It is important when biohazards are involved that tasks and responsibilities be clearly delineated, because mistakes can lead to serious consequences. The potential for problems increases as the potential risk increases. The institution's administration should take a strong role in worker safety, and institutional biosafety personnel should work closely with the animal facility director and veterinary staff to coordinate safety training and oversight in the animal facilities. There should be standard operating procedures (SOPs) for working with each biohazard, and each person having contact should understand the SOPs, be adequately trained in handling exposed animals, and be properly supervised. Regardless of who is designated to supervise the use of biohazards, however, the ultimate responsibility for safety lies with the individual. Each person providing care for the animals or carrying out experimental procedures must be alert to the hazards and understand how to perform the procedures properly and safely. Regular safety meetings with all involved staff during the conduct of a project will help to identify and resolve problems.

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Biohazard Containment

Facilities in which biohazards are used should meet or exceed the requirements in "Biosafety in Microbiological and Biomedical Laboratories" (CDC-NIH, 1999). Table I summarizes the containment equipment and procedures recommended by Centers for Disease Control--National Institutes of Health for research involving infected vertebrates. Four animal biosafety levels (ABSLs) are defined: 1, 2, 3, and 4. The determination of ABSL requires experience and professional judgment. Even different projects involving the same microorganism might have a different level of risk because of the way the project is conducted. For example, many studies in which animals are infected with HIV have been designed so they can be conducted safely at ABSL-2 or at ABSL-2 with ABSL-3 practices. However, if very large quantities of the virus are produced, large amounts of the genome are moved into an infectious vector, or the virus is drawn into a syringe with a hypodermic needle attached, the project might require ABSL-4. When a project is started that involves the use of an agent that has not been used in the facility previously, it is important to consult with people experienced in using the microorganism about safety requirements and SOPs. The principal device used to contain biohazards is the biological safety cabinet. To be effective, these cabinets must be well maintained and regularly tested and certified. Personnel must be

Table I Recommended Animal Biosafety Levels (ABSLs) for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used a Biosafety level

Agents

Practices

Not known to consistently cause disease in healthy adults

Standard animal care and management practices, including appropriate medical surveillance programs ABSL- 1 practices plus Limited access Biohazard warning signs Sharps precautions Biosafety manual Decontamination of all infectious wastes and of animal cages prior to washing ABSL-2 practices plus Controlled access Decontamination of clothing before laundering Cages decontaminated before bedding removed Disinfectant footbath, as needed

Associated with human disease. Hazard: percutaneous exposure, ingestion, mucous membrane exposure

Indigenous or exotic agents with potential for aerosol transmission; disease can have serious health effects

Dangerous/exotic agents that pose high risk of life-threatening disease; aerosol transmission, or related agents with unknown risk of transmission

aFrom CDC-NIH (1999).

ABSL-3 practices plus Entrance through change room where personal clothing is removed and laboratory clothing is put on; shower on exiting All wastes are decontaminated before removal from the facility

Safety equipment (primary barriers) As required for normal care of each species

ABSL-1 equipment plus primary barriers: containment equipment appropriate for animal species; personnel protective equipment (PPE): laboratory coats, gloves, face and respiratory equipment as needed

ABSL-2 equipment plus Containment equipment for housing animals and cage dumping activities Class I or II BSCs available for manipulative procedures (inoculation, necropsy) that might create infectious aerosols. PPE: appropriate respiratory protection ABSL-3 equipment plus Maximum containment equipment (i.e., Class III Biological Safety Cabinet (BSC) or partial containment equipment in combination with full body, air-supplied positive-pressure personnel suit) used for all procedures and activities

Facilities (secondary barriers) Standard animal facility No recirculation of exhaust air Directional air flow recommended Hand-washing sink recommended ABSL- 1 facility plus Autoclave available Hand-washing sink available in animal room Mechanical cage washer used

ABSL-2 facility plus Physical separation from access corridors Self-closing, double-door access Sealed penetrations Sealed windows Autoclave available in facility

ABSL-3 facility plus Separate building or isolated zone Dedicated supply and exhaust, vacuum, and decontamination systems Other requirements outlined in CDC-NIH (1999)

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trained to use them properly. Excellent training courses and videos are available (Eagleson Institute, P.O. Box 954, Sanford ME 04073). There are three classes of biological safety cabinets: I, II, and III. A full description of these cabinets can be found in "Biosafety in Microbiological and Biomedical Laboratories" (CDC-NIH, 1999) and "Primary Containment of Biohazards: Selection, Installation, and Use of Biological Safety Cabinets" (CDC, 1995). Class I cabinets (Fig. 1) are designed for research with lowand moderate-risk agents (ABSL-2 and ABSL-3.). They are negative-pressure, ventilated cabinets, with an inward airflow of at least 75 linear feet per minute (lfpm). Air is exhausted through a HEPA filter into the laboratory or to the outside. Class I cabinets should not be used for handling research materials that are vulnerable to airborne contaminants, because the inward flow of air is unfiltered, and airborne microbial contaminants can be carried into the cabinet. Class I cabinets are gradually being replaced by class II cabinets. Class II biological safety cabinets are appropriate for research requiring ABSL-2 and ABSL-3 containment. There are two types of class II biological safety cabinets, which differ in construction, airflow velocities and patterns, and exhaust systems. Fig. 1. ClassI biological safety cabinet (side view). (A) Front opening, (B) Type B cabinets are appropriate for microbiological research sash, (C) exhaust HEPA filter, and (D) exhaust plenum. (From CDC-NIH, involving volatile or toxic chemicals or radionuclides; type A 1999.) cabinets are not. Class II cabinets have inward airflow velocities of 75-100 lfpm and HEPA-filtered downward vertical laminar airflow and exhaust air. Air in Type A cabinets (Fig. 2) can be exhausted into the laboratory or connected by a thimble connection to the building exhaust system. Air in type B cabinets is exhausted by a duct to the building exhaust system, and the cabinets contain negative-pressure plena. Type B cabinets are further subtyped as B 1, B2, and B3 (Figs. 3, 4, and 5), based on design differences (see CDC-NIH, 1999). Class III biological safety cabinets (Fig. 6) are totally enclosed, ventilated, and gas-tight. They are designed for work with biohazardous agents that require ABSL-3 or ABSL-4 containment. Class III cabinets are operated under negative pressure with HEPA-filtered supply air. Exhaust air is filtered either through two HEPA filters in series or through a HEPA filter followed by incineration before it is discharged outside the facility. Work is done through attached, arm-length rubber gloves or half-suits. Required equipment must be an integral part of the cabinet, and all materials and supplies entering or exiting the cabinet must be sterilized or disinfected. Therefore, several class III cabinets are usually set up as an interconnected system to allow for the required autoclave, chemical dunk tank, refrigerator, and other necessary equipment. A positive-pressure personnel suit worn in a "suit area," combined with the use of a class I or II biosafety cabinet, will provide the same protection as a class III cabinet. The one-piece, Fig. 2. Class II, type A, biological safety cabinet (side view). (A) Front opening, (B) sash, (C) exhaust HEPA filter, (D) rear plenum, (E) supplyHEPA ventilated suit is maintained under positive pressure with a lifesupport system to prevent leakage into the suit. The worker enfilter, and (F) blower. (FromCDC-NIH, 1999.)

24. CONTROL OF BIOHAZARDS

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Fig. 3. ClassII, type B 1, biological safety cabinet. (A) Front opening, (B) sash, (C) exhaust HEPA filter, (D) supply HEPA filter, (E) negative-pressure exhaust plenum, (F) blower, and (G) additional HEPA filter. The cabinet exhaust must be attached to the building exhaust system. (From CDC-NIH, 1999.)

ters the suit area through an aiflock fitted with airtight doors and leaves through a chemical shower, in which the surfaces of the suit are decontaminated. The suit area is maintained under negative pressure, and exhaust air is filtered through two HEPA filter units in series.

B.

Standard Practices

In addition to proper biocontainment equipment, it is necessary to use standard microbiological practices and personnel protective equipment (PPE) appropriate to the ABSL (see Table

I; Richmond et al., 1997; C D C - N I H , 1999). It is essential that personnel be trained in those practices and in wearing the correct PPE. They should wash their hands thoroughly after handling experimental animals or cultures and before leaving the animal room or laboratory. A hand-washing sink should be available in animal and procedure rooms. Personnel should also understand the importance of not eating, drinking, smoking, applying cosmetics, inserting contact lenses, taking medicines, or doing anything that would bring their hands in contact with their noses, mouths, and eyes. Training should be provided on dealing with accidental exposures, including administering first aid, obtaining medical assistance, and reporting exposures.

Fig. 4. ClassII, type B2, biological safety cabinet. (A) Front opening, (B) sash, (C) exhaust HEPA filter, (D) supply HEPA filter, (E) negative pressure exhaust plenum, and (F) filter screen. The cabinet exhaust must be attached to the building exhaust system. The carbon filter in that exhaust system is not shown. (From CDC-NIH, 1999.)

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Fig. 5. ClassII, type B3, biologicalsafety cabinet (tabletopmodel). (A) Front opening, (B) sash, (C) exhaustHEPA filter, (D) supplyHEPAfilter, (E) positivepressure plenum, and (F) negative-pressureplenum. The cabinet exhaust must be attached to the building exhaust system. (FromCDC-NIH, 1999.)

1. Personnel Protective Equipment Personnel protective equipment (PPE) needed for each ABSL is summarized in Table I. Federal regulations (29 CFR 1910.132-1910.139) require training for each person using PPE. The regulations require that each worker be trained to know at least when PPE is necessary; what PPE is necessary; how to properly don, doff, adjust, and wear the PPE; the limitations of the PPE; and the proper care, maintenance, useful life, and disposal of the PPE. Each worker must demonstrate that he or she can use PPE properly before being allowed to perform work in which it is required. Employers must also provide written certification that their employees have received and understood the required training by recording the name of each employee and date and subject of training.

Personnel must use appropriate eye or face protection when there is a chance of disease transmission through contact with body fluids. Many institutions require full-face shields when working with nonhuman primates. While engaged in operations that involve eye hazards, each worker who wears prescription lenses must wear eye protection that incorporates the prescription in its design or that can be worn over the prescription lenses. An eyewash station might be required and should be tested monthly. Gloves should be worn when handling animals or contaminated materials. Gloves are made in a variety of materials, each of which has been designed to work under slightly different conditions. The glove material selected must provide protection from the biohazardous agent. Latex gloves are commonly used in laboratories, but there is a significant allergic reaction prob-

Fig. 6. ClassIII biological safety cabinet. (A) Glove ports with O-ring for attaching arm-length gloves to cabinet, (B) sash, (C) exhaust HEPA filter, (D) supply HEPAfilter, and (E) double-endedautoclaveor pass-throughbox. A chemicaldunk tank can be installedbeneath the worksurface with access from above.The cabinet exhaust must be attached to the building exhaust system. (FromCDC-NIH, 1999.)

24. CONTROL OF BIOHAZARDS lem related to the use of latex. NIOSH (1997) has published information to help deal with this problem. 2. Needles and Syringes

Hypodermic needles and syringes should be used only for parenteral injection of experimental animals or for withdrawal of fluids from experimental animals or diaphragm bottles (BarNey and Richardson, 1984). In those instances, needlelocking syringes or syringes with permanently attached needles should be used. Animals should be restrained or tranquilized to reduce the chance of accidental autoinoculation (BarNey and Richardson, 1984). In some instances, the risk of accidental exposure from using syringes and needles might be great enough (e.g., using a needle and syringe to draw up HIV and inject an animal) to necessitate selection of another method of administration. OSHA has promulgated standards to minimize exposure to blood-borne pathogens (29 CFR 1910.1030). Those standards are for human blood, but the concepts are good and, if applied to all animal blood, will result in increased safety. Syringes and needles should be placed in puncture-resistant sharps disposal containers that are decontaminated, preferably by autoclaving, before they are discarded. Needles should never be bent, removed from the syringe, or replaced in the guard. NIOSH (1998a) has published guidelines for selecting, evaluating, and using sharps disposal containers. 3. Waste Disposal

Before work is begun, a plan must be developed for safe disposal of all items contaminated by the experimental procedures. Table I outlines the disposal methods required when working with infectious agents. The National Research Council (NRC, 1989) has also published guidelines for the disposal of infectious materials. Autoclaves should be large enough to handle the volume of material to be decontaminated. A common problem with the use of autoclaves is the generation of odors that permeate hallways or enter the ventilation system when the autoclave doors are opened. High-volume exhaust hoods that completely cover the swing area of both doors and aiflocks between an autoclave and the surrounding areas will reduce the escape of autoclave odors into a facility. 4. Sanitation Procedures

Sanitation procedures should be designed to minimize aerosol formation. Surfaces should be decontaminated before using water under pressure or a hose and nozzle. SOPs for handling spills should be prepared for every hazardous agent used in the institution and should be included in spill kits. Spill kits containing the materials needed to contain and decontaminate the spill

1053 should be readily available, and workers should be trained to use them properly.

C.

Security and Access to Biohazardous Areas

A biohazard sign, incorporating the universal biohazard symbol, must be posted on the entrance of any room in which there are biohazardous agents. The sign should identify the agent or agents, provide the name of the principal investigator and room supervisor, list emergency telephone numbers, and indicate any special requirements for entering (see Fig. 7 for a sample sign). Personnel must be trained not to give keys or key cards to others and to report lost keys or key cards immediately. In some cases, such additional security measures as retina or fingerprint identification, camera surveillance, or security personnel might be appropriate.

IV.

A.

OTHER CONSIDERATIONS

Public Concerns about Hazardous Projects

Few laboratory-acquired infections have occurred during the conduct of biohazardous research, and disease transmission to nonlaboratory personnel and the public has been rare (Sulkin, 1949, 1951; Collins, 1983; Sewell, 1995). Nonetheless, building a new facility or renovating a facility for research with biohazards might cause public concern. That concern can be handled by informing local officials about the proposed facility and the research that will be conducted there. Preparation of an environmental impact statement should be considered during the planning phase whether or not it is required by local ordinances. A "good neighbor" committee might be formed to involve community members in the review of the environmental impact statement. Taking that committee on periodic tours of the construction site or holding briefings can help allay public fears.

B.

Experiments Involving Recombinant DNA

The ruleS for using recombinant DNA can be obtained from the NIH Office of Recombinant DNA Activities (NIH Office of Recombinant DNA Activities, 1998a). Institutions proposing to work with recombinant DNA are required to establish a committee of no fewer than five members who have experience and expertise in recombinant DNA technology. At least two members must not be affiliated with the institution and must represent the interest of the surrounding community with respect to

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BIOHAZARD

ADMITTANCE TO AUTHORIZED PERSONNEL ONLY

Biohazardous Agents:

Special Requirements for Entry: Responsible Investigator: In Case of Emergency Call:

Daytime Phone

Home Phone

Authorization for Entrance Must Be Obtained from the Responsible Investigator

Fig. 7. Sample biohazard warning sign.

health and protection of the environment. At least one member with expertise in animal containment principles is required if whole-animal experiments are done. Appendix B of the NIH regulations contains lists of biological agents known to infect humans and selected animal agents that pose theoretical risks if inoculated into humans. Other sections describe the required containment practices (NIH Office of Recombinant DNA Activities, 1998b). Special care should be used in evaluating containment conditions for some experiments with transgenic animals, because it is possible that such experiments wi.ll lead to the creation of novel organisms or increased transmission of a recombinant pathogen. The purchase or transfer of transgenic rodents for experiments that require ABSL-1 containment is exempt from the NIH recombinant DNA guidelines.

C.

Allergies to Animals

One of the most important health hazards encountered by laboratory animal workers is allergy resulting from animal contact. Surveys have shown that up to 56% of animal care workers are

affected, and there are no reliable criteria to predict which workers will develop allergies (NIOSH, 1998). It is known that workers who have become sensitized to such domestic animals as cats and dogs are more likely to develop sensitivity to laboratory animals, and those who have symptoms or signs of allergies before they are employed in animal facilities are more likely to develop animal-induced asthma (NIOSH, 1998). Allergy symptoms can be severe and might require affected workers to change jobs. Allergies to animals should be addressed in institutional health and safety programs. Two excellent sources of information are "Occupational Health and Safety in the Care and Use of Research Animals" (NRC, 1997) and "Preventing Asthma in Animal Handlers" (NIOSH, 1998b). Inhalation is the most important route of exposure to allergens, so inhalation protection is the most important way to reduce the problem. Medical screening can identify workers with early symptoms of asthma. Workers who report symptoms should be referred for more extensive evaluation. NIOSH (1998) recommends that workers with early symptoms of occupational asthma be removed from exposure to allergens, because prolonged exposure can lead to irreversible disease.

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24. CONTROL OF BIOHAZARDS

D.

Zoonoses

Whenever a research project is planned, zoonoses should be considered and guidelines for the prevention of disease transmission implemented. Tetanus is a ubiquitous hazard in animal programs, and animal care staff should be encouraged to have tetanus toxoid injections at 10-year intervals to prevent this disease (CDC, 1991a). Rodents obtained from reliable sources generally are not sources of zoonoses, but other animals are commonly a zoonotic risk. For example, Old World monkeys can carry Mycobacterium tuberculosis (CDC, 1993a), randomsource dogs and cats can carry rabies virus, wild-caught rodents are a potential source of hantavirus (CDC, 1993b), and pregnant sheep can harbor Q fever virus (CDC, 1979). All Old World monkeys can harbor Herpesvirus simiae (B virus), which can be transmitted through bite or scratch wounds (CDC, 1989a,b, 1998a). Guidelines for preventing human B virus infection are available (Holmes et al., 1995; CDC, 1987, 1998b). A special first aid kit should be kept in all nonhuman primate housing areas for use when bites or scratches occur. The kit should have materials and SOPs for first aid and for obtaining samples to be sent for analysis (Georgia State University, 1998). Experience has shown that some people are allergic to some of the disinfectants included in these kits, which should be considered in assembling them. Each organization should provide access to a physician who is knowledgeable about zoonotic diseases to administer care in case of an accident. An information sheet should be included in the first aid kit in case emergency room care is needed, because the physicians on duty at the time of the accident might not have information on dealing with primate zoonoses. Guidelines for immunization of personnel against rabies are available (CDC, 199 lb), and immunization should be offered to those working with random-source dogs and cats. Livestock can also be exposed to rabies in the field, and consideration should be given to vaccinating sheep, cattle, and horses that are particularly valuable, are likely to be exposed, or have frequent contact with humans. Recommendations for handling animals exposed to rabies are available (CDC, 1998a). Guidelines are available for the prevention of hantavirus infection when working with wild rodents (CDC, 1993b, 1994) and for the prevention of Q fever (Bernard et al., 1982). "Managing Health Hazards Associated with Bird and Bat Excrement" (U.S. Army, 1992) is an excellent resource for facilities that have problems with the accumulation of bird excrement in ceilings and other areas. Personnel can have special conditions that make them more susceptible to zoonotic agents. Pregnant women usually are considered in this category, because the zoonotic agents can pose an additional threat to an unborn child. Moore et al. (1993) is an excellent source of information about zoonotic risks to pregnant women. Zoonotic diseases that are known to produce

teratogenic or abortifacient effects in pregnant women include brucellosis, tuberculosis, cryptococcosis, listeriosis, lymphocytic choriomeningitis, Q fever, toxoplasmosis, and Venezuelan equine encephalitis. The two most important zoonoses for pregnant women are toxoplasmosis and listeriosis. People are generally aware of the danger of toxoplasmosis from cat feces, but toxoplasmosis can also be transmitted during necropsy or handling the meat of cats, sheep, horses, and pigs. Transmission of listeriosis is also possible from necropsies or handling meat, especially of sheep, goats, and cattle (Moore et al., 1993). Some zoonoses can affect male fertility, and immunosuppressed people are more susceptible to some hazards. Facilities must be managed so that excellent universal precautions are used. Employees must be fully informed of the possible risks so that they can make an informed decision about working in the facility.

E.

Snake Venom

When working with venomous snakes, personnel must exercise special precautions. Antivenom is not available for all venomous snakes (e.g., the African twig snake, Thelotornis sp., and the boomslang, Dispholidus typus). If it is the venom that is needed and antivenom is not available, consideration should be given to purchasing the venom rather than maintaining the snakes. Snakes also might be considered too dangerous to maintain in an animal facility, even if antivenom is available, because of their aggressive behavior and lethal venom (e.g., the fierce snake, Parademansia sp., and the taipan, Oxyuranus sp.). Before bringing venomous snakes into an animal facility, arrangements should be made with an emergency room, and antivenom should be on hand. The University of Arizona Poison Control Center (1501 N. Campbell, Tucson, AZ, 85724, 602626-6016) can help locate antivenom. When the snakes arrive, they should be placed in locked cages in a limited-access, locked room. The room should be marked as hazardous. Access should be allowed only for those properly trained in handling venomous snakes and in emergency procedures to be followed in the event of an accident. A buddy system should be initiated so that a second person capable of assisting is available if required. Experience has shown that it is safer if the buddy is not in the room or stands quietly away from the person handling the snake. Each cage should be labeled with the species, the number of snakes, and the antivenom for that species. A colored card or tag should be placed on the cage of the snake that is being handled so that if there is a bite, emergency helpers will know immediately which species was involved and which antivenom is needed. Each room should have a bite kit containing SOPs for dealing with bites and the telephone number of the emergency room. The kit should contain any supplies specified in the SOPs (e.g., extractors, bandages, and splints), and the SOPs should

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accompany the patient to the emergency room. Detailed information about first aid for snakebites is available (http://www .xmission.com/--gastown/herpmed/snbite.htm).

V.

RESPONSIBILITY FOR REVIEWING AND APPROVING PROTOCOLS INVOLVING BIOHAZARDS

The principal investigator is responsible for presenting in his or her protocol information about any biohazards that will be used, including radioisotopes, viable organisms, carcinogens, transplantable tumors, and such biological materials as tissue, sera, and recombinant DNA. He or she also should state the precautions that will be taken to protect the people who will be exposed to the biohazard. A m e m b e r of the facility veterinary staff should be consulted to determine that appropriate containment is available for housing the animals and conducting experimental procedures. M a n y institutions have biosafety committees or departments that must approve any use of biohazardous agents. If there are no in-house biosafety personnel, investigators should have access to consultation with appropriate biosafety professionals to assist in preparing protocols and, when appropriate, in conducting experiments. The ultimate responsibility for review and approval of experimental protocols lies with the Institutional Animal Care and Use C o m m i t t e e (IACUC). However, the m e m b e r s of the I A C U C are usually not qualified to provide adequate review of all possible biohazards. Therefore, the committee should have access to a biosafety professional who is knowledgeable about the biohazard that will be used. M a n y institutions have someone from the institutional biosafety program serve as a m e m b e r of the IACUC. Alternatively, an appropriate biosafety professional could serve as an advisor to the committee.

VI.

SUMMARY

This chapter has touched on the important points for working with biohazards, including risk assessment, containment (i.e., containment facilities and standard practices), and oversight of protocols involving biohazards. Guidelines from the Centers for Disease Control and Prevention and the National Research Council are particularly important. The references in this chapter, combined with assistance from people with experience in working with biohazards, can be used to develop a comprehensive program for controlling biohazards.

REFERENCES

ACGIH (American Conference of Governmental Industrial Hygienists) (1998). "TLV's and BEI's." Cincinnati. APHA (American Public Health Association) (1995). "Control of Communicable Diseases Manual," 16th ed. (A. S. Beneson, ed). Washington, D.C. Barkley, W. E., and Richardson, J. H. (1984). Control of biohazards associated with the use of experimental animals. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 595-602. Academic Press, Orlando. Bernard, K. W., Parham, G. L., Winkler, W. G., and Helmick, C. G (1982). Q fever control measures: recommendations for research facilities using sheep. Infect. Control 3, 461-465. CDC (Centers for Disease Control and Prevention) (1979). Q fever at a university research centermCalifornia. Morb. Mortal Wkly. Rep. 20(13), 147148. CDC (1987). Guidelines for prevention of Herpesvirus simiae (B virus) infection in monkey handlers. Morb. Mortal. Wkly. Rep. 36(41), 680-682, 687689. CDC (1989a). B-virus infection in humansmFlorida. Morb. Mortal. Wkly. Rep. 36, 289-296. CDC (1989b). B-virus infection in humansmMichigan. Morb. Mortal Wkly. Rep. 38, 453-454. CDC (199l a). Diphtheria, tetanus, and pertussis: recommendations for vaccine use and other preventive measures--recommendations of the Immunization Practices Advisory Committee (ACIP). Morb. Mortal. Wkly. Rep. 40(RR-10), 2-8. CDC (1991b). Rabies prevention--United States, 1991 recommendations of the Immunization Practices Advisory Committee. Morb. Mortal Wkly. Rep. 40(RR-3), 1-19. CDC (1993a). Tuberculosis in imported nonhuman primates, United States. Morb. Mortal Wkly. Rep. 42, 572-575. CDC (1993b). Hantavirus infection~southwestern United States: interim recommendations for risk reduction. Morb. Mortal Wkly. Rep. 42(RR- 11), 1-13. CDC (1994). Laboratory management of agents associated with hantavirus pulmonary syndrome: interim biosafety guidelines." Morb. Mortal. Wkly. Rep. 43(RR-7), 1-7. CDC (1995). "Primary Containment for Biohazards: Selection, Installation, and Use of Biological Safety Cabinets." U.S. Dept. of Health and Human Services, Washington, D.C. CDC (1998a). Compendium of animal rabies control. Morb. Mortal Wkly. Rep. 47(RR-9), 1-10. CDC (1998b). Fatal cercopithecine herpesvirus 1 (B virus) infection following a mucocutaneous exposure and interim recommendations for worker protection. Morb. Mortal. Wkly. Rep. 47(49), 1073-1076, 1083. CDC-NIH (Centers for Disease Control and Prevention-National Institutes of Health) (1999). "Biosafety in Microbiological and Biomedical Laboratories," 4th ed. U.S. Dept. of Health and Human Services, Washington, D.C. Collins, C. H. (1983). "Laboratory-Acquired Infections, History, Incidence, Causes, and Prevention." Butterworths and Co., London. Georgia State Univ. (1998). "NIH B Virus Resource Laboratory." Viral Immunology Center, Atlanta. Holmes, G. P., Chapman, L. E., Stewart, J. A., Straus, S. E., Hilliard, J. K., Davenport, D. S., and the B Virus Working Group (1995). Guidelines for the prevention and treatment of B-virus infections in exposed persons. Clin. Infect. Dis. 20, 421-439. Knudsen, R. C. (1998). Risk assessment for biological agents in the laboratory, J. Am. Biol. Safety Assoc. 3(3), 99-104. Moore, R. M, Jr., Davis, Y. M., and Kaczmarek, R. G. (1993). An overview of occupational hazards among veterinarians with particular reference to pregnant women.Am. Ind. Hyg. Assoc. J. 54(3), 113-120.

24. CONTROL OF BIOHAZARDS NIH (National Institutes of Health) Office of Recombinant DNA Activities (1998a). "Guidelines for Research Involving Recombinant DNA Molecules." U.S. Dept. of Health and Human Services, Washington, D.C. NIH Office of Recombinant DNA Activities (1998b). "Appendix Q, Physical and Biological Containment for Recombinant DNA Research." U.S. Dept. of Health and Human Services, Washington, D.C. NIOSH (National Institute for Occupational Safety and Health) (1997). "NIOSH Alert, Preventing Allergic Reactions to Natural Rubber Latex in the Workplace." NIOSH Publication 97-135. Cincinnati. NIOSH (1998a). "Selecting, Evaluating, and Using Sharps Disposal Containers." DHHS (NIOSH) Publication 97-111. Cincinnati. NIOSH (1998b). "NIOSH Alert, Preventing Asthma in Animal Handlers." NIOSH Publication 97-116. Cincinnati. NRC (National Research Council) (1989). "Biosafety in the Laboratory: Prudent Practices for the Handling and Disposal of Infectious Materials." National Academy Press, Washington, D.C.

1057 NRC (1997). "Occupational Health and Safety in the Care and Use of Research Animals." National Academy Press, Washington, D.C. Richmond, J. Y., Ruble, D. L., Brown, B., and Jaax, G. P. (1997). Working safely at animal biosafety levels 3 and 4: facility design implications. Lab. Anim. 26(4), 28-36. Sewell, D. L. (1995). Laboratory associated infections and biosafety. Clin. Microbiol. Rev. 8(3), 389-405. Sulkin, S. E., and Pike, R. M. (1949). Viral infections contracted in the laboratory. N. Engl. J. Med. 241, 205-213. Sulkin, S. E., and Pike, R. M. (1951). Survey of laboratory acquired infections. Am. J. Public Health 41, 769-781. U.S. Army (1992). "Managing Health Hazards Associated with Bird and Bat Excrement." USAEHA TG 142. U.S. Army Center for Health Promotion and Preventive Medicine, Aberdeen, Maryland.

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Chapter 25 Selected Zoonoses James G. Fox, Christian E. Newcomer, and Harry Rozmiarek

I. II.

III.

IV. V.

VI. VII.

VHI. IX.

LABORATORY ANIMAL MEDICINE, 2nd edition

Introduction .................................................

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Viral D i s e a s e s

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A.

Poxviruses

B.

H e m o r r h a g i c Fevers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C.

L y m p h o c y t i c C h o r i o m e n i n g i t i s Virus ( L C M ) . . . . . . . . . . . . . . . . . . .

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D.

B Virus I n f e c t i o n ( C e r c o p i t h e c i n e H e r p e s v i r u s 1, C V H - 1 )

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E.

Rabies

E

Viral H e p a t i t i s I n f e c t i o n s

G.

Retroviruses .............................................

H.

M e a s l e s Virus (Rubeola, Giant Cell P n e u m o n i a )

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1069 1070 1072

................

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I.

N e w c a s t l e D i s e a s e Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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J.

I n f l u e n z a Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Rickettsial D i s e a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A.

Murine Typhus (Endemic Typhus)

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B.

Rickettsial Pox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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C.

Coxiella burnetii I n f e c t i o n (Q Fever) . . . . . . . . . . . . . . . . . . . . . . . . .

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Chlamydial Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A.

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C h l a m y d i o s i s (Psittacosis, O r n i t h o s i s or Parrot Fever) . . . . . . . . . . . .

Bacterial D i s e a s e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Trauma-Associated Bacterial Diseases . . . . . . . . . . . . . . . . . . . . . . . .

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B.

Systemic Diseases

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C.

Enteric Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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D.

Respiratory Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Fungal Diseases ..............................................

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Protozoal Diseases ...........

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A.

Enteric Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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B.

Systemic Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Helminth Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1093

A r t h r o p o d Infestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References ..................................................

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JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

I.

INTRODUCTION

Human risks of acquiring a zoonotic disease from animals used in biomedical research have declined over the past decade because higher quality research animals have defined microbiologic profiles. Even with diminished risks, the potential for exposure to infectious agents still exists, especially from larger species such as nonhuman primates, which are frequently obtained from the wild, and from livestock, dogs, ferrets, and cats, which are generally not raised in barrier facilities and are not subject to the intensive health monitoring performed routinely on laboratory rodents and rabbits. Additionally, when laboratory animals are used as models for infectious disease studies, exposure to microbial pathogens presents a threat to human health. Also, with the recognition of emerging diseases, some of which are zoonotic, constant vigilance and surveillance of laboratory animals for zoonotic diseases are still required. Transmission of zoonotic agents between animals and personnel is either by direct contact with the infected animal or indirect contact by exposure to contaminated equipment or supplies. Many activities performed in laboratories and animal facilities result in the formation of aerosols. Aerosolization of infectious material remains the principal means of disease transmission. However, direct inoculation through bites and scratches, exposure to contaminated equipment, and accidental ingestion supplement spread of agents by aerosol. As in a microbiologic laboratory or an infectious disease ward of a hospital, safety procedures can minimize potential zoonotic disease transmission to associated personnel in the biomedical laboratory. Some examples of sound procedures to follow in the control of exposure to zoonotic pathogens are (1) purchase of pathogen-free animals, (2) quarantine of incoming animals to detect any zoonotic pathogens, (3) appropriate treatment of infected animals or their removal from the facility, (4) vaccination of animal carriers and high-risk contacts where vaccines are available, (5) use of specialized containment caging or facilities and protective clothing, and (6) regular surveillance. It is not within the scope of this chapter to discuss these issues in detail. A number of sources are available that offer additional information. In particular, the Centers for Disease Control and Prevention (CDCP) in conjunction with the National Institutes of Health (NIH) has published a monograph, "Biosafety in Microbiological and Biomedical Laboratories (CDCP-NIH, 1999). The National Academy of Sciences (NAS) has published "Occupational Health and Safety in the Care and Use of Research Animals" (National Research Council, 1997). "Occupational Medicine: State of the Art Reviews," dealing with animal handlers (Langley, 1999), is also available. All of these are important resources for personnel involved in biomedical research using animals. The discussion that follows is a brief overview of select viral, rickettsial, chlamydial, bacterial, fungal, protozoal, and para-

sitic diseases shared by humans and the animals that are commonly used in biomedical laboratories.

II.

VIRAL DISEASES

A.

Poxviruses

Although there are numerous poxviruses capable of zoonotic transmission from laboratory animals to humans, with the possible exception of off virus transmission from small ruminants, these infections appear to be mostly of historical interest (Fenner, 1990). The poxviruses involved in zoonotic transmission in the laboratory animal facility represent three genera, Orthopoxvirus, Parapoxvirus, and Yatapoxvirus, with the nonhuman primate serving as host for the majority of the potentially zoonotic poxviruses species. In humans, these infections usually are characterized by the development of proliferative cutaneous or subcutaneous self-limiting lesions. Humans occasionally manifest the clinical signs of systemic disease in these zoonotic infections, particularly when the poxviruses of nonhuman primates are involved. 1.

Nonhuman Primate Poxvirus Infections

There are five nonhuman primate poxvirus infections that are either known to be zoonotic or are naturally occurring in humans. There is no evidence that smallpox, an Orthopoxvirus that is closely related to monkeypox virus, is naturally occurring in nonhuman primates, although it has been transmitted to nonhuman primates experimentally. Since 1980, smallpox has been considered to be eradicated on a worldwide basis by the World Health Organization, and the concomitant outbreaks of a smallpox-like disease in monkeys and humans have now been ascribed to monkeypox virus (Breman et al., 1980).

a.

Monkeypox

i. Reservoir and incidence. Monkeypox is an Orthopoxvirus causing sporadic cases of a human disease in Africa that mimics smallpox. Natural outbreaks of monkeypox have also been recorded in nonhuman primates in the wild and in laboratory settings (Fenner, 1990; CDCP, 1997). The virus appears to be naturally occurring in animals only on the continent of Africa, although Asian, African, and South American nonhuman primates are susceptible to infection (Fenner, 1990). Most of the infections in captive nonhuman primates have involved Asian macaques (Fenner, 1990). Recently, squirrels of the genera Funisciurus and Heliosciurus and nonhuman primates have been identified as hosts and significant reservoirs of the disease (Jerzek and Fenner, 1988).

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ii. Mode of transmission. Within susceptible nonhuman primate populations, the disease spreads rapidly with high morbidity and variable mortality. However, the transmission of monkeypox from captive nonhuman primate populations to humans has not been recorded. Human-to-human transmission of this agent has occurred presumably through close contact with active lesions, recently contaminated fomites, or respiratory secretions (Fenner, 1990; CDCP, 1997). The possibility of zoonotic spread should be considered. iii. Clinical signs. Clinical signs in the nonhuman primate host include fever followed in 4 - 5 days by cutaneous eruptions, usually on the limbs and less frequently on the trunk, face, lips, and buccal cavity. Monkeypox in humans is primarily of interest and importance because it produces a disease similar to smallpox, characterized by fever, malaise, headache, severe backache, prostration, and occasional abdominal pain (Chin, 2000). Lymphadenopathy and a maculopustular skin rash develop subsequently. Some individuals develop a severe fulminating disease with fatality. iv. Control and prevention. Smallpox vaccination will protect against monkeypox in humans and has also been used for the control of this disease in monkeys. b.

Benign Epidermal Monkeypox

Benign epidermal monkeypox (BEMP) is a Yatapoxvirus that has been zoonotic in the laboratory environment on numerous occasions.

i. Reservoir and incidence. Benign epidermal monkeypox, or tanapox, affects monkeys of the genus Presbytis in Africa and captive macaques in the United States. The African nonhuman primate genera Cercopithecus and Cercocebus and South American monkeys are apparently unaffected. Tanapox continues to be endemic in regions of Africa, and many cases of the disease in humans have also been detected in Africa during the course of surveillance for monkeypox (Jezek et al., 1985). ii. Mode of transmission. The rapid spread of BEMP among nonhuman primates housed in gang cages suggested direct viral transmission. The infections in animal handlers were attributed to viral contamination of skin abrasions. iii. Clinical signs. Benign epidermal monkeypox is characterized by the development of circumscribed, oval to circular, elevated red lesions usually on the eyelids, face, body, or genitalia. These lesions regress spontaneously in 4 - 6 weeks. The localization of BEMP lesions in the epidermis and adnexal structures differentiates them from Yaba lesions histologically, but similar to Yaba, eosinophilic, intracytoplasmic inclusion bodies are present (Kupper et al., 1970).

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iv. Control and prevention. Appropriate personal protective equipment should be sufficient to prevent the zoonotic transmission of this agent.

c.

Yaba

Yaba monkey tumor poxvirus is a member of the genus Yatapoxvirus that was reported initially in a colony of rhesus monkeys (Macaca mulatta) housed outdoors in Yaba, Nigeria (Bearcroft and Jamieson, 1958). There have been subsequent outbreaks and experimental studies of the agent, as well as sporadic incidental cases of the disease in laboratory-housed nonhuman primates.

i. Reservoir and transmission. Natural cases of the disease have been reported in the rhesus monkey and the baboon (Papio spp.), and experimental studies have expanded the host range to include pigtail macaques (Macaca nemestrina), stumptail macaques (Macaca arctoides), cynomolgus (Macaca fascicularis), African green (Cercopithecus aethiops), sooty mangabey (Cercocebus atys) and patas monkeys (Erythrocebus patas) (Ambrus and Strandstrom, 1966; Ambrus et aL, 1969). Many African monkeys apparently originate from areas with endemic infection and are immune to the agent, and New World nonhuman primate species are resistant to infection (Ambrus and Strandstrom, 1966). The role of insect vectors in the natural spread of this disease is unproven. Experimental studies have demonstrated the spread of the agent by aerosol transmission; thus, aerosolized Yaba virus must be considered a potential hazard to humans. ii. Clinical signs. Infected animals consistently have developed subcutaneous benign histiocytomas that reached a maximum size approximately 6 weeks postinoculation and regressed approximately 3 weeks thereafter. Natural tumor regression conferred immunity to reinfection (Niven, 1961), and the surgical removal of a Yaba tumor in a baboon prior to natural tumor regression was associated with subsequent susceptibility and reinfection with Yaba virus. Six human volunteers have been inoculated experimentally with Yaba virus and developed similar, but smaller, tumors than those seen in monkeys; tumor regression was also earlier. Yaba tumor induction has also been recorded as a result of accidental self-inoculation (needlestick) in a laboratory worker using the virally infected tumor; complete tumor resection was curative. 2.

Orf Virus (Contagious Ecthyma)

Off virus is a Parapoxvirus disease of sheep, goats, and wild ungulates and is characterized by epithelial proliferation and necrosis in the skin and mucous membranes of the urogenital and gastrointestinal tracts.

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i. Reservoir and incidence. Off virus disease is an endemic infection in many sheep flocks and goat herds throughout the United States and worldwide. The disease affects all age groups although young animals are most frequently and most severely affected. In sheep, off virus infection does not reliably confer protection against reinfection with different strains of virus, aiding in viral persistence within a population (Haig et al., 1997). ii. Mode of transmission. Off virus is transmitted to humans by direct contact with scabs and exudates from viralladen lesions. External lesions are not always readily apparent. Transmission of this agent by fomites or other animals contaminated with the virus is also possible due to the extended environmental persistence of this double-stranded DNA virus. Although the virus requires a break in skin for entry, rare cases of person-to-person transmission have been recorded (Chin, 2000). iii. Clinical signs. Orfvirus produces proliferative, pustular encrustations on the lips, nostrils, and mucous membranes of the oral cavity and urogenital orifices of infected animals.

The disease in humans is usually characterized by the development of a solitary lesion located on the hands, arms, or face (Fig. 1). The lesion is maculopapular or pustular initially and progresses to a weeping proliferative nodule with central umbilication. Occasionally, several nodules are present, each measuring up to 3 cm in diameter and persisting for 3 - 6 weeks, followed by spontaneous regression with minimal residual scarring. Regional adenitis is uncommon, and progression to generalized disease is considered a rare event (Chin, 2000). iv. Control and prevention. Personnel should wear gloves and wash hands, as well as launder clothing and disinfect boots, after contact with sheep. Current herd-management practices often involve the use of live unattenuated off virus vaccines that contribute to the perpetuation of environmental contamination and entail some risk to the individuals handling the vaccine product. Efforts are underway to explore the development of a subunit vaccine for off virus to eliminate these problems (Mercer et al., 1997).

Fig. 1. Humanoff. Firm, raised, centrallynecrotic lesion on the thumb of an animaltechnicianwho handled an infected goat. (Courtesyof Dr. J. Griffith.)

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Hemorrhagic Fevers

The hemorrhagic fever viruses constitute a group of RNA viruses that produce a clinical syndrome in humans characterized by high fever, epistaxis, ecchymoses, diffuse hemorrhage in the gastrointestinal tract and other organs, hypotension, and shock. These diseases often are spread to humans by mosquitoes, ticks, or other arthropod vectors (hence, the term arthropod-borne or arboviruses for the causative agents); by direct contact with the excreta of infected rodents; or by the contaminated blood and bodily fluids of other infected animals. These viral agents have taken on increased importance in recent years and are receiving considerable attention within the context of emerging infections potentially impacting the United States and other regions of the globe. Contemporary society has catalyzed the process of emerging infections by introducing ecological disturbances affecting host and vector availability and distribution and by developing rapid means of international transportation, enhancing the potential dissemination and dispersion of these agents (Committee on Emerging Microbial Threats to Health, 1992; Le Guenno, 1997). Nonhuman primates serve as reservoirs and are susceptible to numerous zoonotic viral hemorrhagic diseases, including yellow fever, dengue, Marburg virus disease, and Ebola, as well as to viral hemorrhagic diseases such as simian hemorrhagic fever, which is not considered zoonotic. However, these diseases are not of high concern in programs that follow an appropriate quarantine/importation process and are involved in the conventional care of nonhuman primates in indoor facilities. The salient features of natural and experimental infections by these agents in nonhuman primates have been reviewed in detail and will be discussed only briefly in this section (Adams, 1995; Mansfield and King, 1998). Rodent hantavirus infections have resulted in serious and fatal human zoonotic infection in association with laboratory animal studies and field studies involving wild animals and are covered in more detail. 1. FlavivirusesmYellow Fever and Dengue

i. Reservoir, incidence, and transmission. Yellow fever is caused by an RNA flavivirus and is maintained in a monkeymosquito-monkey cycle in the sylvatic or jungle form and in a monkey and human-mosquito transmission cycle in the urban or rural form of the disease (Adams, 1995). The main vectors are Aedes aegypti in the urban setting and A. africanus or A. leucoelaenus in the African or South American jungle settings, respectively. Cases of the disease in persons result from human forestry activities that disrupt the forest canopy, bringing mosquitoes to the ground, or from nonhuman primate epizootics, achieving a similar result. There are four serotypes of dengue virus, any of which can cause dengue hemorrhagic fever. Dengue is endemic in tropical and subtropical Asia, Africa, Oceania, Australia, and the Americas and is widespread in the Caribbean basin. The virus persists

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in a nonhuman-mosquito cycle involving A. aegypti and A. albopictus; both of these vectors are now established in the United States (Committee on Emerging Microbial Threats to Health, 1992). Both dengue and yellow fever viruses are passed transovarially in the mosquito vector (Chin, 2000). ii. Clinical signs. African monkeys apparently acquire yellow fever infection as young animals and develop a mild form of the disease with subsequent immunity as indicated by antibody titers. The disease in New World nonhuman primates and humans is fulminating and severe, characterized by fever, vomiting, anorexia, yellow to green urine, icterus, and albuminuria. At necropsy, the internal organs are hemorrhagic, necrotic, and bile-stained. The classic lesion is massive, midzonal necrotizing hepatitis with necrotic hepatocytes containing characteristic eosinophilic, intracytoplasmic inclusion bodies, or "Councilman bodies." Human dengue infection is characterized by the abrupt onset of fever, intense headache, myalgia, arthralgia, retro-orbital pain, anorexia, gastrointestinal disturbances, and rash. The clinical presentation of dengue virus infection can be more severe, involving a generalized hemorrhagic syndrome with increased vascular permeability, thrombocytopenia, and unusual bleeding manifestations. iii. Diagnosis and control. The variable expression of yellow fever in African versus New World nonhuman primates decreases the reliability of clinical signs as indicators of active infection. Consequently, imported monkeys should have a certificate that they have originated from a yellow fever-free area; have been maintained in double-screened, mosquito-proof enclosures; or have been vaccinated for yellow fever. The same general principles apply to the prevention of introduction of dengue virus in newly imported nonhuman primates. The Center for Disease Control and Prevention (CDCP), which regulates nonhuman primate importation facilities, stipulates specific record-keeping procedures and requires the prompt (within 24 hr) reporting of any disease in a nonhuman primate suspected of being infected with yellow fever, Marburg, monkeypox, or Ebola disease (filovirus). This reporting requirement also applies to any illness among staff members that may have been acquired from nonhuman primates (Johnson et al., 1995). Also, imported nonhuman primates that die within 10 days of arrival should be carefully examined at necropsy for the lesions suggestive of viral hemorrhagic fever. 2. Marburg Virus Disease (Vervet Monkey Disease)

The Marburg virus has been responsible for several highly fatal episodes of disease. The first outbreak occurred in Germany in 1967 and serves as the basis for most of the descriptive information about clinical disease (Siegart, 1972). The most recent case occurred in 1980 in an individual who was believed to have been exposed while visiting a site on the Kenya-Uganda

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border located only 80 kilometers from the site where the vervet monkeys associated with the 1967 outbreak were maintained prior to shipment to Europe (Jahrling, 1989). i. Reservoir and incidence. Originally classified as a rhabdovirus, the Marburg agent now has been reclassified into the genus Filovirus along with Ebola virus. In the initial outbreak of the disease, 31 persons became severely ill from infection and 7 died subsequently. Most of these individuals had handled fresh tissues or primary cell cultures from African green monkeys (Cercopithecus aethiops). Secondary infection occurred in 4 persons who had contact with the patients who had been exposed originally through blood, tissue, or cell cultures. The natural reservoir for the Marburg disease agent has never been identified nor has the source of infection for the index case in any of the outbreaks (Jahrling, 1989). Experimental studies in nonhuman primates and other laboratory animals have shown that the virus produces a 100% fatal infection in African green monkeys, rhesus monkeys, squirrel monkeys, guinea pigs, and hamsters. Although African green monkeys were clearly incriminated in the original outbreak, their high fatality rate when infected experimentally with Marburg virus suggested that they would not be a likely reservoir (Jahrling, 1989). ii. Mode of transmission. Transmission appears to be from direct contact with infected tissues and close contact with infected patients. Experimental infections in nonhuman primates suggest the possibility of airborne transmission because uninoculated monkeys housed in the same room as experimentally infected animals contracted the disease. iii. Clinical signs. The incubation period for Marburg disease is 4 - 1 6 days. The disease in humans is manifested by the abrupt onset of fever, chills, myalgia, headache, anorexia, and conjunctival suffusion. Later, progressive involvement of the gastrointestinal tract with severe pain and gastrointestinal bleeding, maculopapular rash, and systemic bleeding disorders often occurs. Abnormalities in the coagulation pattern indicative of disseminated intravascular coagulation occur and may be the proximate cause of death in approximately a quarter of the cases. Treatment of Marburg virus disease consists of intensive supportive therapy directed at controlling the progressive pathophysiological events and the use of specific immune plasma if available (Jahrling, 1989). iv. Diagnosis and prevention. Diagnostic studies for these agents must be conducted under biosafety level 4 conditions. These entail strict engineering controls, the use of primary barriers and personal protective equipment in conjunction with differential air-pressure gradients to ensure isolation, and complete standard operating procedures covering all aspects of facility function. The diagnosis of Marburg virus disease involves isolation of the virus from blood, other tissues, or bodily fluids in Vero cell culture or the detection of serum antibodies.

3.

Ebola and Other Filovirus Infections

i. Reservoir and incidence. Ebola hemorrhagic fever is a rare disease caused by a filovirus that is morphologically identical to but antigenically distinct from Marburg disease virus. Human cases of Ebola have been confined to the continent of Africa. Although a case of the disease in a human has resuited from contact with infected chimpanzees (Formenty et al., 1999b) and natural outbreaks of the disease have been recorded in a community of wild chimpanzees, the chimpanzee is not regarded as the natural reservoir (Formenty et al., 1999a). Also, the Sudan and Zaire Ebola virus strains have been shown experimentally to produce lethal infection in nonhuman primates in about 8 days, but paralleling the situation with Marburg virus, monkeys have not been demonstrated to be the natural reservoir (Dalgard et al., 1992). The natural reservoir for this agent has not been identified, although the bat, especially solitary microchiropteran species, has been suggested as a leading reservoir candidate (Monath, 1999). The first and only non-African Ebola-like filovirus, EbolaReston, was isolated and identified from macaques recently imported into the United States from the Philippines during 1989. The infected monkeys died of an acute hemorrhagic disease and prompted the revision and implementation of nonhuman primate importation and handling guidelines (Centers for Disease Control [CDC], 1989, 1990). The natural reservoir for the Ebola-Reston strain is also unknown. Despite the novel epidemiological findings in this case, related to geography and natural reservoir, the deaths of imported monkeys were caused by another viral agent, simian hemorrhagic fever of a non-African Ebola-like filovirus, which is nonpathogenic for humans. ii. Mode of transmission. Transmission appears in most cases to be from direct contact with infected tissues and close contact with humans or animals shedding the organism. Oral and conjunctival transmission of Ebola-Zaire in macaques has also been confirmed experimentally (Jaax et al., 1996). However, in the natural outbreak of Ebola-Reston infection in a laboratory colony of nonhuman primates, transmission occurred among animals without apparent direct intimate contact, suggesting the possibility of airborne or aerosol transmission. Three of six animal technicians working with these animals developed antibody response to Ebola-Reston virus, but the details of transmission were not determined in all cases. One of these individuals was infected during postmortem examination of an infected monkey (Ksiazek et al., 1999). Epidemiologic findings in animal caretakers working in the Philippine-source colony for Ebola-Reston suggest that the transmission of Ebola-Reston to humans is rare (Miranda et al., 1999). iii. Clinical signs. In experimental nonhuman primate infections with Ebola-Zaire or Sudan, animals rapidly develop a febrile, debilitating illness characterized by high titer viremia; virus dissemination and replication in multiple organs, produc-

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ing tissue necrosis, effusions, coagulopathy, and hemorrhage; and death. Humans develop a similar pattern of infection, manifesting acute illness, fever, chills, headache, myalgia, and anorexia with progressive deterioration to vomiting, abdominal pain, sore throat, and bloody diarrhea. Although less virulent than the Sudan or Zaire strains of Ebola virus in nonhuman primates, Ebola-Reston produced a hemorrhagic disease in macaques involving multiple organ systems, resulting in death in 8-14 days. Clinical disease was not recognized in animal technicians who developed filovirus-specific serum antibodies associated with the disease outbreak in macaques due to EbolaReston (CDC, 1990; Dalgard et aL, 1992).

iv. Diagnosis and prevention. The gross and histopathologic findings of Ebola infection have been reported in numerous nonhuman primate species, including chimpanzees (Wyers et al., 1999), baboons and African green monkeys (Ryabchikova et al., 1999), and macaques (Dalgard et al., 1992). In macaques, intracytoplasmic inclusion bodies associated with hepatocellular necrosis, adrenal necrosis, and patchy pulmonary interstitial infiltrates were noted in cases of Ebola-Reston infection and considered useful for the differentiation of this disease from simian hemorrhagic fever (Dalgard et al., 1992). Many techniques have been used to detect Ebola virus or the viral antigen (Jahrling, 1989). Serologically, the infection is diagnosed by rising antibody titer using indirect immunofluorescence assay (IFA), radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA) (Jahrling, 1989; Ksiazek et al., 1999). The immunoglobulin M (IgM) capture assay proved useful for the detection of antibodies early in the course of infection (day 6) for both nonhuman primates and humans infected with Ebola-Reston. Also, the long-term persistence of IgG antibodies (> 400 days in nonhuman primates and up to 10 years in two humans) suggested that the ELISA would be very useful for field investigations. Due to effective importation procedures mandated by CDC (1990), only those personnel employed in facilities involved in the importation of nonhuman primates have the potential for the occurrence of Ebola. These personnel should become familiar with the equipment and procedures used to minimize the potential for Ebola virus transmission in the event of an outbreak. Neither vaccination nor antiviral pharmaceuticals are available for the treatment of Ebola virus infection. The Subcommittee on Arbovirus Laboratory Safety of the American Committee on Arthropod-Borne Viruses recommends that biosafety level 4 procedures be employed in studies using Ebola virus (CDCPNIH, 1999). 4.

Hantaviruses (Hemorrhagic Fever with Renal Syndrome; Hantavirus Pulmonary Syndrome)

i. Reservoir and incidence. Hantavirus is one of several genera within the family Bunyaviridae that can cause severe hemorrhagic fever with renal syndrome (HFRS). The hantaviruses

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are widely distributed in nature among wild rodent reservoirs, but unlike other members of the family, they are usually not transmitted by insect vectors (Schmaljohn and Hjelle, 1997). At least 14 viruses are recognized in the genus, and the severity of the disease produced depends on the virulence of the virus involved (LeDuc, 1987; Schmaljohn and Hjelle, 1997). Viruses producing HFRS are prevalent in southeastern Asia, Japan, and focally throughout Eurasia; whereas variants producing a less severe form of the disease, known as nephropathia endemica, occur throughout Scandinavia, Europe, and western portions of the former Soviet Union. An outbreak of hantavirus infection resulting in numerous deaths in adults from fatal hantavirus pulmonary syndrome (HPS) was first recognized in the United States (Schmaljohn and Hjelle, 1997; CDCP-NIH, 1999). Since this initial outbreak, cases of HPS have been reported from 30 states, and about three-quarters of the patients have been from rural areas. Rodents from numerous genera have been implicated in foreign outbreaks of the disease, including Apodemus, Clethrionomys, Mus, Rattus, Pitymys, and Microtus. In the United States, serological surveys have detected evidence of hantavirus infection in urban and rural areas involving Rattus norvegicus, Peromyscus maniculatus, P. leucopus, Microtus pennsylvanicus, Tamias spp., Sigmodon hispidus, Reithrodontomys megalotis, Oryzomys palustris, and Neotoma spp. (Tsai et al., 1985; Schmaljohn and Hjelle, 1997; CDCP-NIH, 1999). Numerous cases of hantavirus infection have occurred among laboratory animal facility personnel from exposure to infected rats (Rattus), including outbreaks in Korea, Japan, Belgium, France, and England (LeDuc, 1987), although there have not been any cases reported in U.S. laboratories. Hantavirus pulmonary syndrome has been reported in the United States in persons associated with outdoor activities and occupations that place them in close proximity with infected wild rodents and their excrement (Hjelle et al., 1996; Jay et al., 1996; Schmaljohn and Hjelle, 1997). Several cases have involved individuals from academic institutions involved in field studies. There is also epidemiologic evidence that cats may become infected through rodent contact and serve as a potential reservoir.

ii. Mode of transmission. The transmission of hantavirus infection is through the inhalation of infectious aerosols, and extremely brief exposure times (5 min) have resulted in human infection. Rodents shed the virus in their respiratory secretions, saliva, urine, and feces for many months (Tsai, 1987). Transmission of the infection can also occur through an animal bite or from disturbing dried materials contaminated with rodent excreta, allowing wound contamination, conjunctival exposure, or ingestion to occur (CDCP-NIH, 1999). The recent cases that have occurred in the laboratory animal facility environment have involved infected laboratory rats. In this environment, the possibility of transmitting the infection from animal to animal by the transplantation of cells or tissues should also be considered (Kawamata et al., 1987). Person-to-person transmission

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apparently is a very rare feature of hantavirus infection (Schmaljohn and Hjelle, 1997). iii. Clinical signs. The clinical signs are related to the hantavirus species involved. The form of the disease known as nephropathia endemica is characterized by fever, back pain, and nephritis and causes only moderate renal dysfunction from which the patient recovers. Recent cases of HPS in the United States developed a febrile prodrome, thrombocytopenia, and leukocytosis in common with HFRS. Patients proceeded rapidly to respiratory failure due to capillary leakage into the lungs, followed by shock and cardiac complications (Schmaljohn and Hjelle, 1997; CDCP-NIH, 1999). The form of the disease that has been noted following laboratory animal exposure fits the classical pattern for HFRS, characterized by fever, headache, myalgia, petechiae, and other hemorrhagic manifestations, including anemia and gastrointestinal bleeding, oliguria, hematuria, severe electrolyte abnormalities, and shock (Lee and Johnson, 1982). iv. Diagnosis and prevention. Both antigenic and genetic methods have been used for the characterization of the hantaviruses. Routine serological tests include the IFA and ELISA for the demonstration of specific antibody, while plaque reduction neutralization is the most sensitive serological assay for virus differentiation (Committee on Emerging Microbial Threats to Health, 1992; Schmaljohn and Hjelle, 1997). Additional information about hantavirus serological testing is available through the Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention. Hantavirus infections should be prevented through the detection of infection in incoming rodents and rodent tissues prior to their introduction into resident laboratory animal populations. Rodent tumors and cell lines can be tested for hantavirus contamination using a modified rat antibody production test as well as polymerase chain reaction (PCR)-based assays. Animal biosafety level 4 guidelines are recommended for animal studies involving hantavirus infections in permissive hosts such as Peromyscus maniculatus, and wild-caught rodents brought into the laboratory that are susceptible to hantaviruses producing HPS or HFRS should also be handled according to these guidelines (CDCP, 1994). Animal biosafety level 2 practices are sufficient for handling rodent strains known not to excrete the virus.

C.

Lymphocytic Choriomeningitis Virus (LCM)

Of the many viruses present in the mouse, only LCM virus naturally infects humans. Recent cases and a review of the literature attest to the ease with which LCM virus can be transmitted from animals to humans (Dykewicz et al., 1992).

i. Reservoir and incidence. Lymphocytic choriomeningitis virus (LCMV) is a member of the family Arenaviridae, which are single-stranded RNA viruses with a predilection for rodent reservoirs. Other members of the family are also important zoonoses that produce a hemorrhagic fever syndrome, including Lassa fever (in Africa) and Argentine and Bolivian hemorrhagic fevers (in South America). In addition, a new zoonotic arenavirus that produced fatal infection in three persons, characterized by acute respiratory distress syndrome, liver failure, and hemorrhagic manifestations, has been identified in North America, specifically in California (CDCP, 2000). This agent shared 87% identity with the Whitewater Arroyo prototype strain isolated from Neotoma albigula (white-throated wood rats) in New Mexico in 1990, and one of these patients had cleaned rodent excreta in her home during the 2 weeks prior to illness. In parallel with the persistent and emerging importance of arenaviruses for humans with wild rodent contact, LCMV has remained an important natural infection of laboratory animals (Bowen et al., 1975; Dykewicz et al., 1992; Jahrling and Peters, 1992; Rousseau et al., 1997). The LCM virus is widely distributed among the wild mouse population throughout most of the world and presents a zoonotic hazard (Childs et al., 1992; Smith et al., 1993; Morita et al., 1996). Mice, hamsters, guinea pigs, nonhuman primates, swine, and dogs are among the laboratory animal hosts that sustain natural infections. However, LCMV is especially well adapted to the mouse, living in a symbiotic relationship characterized by latent infection of the mouse for extended periods. The mouse, and in certain well-defined outbreaks, the hamster, have remained the species of primary concern as zoonotic reservoirs in the laboratory, as evidenced by a recent outbreak of LCMV in humans (Dykewicz et al., 1992). Also, athymic and other immunodeficient mouse strains may pose a special risk of harboring silent, chronic infections and present a hazard to personnel (Dykewicz et al., 1992; CDCP-NIH, 1999). There have been numerous reports of epizootic infectious hepatitis (callitrichid hepatitis) with a high mortality rate in marmosets and tamarins in zoological parks in both the United States and England during the past 2 decades (Montali et al., 1989). Early serological studies indicated that the same, or a closely related virus, was involved in five of six outbreaks studied and that the etiologic agent had a close antigenic relationship with LCMV (Stevensen et al., 1990, 1991). Subsequently, LCMV was shown to be the etiologic agent for callitrichid hepatitis through cDNA genome analysis (Stevensen et al., 1995). Rodent infestations are common in zoos, and mice, as known carriers of LCMV, are the probable source of infection in these outbreaks. Moreover, it had been a common practice in some facilities to supplement the diets of tamarins and marmosets with suckling mice (Richter et al., 1984), a prime source for the infectious agent. Two veterinarians involved in the care of infected callitrichids became seropositive to the agent but did

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not develop clinical signs of disease (Adams, 1995). Nevertheless, the wide geographic distribution of the outbreaks of callitrichid hepatitis underscores the need to better understand the public health implications of LCMV as a human pathogen as well. ii. M o d e o f transmission. There are many forms of infection produced by LCMV in the laboratory mouse depending on host factors and variation in LCMV strain organotropism (Lehmann-Grube, 1982). Under some circumstances LCMV produces a pantropic infection and may be copiously present in blood, cerebrospinal fluid, urine, nasopharyngeal secretions, feces, and tissues of infected natural hosts and possibly humans. In endemically infected mouse and hamster colonies, the infection is transmitted in utero or early in the neonatal period, producing a tolerant infection characterized by chronic viremia and viruria without significant clinical disease. Thus, bedding material and other fomites contaminated by LCMV-infected animals can also be important sources of infection for humans, as demonstrated in numerous outbreaks among laboratory animal technicians (Lehmann-Grube, 1982; Dykewicz et al., 1992). The experimental passage of tumors and cell lines appears to pose one of the biggest threats for the introduction of LCMV into animal facilities at the present time. Spread of LCMV among animals by contaminated tumors and cell lines has been widely recognized (Bhatt et al., 1986; Dykewicz et al., 1992; Nicklas et al., 1993). Bhatt et al. (1986) reported 17 of 63 rodent transplantable tumors screened were positive for LCMV, and Nicklas et al. (1993) identified contamination in 4 of 14 hamster tumors and 2 of 81 mouse tumors that had been propagated in animals. Transmission by infected, bloodsucking ectoparasites has been demonstrated experimentally, and LCMV has been recovered from cockroaches. However, these sources for LCMV infection have not been shown to play a significant role in any of the LCMV infections (human or animal) in laboratory animal facilities to date. Infection in humans may be by parental inoculation, inhalation, or contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals. Airborne transmission is well documented. In human LCMV infections associated with infected pet hamsters, the infection rate correlated with cage type and location in the household. Open wire cages were correlated with the highest rate of infection, whereas deep boxes and aquariums were associated with a lower human infection rate. Similarly, cage placement in an area of high human activity was associated with infection, but remotely located cages away from areas of frequent human activity (e.g., the basement) did not result in infection of occupants (Biggar et al., 1975). Also, infections are known to occur in individuals who have not had direct physical contact with infected hamsters but who had simply entered the room housing the animals (Hinman et al., 1975). These findings suggest that airborne transmission plays an important role in human infection.

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Table I

Symptomsof Persons with PositiveTiters for LymphocyticChoriomeningitis Number of cases Symptom

49a

11b

None recognized Fever Headache Myalgia Pain on movingeyes Nausea Vomiting Biphasic illness Sore throat Photophobia Cough Swollen glands Diarrhea Rash Upper respiratory tract symptoms Orchitis

3 44 42 39 29 26 17 12 12 12 9 8 8 6 6 1

1 9 7 8 7 9 9 NRc NR 7 1 NR 1 1 NR NR

aFrom Biggar et al. (1975). bFromMaetz et al. (1976). cNR, None recognized.

iii. Clinical signs. Humans usually develop a flulike illness characterized by fever, myalgia, headache, and malaise following an incubation period of 1-3 weeks (Table I). However, there can be more serious manifestations of the disease in patients, including maculopapular rash, lymphadenopathy, meningoencephalitis, and rarely, orchitis, arthritis, and epicarditis (Johnson, 1990). Central nervous system involvement has resulted in death in several cases. The virus may pose a special risk during pregnancy because of potential infection of the human fetus (Wright et al., 1997). Wright et al. (1997) reported 26 cases in human infants, with LCMV confirmed serologically over a 2year period in a major U.S. medical center. These infants presented with ocular abnormalities, macrocephaly, and microcephaly. Fifty percent of the mothers reported having had illnesses compatible with LCMV infection, and over half reported exposures to rodents during their pregnancies. Intravenous ribavirin therapy significantly reduces mortality in patients infected with Lassa fever virus and may be of some benefit in patients with severe LCMV infections (Andrei and DeClerq, 1993). iv. D i a g n o s i s a n d prevention. Virus isolation from blood or spinal fluid in conjunction with the use of immunofluorescence assay (IFA) of inoculated cell cultures is the primary method of diagnosing acute disease. Antibody is detectable using an IFA approximately 2 weeks following the onset of illness. Prevention of this disease in the laboratory is achieved through the periodic serological surveillance of new animals

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with inadequate disease profiles and resident animal colonies at risk, using ELISA and IFA tests. Screening all tumors and cell lines intended for animal passage for the presence of LCMV is another crucial element in the program to prevent the introduction of LCMV into established animal colonies. The elimination of ectoparasites and insect vectors in animal facilities as part of the overall scheme for disease prevention and control would also be prudent measures for the control of LCMV.

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B Virus Infection (Cercopithecine Herpesvirus 1, CHV-1)

There are many herpesviruses of nonhuman primates and other animal species that might be studied as laboratory animals, but CHV-1 stands alone as a documented hazard with devastating potential for humans working in the laboratory animal facility environment. Among the many other nonhuman primate herpesviruses, Herpesvirus saimiri will replicate in human tissues and is classified as an oncogenic virus by the National Cancer Institute, and H. tamarinus has been shown to produce skin pustules, fever, and nonfatal encephalitis in humans (Adams, 1995; Mansfield and King, 1998). The reader is referred to the sources cited for more information on these latter two agents. i. Reservoir and incidence. First described in 1933 by Gay and Holden, B virus produces a life-threatening disease of humans that has resulted in several fatalities within the past decade (CDC, 1987, 1989, 1991; CDCP, 1998). In macaques, B virus produces a mild clinical disease similar to human H. simplex virus infection. Duringprimary infection, macaques develop lingual and/or labial vesicles or ulcers that generally heal within a 1- to 2-week period; keratoconjunctivitis or corneal ulcer may also be noted. The virus persists latently in the trigeminal and genital ganglia of the macaque, and reactivation of viral shedding from peripheral sites in asymptomatic animals subjected to physical or psychological stressors or treated with immunosuppressive agents is known to occur. The infection is transmitted between macaques by virus-laden secretions through close contact involving primarily the oral, conjunctival, and genital mucous membranes (Weigler et aL, 1995). In an endemically infected domestic macaque production colony, an age-related increase in the incidence of B virus infection occurred during adolescence as exposure to the agent continued, with the incidence approaching 100% in colonyborn animals by the end of their first breeding season (Weigler et al., 1993). Seroconversion to B virus among wild-caught rhesus monkeys also indicates that eventually 100% of the newly trapped individuals acquire the infection. Consequently, B virus should be considered endemic among Asian monkeys of the genus Macaca unless these animals have been obtained from specific breeding colonies known to be B virus-free. Several

species of New World monkeys and Old World monkeys other than macaques are known to succumb to fatal B virus infection, but only macaques are known to harbor B virus naturally (Holmes et al., 1995). Eight of 25 langurs and 4 of 6 proboscis monkeys were seropositive to B virus in a survey, indicating that these Asian Old World monkey species may also be potential reservoirs (Kalter et al., 1997). Many of the human B virus infections have resulted from exposure to rhesus macaques (Macaca mulatta), leading to the supposition that there may be strain-specific pathogenicity of CHV-1. Several strains have now been identified by antigenic, restriction enzyme digest patterns, polymerase chain reaction (PCR), and phylogenetic analyses of B virus (Slomka et al., 1993; Smith et al., 1998). Three distinct genotypes have been described and associated with the macaque species of origin (Smith et al., 1998). These B virus genotypes were composed of (1) isolates from the rhesus and Japanese macaques, (2) isolates from cynomolgus monkeys (M. fascicularis, and (3) isolates from pigtail macaques (M. nemestrina). However, it remains to be determined whether these strains will correlate with differences in pathogenicity for nonmacaque species. ii. Mode of transmission. The transmission of B virus to humans primarily occurs through exposure to contaminated saliva through bites and scratches. Exposure by the airborne route was believed to have played a role in several human cases (Palmer, 1987), and exposure of ocular mucous membranes to biological material, possibly fecal, has been confirmed in a human fatality (CDCP, 1998). Other types of B virus transmission to humans that have been confirmed are needlestick injury (Benson et al., 1989) and exposure to infected nonhuman primate tissues (Wells et al., 1989). The possibility of fomite transmission through an injury obtained in handling contaminated caging may be less likely but warrants consideration in an institution's hazard assessment and risk analysis. One case of humanto-human transmission has also been documented (CDC, 1987). In this case, the spouse of an infected animal handler, who applied ointment to herpetic skin lesions on her husband and subsequently to an area of dermatitis on her own hand, contracted B virus infection (Holmes et al., 1990). iii. Clinical signs. The incubation period between the initial exposure and onset of clinical signs ranges from 2 days to, more frequently, 2 - 5 weeks. However, in one case, an individual developed severe clinical disease from B virus 10 years following his last known exposure to the agent. Researchers in the field have also suggested that asymptomatic human B virus infection may occur (Benson et al., 1989), but it is not known whether viral reactivation resulting in severe clinical disease can occur later. In most cases, following exposure by bite, scratch, or other local trauma, humans may develop a herpetiform vesicle at the site of inoculation. In the B virus fatality resulting from ocular

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exposure, the patient did not develop a dendritic corneal lesion typical of ocular herpes infections; rather, she developed a swollen, painful orbit With conjunctivitis (CDCP, 1998). As the clinical signs in this patient progressed, she developed retroorbital pain, photophobia, anorexia, nausea, and abdominal pain. Other early clinical signs of B virus include myalgia, fever, headache, and fatigue, which are later followed by progressive neurological disease Characterized by numbness, hyperesthesia, paresthesia, diplopial ataxia, confusion, urinary retention, convulsions, dysphagia, and an ascending flaccid paralysis. iv. Control and prevention. A key provision to prevent B virus exposures within an institution's animal care and use program concerns the decision of whether or not the studies proposed warrant the Use of macaques. Macaques should be used only when there are no suitable alternative animal models, and efforts to acquire macaques that are free of B virus infection and to maintain them appropriately to preserve this status should be pursued whenever feasible. In B virus endemically infected colonies with individually housed macaques, culling seropositive animals and replacement with seronegative stock might be useful in establishing a B virus-negative colony over time (Weir et al., 1993). However, all macaques must be handled as though they are potentially infected because viral shedding is intermittent and viral serology does not adequately reflect the viral status of the animal. After the outbreak of B virus infection in monkey handlers that occurred in 1987, guidelines were developed to prevent B virus infection in humans (CDC, 1987). Additional provisions for protection against B virus exposure via ocular splash were adopted following the death of a young woman exposed by this route (CDCP, 1998). Readers should refer to these sources or other detailed reviews before engaging in studies involving macaques or developing institutional programs for the prevention and control of B virus among monkey handlers (Adams, 1995; Holmes et al., 1995). Briefly, these recommendations emphasize the need for nonhuman primate handlers to conform fully with a written comprehensive personal protective equipment (PPE) program based on a thorough hazard assessment of all work procedures, potential routes of exposure, and potential adverse health outcomes (CDCP, 1998). Approaches to hazard assessment and the development of occupational health and safety programs for research animal facilities have been reviewed extensively in other sources (Committee on Occupational Health and Safety in Research Animal Facilities, 1997). Protective clothing, including leather gloves or long-sleeved garments for hand and arm protection and protective goggles designe d for splash protection, along with a mask, are considered essential equipment to protect other mucous membranes from exposure to macaque secretions. The use of a face shield is insufficient as the sole method for protection against ocular exposure because droplet splashes to the head may drain into the eyes and infectious materials may enter via the gap along the

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margins of the shield. Importantly, personal eyeglasses are not considered PPE. The use of latex gloves alone for hand protection should be reserved for the handling of monkeys under full chemical restraint. Chemical restraint or specialized restraining devices should be used whenever possible for nonhuman primates to reduce personnel injuries. The CDC recommendations further specify that institutions should be prepared to handle patients with a suspected B virus exposure promptly, and the patient should have direct and immediate access to a local medical consultant knowledgeable about B virus. This consultant should be available at any time the worker is concerned that potential occupational exposure to B virus may be relevant to worker symptoms. The wound should be cleansed thoroughly, and serum samples and cultures should be obtained for serology and viral isolation from both the patient and the monkey. The initiation of antiviral therapy with acyclovir or gangcyclovir may also be warranted if the history and symptoms are compatible with B virus infection. The management of antiviral therapy in B virus-infected patients is controversial because increasing antibody titer has been demonstrated in a patient following the discontinuation of acyclovir therapy (Holmes et al., 1995). Physicians should consult the Viral Exanthems and Herpesvirus Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, for assistance in case management. Additional information about the B virus diagnostic resources is available through the B Virus Research and Resource Laboratory, Georgia State University, Atlanta, Georgia.

E.

Rabies

Rabies is an acute, almost invariably fatal disease caused by a virus in the genus Lyssavirus of the family Rhabdoviridae (Johnson, 1989). i. Reservoir and incidence. Rabies occurs worldwide with the exception of a few countries, generally island nations, and other regions that have excluded the disease through animal importation and control programs and the aid of geographic barriers. Rabies virus infects all mammals; however, the main reservoirs are wild and domestic canines, cats, skunks, raccoons, bats, and other biting animals. In the United States the dog, cat, and other domestic animals have declined in importance as sources of exposure in confirmed human cases of the disease (CDCP, 1999). According to one study however, 18.4% of 3329 domestic animals that were reportedly exposed to rabiespositive wild animals are unvaccinated (Wilson et al., 1997). The disease historically has not been a problem in the laboratory animal facility setting. Among the rodent and lagomorph species maintained in the laboratory, the wild-caught groundhog and rabbit appear to represent a risk of transmitting rabies (Childs et al., 1997; Karp et al., 1999). However, because the

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JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

incidence of rabies in wildlife in the United States has risen, the possibility of rabies transmission to dogs, cats, or other species having uncertain vaccination histories and originating from an uncontrolled environment must be considered. In addition, rabies-susceptible wildlife species studied in the field or introduced into the laboratory for special research investigations could also have the potential to harbor rabies virus. In the skunk and some bat species, rabies virus strains appear to have adapted to growth in the lung, producing respiratory tract infections, and the virus is spread via the aerosol route (Johnson, 1989). Of the human cases of rabies in the United States reported since 1990, most have involved bat-associated viral variants; and the predominant strain has been associated with two insectivorous bat species, Eastern pipistrelle bats (Pipistrellus subflavus) and silver-haired bats (Lasionycteris noctivagans) (CDCP, 1999). Another rabieslike lyssavirus causing fatal human encephalitis has also been recently isolated from bats in Australia, a country believed to have a rabies-free status, further emphasizing the bat as a potential reservoir for these agents (Anonymous, 1997; Gerrard, 1997).

ii. Mode of transmission. Rabies virus is transmitted by the bite of a rabid animal or by the introduction of virus-laden saliva into a fresh skin wound or an intact mucous membrane. Airborne transmission can occur in a laboratory setting and in caves where rabid bats roost (Johnson, 1989). In the bat-associated cases reported in the United States since 1990, a majority of patients handled bats but denied being bitten, and many had no known bat exposure (CDCP, 1999). The virus has also been transmitted through corneal transplants from individuals with undiagnosed central nervous system disease. iii. Clinical signs. Humans, as well as all other mammals, are generally regarded as susceptible to this disease. The course of the disease proceeds through several phases: incubation, prodromal, acute neurologic, coma, and rarely, recovery (Johnson, 1989). The incubation period in humans is ordinarily 13 months but may vary from 9 days to over 8 months. During the prodromal stage lasting 2 - 4 days, patients experience a period of apprehension and develop headache, malaise, and fever. An abnormal, indefinite sensation at the site of a prior animal bite wound is the first specific symptom. Patients may also develop intermittent periods of excitation, nervousness, or anxiety interspersed with quiet periods when the mental state appears normal. Further progression of the disease is marked by paresis or paralysis, inability to swallow, and the related hydrophobia, delirium, convulsions, and coma. Rabies produces an almost invariably fatal acute viral encephalomyelitis, with death due to respiratory paralysis. iv. Diagnosis and prevention. Rabies should be considered as a differential diagnosis in any wild-caught or random-source laboratory animal of unknown vaccination history exhibiting

encephalitic signs. Any wild animal that has bitten someone should be submitted for rabies examination in a manner that permits definitive identification of the species for epidemiologic purposes if the species is not already known. The postmortem diagnosis of rabies virus infection can be based on the demonstration of Negri bodies, rabies virus antigen, or infectious virus from the brain of the infected animal (Johnson, 1989). Ammon's horn of the hippocampus is the best site for the demonstration of Negri bodies or rabies antigen by direct fluorescent antibody (DFA) test, but other brain areas should be sampled. Corneal impression smears or scrapings may also be a reliable site for the demonstration of antigen by DFA; mucosal scrapings and frozen skin biopsy specimens may also be used but are regarded as less reliable and have not been widely adopted by diagnostic laboratories. Many tissues are useful for virus isolation, especially brain and submaxillary salivary gland. Vigorous first-aid and wound-care procedures for bites and scratches inflicted by animals are a crucial first step against the transmission of rabies virus to humans. General guidelines for proper administration of rabies postexposure prophylaxis (RPEP) treatments have been published by the Advisory Committee on Immunization Practices. Guidelines stipulate that unvaccinated persons potentially exposed to rabies should be treated with human rabies immune globulin and a 5-dose series of rabies vaccine administered (CDCP, 1999). Surprisingly, one study suggests that the use of RPEP is often inappropriate (Moran et al., 2000). The authors urge the routine use of current published guidelines, physician education, and improved coordination with public health officials. The control of rabies through vaccination programs, animal control measures, and rabies surveillance efforts is an equally important factor in the prevention of rabies cases in the domestic animal population and therefore in humans. Whenever possible, animals brought into the laboratory should have histories that preclude their exposure to rabies or assure that they have been vaccinated for this disease. However, due to the potential for a long incubation in the natural history of the disease, the types of animal models utilized, or the prospect of vaccination failures, most institutions are likely to encounter situations where a rabies-free environment cannot be assured adequately. Thus, preexposure immunization to rabies should be available to personnel working in high-risk categories, such as veterinarians, people who are working with or involved in the care of high-risk or inadequately characterized animals, and field biologists who work in rabies-endemic areas.

F.

Viral Hepatitis Infections

Many of the nonhuman primate zoonoses causing systemic infections in humans include hepatitis as one component of the disease. However, of the viral infections that target the liver

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as the primary site of involvement, only hepatitis A virus has proven to be a significant zoonotic pathogen in the laboratory animal facility environment. Nonhuman primates are important experimental hosts in viral hepatitis research and have been used to study hepatitis A, B, C, D, and E infections (Adams, 1995). The pig and wild rat are also natural hosts for hepatitis E infection and have been incriminated as zoonotic reservoirs for this agent, but no instances of transmission have been reported in the laboratory animal facility setting (Kabrane-Lazizi et al., 1999). Mouse hepatitis virus, a coronavirus, and infectious canine hepatitis, an adenovirus, are not transmissible to humans. 1.

Hepatitis A

i. Reservoir and incidence. Hepatitis A virus (HAV) is a human enterovirus belonging to the family Picornaviridae (Purcell et al., 1989). The primary reservoirs for HAV infection are humans, with nonhuman primate infections resulting from contact with infected humans or other infected nonhuman primates. However, more than 100 cases of HAV infection in humans have been associated with newly imported chimpanzees. There are also many other nonhuman primate species that are naturally susceptible to HAV, including the other great apes, marmosets, owl monkeys, cynomolgus monkeys, and patas monkeys, and could serve as sources for human HAV infection (Shevtsova et al., 1988; Purcell et al., 1989; Lemon et al., 1990; Adams, 1995). A suspected outbreak of HAV infection in young domestically reared rhesus monkeys has demonstrated the need for continued attention to this zoonotic agent in the laboratory animal facility environment (Lankas and Jensen, 1987). ii. Mode o f transmission. Hepatitis A virus is transmitted by the fecal-oral route, and some outbreaks can be related to poor hygienic conditions or contaminated food and water. iii. Clinical signs. The disease in nonhuman primates is much less severe than the disease in humans and is frequently subclinical. Clinical disease develops in the chimpanzee, owl monkey, and several marmoset species and is characterized by malaise, vomiting, jaundice, and elevated serum levels of hepatic enzymes. The disease in humans varies from a mild illness lasting 12 weeks to a severely debilitating illness lasting several months (Hollinger and Glombicki, 1990). Following an incubation period of approximately 1 month, patients experience an abrupt onset of fever, malaise, anorexia, nausea, and abdominal discomfort followed within a few days by jaundice (Fig. 2). Children often have mild disease without jaundice, whereas HAV infections in older patients may be fulminant and protracted with prolonged convalescence. However, protracted HAV infection is considered an acute infection that is ultimately resolved by the patient; a chronic hepatitis A carrier state has never been shown to exist.

Fig. 2. Humanliver with hepatitis A infection. Leukocyticinfiltration of portal areas with hepatocellular necrosis in the peripheral areas of the lobules. Hematoxylinand eosin, x 250. (Courtesyof Dr. K. Ishak, ArmedForcesInstitute of Pathology.)

iv. Diagnosis and prevention. Enzyme immunoassay (EIA) and radioimmunoassay (RIA) for the demonstration of IgMspecific anti-HAV in the serum or plasma is useful early in the course of the infection. Later in the course of the infection (6 weeks), a fourfold rise in IgG antibody detected by the immune adherence hemagglutination assay can also be used to diagnose HAV infection. Alternatively, fecal samples can be tested for virus particles, viral antigen, or viral RNA to secure a definitive diagnosis (Purcell et al., 1989). A safe, effective hepatitis A vaccine is now available in the United States and is recommended for individuals at high risk for exposure to HAV infection, such as persons involved with the care of nonhuman primates being used in experimental HAV-infection studies. Passive protection of such persons can also be undertaken through the intramuscular administration of specific immune serum globulin (ISG). Passive protection should be given before experimental animal HAV-infection studies begin because infected animals start shedding HAV at 7-11 days postinoculation and continue shedding for several weeks. The recommended dose of 0.02 ml/kg ISG provides

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2 - 3 months of protection that is of sufficient duration for the period of nonhuman primate HAV shedding (Adams, 1995). Postexposure prophylaxis with ISG at the dose of 0.02 ml/kg within 2 weeks of exposure is also recommended (CDC, 1991). The use of protective clothing, personal hygiene, and appropriate sanitation practices for equipment and facilities will also minimize the potential for zoonotic transmission. 2.

Other Viral Hepatitis Agents

Humans are considered natural hosts for viral hepatitis types B, C, D, and E. In the cases of hepatitis B, C, and D viruses, these agents are transmitted parenterally by exposure to blood or other bodily fluids. Hepatitis B virus (HBV), caused by human hepadnavirus, has been widely studied experimentally in the chimpanzee although the gibbon is known to be susceptible (Adams, 1995), and there has been one report of natural infection in a cynomolgus monkey (Kornegay et al., 1985). In the presumed natural HBV infection in cynomolgus monkeys, HBV infection was suggested by the demonstration of HBV surface antigen in hepatic cells but was not associated with zoonotic disease transmission. These animals developed mild clinical disease characterized by anorexia, elevated hepatic enzyme levels, and hyperbilirubinemia. Also, there are other natural hepadnavirus infections of animals (woodchuck, ground squirrel, and duck) that are used as animal models of HBV infection, but none are transmissible to humans (Adams, 1995). The chimpanzee has been used as an experimental model for the study of hepatitis C and D viruses. Thus, the concern for hepatitis B, C, and D as zoonoses is minimal in the laboratory animal facility environment except where these agents are being used in experimental animal studies. In these cases, personnel should adhere to appropriate precautions when handling nonhuman primates. Hepatitis E virus (HEV) is an enterically transmitted calicivirus that causes acute, icteric, self-limiting disease that may have a high mortality. It has been experimentally transmitted to tamarins, owl monkeys, and cynomolgus monkeys, but there have not been any reports of natural HEV infections in monkeys (Adams, 1995). Other domestic and wild animals are also susceptible to HEV infection (Balayan, 1997). Reports have indicated that domestic pigs and wild rats may serve as reservoirs for this infection (Clayson et al., 1995), with infection in a majority of pigs over 3 months of age in some U.S. Midwestern herds (Meng et al., 1997). These pigs have been reported to appear clinically normal but developed viremia, positive serology, and microscopic signs of hepatic disease. Phylogenetic analyses have shown that the swine HEV is closely related to the human HEV strains in the United States forming a distinct phylogenetic branch (Meng et al., 1997, 1998). Although there have not been any recorded cases of HEV transmission in the laboratory animal facility environment from swine, nonhuman primates, or other laboratory animals, personnel should be instructed to observe proper PPE practices to prevent possible HEV transmission.

G.

Retroviruses

In the wake of the human AIDS epidemic there has been an intense, multifaceted interest in the study of human and comparative retrovirology, and the zoonotic potential for animal retroviruses has clearly been identified. Notably, both HIV-1 and HIV-2 (human immunodeficiency virus) are known to have originated as zoonotic infections on the continent of Africa from the chimpanzee (Pan troglodytes troglodytes) and sooty mangabey (Cercocebus atys), respectively (Chen et al., 1996; Gao et al., 1999). These findings have heightened the concerns about zoonotic retroviral transmission, particularly in connection with the use of nonhuman primates as potential xenograft donors to humans requiring organ transplantation. Similar concerns have been raised about the pig as a donor for xenotransplants to humans because the porcine endogenous retrovirus has been demonstrated to grow in human cells in vitro (Wilson et al., 1998). However, although there are numerous retrovirus infections of wild, laboratory, and domestic animal species, the transmission of these agents from their natural host to humans under laboratory conditions has been documented in only a few cases, both involving nonhuman primates as source species. Further discussion herein is limited to these cases, and the reader should refer to Mansfield and King (1998) for an authoritative review of the retroviral diseases of nonhuman primates. 1.

Simian Immunodeficiency Virus (SIV)

i. Reservoir and incidence. Simian immunodeficiency virus (SIV) is a lentivirus that infects a variety of Old World nonhuman primates and produces a clinical syndrome in rhesus monkeys and other susceptible macaque species with many important parallels to AIDS. Although the seroprevalence of SIV in Asian macaques is low and most SIV infections in these species are related to their use as animal models of HIV infection, the seroprevalence is much higher among wild-caught African nonhuman primate species with host-adapted SIV infections (Hayami et al., 1994). ii. Mode of transmission. Transmission of SIV between monkeys is likely both horizontal and vertical. Vertical transmission may be by sexual contact, as seroconversion to SIV parallels sexual maturity. Direct inoculation of open wounds or mucous membranes with infectious secretions during other activities such as fighting may also play a role, but airborne transmission is regarded as unlikely (Lairmore et al., 1989). The blood, secretions, and tissues of SIV-infected monkeys should be presumed to be infectious for persons potentially exposed to these materials. Two human cases of seroconversion to SIV associated with known exposure incidents have been recognized (CDC, 1992b; Khabbaz et al., 1992), and a blind serological survey of other personnel working with SIV has identified perhaps an additional three seropositive individuals (CDC, 1992a). The possible inclusion of the aforementioned cases of known

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SIV exposure and the cross-reactivity of SIV and HIV-2 in the assay employed confounded the interpretation of this survey (CDC, 1992a). In the first case, the individual's skin was punctured accidentally with a needle contaminated by the blood of an infected macaque. In the second case, a laboratory worker who had dermatitis on the hands and forearms and was under treatment with corticosteroids handled SIV-infected blood specimens without wearing gloves. The pattern of seroreactivity suggested the possibility of infection in the second case, and SIV infection was documented subsequently by PCR techniques (Khabbaz et al., 1994).

iii. Clinical signs. Clinical signs have not been recorded in these cases of human SIV exposure. iv. Diagnosis and prevention. Serological techniques and viral isolation are available for the diagnosis of SIV in Asian macaques, which invariably seroconvert following natural infection (Mansfield and King, 1998), but African species may harbor the virus and not seroconvert. Personnel should be instructed to observe the requirements for the use of PPE when working with potentially infected nonhuman primates and to follow safe syringe/needle handling practices. Potential SIV exposures should be cleansed immediately with soap and water, and supervisory personnel should be informed of the incident. Personnel should be advised to report and to seek medical attention for any acute febrile illness occurring within 12 weeks of exposure. The medical evaluation should include periodic monitoring for serum antibody to SIV at appropriate intervals. Written institutional policies should be in place to address confidentiality, counseling, and other issues related to potential SIV exposure. The absence of data related to the potential for SIV transmission between humans prevents any specific recommendations about the need for modifying personal behavior in the SIV-seropositive individual. However, the SIV-seropositive individual should not donate blood. 2. Simian Foamy Virus Infection The simian foamy viruses are complex retroviruses that have been isolated from a number of New and Old World nonhuman primates and share considerable homology to the human foamy viruses (Mansfield and King, 1998). Although foamy viruses have not been demonstrated as the cause of any disease entity in humans or in the nonhuman primate, they have been linked to a number of human disorders. The transmission of simian foamy viruses to humans accidentally exposed or occupationally exposed to nonhuman primates has been reported (NeumanHaeflin et al., 1993; Heneine et al., 1998). In the case of occupational exposure, 4 of 231 individuals surveyed serologically were positive, and proviral DNA detection and viral isolation were used to confirm the infection (Heneine et al., 1998). In 1 human case, the simian foamy virus originated from African green monkeys; and in the other 3 cases, baboons served as the

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source. Clinical disease in humans has not resulted in any of these cases, and it is suggested that the virus persists in humans in a state of latency following accidental infection (Schweizer et al., 1997). The use of PPE as described in connection with the prevention of SIV transmission to humans should also be applied when handling simian foamy virus-infected nonhuman primates.

H.

Measles Virus (Rubeola, Giant Cell Pneumonia)

i. Reservoir and incidence. Measles virus is a member of the genus Morbillivirus, which is known to cause infection in a wide variety of Old and New World nonhuman primate species (Mansfield and King, 1998). Humans serve as the reservoir with nonhuman primates becoming infected through contact with human populations with endemic measles. The disease spreads rapidly through infected nonhuman primate colonies, often with devastating consequences, and a 100% seroconversion rate to measles is common in wild-caught nonhuman primate populations within several weeks of capture. A recent serological survey in nonhuman primates from miscellaneous sources has indicated that in three macaque species commonly used in biomedical research, between 45 and 73% of the animals were seropositive for measles virus, but that less than 10% of the squirrel monkeys and marmosets surveyed were seropositive (Kalter et al., 1997). This finding possibly reflects the fact that these New World monkey species are more vulnerable and less likely to survive the disease than macaques. With the current emphasis on and success of domestic nonhuman primate production, it has become more likely that institutions will develop large populations of susceptible nonhuman primates that could contract measles and then transmit the disease to susceptible humans, as previously reported (Roberts et al., 1988). ii. Mode of transmission. Measles is a highly communicable disease that is transmitted by infectious aerosols, contact with nasal or throat secretions, or contact with fomites freshly contaminated with infectious secretions. The virus is shed beginning in the prodromal stage and continuing through the exanthematous stage of the infection. iii. Clinical signs. The clinical signs of measles are similar in both nonhuman primates and humans. In humans, after an incubation period of about 10 days, fever develops followed by conjunctivitis, coryza, cough, and Koplik's spots on the buccal mucosa. Subsequently, a characteristic exanthematous rash develops beginning on the face, becoming generalized over the body, and progressing to a dry and scaly desquamative dermatitis. Complications of viral replication or secondary bacterial infection can result in bronchopneumonia, otitis media, diarrhea, or rarely, encephalitis. iv. Diagnosis and prevention. Characteristic clinical signs generally obviate the need for other diagnostic methods for

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measles, but serology, immunofluorescent antibody, or PCR screening for virus in clinical specimens or viral isolation can be used. Vaccination of susceptible nonhuman primates with a modified live vaccine is effective in preventing the disease, and treatment of exposed animals with human gamma globulin may be useful in controlling disease during epizootics (Roberts et al., 1988). Vaccination for measles should be assured for all handlers of nonhuman primates. The reader should refer to an excellent overview of the characteristics of a measles epizootic and the discussion of disease management in a nonhuman primate colony for additional information on factors relevant to the control and prevention of this disease (Willy et al., 1999).

I.

Newcastle Disease Virus

i. Reservoir and incidence. Newcastle disease is caused by a paramyxovirus and is seen among wild, pet, and domestic birds. Wild birds transmit the infection to domestic bird populations (Mufson, 1989). The zoonotic potential of this agent in the laboratory environment has been realized on numerous occasions (Barkley and Richardson, 1984). ii. Mode of transmission. Aerosol transmission is the important means of spread to humans, but contaminated food, water, and equipment also transmit infection within bird populations. iii. Clinical signs. The severity of the disease in birds depends on the pathogenicity of the infecting strain. Highly pathogenic strains have largely been excluded from flocks within the United States. Moderately pathogenic strains produce anorexia and respiratory disease in adult birds and neurologic signs in young birds. In humans, the disease is characterized by a follicular conjunctivitis that resolves without complications and without therapy. Mild fever and respiratory involvement ranging from cough to bronchiolitis and pneumonia can also be seen in humans.

Animal reservoirs are thought to contribute to the emergence of new human strains of influenza infection by the passage of avian influenza viruses through pigs, which act as intermediate hosts. This is believed to involve multiple mutational or reassortment events, and once established in the pig, transmission occurs by the airborne route (Webster, 1997). In the laboratory, ferrets are highly susceptible to human influenza and often are used as experimental models of influenza infection (Harmon and Kendal, 1989). ii. Mode of transmission. Transmission occurs by airborne spread of the virus and by direct contact through droplet spread. The transmission of animal influenza strains from animals to humans is an uncommon occurrence. However, a study has shown that pigs experimentally infected with influenza virus in the laboratory can directly and readily spread this agent to persons working with these animals (Wentworth et al., 1997). Also, ferrets housed in the laboratory will develop epizootic infection concomitant with human outbreaks of the disease. Ferret-tohuman transmission of the virus has also been documented (Marini et al., 1989). iii. Clinical signs. Influenza is an acute disease of the respiratory tract and is characterized by fever, headache, myalgia, prostration, coryza, sore throat, and cough. Viral pneumonia and gastrointestinal involvement manifested by nausea, vomiting, and diarrhea may also develop. iv. Diagnosis andprevention. Personnel shouldwearproper protective clothing and practice appropriate personal hygiene measures if contact is unavoidable with experimentally infected animals or with ferrets suspected of having natural influenza infection.

llI.

A. iv. Diagnosis and prevention. This disease can be prevented in the laboratory environment by immunizing birds susceptible for this disease or obtaining birds from flocks known to be free of this agent. Personal hygiene practices should also be in place to prevent zoonotic transmission.

J.

Influenza Virus

i. Reservoir and incidence. Humans are considered the reservoir for human influenza virus infections. However, influenza virus infections from different antigenic strains occur naturally in many animals, including avian species, swine, horses, ferrets, mink, and seals (Harmon and Kendal, 1989).

R I C K E T T S I A L DISEASES

Murine Typhus (Endemic Typhus)

Murine typhus is caused by Rickettsia typhi. Although this disease has been recognized for centuries, not until the 1920s was it distinguished from epidemic typhus. The absence of louse infestation in humans, seasonal occurrence, and sporadic nature help differentiate it from louse-borne typhus (i.e., epidemic typhus). Epidemic typhus is seen only in the eastern United States in association with flying squirrels (Duma et al., 1981). i. Reservoir and incidence. Murine typhus is worldwide, and in the United States it is usually diagnosed in southeastern or Gulf Coast states and in areas along the northern portion of the Mississippi River and southern California. It is also associ-

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ated with human populations subjected to areas of high-density wild rat colonies, such as ports, granaries, farms, or rat-infested buildings in inner cities. Laboratory personnel have been infected with this agent when inoculating rodents and handling infected animals. Since the 1970s, there has been a shift in the distribution of human cases of murine typhus to more rural locales in southern California and central and south Texas (Adams et al., 1970). Southern California was considered an unusual locale because Orange County was considered a wealthy suburb where rat infestation was uncommon. Epidemiologic studies indicated that opossums had a high seropositivity to murine typhus, and the cat fleas infesting the opossums were infected with either R. typhi or a newly recognized rickettsia first called ELB agent and later R. felis (Adams et al., 1990; Williams et al., 1992). Findings extended to a survey of fleas on dogs, cats, and opossums in California, Texas, and Georgia also confirmed that fleas were infected with R. typhi or R. felis, helping explain the spread of murine typhus into rural areas in the United States. Also, human cases of typhus caused by R. felis, based on PCR, have been recorded (Schriefer et al., 1994). Determining exact taxonomic specifications of R. felis has not been possible because no isolates have been obtained for detailed comparative analysis.

ii. Mode of transmission. Murine typhus is primarily a disease of rats, with its principal vectors being the oriental rat flea, Xenopsylla cheopis, and the flea Nasopsyllus fasciatus. These fleas will also naturally colonize the mouse Mus musculus. The cat flea, Ctenocephalidesfelis, (as well as seven other species of fleas) has also been implicated in the spread of the disease. Rickettsiae are ingested by a blood meal of the flea, where they multiply in the gut, and are subsequently passed out in the dejecta of the flea. Infection in the rat and the human is the result of contamination of the puncture wound by flea feces (FarhangAzad et al., 1985). Experimental evidence indicates that a flea bite can also directly transmit the infection (Farhang-Azad et al., 1985). Rickettsia typhi are resistant to drying and remain infectious for up to 100 days in rat feces. iii. Clinical signs. After infection with rickettsia, the incubation period is 7-14 days. Because murine typhus is difficult to differentiate either clinically or anatomically from other rickettsial diseases, specific serological tests or PCR-based assays are extremely important in making the correct diagnosis (Farhang-Azad et al., 1985). The acute febrile disease is usually characterized by general malaise, headache, rash, and chills, with signs ranging from mild to severe. An encephalitic syndrome can also occur (Mushatt and Hyslop, 1991). In one report, 25% of 180 patients with the disease had delirium, stupor, or coma. Fortunately, these findings resolve with lowering of the febrile response. Fatality rate for all ages is about 2% but increases with age. Proper antibiotic therapy is the most effective measure to prevent morbidity or mortality due to rickettsial infections. Tetracycline and chloramphenicol have proven to be

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effective in hastening recovery and preventing neurologic sequelae, such as deafness due to eighth cranial nerve involvement (Mushatt and Hyslop, 1991).

iv. Diagnosis and control. Recovery of rickettsial organisms or antigens from biological specimens is inconsistent and is not routinely done except in labs equipped to process and identify these samples. It must be stressed that manipulation of rickettsia in the laboratory is hazardous and has accounted for numerous infections of laboratory personnel. Currently, serological diagnosis is accomplished by ELISA and RIA; however, the IFA technique remains the most commonly used. Unfortunately, this test cannot distinguish epidemic from endemic typhus. The CDC considers a fourfold rise in titer detected by any technique (except Weil-Felix) as evidence of rickettsial infection. Complement fixation titer of 1:16 or greater in a single serum sample from a patient with clinically compatible signs is also considered diagnostic (McDade and Fishbein, 1988). Newer PCR techniques specific for rickettsial species are increasingly being used, which in time may replace serological tests. Fleas can be controlled by applying insecticides (organochlorines, as well as others) as residual powders or sprays in areas where rats nest or traverse. It is imperative that insecticides be applied prior to using rodenticides; this will prevent fleas from leaving the dead rodents and feeding on human hosts (Beaver and Jung, 1985). This disease should not be encountered in rat colonies in well-maintained research vivaria. However, with the cat flea being a newly recognized vector, its presence on random-source dogs, cats, and opossums raises the risk of transmission of murine typhus to personnel working with these fleainfested animals.

B.

Rickettsial Pox

A variety of rodents are infected with other rickettsial diseases. Mus musculus is the natural host for the causative agent of rickettsial pox, R. akari, a member of the spotted fever group of rickettsia (Chin, 2000). This organism is also isolated from Rattus rattus and R. norvegicus, and the rat under certain circumstances may transmit the disease to humans. The disease is transmitted by the mite Liponyssoides (Allodermanyssus) sanguineus and has been diagnosed in New York City and other eastern cities, as well as in Russia, Egypt, and South Africa (Chin, 2000). The incubation period is approximately 1024 days, and the clinical disease is similar to that noted in murine typhus. The rash of rickettsial pox commences as a discrete maculopapular rash, which then becomes vesicular. The palms and soles are usually not involved. About 90% of affected persons develop an eschar, with a shallow ulcer covered by a brown scab (Farhang-Azad et al., 1985; Chin, 2000). Although headaches are common and may be accompanied by stiff necks, lumbar cerebrospinal fluid (CSF) samples are normal. Pulmonary

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JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

and gastrointestinal involvement also are almost never encountered. Diagnosis, treatment, and control are similar to those described for murine typhus and Yersinia pestis.

C.

Coxiella burnetii Infection (Q Fever)

i. Reservoir and incidence. Coxiella burnetii, the causative agent of Q fever, has a worldwide distribution perpetuated in two intersecting cycles of infection composed of domestic or wild animals and their associated ticks (Babudieri, 1959; Marrie, 1990). The domestic animal cycle involves mainly sheep, goats, and cattle. The prevalence of the infection among sheep is high throughout the United States, and sheep have been the primary species associated with disease outbreaks, including those occurring in research animal facilities (Asher, 1989). However, human cases of the disease have also been associated with nonruminants, such as pregnant cats (Langley et al., 1988) and wild rabbits (Marrie et al., 1986). Thus, a broad range of domestic and wild animal species, including birds, should be given consideration as potential sources for Q fever infection in animal care and use activities (To et al., 1998). ii. Mode of transmission. Coxiella burnetii are shed in the urine, feces, milk and especially placental tissues of domestic ungulates that generally are asymptomatic. The placenta of infected ewes can contain up to 109 organisms per gram of tissue, and milk may contain 105 organisms per gram (CDCP-NIH, 1999). The organism is highly infectious with possibly as few as 10 organisms inducing infection (CDCP-NIH, 1999). The primary method of transmission is through infectious aerosols. The organism produces a sporelike form that is resistant to desiccation and persists in the environment for long periods of time, contributing to the widespread dissemination of infectious aerosols and resulting in infections miles from the original organism source (Franz et al., 1997; Tissot-Dupont et al., 1999). The importance of these factors was illustrated in outbreaks of the disease associated with the use of pregnant sheep in research facilities in the United States (Bernard et al., 1982). In these outbreaks, personnel who did not have direct contact with infected sheep but who worked along the transport routes for these animals became serologically positive for Q fever (Bernard et al., 1982; Reimer, 1993). Also, five of nine laundry workers without direct sheep contact but who processed linens soiled during sheep surgery developed serological evidence of infection.

nephritis, epicarditis, and endocarditis, may also occur. Individuals with valvular heart disease should not work with C. burnettii due to the prospect of serious, chronic, relapsing infection (Asher, 1989; CDCP-NIH, 1999). iv. Diagnosis, prevention, and control. Whenever possible, male or nonpregnant sheep should be used in research programs; however, many research applications specifically call for the use of pregnant animals. Several commercial vendors now supply sheep from flocks that have not had serological evidence of infection for an extended period. Although serological status is not a useful indicator of organism shedding in individual sheep, many institutions have elected to use these animals, reasoning that cumulative and consistent negative Q fever serology on a herd basis provides a reasonably strong assurance of Q fever-free status. Advances in polymerase chain reaction (PCR) methods have improved the sensitivity of C. burnetii detection over that of the antigen capture ELISA, as well as improved the speed, safety, and convenience of the assay. The PCR method may offer some hope that the potential for organism shedding could be assessed on an individual animal basis to minimize the potential risk of Q fever outbreaks in animal facilities (Lorenz et al., 1998; Yanase et al., 1998). Sheep and other animals harboring Q fever infections should be maintained under animal biosafety level 3 (ABSL-3) conditions to prevent the transmission of the organism in the research animal facility environment (CDCP-NIH, 1999). Additional detailed recommendations have been published concerning sheep handling in biomedical research programs (Bernard et al., 1982). In many institutions, ABSL-3 conditions would prove to be unachievable for sheep held under agricultural conditions for food and fiber production or for instructional exercises. The use of personal protective equipment conforming to ABSL-3 practices is important in these settings even though the facilities may be deficient in meeting ABSL-3 criteria. An effective Q fever vaccine is licensed in Australia (Q-Vax), and an investigational new phase 1 Q fever vaccine (IND) is available from the Special Immunizations Program, U.S. Army Medical Research Institute for Infectious Disease (USAMRIID), Fort Detrick, MD 21701 (Franz et al., 1997; CDCP-NIH, 1999). The use of this vaccine should be limited to personnel at high risk of exposure and who have no demonstrated sensitivity to Q fever antigen.

IV. iii. Clinical signs, susceptibility, and resistance in humans. Q fever in humans varies in duration and severity, and asymptomatic infection may occur. The disease often presents as a flulike illness with fever, frontal headache with retro-orbital pain, and chest pain with a nonproductive cough and pneumonia, resolving within 2 weeks of infection. However, serious extrapulmonary complications, such as acute or chronic hepatitis,

CHLAMYDIAL INFECTIONS

AO Chlamydiosis (Psittacosis, Ornithosis, or Parrot Fever) i. Reservoir and incidence. The taxonomy for the order Chlamydiales has recently been revised based upon ribosomal

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operon genes and phenotypic characteristics of the chlamydial organisms, and many of the new species may be unfamiliar to the reader (Everett et al., 1999). Chlamydial agents are widely distributed among birds and mammals worldwide and occur naturally among many laboratory animal species, including birds, mice, guinea pigs, hamsters, rabbits, ruminants, swine, cats, ferrets, muskrats, and frogs (Storz, 1971; Newcomer et al., 1982). Of these host species, birds with Chlamydophila psittaci comb. nov. (previously Chlamydia psittaci) infection, particularly psittacines, have proven to be the most frequent sources of virulent human infection (CDCP, 1997); however, infections in ruminants (Hyde and Benirschke, 1997; Jorgesen, 1997) and cats (Cotton and Partridge, 1998) have also been involved in serious human cases of the disease. The most common human chlamydial infection, Chlamydia trachomatis, is not naturally transmissible to animals but is used to produce experimental infections in nonhuman primates. Chlamydia muridarium occurring in the mouse and C. suis occurring in the pig are closely related to C. trachomatis but are not infectious for humans. Also, Chlamydophila pecorum comb. nov. (previously Chlamydia pecorum) produces intestinal infection in ruminants and other animals but not in humans, and C.hlamydophila pneumoniae comb. nov. (previously Chlamydia pneumoniae) produces respiratory infections in humans and has been isolated from only the koala, horse, and frog (Berger et al., 1999). Zoonotic infections from Chlamydiophila pneumoniae have not been recorded.

ii. Mode of transmission. The organism is spread to humans from infectious material present in exudates, secretions, or desiccated fecal material by direct contact or the aerosol route. Latent infection is an important feature of epizootology of the Chlamydiophila psittaci infection in birds; stress can reactivate enteric shedding of the organism and clinical signs (Storz, 1971). iii. Clinical signs, susceptibility, and resistance in humans. Chlamydiae produce a diverse spectrum of animal disease, including conjunctivitis, pneumonitis, air sacculitis, pericarditis, hepatitis, enteritis, arthritis, meningoencephalitis, urethritis, endometritis, and abortion. Zoonotic infections in humans are characterized mainly by upper and lower respiratory tract complaints; however, conjtinctivitis, thrombophlebitis, myocarditis, hepatitis, and encephalitis have also been reported (Smith, 1989; Leitman et al., 1998). Although the avian strains of the organism are considered to be more pathogenic for humans than are the mammalian strains, ovine strain-related (Chlamydophila abortus gen. nov. sp. nov.) human gestational infections (Hyde and Benirschke, 1997; Jorgesen, 1997) and feline pneumonitis strain-related (Chlamydophila felis gen. nov. sp. nov.) conjunctivitis, pneumonia, and extrapulmonary infection (Cotton and Partridge, 1998) emphasize the relevance of diverse reservoir hosts in the human disease.

iv. Diagnosis and control. The diagnosis of C. psittaci in birds is based upon positive complement fixation serology (titer

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> 1 : 16), ELISA-based fecal antigen tests, identification of inclusions in tissue specimens, or impression smears or organism isolation. Birds used in research animal facilities should be acquired from flocks free from C. psittaci infection. Antibiotic treatment should be provided to wild-caught birds or birds of unknown disease status, as well as to mammalian or amphibian species with chlamydial infection if these are used in research programs. Personnel protection adhering to ABSL-2 procedures along with respiratory protection is generally adequate, but ABSL-3 procedures are warranted for activities with the high potential for droplet or infectious aerosol production (CDCP-NIH, 1999).

V.

A. 1.

B A C T E R I A L DISEASES

Trauma-Associated Bacterial Diseases

Bites and Scratches

Several million Americans annually suffer animal bites, which continues to be a major health problem in the United States and accounts for approximately 1% of emergency room visits. Dogs and cats are responsible for 90% of the recorded bites (Weber and Hansen, 1991; Talan et al., 1999), and each year dog attacks account for 10 to 20 deaths in the United States. Veterinarians, animal control officers, and presumably animal care personnel in research facilities as well as in municipal pounds are at higher risk of bites than the general population. Although rabies is the most serious public health threat from bites and scratches, the risk of bacterial infection from dog bites is lower (approximately 3-18%) than that from cat bites, which is reported to be approximately 28-80% (Weber and Hansen, 1991). It is estimated that 400,000 persons in the United Statesare bitten or scratched by cats annually. According to one report, approximately 40,000 rat bites are recorded annually (Committee on Urban Pest Management, 1980). As with bites from dogs and cats, the majority of rat bites occur in children. It is estimated that 2% of rat bites become infected (Ordog, 1985). Animals in general have a complex oral microflora consisting of numerous bacterial species; both aerobic and anaerobic bacteria are therefore routinely isolated from traumatic bite wounds inflicted by domestic and wild animals. Common organisms isolated from dog bites include Staphylococcus species, Streptococcus species, a variety of anaerobes, and Pasteurella multocida. In a comprehensive multicenter study, 60% of dog-bite wounds were punctures, 10% were lacerations, and 30% were a combination of both. This compared to 85% of cat-bite wounds being punctures, 3% lacerations, and 12% a combination of both. In this study, 39% of 57 patients with cat bites presented as purulent wounds, whereas abscesses were present in 19% of the cases reviewed (Talan et al., 1999). Of the

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JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

50 patients with dog bites, 58% had purulent wounds, 30% were nonpurulent, and 12% were noted to have abscesses. Dog and cat bites had a mean of 5 bacterial species per wound; 63% of the cat bites analyzed compared to 48% of dog bites had a mixed anaerobic and aerobic population (Talan et al., 1999). Only aerobes grew in 36% of the cases (42% of dog bites and 32% cat bites), whereas anaerobes were the only species grown in 1% of the cases. Capnocytophaga canimorsus, an invasive organism, was recovered from 4.7% of the wounds. It should be noted that if fever occurs in immuncompromised patients after a bite wound, this organism should be considered in the differential diagnosis. Erysipelothrix rhusiopathiae was isolated from two cat-bite wounds, whereas Pasteurella spp. were present in the wounds 75% of the time in cats and 50% in dogs. Geographic locale is also important in defining bacterial flora of bites and scratches. In a study conducted in the southwestern and central United States, 17 of 1041 (1.6%) of the cases of tularemia in humans diagnosed from 1981 to 1987 were associated with cat scratches or bites (Taylor et al., 1991). Several bacterial pathogens have been isolated from rat bites, including Leptospira interrogans, Pasteurella multocida, and Staphylococcus species; however, the most commonly isolated pathogens are Streptobacillus moniliformis and Spirillum minus (Fox, 1999). Bite wounds from primates and ferrets (and other laboratory animals) can also result in bacterial infection. For example, a chronic Mycobacterium bovis infection on the hand of a human resulted from a ferret bite that had occurred 22 years previously (Jones et al., 1993). The greatest concern from macaque bites still remains the threat of B virus infection.

2.

Atypical Mycobacteriosis

i. Reservoir and incidence. The rapidly growing mycobacteria (RGM) Mycobacterium fortuitum, M. chelonae, and M. abscessus are ubiquitous, being found in soil throughout the world. Mycobacterium chelonae was first isolated from sea turtles; M. fortuitum from frogs (originally called ranae); and M. abscessus, as the name implies, from soft tissue abscesses of a patient. Of the nontuberculosis mycobacterium belonging to Runyon group I, M. marinum is by far the most common. The organism was first isolated from cutaneous lesions in 1826 and was responsible for the death of saltwater fish in a Philadelphia aquarium 100 years later; the authors named the mycobacterium M. marinum.

ii. Mode of transmission. The RGM most commonly are associated with a traumatic injury with potential soil contamination and result in skin, soft tissue, or bone disease. Mycobacterium marinum is pathogenic only on abraded skin; a disruption of the epidermis must be present for development of disease. Because this organism is recognized as a pathogen in zebrafish, it can be a source of infection in personnel working with this species in a research environment.

iii. Clinical signs. Mycobacterium marinum is a free-living mycobacterium that causes disease in fresh-water and saltwater fish and occasionally in humans. It is often called swimming pool granuloma or fish tank granuloma because of the association with these two environmental exposures and human infections. Importantly, M. marinum, because of its optimum growth at 300-32 ~C, is primarily localized to skin infections. However, it can extend to deeper tissues, including joints and tendons. For individuals exposed to diseased fish and/or their environment, the lesions are in general located on the backs of hands or fingers or forearms. Infections have also resulted from the bite of a dolphin (Flowers, 1970). iv. Diagnosis and control. Identification for the common RGMs and M. marinum has been given low priority and is only performed routinely in reference laboratories. Fortunately however, PCR-based assays have become available for rapid diagnosis of atypical mycobacteria. 3.

Rat-Bite Fever

Rat-bite fever (RBF) can be caused by either of two microorganisms: Streptobacillus moniliformis or Spirillum minus. Streptobacillus moniliformis causes the diseases designated as streptobacillary fever, streptobacillary rat-bite fever, or streptobacillosis (McEvoy et al., 1987; Rupp, 1992; Chin, 2000). Haverhill fever and epidemic arthritic erythema are diseases associated with ingestion of water, food, or raw milk contaminated with S. moniliformis. Sodoku is derived from the Japanese words for rat (so) and poison (doku), and is used to designate infection with Spirillum minus. Spirillosis and spirillary rat-bite fever are other names given to the infections caused by Spiril-

lum minus. i. Reservoir and incidence. These organisms are present in the oral cavity and upper respiratory passages of asymptomatic rodents, usually rats (Wilkins et al., 1988). Streptobacillus moniliformis has been isolated as the predominant microorganism from the upper trachea of laboratory rats in one study (Paegle et aL, 1976). Other surveys indicate isolation of the organism in 0/15, 7/10, 2/20, 7/14 laboratory rats and 4/6 wild rats (Geller, 1979). The incidence of S. moniliformis is probably lower in high-quality, commercially reared specific pathogenfree rats. Surveys in wild rats indicate 0 - 2 5 % infection with Spirillum minus (Hull, 1955) or 50-100% for Streptobacillus moniliformis. ii. Mode of transmission. The bite of an infected rat is the usual source of infection. In some cases, bites from other animals, including mice, gerbils, squirrels, weasels, ferrets, dogs, and cats, or rare traumatic injuries unassociated with animal contact cause the infection. iii. Clinical signs. Rat-bite fever is not a reportable disease, which makes its prevalence, geographic location, racial data,

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and source of infection in humans difficult to assess. The disease, though uncommon in humans, has nonetheless appeared among researchers or students working with laboratory rodents, particularly rats (Anderson et al., 1983). Historically, bites from wild rats and subsequent illness (usually in small children) relate to poor sanitation and overcrowding (Hull, 1955). One survey of rat bites in Baltimore tabulated rat-bite fever in 11 of 87 cases (Brooks, 1973). The disease can also occur in individuals who have no history of rat bites but reside or work in ratinfested areas. Exposure to dogs and cats who prey on wild rodents may also be the source of the organism. Ingestion of milk, food, or water contaminated with rat feces can result in RBF (CDC, 1995). The incubation period for S. moniliformis infection varies from a few hours to 2 - 1 0 days, whereas the incubation period for Spirillum minus infection, most commonly seen in Asia, ranges from 1 to 6 weeks (Table II). Fever is present in either form. Inflammation associated with the bite and lymphadenopathy are frequently accompanied by headache, general malaise, myalgia, and chills. The discrete macular rash that often appears on the extremities may generalize into pustular or petechial sequelae. Arthritis occurs in 50% of all cases of Streptobacillus moniliformis but is less common in Spirillum minus. Streptobacillus moniliformis may be cultured from serous to purulent effusion that is recovered from affected larger joints. Most cases of RBF resolve spontaneously within 14 days; however, 13% of untreated cases are fatal (Sens et al., 1989). Prophylactic efficacy of antibiotic treatment following rat bites has not been thoroughly investigated. If antibiotic treatment (intravenous penicillin for 5 - 7 days, followed by oral penicillin for 7 days) is not instituted early, complications such as pneumonia, hepatitis, pyelonephritis, enteritis, and endocarditis may develop (Anderson et al., 1983). If endocarditis is present, the penicillin should be given parenterally at doses of 15-20 million units daily for 4 to 6 weeks. Streptomycin and tetracyclines are also effective antibiotics for those individuals with penicillin-

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associated allergies. Death has occurred in cases of Streptobacillus moniliformis involving preexistent valvular disease.

iv. Diagnosis and prevention. Spirillum minus does not grow in vitro and requires inoculation of culture specimens into laboratory animals, with subsequent identification of the bacteria by dark-field microscopy. Streptobacillary rat-bite fever can be diagnosed only by blood culture. StreptobaciUus moniliformis grows slowly on artificial media, but only in the presence of 15% blood and sera, usually 10 to 20% rabbit or horse serum incubated at reduced partial pressures of oxygen (Fox and Newcomer, 1990). Because of its properties as a bacterial growth promoter, sodium polyanethol sulfonate, which is sometimes found in blood-based media, should not be used due to its inhibitory effects on S. moniliformis. Growth on agar consists of 1-2 mm gray, glistening colonies. The API-ZYM diagnostic system can be used for rapid biochemical analysis and diagnosis. Unfortunately, no serological test is available. Acute febrile diseases, especially if associated with animal bites, are routinely treated with penicillin or other antibiotics. 4.

Cat Scratch Disease

Histopathologic examination of lymph nodes from 39 patients with clinical criteria for cat scratch disease (CSD) revealed pleomorphic, gram-negative bacilli in 34 of the 39 nodes. Organisms in lymph node sections exposed to convalescent serum from 3 patients and to immunoperoxidase stained equally well with all three samples. The authors concluded that the bacilli appear to be the causative agents of CSD. Bartonella (formerly Rochalimaea) henselae, a recently described fastidious gram-negative bacteria, is now recognized as the primary cause of cat scratch disease. Bartonella henselae has been isolated from lymph nodes of CSD patients, and elevated serological titers to B. henselae are also noted in these individuals (Dolan et al., 1993; Zangwill et al., 1993). A second organism,

Table II

Clinical Signs of Rat-Bite Fevera Clinical features Incubation period Fever Chills Myalgia Rash Lymphadenitis Arthralgia, arthritis Indurated bite wound Recurrent fever/ constitutional signs (untreated) a

Modified from Lipman (1996).

9

Streptobacillary fever (Streptobacillus moniliformis)

Spirillosis (Spirillum minus)

2-10 days +++ +++ +++ ++ Morbilliform, petechial + ++

1-6 weeks +++ +++ +++ ++ Maculopapular ++ _+_ +++

Irregular periodicity

Regular periodicity

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Afipia felis, has also been isolated from CSD lesions but is not considered the common etiologic agent of CSD. i. Reservoir and incidence. An estimation of 22,000 cases of cat scratch disease in the United States, of which approximately 2000 require hospitalization, is based on an analysis of three databases (Jackson et al., 1993). Almost all B. henselae infections are associated with exposure or ownership of cats; however, not all cases of CSD are associated with a scratch or bite. ii. Mode of transmission. Patients with CSD commonly have a history of exposure to a cat and of these patients, the majority have either been bitten or scratched. Most of the patients are under 20 years of age. It is now known that cat fleas are infected with B. henselae. It is suspected that the organism is shed in the feces of the flea and can result in the transmission of the organism from cat to cat and from cat to human via mucous membrane or skin contact. Subsequently there is self-inoculation by scratching the flea bite, or alternatively by having the contaminated claws or teeth of cats inoculate the organism into traumatized skin. Importantly, several surveys have shown that cats can be chronically infected with B. henselae, with the organism capable of being isolated from blood of asymptomatic cats over an extended period of time (Koehler et al., 1994). iii. Clinical signs. The natural course of CSD, which consists of a mild or absent fever, few systemic sequelae, and localized lymphadenitis with little or no discomfort, probably resuits in a large number of unrecognized cases. A primary lesion will develop in 50% of the cases about 10 days after a cat bite or scratch; the erythematous pustule will usually persist for 12 weeks (Fig. 3). A regional lymphadenopathy develops 14 days after the initial lesion in most cases. Lymphadenitis regresses in about 6 weeks, with 30-50% of the nodes becoming suppurative. Of the approximately 65% of people who develop systemic illness, fever and malaise are the symptoms most often noted. Occasionally observed are generalized lymphadenopathy, thrombocytopenia, encephalitis, osteolytic lesions, and erythema nodosum. The disease is benign, and most patients recover spontaneously without sequelae within 2 months, although lymphadenopathy can persist up to a year. In immunocompromised individuals, CSD is manifested by an unusual vascular growth seen on the skin and given the name bacillary epithelioid angiomatosis (LeBoit et al., 1988; Kemper et al., 1990). Systemic disease involving spleen and liver also occurs in these patients. iv. Diagnosis and control. If lymphadenitis is present, three of the four following criteria should be fulfilled to diagnose CSD: (1) positive serology for B. hensalae; a positive titer of 1:64 or greater by IFA assay is considered positive, (2) history of contact with a cat, (3) characteristic histopathologic changes present on involved lymph node biopsy, (4) absence of

other disease, and (5) growth of the organism on rabbit blood agar in 5% CO2. Prevention is based on flea control as well as thorough cleansing of cat bites and scratches. 5.

Pasteurella spp.

i. Reservoir and incidence. Pasteurella spp. colonize the respiratory and intestinal tracts of a variety of domestic and wild animals, including birds. The bacteria are gram-negative rods that grow readily on blood agar. ii. Mode of transmission. Human infection caused by P. multocida is commonly the result of contact with animals, particularly when bitten or scratched. iii. Clinical signs. Traumatic wounds resulting from bites or scratches are clinically recognized by acute onset of pain, erythema, cellulitis, and purulent discharge. Osteomyelitis can also occur in bone underlying the wound, and septicemia can result on occasion (Hombal and Dincsoy, 1992). Cat scratches have also resulted in P. multocida-associated corneal ulceration and keratitis (Ho and Rapuan, 1993). iv. Diagnosis and control Bacterial culture of the wound is undertaken prior to local cleansing and antisepsis of the traumatic site of injury. 6. Streptococcus iniae i. Reservoir and incidence. Streptococcus iniae is now recognized as a cause of high mortality in rainbow trout and tilapia (members of the cichlid group of fish) being raised in fish farming environments. Streptococcus iniae was recognized as a pathogen in 1976 when the bacteria was first cultured from cutaneous abscesses in aquaria-maintained Amazon freshwater dolphins (Pier and Madin, 1976). ii. Mode of transmission. Many infected patients sustain an injury to the hand when preparing infected fish for consumption. The organism can be readily cultured from these infected fish (Goh et al., 1998). iii. Clinical signs. Streptococcus iniae was identified as a zoonotic agent in 1995-1996 when a cluster of cases presented with fever and lymphangitis in individuals handling whole or live fish purchased in Toronto, Canada (CDCP, 1996; Weinstein et al., 1997). Streptococcus iniae was cultured from the blood of each of these patients. iv. Diagnosis and control. The organisms are grampositive cocci, [3-hemolytic on 5% sheep blood agar and are nonreactive in the Lancefield sero-grouping system. A nested PCR assay specific for the 16S-23S ribosomal intergenic spacer,

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Fig. 3. Cat scratch disease. Ulcerated, circular lesion adjacent to cat scratch. (Courtesy of Dr. J. H. Graham, Armed Forces Institute of Pathology.)

or alternatively, a chaperonin 60 (cpu 60) gene identification method, are two molecular techniques that provide accurate, rapid, and specific diagnosis of this organism (Berridge et al., 1998; Goh et al., 1998). Infected individuals respond to parenteral antibiotics within 2 - 4 days after initiation of treatment. B.

Systemic Diseases

1. Brucellosis i. Reservoir and incidence. Of the Brucella spp., Brucella canis is the most likely zoonotic agent in the laboratory animal facility due to the extensive use of random-source and

laboratory-bred dogs in comparison to other large domestic animals known to be infected with other Brucella spp. ii. Mode o f transmission. In one study, investigators considered the zoonotic transmission of B. canis unlikely, as evidenced by negative serological tests among 12 individuals exposed to five infected dogs. Since 1967, when the first human B. canis infection was identified, more than 35 natural and laboratory-acquired infections have been reported; most resulted from contact with aborting bitches. Fortunately, humans are relatively resistant to infection; however, B. canis is not a reportable disease, and prevalence data are not available. Although B. canis is particularly well adapted to dogs and is not readily transmitted

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to other species, susceptibility has been reported in several wild species of Canidae (Carmichael and Greene, 1998). iii. Clinical signs. Bacteremia occurred in several infections; other systemic involvement included painful generalized lymphadenophathy and splenomegaly. Additional signs include fever, headache, chills, sweating, weakness, malaise, myalgia, nausea, and weight loss. Rare complications include endocarditis, meningitis, hepatitis, and arthritis. Although B. canisproduced clinical disease in humans is similar to that caused by other Brucella spp., it is generally not as severe. Seroconversion to B. canis has been reported in 0.5% of asymptomatic military personnel who had contact with infected dogs, indicating that inapparent infection may occur (Polt et al., 1982). iv. Diagnosis and control. When a canine's history includes abortions, infertility, testicular abnormalities, and poor semen quality, infection should be considered. A rapid slide agglutination test that produces presumptive diagnostic information is commercially available. To confirm the results of the slide test, one should perform blood cultures and additional serological tests, such as the tube agglutination test (Polt et al., 1982; Serikawa et al., 1989). There have not been any largescale efforts to eradicate B. canis in the general canine population as there have been with Brucella spp. of large domestic animals (Forbes and Pantekoek, 1988). Because of the intracellular location of B. canis, efficacy of antibiotic therapy is variable, and failures or relapses after therapy are reported in dogs. Ultimate control of B. canis in humans relies on elimination of dogs with the disease. 2.

Plague

(Kaufman et al., 1980; Rosner, 1987). Cricetid rodents, such as the wood rat, are occasionally cited as reservoir hosts. The oriental rat flea, Xenopsylla cheopis, the common vector of plague, is well established throughout the United States, particularly in the southern United States and southern California. It is important to remember that more than 1500 species of fleas and 230 species of rodents are infected with Yersinia pestis. Only 30 to 40 rodent species, however, are permanent reservoirs of the infection (Macy, 1999). Plague is infrequently reported in the United States, with a low of 1 case in 1972 and a high of 40 cases in 1983 (Craven and Barnes, 1991). Ninety percent of the cases have been diagnosed in New Mexico, Colorado, and California. Urban development (particularly in New Mexico) encroached into plague-enzootic rodent habitats, placing human populations at increased risk of contracting the disease. In addition to rodent epizootics, dogs, and increasingly cats, either have served as passive transporters of the disease or have been actively infected (Rosner, 1987). The disease has seasonal peaks, with the highest proportion occurring May through September. ii. Mode of transmission. An individual is usually infected by the bite of an infected flea, but infection can also occur via cuts or abrasions in the skin or via infected aerosols coming in contact with the oropharyngeal mucous membrane. Primary pneumonic plague historically occurred by inhalation of infectious droplets from a pneumonic plague patient. However, in the last several decades, this form of the disease has occurred from exposure to infected animals (usually cats) that have developed secondary pneumonia due to septicemic spread of the organism (Rosner, 1987; Craven and Barnes, 1991). Personnel attending these sick animals are then infected by inhaling infected aerosols.

Human infections due to Yersinia pestis, a gram-negative coccobacillus, in the United States are sporadic and limited, usually resulting from infected flea or rodent contact. Since 19241925, when a plague epidemic ravaged Los Angeles, neither urban plague nor rat-borne plague has been diagnosed in the United States (Craven and Barnes, 1991). All reported cases since then have occurred in states located west of the 101st meridian.

iii. Clinical signs. Bubonic plague in humans is usually characterized by fever (2-7 days postexposure) and the formation of large, tender, swollen lymph nodes, or buboes. If untreated, the disease may progress to severe pneumonic or systemic plague. Inhaled infective particles, particularly from animals with plague pneumonia, may also result in the pneumonic form of the disease.

i. Reservoir and incidence. Although plague has occurred repeatedly in recorded history, by the fourteenth century the disease had appeared in the Far East, spread to Asia Minor, and followed the trade routes to Europe. Plague, however, did not make its arrival in the United States until the disease appeared in California in the early 1900s, where it still exists endemically in the ground squirrel and chipmunk. Wild rat populations still act as the primary reservoir in many parts of the world and remain a continued threat in the United States. Sciurid rodents (rock squirrels, California ground squirrels, chipmunks, and prairie dogs) account for the primary plague reservoir in the western parts of the United States

iv. Diagnosis and control. A presumptive diagnosis can be made by visualizing bipolar-staining, ovoid, gram-negative rods on the microscopic examination of fluid from buboes, blood, sputum, or spinal fluid; confirmation can be made by culture. Complement fixation, passive hemagglutination, and immunofluorescence staining of specimens can be used for serological confirmation. Mortality without antibiotic therapy, particularly in cases of pneumonic plague, exceed 50% in untreated cases. Although Y. pestis is susceptible to a wide variety of antibiotics, multiple antibiotic-resistant strains are being isolated with increasing frequency (Dennis and Hughes, 1997). Aminogylcosides, such

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as streptomycin and gentamicin, are the most effective antibiotics in vivo against Y pestis. Chloramphenicol is the drug of choice for treating plague meningitis and endophthalmitis (Craven and Barnes, 1991; Mushatt and Hyslop, 1991). In people exposed to Y pestis, prophylactic therapy with tetracycline for a 7-day period is often prescribed. An inactivated plague vaccine is available for laboratory personnel working with the organism and in high-risk individuals working in areas where the disease is endemic (e.g., wildlife management employees, Peace Corps volunteers) and where they are exposed to plague reservoirs. Rodent and flea control, particularly in endemic areas, is an indispensable part of containing exposure to plague, as is restricting certain locales for recreational use. Animal facilities should be constructed and maintained to prevent wild rodent egress. Furthermore, feral or random-source animals acquired from plague-endemic areas should be quarantined and treated with appropriate insecticides to kill fleas.

3. Leptospirosis Leptospirosis is solely a zoonotic disease of livestock, pet and stray dogs, and wildlife, including wild rodents. Human-tohuman transmission is extremely rare. Leptospira interrogans (comprising > 200 serovars) has been isolated worldwide. Although particular serotypes usually have distinct host species, most serotypes can be carried by several hosts. Leptospira spp. are well adapted to a variety of mammals, particularly wild animals and rodents.

i. Reservoir and incidence. Leptospira icterohaemorrhagiae was first recovered in 1918 in the United States from wild rats sampled in New York City. In the 1950s, in a study conducted in Baltimore, 45.5% of 1643 rats were infected with Leptospira; higher prevalence rates occurred in older rats (approximately 60%). In the late 1970s, more than 90% of adult Brown Norway rats sampled in Detroit were infected with L. icterohaemorrhagiae (Thiermann, 1977). Other studies confirm the high prevalence of this organism in wild rats inhabiting U.S. cities (Alexander, 1984; Sanger and Thiermann, 1988). Rodent reservoir hosts of leptospirosis, in addition to rats, include mice, field moles, hedgehogs, gerbils, squirrels, rabbits, and hamsters (Torten, 1979; Fox and Lipman, 1991). Livestock serve as a significant source of primary long-term shedding of at least three serovars. Cattle are the natural carriers of the serotype L. hardjo, whereas swine carry L. pomona and L. bratislava; each animal can shed the organism for extended periods in their urine. Dogs also commonly harbor two other serotypes; feral dogs harbor L. icterohaemorrhagiae as well as serve as natural carrier hosts of L. canicola. Sheep, goats, and horses can also be infected with a variety of serotypes. Raccoons are reservoirs of L. antumnalis, whereas rats, mice, and otherwild rodents are common animal hosts for another serotype, L. ballum. In wild mice, the infection can persist unnoticed for the ani-

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mal's lifetime and can also be harbored by laboratory mice, although their carrier rates in the United States are unknown (Torten, 1979). There was, however, a report of leptospirosis in a research colony of mice in the United States in the early 1980s (Alexander, 1984). In several European laboratories, personnel have contracted leptospires from laboratory rats (Geller, 1979).

ii. Mode of transmission. Infection with Leptospira most frequently results from handling infected animals (contaminating the hands with urine) or from aerosol exposure during cage cleaning. Skin abrasions or mucous membrane exposure may serve as the portal of entry in humans. All secretions and excretions from infected animals should be considered infective. In one instance, a father apparently was infected after his daughter used his toothbrush to clean a contaminated pet mouse cage. Handling infected wild rats increases the risk of contracting leptospires (Luzzi et al., 1987). Also, a young man died of acute leptospirosis by falling into a heavily polluted river contaminated with L. icterohaemorrhagiae (Sanger and Thiermann, 1988). In addition, rodent bites can transmit the disease. Children living in rat-infested tenements may be at increased risk of infection. For example, children from inner-city Detroit had significantly higher L. icterohaemorrhagiae antibody titers when compared to those of children living in the Detroit suburbs (Demers et al., 1983). Outbreaks of leptospirosis in humans with varying mortality in underdeveloped countries were documented in 1995-1998. iii. Clinical signs. The disease may vary from inapparent to severe infection and death. Infected individuals experience a biphasic disease (Stoenner and Maclean, 1958; Sanger and Thiermann, 1988; Faine, 1991). They become suddenly ill with weakness, headache, myalgia, malaise, chills, and fever and usually exhibit leukocytosis. During the second phase of the disease, conjunctival suffusion and a rash may occur. On examination, renal, hepatic, pulmonary, and gastrointestinal findings may be abnormal. Penicillin is the drug of choice in treating early onset of leptospirosis infection (Faine, 1991). Ampicillin and doxycycline have also been effective in treating people with leptospirosis. iv. Diagnosis and control. Leptospirosis in humans is often difficult to diagnose; therefore, the low incidence of reported infection in humans may be misleading. Outbreaks have been documented in the United States from personnel working with laboratory mice (Stoenner and Maclean, 1958; Barkin et al., 1974). In one study, 8 of 58 employees handling infected laboratory mice (80% of breeding females were excreting L. ballum in their urine) contracted leptospirosis (Stoenner and Maclean, 1958). Because of the variability in clinical symptoms and lack of pathognomonic findings in humans and animals, serological diagnosis or actual isolation of leptospires is imperative (Faine,

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1991). As an aid to diagnosis, leptospires can sometimes be observed by examination or direct staining of body fluids or fresh tissue suspensions. The definitive diagnosis in humans or animals is made by culturing the organisms from tissue or fluid samples, or by animal inoculation (particularly in 3- to 4-weekold hamsters) and subsequent culture and isolation. Culture media with long-chain fatty acids with 1% bovine serum albumin are routinely used as a detoxicant (Faine, 1991). Serological assessment is accomplished by indirect hemagglutination, agglutination analysis, complement fixation, microscopic agglutination, and fluorescent antibody techniques (Faine, 1991). The serological test most frequently used is the microscopic agglutination test, which employs dark-field microscopy. Titers of 1"100 or greater are considered significant. Personnel hygiene and protective garments that minimize exposure to infected urine and other infected animal tissue are important for control of zoonotic infection with leptospires.

C. 1.

Enteric Diseases

Campylobacteriosis

Campylobacter has been known as a pathogenic and commensal bacterium in domestic animals for decades. During the last several years, C. jejuni and C. coli have gained recognition as a leading cause of diarrhea in humans. i. Reservoir and incidence. Campylobacter jejuni, C. coli, C. upsaliensis, and C. helveticus have been isolated from a variety of laboratory animals, including dogs, cats, guinea pigs, hamsters, ferrets, nonhuman primates, poultry, and rabbits (Fox, 1982a) and also from healthy swine, sheep, and cattle. Campylobacter spp. commonly cause abortion in livestock. Campylobacter spp. can be shed in the stool for variable periods of time in asymptomatic carriers, and multiple species of Campylobacter as well as Helicobacter spp. can be isolated from the feces of a single individual or animal (Allos et al., 1995; Shen et al., 1999). ii. Mode of transmission. In most reports citing pet-tohuman transmission of C. jejuni, diarrheic puppies or kittens recently obtained from animal pounds were the source of the infection (B laser et al., 1980; Deming et al., 1987). In a laboratory animal setting, personnel performing husbandry chores have become infected when handling Campylobacter-infected animals (Fox et al., 1989b). Prevalence studies of dogs, cats, newly imported primates, or animals housed in groups suggest that younger animals more easily acquire the infection and, hence, commonly shed the organism. More recently, C. upsaliensis and C. helveticus have been isolated from dogs and cats. Campylobacter upsaliensis has also been associated with diarrheal disease in humans (Fox et al., 1989a).

iii. Clinical signs. The clinical features of campylobacter enteritis in humans are usually consistent with an acute gastrointestinal illness. Diarrheamsometimes watery m with or without blood and leukocytes, abdominal pain, and constitutional symptoms, especially fever, occur routinely. The severity of the illness can be variable, but in most cases it is brief and self-limiting. In protracted or severe cases, antimicrobial therapy (e.g., erythromycin) is instituted (Blaser, 1985). iv. Diagnosis and control. There are multiple C. jejuni/coli serotypes; the use of serotyping schemes and restriction enzyme analysis of isolates aids in confirming zoonotic spread of the organism (Russell et al., 1990). Because animals can be asymptomatic carriers of campylobacters, protective measures preventing fecal contamination and inadvertent oral ingestion are important for prevention of infection. 2.

Enteric Helicobacteriosis

i. Reservoir and incidence. Helicobacter cinaedi is primarily recovered from immunocompromised individuals; the organism is also recovered from chronic alcoholics as well as immunocompetent men and women. The hamster is suspected to be the reservoir host for H. cinaedi (Gebhart et al., 1989). Even though H. canis, H. cinaedi, H. fennelliae, and H. rappini have been isolated from both dogs and humans and H. canis and H. cinaedi from cats, additional investigations will be required to ascertain whether these enteric helicobacters in dogs, cats, and other unrecognized mammalian hosts constitute a potential reservoir for zoonotic transmission to people. ii. Mode of transmission. Fecal-oral transmission is the likely route of infection. Helicobacter cinaedi, a fastidious microaerophile, has been recovered from blood and fecal specimens of children and of a neonate with septicemia and meningitis. The mother of the neonate had cared for pet hamsters during the first two trimesters of her pregnancy (Orlicek et al., 1993). Because H. cinaedi has been isolated from normal intestinal flora of hamsters, it was suggested that the pet hamsters served as a reservoir for transmission to the mother. The mother had a diarrheal illness during the third trimester of pregnancy; the newborn was likely to have been infected during the birthing process, although this was not proven (Orlicek et al., 1993). Furthermore, the hamster has been suggested as possibly infecting other humans with Helicobacter cinaedi (Gebhart et al., 1989). Studies are needed to confirm zoonotic risk of handling H. cinaedi-infected hamsters (Gebhart et al., 1989). Also of interest is the isolation, based on cellular fatty acid and biochemical identification analysis, of H. cinaedi from the feces of dogs and a cat and from a rhesus monkey with chronic colitis (J.G. Fox, unpublished observations) (Kiehlbauch et al., 1995). iii. Clinical signs. Helicobacter cinaedi (previously Campylobacter cinaedi) was first isolated from the lower bowel of

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homosexuals with proctitis and colitis. It has also been isolated from the blood of homosexual patients with human immunodeficiency virus (HIV) as well as children and adult women (Orlicek et al., 1993). In a retrospective study of 23 patients with H. cinaedi-associated illness, 22 of the cases had the organism isolated from blood by using an automated blood culture system in which a slightly elevated growth index was noted (Kiehlbauch et al., 1994). This study also described a new H. cinaedi-associated syndrome consisting of bacteremia and fever, and accompanied by leukocytosis and thrombocytopenia. Recurrent cellulitis and/or arthritis are also noted in a high percentage of infected immunocompromised patients (Kiehlbauch et al., 1994; Burman et al., 1995). Other enteric helicobacters have been isolated from diarrheic patients as well as bacteremic immunocompromised individuals. iv. Diagnosis and control. It should be stressed that many hospital and v.eterinary laboratories have difficulty isolating this organism. Because of the slow growth of H. cinaedi and other enteric helicobacters, laboratory diagnosis is unlikely if blood culture procedures that rely on visual detection of the culture media are used (Kiehlbauch et al., 1994; Burman et al., 1995; Kiehlbauch et al., 1995). Use of dark-field microscopy or acridine orange staining of blood culture media, rather than gram staining, increases likelihood of seeing the organism. Likewise, fecal isolation is difficult; selective antibiotic media are required, and recovery is facilitated by passing fecal homogenates through a 0.45 ~tm filter (Gebhart et al., 1989). In one study, several strains of both H. cinaedi and H. fennelliae were inhibited by concentrations of cephalothin and cetazolin used frequently in selective media for isolation of enteric microaerophilic bacterium. These organisms also require an environment rich in hydrogen for optimum in vitro growth. Until diagnostic laboratories embark on routine isolation attempts of Helicobacter spp. from feces, the extent of their presence in companion and pocket pets and their zoonotic potential will be unknown. 3.

Gastric Helicobacter Infections

i. Reservoir and incidence. Because gastric helicobacterlike organisms (GHLO) (i.e., "H. heilmannii" or H. felis, currently referred to as H. bizzozeronii in dogs) cause a small percentage of gastritis in humans and no environmental source for these bacteria has been recognized, various animals, particularly dogs and cats, have been implicated in zoonotic transmission. In colony-reared animals, GHLO infection may approach 100%. Helicobacter pylori, the primary gastric pathogen in humans, has been isolated from only one colony of commercial cats and macaque species. If H. pylori, as demonstrated in commercially reared cats (Handt et al., 1994; Fox etal., 1996), is isolated from pet cats, the zoonotic potential of helicobacteriosis from cats would obviously increase substantially. Heli-

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cobacter pylori infection is an important cause of human gastritis; however, most epidemiologic studies do not incriminate animal contact as a cause of human infection. An epidemiologic survey conducted in Germany did not show an increased risk of H. pylori because of cat ownership. In a serological survey measuring antibodies to H. pylori, lower socioeconomic status, and not pet ownership or day care, was associated with seropositivity (Staat et al., 1996). ii. Mode of transmission. Oral-oral transmission is likely, but fecal-oral transmission may also occur. In one case study, a researcher performing physiologic studies with cat stomachs developed an acute gastritis, presumably resulting from H. felis on the basis of electron microscopy (EM) (Lavelle et al., 1994). Gastric spiral bacteria were demonstrated in gastric mucosa of cats being used by this scientist. In Germany, a survey of 125 individuals infected with GHLOs provided information in a questionnaire regarding animal contact. Of these patients, 70.3% had contact with one or more animals compared with 37% in the clinically healthy control population (Stolte et al., 1994). iii. Clinical signs. Infection with GHLOs and H. pylori in animals (although associated with gastritis in the majority of humans) does not cause characteristic clinical illness with any consistency or reproducibility. In people with GHLO infections, bismuth subsalicylate, amoxicillin, tetracycline, and metronidazole in various combinations successfully eradicated GHLOs from the gastric mucosa with resolution of gastritis (Heilmann and Borchard, 1991). No systematic antibiotic trials have been conducted in dogs and cats to test for efficacy in eradicating either "H. heilmannii" or H. felis from gastric mucosa. iv. Diagnosis and control. A diagnosis of chronic gastritis in animals, as in humans, cannot be made by gross visual examination of the gastric mucosa by endoscopy. Histologic evaluation of gastric biopsy samples is required, utilizing a special silver stain or modified Giemsa stain to reveal the presence of GHLOs. Unfortunately, H. bizzozeronii is the most common spiral organism in dogs and cats, and it has been extremely difficult to culture on artificial media (Hanninen et al., 1996). "Helicobacter heilmannii," also common in primates, has not been cultured. Helicobacter felis is also difficult to isolate. In practice, histological findings of inflammatory changes accompanied by gastric spiral organisms on the gastric mucosa or in the gastric mucous layer have been used for diagnosis. Helicobacterfelis cannot be distinguished from "H. heilmannii" by histologic examination; EM evaluation is necessary. Because oral bacteria and bacteria refluxed from the duodenum may overgrow the fastidious Helicobacter species, selective antibiotic media are available for isolation. Helicobacters, like campylobacters, require special environmental and cultural conditions for their growth. The organisms are thermophilic and grow at 37 ~C, and some species at 42~ Growth on chocolate

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or blood agar takes 3 to 5 days (Hanninen et al., 1996). For H. bizzozeronii isolation, incubation requires 5 to 10 days. A provisional diagnosis of gastric helicobacters takes advantage of a biochemical feature of these organisms: the ability to produce large quantities of urease. Gastric biopsy samples can be placed in a urea broth containing a pH indicator (phenol red) and a preservative (sodium azide). A similar test is available commercially. Serological assays are being employed to diagnose H. pylori in humans (Staat et al., 1996; Versalovic and Fox, 1999). However, serological tests currently do not provide a reliable, noninvasive diagnostic test for gastric helicobacter infection in dogs and cats or primates.

4.

Salmonellosis

The genus Salmonella are gram-negative bacteria with approximately 2400 serotypes that require antigenic analysis for identification. Nontyphoidal salmonellosis is caused by any of these serotypes. Salmonella are flagellated, nonsporulating, aerobic gram-negative bacilli that can be readily isolated from feces on selective media designed to suppress bacterial growth of other enteric bacteria. i. Reservoir and incidence. Salmonellosis occurs worldwide and is important in humans and animals. Salmonella isolates, because of molecular taxonomics, are now classified under a single species, S. choleraesuis. This species is further subclassified into seven subgroups. References to serotypes, however, are abbreviated such that "choleraesuis" is dropped, e.g., S. choleraesuis serotype typhimurium is called S. typhimurium. Salmonella typhimurium is the serotype most commonly associated with disease in both animals and humans. Other serotypes most commonly reported from humans and animals are S. heidelberg, S. agona, S. montevideo, and S. newport. Salmonellae are pathogenic to a variety of animals. Although the reported prevalence of Salmonella spp. in laboratory animals has decreased in the last several decades because of management practices (e.g., pasteurizing animal feeds), environmental contamination with Salmonella spp. continues to be a potential source of infection for these animals and for the personnel handling them. Until all animal feeds in the United States and Europe are Salmonella-free and animals are procured from Salmonella-free sources, laboratory animal-associated cases of salmonellosis in humans will continue. Endemic salmonellosis in commercially raised guinea pigs as well as dogs, cats, and nonhuman primates has also been a source of infection in personnel working with these animals. Prevalence data from eight studies conducted worldwide indicated that a wide range (0.6-27%) of cats were culture-positive for Salmonella, and a conservative estimate for the U.S. canine population would be 10%. Rats are extremely susceptible to infection with Salmo-

nella spp. In studies performed in the 1920s through 1940s, prevalence of Salmonella in wild rats surveyed in the United States varied from 1 to 18%, compared to 19% in Europe (Geller, 1979; Weisbroth, 1979; Alexander, 1984). In experimental studies, when rats were dosed orally with Salmonella, 10% shed the organism in the 2 months after inoculation, and a few remained carriers when examined 5 months after experimental challenge. These rats, when placed with other naive rats, were capable of initiating new epizootics. Fortunately, the disease in laboratory rats, although common prior to 1939, has been isolated rarely in U.S. commercially reared rats since that time. Birds and reptiles are particularly dangerous sources of Salmonella; as much as 94% of all reptiles harbor Salmonella spp. (Chiodini and Sundberg, 1981). Turtles have received a great deal of zoonotic attention and in 1970 alone may have caused 280,000 human cases of salmonellosis. In the late 1960s, with annual sales of 15 million turtles, zoonotic salmonellosis became a growing problem. In 1972, the U.S. Food and Drug Administration (FDA) banned importation of turtles and turtle eggs and the interstate shipment of turtles that were not certified as free of Salmonella or Arizona hinshawii in their state of origin. However, the unreliable effectiveness of this method forced the FDA in 1975 to rule against the sale of viable turtle eggs or live turtles with a carapace length less than 10.2 cm, with exceptions made for educational or scientific institutions and marine turtles. Subsequently, there was a substantial decrease in turtle-associated salmonellosis, indicating the efficacy of this regulation. These restrictions are difficult to enforce, and other reptiles, e.g., iguanas, are increasingly cited in zoonotic outbreaks of salmonellosis, particularly in children. Also of note, because of repeated reports of chick- and ducklingassociated salmonellosis, some states have also restricted their sale as pets. ii. Mode of transmission. Salmonella spp. are ubiquitous in nature and are routinely found in water or food contaminated with animal or human excreta. Fecal-oral transmission is the primary mode for spread of infection from animal to animal or to humans. Rat feces can remain infective for 148 days when maintained at room temperature. Salmonella is routinely associated with food-borne disease outbreaks, is a contaminant of sewage, and is found in many environmental water sources. Transmission is enhanced by crowding and poor sanitation. Both humans and animals can be asymptomatic carriers and periodic shedders; they may have mild, unrecognized disease, or they may be completely asymptomatic. In the biomedical laboratory, asymptomatic animals can easily infect other animals, technicians, and investigators. Personnel at veterinary hospitals are at increased risk because of outbreaks of salmonellosis in hospitalized animals (Ikeda et al., 1986). The prevalence of human salmonellosis acquired from laboratory animals or vice versa is unknown; however, the literature is replete with

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examples of cases of this infection obtained from pets; this is particularly true for exotic pets such as iguanas, turtles, sugar gliders, and hedgehogs (Woodward et al., 1997).

to humans and has caused epizootic gastroenteritis and fatal bacteremia in dairy cattle (Besser et al., 1997).

iii. Clinical signs. Clinical signs of salmonellosis in humans include acute sudden gastroenteritis, abdominal pain, diarrhea, nausea, and fever. Diarrhea and anorexia may persist for several days. Organisms invading the intestine may create septicemia without severe intestinal involvement; most clinical signs are attributed to hematogenous spread of the organisms. As with other microbial infections, the severity of the disease relates to the serotype of the organism, the number of bacteria ingested, and the susceptibility of the host. In experimental studies with volunteers, several serovars induced a spectrum of clinical disease, from brief enteritis to serious debilitation. Incubation varied from 7 to 72 hr. Cases of asymptomatic carriers, persisting for several weeks, were common (Hull, 1955). Salmonella gastroenteritis is usually mild and self-limiting. With careful management of fluid and electrolyte balance, antimicrobial therapy is not necessary. In humans, antimicrobial therapy may prolong rather than shorten the period that Salmonella spp. is shed in the feces (Nelson et al., 1980; Pavia and Tauxe, 1991). In one double-blind placebo study of infants, oral antibiotics did not significantly affect the duration of Salmonella spp. carriage. Bacteriological relapse after antibiotic treatment occurred in 53% of the patients, and 33% of these suffered a recurrence of diarrhea, whereas none of the placebo group relapsed (Nelson et al., 1980). Also of interest is the fact that in recent outbreaks of DT104 Salmonella typhimurium infection, a high percentage of patients had been recently on antibiotics before becoming infected with the Salmonella typhimurium strain DT104 (Molba et aL, 1999).

5. Shigellosis

iv. Diagnosis and control. As with other fecal-oral transmitted diseases, control depends on eliminating contact with feces, food, or water contaminated with Salmonella or animal reservoirs excreting the organism. Salmonella survive for months in feces and are readily cultured from sediments in ponds and streams previously contaminated with sewage or animal feces. Fat and moisture in food promote survival of Salmonella. Pasteurization of milk and proper cooking of food (56~ for 10 to 20 min) effectively destroy Salmonella. In the laboratory, control and prevention of salmonellosis depends on the rapid detection, removal, or treatment of both acute and chronic animal infections, particularly during the quarantine period. Multiple antibiotic resistance is commonly encountered in Salmonella strains. For example, multiple-resistant S. typhimurium strain DT104 has been increasingly cited (in Europe and recently in the United States) as a cause of human infections (Tauxe, 1999). Importantly, this organism has been isolated from farm animals, cats, wild birds, rodents, foxes, and badgers. It definitely has been transmitted from cattle and sheep

i. Reservoir and incidence. Shigellosis is a significant zoonotic disease in nonhuman primates (Fox, 1975; Richter et al., 1984). Shigella flexneri, S. sonnei, and S. dysenteriae are the most common species found in nonhuman primates. Humans are the main reservoir of the disease, which occurs worldwide. Nonhuman primates acquire the disease following capture and subsequent contact with other infected primates or contaminated premises, food, or water. Shigellosis is one of the most commonly identified causes of diarrhea in nonhuman primates. ii. Mode of transmission. Shigella organisms may be shed from clinically ill as well as asymptomatic humans and nonhuman primates. In humans, transmission occurs by ingestion of fecally contaminated food or water, or by direct contact (even if only minimal) with infected animals. Pet monkeys shedding Shigella are a particular threat to owners, and pet store proprietors, unless cautious, can contract the disease (Fox, 1975). iii. Clinical signs. Humans are generally susceptible to shigellosis, although it is much more severe in children than in adults. The disease varies from completely asymptomatic to a bacillary dysentery syndrome characterized by blood and mucus in the feces, abdominal cramping, tenesmus, weight loss, and anorexia. Usually, the disease presents only as a clinically mild diarrhea. However, fatal shigellosis has been reported in children and adults who have had contact with infected pet or zoo monkeys (Fox, 1975); survivors can remain asymptomatic carriers. The clinical disease in nonhuman primates is similar to that in humans but may be associated with higher mortality rates. iv. Diagnosis and control. When humans or nonhuman primates experience acute diarrhea (especially if traced with blood or mucus), Shigella spp. may be the cause (Richter et al., 1984; Dupont, 2000). A definitive diagnosis requires the isolation of the organism from inoculation of fresh feces onto selective media. An identification can be confirmed by agglutination with polyvalent Shigella antisera. Because many Shigella spp. from nonhuman primates have plasmid-mediated antibiotic resistance markers, determination of antibiotic sensitivities of these isolates is mandatory before instituting treatment (Fox, 1975). To prevent shigellosis in the laboratory, quarantine and screening of all newly arrived primates to detect microbial carriers are required. As in the treatment of the disease in humans, trimethoprim and sulfamethodoxazole can be effective in eliminating the

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Shigella spp. carrier state in rhesus monkeys. Enrofloxacin is also used to eliminate subclinical Shigella in macaques. D.

Respiratory Infections

coughing of clinically affected animals. The disease may also be contracted by direct ingestion of bacilli. Reports have documented an increase of tuberculin skin conversion in personnel working with primates infected with Mycobacterium spp. (Kalter et al., 1978).

1. Bordetella bronchiseptica Bordetella bronchiseptica is commonly recovered from the respiratory tract of dogs, cats, rabbits, and a variety of laboratory rodents. Despite its widespread occurrence in animals, it is seldom cultured from diseased tissues of humans, with fewer than 50 cases reported in the literature. Its isolation is often from immunocompromised patients (Woolfrey and Moody, 1991) who have pneumonia and/or bacteremia. It has also been isolated from AIDS patients (Ng et al., 1992). In children with respiratory infection due to B. bronchiseptica, a "whooping cough"-like syndrome is described. This is not surprising given that B. bronchiseptica produces a dermatonecrotoxin, tracheal cytotoxin, and adenylate cyclase similar to that isolated from B. pertussis. In one interesting report, three children with B. bronchiseptica infection developed whooping cough-like symptoms; both their pet rabbits and cats subsequently died of B. bronchiseptica pneumonia (Kristensen and Lautrop, 1962). 2.

Tuberculosis

i. Reservoir and incidence. Tuberculosis is an important zoonosis associated with laboratory animals. It is caused by acid-fast bacilli of the genus Mycobacterium. Natural reservoir hosts for the etiologic agent of this disease correspond to the three most common species of Mycobacterium: M. bovis, M. avium complex, and M. tuberculosis. Although cattle, birds, and humans are the major reservoir hosts, many animals, including swine, sheep, goats, monkeys, cats, dogs, and ferrets, are susceptible and contribute to the spread of disease (Marini et al., 1989; Fox, 1998). This susceptibility varies according to the immune response of the host and to the particular Mycobacterium sp. infecting the host. In nonhuman primates, outbreaks of tuberculosis still occur, particularly in the Old World species of monkeys. They initially contract the disease in the wild through human contact, and then the organism is transmitted from monkey to monkey (Richter et al., 1984). ii. Mode of transmission. Mycobacterium bacilli are transmitted from infected animals or tissue samples via the aerosol route. The disease is spread beyond the natural host range through animal-to-animal and human-to-human contact, usually by airborne infectious particles. Laboratory workers have the highest risk of contracting the disease when caring for or performing autopsies on infected animals. In the laboratory, certain situations can enhance disease transmission, such as exposure to (1) dusty bedding of infected animals, (2) aerosolized organisms from a high-pressure water sanitizer, and (3) the

iii. Clinical signs. Clinical signs of tuberculosis in humans are dependent on the organ system or systems involved. Most familiar are the signs related to the pulmonary form. Although this form of the disease often remains asymptomatic for months or years, it may eventually produce a cough with sputum and hemoptysis. In addition, general symptoms include anorexia, weight loss, lassitude, fatigue, fever, chills, and cachexia (Division of Tuberculosis Elimination, 2000). iv. Diagnosis and control. A positive diagnosis is often quite difficult to obtain. Three widely used tools for a presumptive diagnosis are the intradermal tuberculin test, radiographic analysis, and positive acid-fast-stained sputum smears. A more definitive diagnosis of the organisms from body fluids or biopsy specimens is obtained by culture, PCR analysis, and confirmation using standard biochemical techniques. Control of tuberculosis infection, particularly within the biomedical research arena, requires a multifaceted approach. This includes personnel education, a regular health surveillance program for personnel and nonhuman primates, isolation and quarantine of suspect animals, and rapid euthanasia and careful disposal of confirmed positive animals. Vaccination or chemoprophylaxis may be considered, but certain precautions are necessary (Division of Tuberculosis Elimination, 2000). Vaccination with Bacillus Calmette-Guerin (BCG), a strain of M. bovis, is an effective means of preventing active tuberculosis. Vaccination is suggested in high-risk groups. However, this vaccine often elicits a positive tuberculin test, thereby negating the best diagnostic indicator of early disease. Vaccination in the United States is therefore reserved for demonstrated high-risk individuals and children in locations where 20% or more of school-age children are tuberculin-positive (Division of Tuberculosis Elimination, 2000). Chemoprophylaxis with effective antituberculosis agents used to treat humans, such as isoniazid, rifampin, and ethambutol, has been used to treat valuable nonhuman primates (Wolf et al., 1988). A well-conceived tuberculosis control program will include some or all of the above methods tailored to the needs and special circumstances of individual animal resource programs.

VI.

FUNGAL DISEASES

The superficial mycoses are commonly referred to as ringworm due to the characteristic circular erythematous lesion

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found on the skin of the host. The most common of the fungi responsible for disease in animals and humans are the three genera of the dermatophytes: Microsporum, Epidermophyton, and Trichophyton. Species of dermatophytes are subcategorized as anthropophilic (primarily infect humans), geophilic (soil inhabitants), and zoophilic (parasitic on animals). The zoophilic dermatophytes are known to infect humans.

i. Reservoir and incidence. Dermatophytes are distributed worldwide, with particular species found more frequently in specific geographic regions. Ringworm in laboratory animals is common, particularly among random-source animals, such as dogs, cats, and livestock. Microsporum canis is the common isolate from dogs and cats, whereas Trichophyton verrucosum is the species usually isolated from livestock, and T. mentagrophytes from laboratory rodents. ii. Mode of transmission. Transmission to humans occurs from direct or indirect contact with symptomatic or asymptomatic carrier animals; contaminated bedding, caging, or other equipment; or fungal contamination of the environment. The resultant disease in humans, tinea, is frequently self-limiting and often goes unnoticed. When lesions occur, they are generally on the extremities, particularly on the arm or hand. Lesions are focal, annular, scaling, and erythematous with central clearing resembling a ring. Occasionally, vesicles or fissures are reported. In contrast with anthropophilic species, zoophilic dermatophytes generally produce more eczematous and inflammatory lesions, which regress rapidly. iii. Clinical signs. Generally, dermatophytes grow only in dead, keratinized tissue. Advancing infection is halted when contact with live cells and inflammation occurs. Dermatophytes are species-adapted and rarely cause severe inflammatory lesions in the specific-host species. When zoophilic species infect humans, the inflammatory response usually restricts the progress of the infection. Contact with the dermatophyte does not necessarily result in infection in the animal or human host. A number of factors, including but not limited to, age; immune, hormonal, and nutritional status; and prior exposure all are important in disease expression. When observed, disease in animals is often mild and goes undetected. Disease in cats, usually seen in kittens, is quite variable. Lesions, generally seen on and around the head, are crusting and mildly erythemic. The areas may be alopecic with numerous broken hairs. In dogs, lesions consist of circular, alopecic, crusting patches. In laboratory rodents, lesions are generally absent. Presence of the organism may not be detected until personnel become infected and manifest lesions (Fig. 4). iv. Diagnosis and control. Diagnosis in humans and animals is similar. Fungal culture is the most effective and specific means of diagnosis. Specialized dermatophyte test media

Fig. 4. Circularringwormlesion on the arm of a man. Contracted from a rodentinfectedwith Trichophytonmentagrophytes (Courtesyof Dr. W.Kaplan.) (DTM) or Sabouraud's agar may be used. A Wood's lamp can be used to screen lesions, scrapings, or cultures, as approximately 50% of Microsporum canis isolates fluoresce when examined with the cobalt-filtered ultraviolet lamp. Direct microscopic examination of hairs and skin scrapings may allow for definitive diagnosis. The risk of zoonotically acquired dermatophytosis can be reduced among laboratory and animal care personnel by wearing protective garments, specifically long-sleeved clothing or laboratory coats; practicing effective personal hygiene; handling random-source animals with disposable gloves; screening newly acquired animals for suggestive lesions; and isolating and treating animals with lesions. Treatment consists of either systemic therapy with griseofulvin or topical therapy with any one of a number of antifungal agents, such as miconazole. Infectious spores will persist on the animal despite successful treatment of active lesions. Eradication of spores is generally unfeasible, as it may require extensive depilation and the use of sporicidal dips.

VII.

PROTOZOAL DISEASES

A.

Enteric Diseases

1. Amebiasis

Amebiasis is a parasitic infection of the large intestine caused by the protozoan parasite Entamoeba histolytica (Ravdin, 1995).

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JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

i. Reservoir and incidence. The disease occurs worldwide in humans, with a greater prevalence in tropical areas. The parasite is found routinely in clinically normal monkeys and anthropoid apes and may cause severe clinical disease in these animals. The reported incidence of E. histolytica has ranged from 0 to 21% in rhesus monkeys, 2 to 67% in chimpanzees, and up to 30% in other nonhuman primates. ii. Mode of transmission. Entamoeba histolytica exists as either resistant cysts or the more fragile trophozoites (Visvesvar and Stehr-Green, 1990). Cysts are the infectious form of the parasite and are usually found in the normal stool of asymptomatic carriers or humans with mild disease (Ravdin, 2000). Cysts may remain viable in moist, cool conditions for over 12 days and in water for up to 30 days. Epidemics of amebiasis in humans usually result from ingestion of fecally contaminated water containing amebic cysts. Laboratory animal workers handling nonhuman primates are potentially exposed to infection from infected fecal matter transferred through the workers' skin or clothing. The infective cyst forms may be subsequently ingested. iii. Clinical signs. Most human infections with E. histolytica have few or no detectable symptoms (Ravdin, 2000). Clinical signs result when trophozoites invade the large bowel wall causing an amebic colitis. Signs begin with a mild, watery diarrhea with bad-smelling stool, which is frequently preceded by constipation in early stages. There may be gas, abdominal cramps, and tenderness progressing to an acute fulminating bloody or mucoid dysentery with fever, chills, and muscle ache. The disease may have periods of remission and exacerbation over months to years (Ravdin, 2000). Rarely, extraintestinal amebic abscesses may form in the liver, lung, pericardium, or central nervous system. Involvement of the liver may lead to tenderness in the right abdomen and can progress to jaundice. iv. Diagnosis and control. The diagnosis of amebiasis requires the microscopic identification of trophozoites or cysts in fresh stool specimens. The organism must be carefully measured to differentiate it from other nonpathogenic amebas. Control measures to prevent amebiasis should include strict adherence to sanitation and personal hygiene practices. Water supplies should be protected from fecal contamination since usual water-purification chlorine levels do not destroy the cysts (Chin, 2000). A chlorine concentration of 10 ppm is necessary to kill amebic cysts (Ravdin, 2000). Cysts may also be killed by heating to 50 ~C. Nonhuman primates should be screened during quarantine to identify carriers of E. histolytica and should be appropriately treated. Nonhuman primates with acute diarrhea or dysentery should also have stool examined for the presence of E. histolytica and should be treated as necessary. Recommended drugs for treatment of E. histolytica infection include metronidazole, paromomycin, emetine, and diiodohydroxyquin (iodoquinol). Both asymptomatic carriers and symptomatic patients should be treated (Ravdin, 2000).

2.

Balantidiasis

Balantidiasis is a zoonotic disease caused by the large ciliated protozoan Balantidium coli.

i. Reservoir and incidence. Balantidium coli is distributed worldwide and is common in domestic swine. It may also be found in humans, great apes, and several monkey species. The incidence in nonhuman primate colonies has ranged from 0 to 63%. These infections are usually asymptomatic in most animals, although clinical disease characterized by diarrhea or dysentery may occur. ii. Mode of transmission. Infection usually results from the ingestion of trophozoites or cysts from the feces of infected animals or humans. Transmission may also occur from ingestion of contaminated food or water. iii. Clinical signs. Balantidiasis may cause ulcerative colitis characterized by diarrhea or dysentery, tenesmus, nausea, vomiting, and abdominal pain. In severe cases, blood and mucus may be present in the stool. Humans apparently have a high natural incidence, and infections are often asymptomatic (Chin, 2000). iv. Diagnosis and control Balantidiasis is diagnosed by the detection of trophozoites or cysts in fresh fecal samples. Control measures to prevent balantidiasis should be directed at maintaining good sanitation and personal hygiene practices in nonhuman primate and swine colonies. Water supplies should be protected from fecal contamination, especially since usual water chlorination does not destroy cysts (Chin, 2000). Nonhuman primates exhibiting acute diarrhea should be examined for the presence of B. coli organisms in the feces. Positive animals should be isolated and the infection appropriately treated. Tetracyclines, metronidazole, paromomycin, and ampicillin have been used successfully to eliminate B. coli infections (Teare and Loomis, 1982). 3.

Cryptosporidiosis

Cryptosporidiosis was first described in the mouse. The genus Cryptosporidium now contains over 10 named species (Levine, 1980), many of which have been incriminated as opportunistic, pathogenic parasites (Angus, 1983). Cryptosporidiosis, once considered an infrequent, inconsequential protozoan infection in mammals and reptiles, is now considered a significant enteric pathogen. Cryptosporidium parvum is considered the human pathogen.

i. Reservoir and incidence. Cryptosporidium spp. are coccidian parasites known to infect a variety of mammals, including humans, monkeys, livestock, ferrets, pigs, guinea pigs, mice, fish, reptiles, and birds. Neonates of mammalian domes-

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tic species are uniquely susceptible to this infection, in comparison to the adults, who are resistant. In humans, however, both children and adults are susceptible. Cryptosporidia isolated from mammals are not host-specific, and zoonotic transmission from calves to humans has been reported (Levine et al., 1988). Bovine cryptosporidia from calves can also cause infection in newborn pigs, lambs, chicks, mice, rats, and guinea pigs. ii. Mode of transmission. The life cycle of cryptosporidia is direct, with infection generally limited to the small intestine; however, infections of the respiratory tract, stomach, and conjunctiva have been reported. The life cycle of cryptosporidia is similar to that of other coccidia except that cryptosporidial oocysts do not require time outside the host to sporulate but are infectious at the time of excretion. Large epidemics have occurred in humans ingesting the organism in contaminated municipal drinking water. Sporulated oocysts can exist in the intestine before being excreted. Disease transmission is through ingestion of infectious oocysts. The organisms are small (4-5 ~tm in diameter) and are located on the apical surface of the parasitized epithelial cell, where they protrude from the brush border. The organisms are intracellular, as the plasma membrane of the host cell envelops the parasite. iii. Clinical signs. Recorded cases of this disease generally occur in children, particularly in developing countries with poor sanitation, and in immunosuppressed (compromised) individuals. Zoonotic disease has been reported among animal handlers and veterinary students working with neonatal ruminants, principally calves, infected before 6 weeks of age (Levine et al., 1988). Another transmission was recorded in an individual who became infected performing a survey of Cryptosporidium spp. in calves (Reese et al., 1982). In this patient, clinical remission occurred by day 13, and oocytes of cryptosporidium were no longer apparent on fecal flotation (Fig. 5). Disease in neonatal ruminants may be subclinical or may present with protracted watery diarrhea, very similar to what occurs in humans. Symptoms in humans occur 1 to 2 weeks after contact with infected calves, and diarrhea may be accompanied by vomiting, severe abdominal cramps, lassitude, fever, and headache. Disease is generally self-limiting except in immunocompromised individuals (Fayer and Ungar, 1986). Most of the recorded cases of protracted human cryptosporidiosis have occurred in immunodeficient individuals, particularly AIDs patients, and are regarded as opportunistic infections (Chin, 2000). Disease in these individuals produced low-grade fever, malaise, anorexia, nausea, abdominal cramps, and a protracted, watery diarrhea. Repeated intestinal biopsies in a patient have documented indigenous cryptosporidial stages for as long as 1 year; clinical signs also persisted in this patient. iv. Diagnosis and control. Diagnosis is made by examination of feces for the characteristic oocysts. Direct wet mounts may be satisfactory in heavy infections; the organism can be

Fig. 5. Cryptosporidialoocystsin an unstained wet mount from calf feces. Note single prominent black dot, which is central or slightly eccentric. Some oocysts are indented, x 1280. (Courtesyof Dr. B. Anderson.)

concentrated by the Sheather sugar flotation or the Formalinethyl acetate method. Histologic evaluation of intestinal and rectal biopsies can also be used for diagnosis. Currently, no pharmaceutic agent is effective in treating cryptosporidiosis. More than 50 antibiotics have been tried without effect. Immunocompromised patients are persistently infected. Some drugs, such as paromomycin, may reduce the symptoms, and new drugs are being tested. The infection persists until the host's immune response clears the parasite. 4.

Giardiasis

Giardiasis is usually a mild intestinal illness, caused by the protozoan parasite Giardia lamblia. The parasite can be found in the feces of infected animals (dogs, cats, beavers, and rodents). i. Reservoir and incidence. Giardia spp. are found worldwide among all classes of vertebrates and occur among numerous laboratory animals. Giardia cysts isolated from a human

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JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

produced infections when fed to eight different species of test animals, including dogs.

ii. Mode of transmission. Historical classification schemes have speciated Giardia based on host origin. Studies conducted over the past decade have demonstrated a lack of host specificity, at least for some species of Giardia. Epidemiologic studies and zoonotic transmission have corroborated the lack of host specificity exhibited by Giardia spp. Contamination of drinking water with Giardia cysts from beavers has been implicated in several human giardiasis outbreaks (Dykes et al., 1980; Keifer et al., 1980), and cysts from this source have infected human volunteers and dogs. Other studies have produced patent infections in dogs, using Giardia cysts isolated from municipal drinking water known to have infected humans (Shaw et al., 1977; Dykes et al., 1980). Nonhuman primates have also been implicated. A clinically ill gibbon was presumed to be the source of infection for three zoo attendants and six apes who subsequently developed clinical giardiasis (Armstrong and Hertzog, 1979). The life cycle of Giardia is direct, with trophozoites, the feeding stage of the organism, residing in the upper gastrointestinal tract. They multiply and develop into infective cysts that are shed in the feces and ingested by subsequent hosts. iii. Clinical signs. The disease in humans and animals is often similar. Giardiasis in humans is characterized by chronic or intermittent diarrhea, bloating, abdominal cramping, anorexia, fatigue, and weight loss. The stool frequently is mucus-laden, light-colored, and soft, but not watery. Symptoms may persist for several weeks and then resolve spontaneously. Fever is usually not present, and many persons infected with Giardia may have no symptoms at all. Individuals with the disease are contagious for the entire period of infection and may recover without treatment. iv. Prevention and control. Although many species of laboratory animals can be infected experimentally with Giardia pathogenic for humans, they have not been demonstrated to harbor these organisms naturally. Giardia infections of dogs, nonhuman primates, and other animals probably present a greater public health risk, and infected animals may warrant treatment. Personnel handling these animals should take appropriate safety measures. Quinacrine is the drug of choice for treating giardiasis. Metronidazole and furazolidone are also used for treatment in the United States.

B.

Systemic Infections

First discovered in 1908, toxoplasmosis is caused by infection with a microscopic parasite called Toxoplasma gondii. Toxoplasmosis has been found in humans and most warm-blooded animals. An estimated 500 million humans have been infected

with the organism, and nearly one-third of all adult humans in the United States and in Europe have antibodies to toxoplasma, which provides evidence that they have been exposed to this parasite.

i. Reservoir and incidence. The life cycle of T. gondii consists of definitive and intermediate hosts. Toxoplasma infection has spread throughout the animal kingdom to include hundreds of species of mammals and birds as its intermediate hosts. Mice, rats, hamsters, guinea pigs and other rodents, rabbits, dogs, sheep, cattle and nonhuman primates include some of the laboratory animals that could serve as intermediate hosts (Teusch et al., 1979; Wright, 1985). These laboratory animal hosts have not been shown to be important in zoonotic infection by T. gondii in the laboratory environment because the organism replicates only asexually in extraintestinal sites (Parker and Holliman, 1992; Herwaldt and Juranek, 1993). Serological surveys conducted in the United States during the early 1980s using the Sabin-Feldman dye test have demonstrated T. gondii infection in 3 0 - 8 0 % of cats (Ladiges et al., 1982). Presumably, all serologically positive cats have shed Toxoplasma oocysts and could again shed organisms by reinfection or by reactivation. ii. Mode of transmission. Domestic and wild felids develop extraintestinal invasion with T. gondii analogous to that of the nonfelid hosts. In addition, as the definitive hosts in the T. gondii life cycle, felines develop intestinal infection, with the shedding of oocysts. Thus, the domestic cat is the primary reservoir for the zoonotic transmission of T. gondii in the laboratory environment. The three common modes of transmission are congenital infection, ingestion of T. gondii-infected tissue, and ingestion of toxoplasma oocytes or from direct exposure and consumption of contaminated food or water (Dubey, 1998). Most postnatally acquired infections in cats are asymptomatic and have a variable prepatent period and pattern of oocyst shedding. The prepatent period can be as brief as 3 days if the cat has ingested mice or meat containing T. gondii cysts, or it can be as long as several weeks if oocysts have been ingested. Shedding of oocysts in the feces occurs for 1-2 weeks, during which time cats are considered a public health risk (Dubey, 1998). Oocysts become infectious after sporulation, which occurs in 1-5 days. Oocysts survive best in warm, moist soil. Oocyst shedding is less likely to occur if the cat was infected by oocysts or tachyzoites than if infection resulted from the ingestion of Toxoplasma cysts. Oocyst shedding can be reactivated by induction of hypercorticism or by superinfection with other feline microorganisms, such as Isosporafelis (Chessman, 1972). Oocysts of T. gondii have been observed infrequently in the feces of naturally infected cats (Ladiges et al., 1982), and shedding usually precedes the development of antibody titers to T. gondii. The oocyst is very hardy and can survive freezing and as much as several months of extreme heat and dehydration. Importantly,

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high IgG titers do not prove recent or active infection (Dubey et al., 1995). iii. Clinical signs. Toxoplasma infection in humans and animals is very common, but clinical disease occurs only sporadically and has a low incidence. Sporadic clinical cases and occasional epidemics do occur. Outbreaks have occurred when humans are exposed to oocyst-contaminated dust, either by inhalation or ingestion. Populations at high risk for infection are pregnant women and immunodeficient individuals. Congenital infection in humans results in systemic disease, frequently with severe neuropathological changes. Postnatal infection results in disease that is less severe and commonly presents as nondescript, consisting of fever, myalgia, and generalized lymphadenopathy that may resolve without treatment in a few weeks. Asymptomatic infection may recrudesce with encephalitis if patients become immunocompromised. Although rare, serious systemic toxoplasmosis can be acquired by older individuals. This is manifested by fever, maculopapular eruption, malaise, myalgia, arthralgia, posterior cervical lymphadenopathy, pneumonia, myocarditis, and meningoencephalitis. Ocular toxoplasmosis, usually chorioretinitis, is commonly seen in postnatal infections but can also occur in infections of older individuals. Clinically severe and progressive illness is most likely to develop in immunocompromised individuals. As high as 10% of AIDS patients have toxoplasmosis (Gill and Stone, 1992). These patients develop neurologic disease and can experience convulsions, paralysis, or coma or even die from toxoplasmosis, even after treatment is administered. iv. Diagnosis and control. Diagnosis can be made by histopathologic demonstration of the organisms, demonstration of serum antibody, testing for antigenemia, or skin test. Chemotherapeutic treatment is indicated in patients with diagnosed clinical disease, active ocular lesions, or congenital infection, and in immunocompromised individuals with disease suggestive of toxoplasmosis. The preferred therapy is pyrimethamine administered in combination with sulfonamide. Laboratoryacquired infections are likely restricted to the use and handling of laboratory cats (DiGiacomo et al., 1990). Rigorous sanitation should effectively prevent human toxoplasmosis from occurring in the laboratory environment. Since oocysts must sporulate before they are infectious, daily cleaning of litter plans will prevent accumulation of infectious oocysts. Personnel should wear gloves when handling litter pans and wash their hands thoroughly before eating. Pregnant women should completely avoid contact with cat feces. Interestingly, there is no correlation between toxoplasmosis in adults and cat ownership. Most cats acquire infection shortly after weaning and shed the oocysts for a short period of time (< 3 weeks). Nevertheless, unsporulated oocysts are more susceptible to proper disinfection, and control of exposure should be centered around disinfection of litter pans at this stage.

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HELMINTH INFECTIONS

Many of the helminth parasites common to animals and humans have an indirect life cycle that is interrupted in the laboratory environment, thus precluding cross-infection of animals and humans. Although numerous helminths of laboratory animals should be regarded as zoonotic (Soulsby, 1969; Flynn, 1973), the risk of human infection from laboratory-housed animals appears to be minimal. One exception may be the dwarf tapeworm of humans, Hymenolepis nana, a common parasite of house mice and occasionally diagnosed in mice used for research. It is conservatively estimated that over 20 million people (mostly children) are infected with this parasite (Markell et al., 1999). Hymenolepis nana is unique among cestodes in that the adult worm develops following ingestion of the egg by humans and does not require an intermediate host for its life cycle (Table III). Nematodes in aberrant hosts are a potential cause of visceral and ocular larval migrans. Ingested eggs of several nematode larvae may be shed in the feces and ingested by humans. These ingested eggs hatch in the abnormal host and migrate into deep tissues, but development proceeds no further. Larvae may persist in the visceral organs or the eyes and cause granulomatous lesions, resulting in hepatosplenomegaly, fever, and eosinophilia (visceral larval migrans) (Edelglass et al., 1982; Davies et al., 1993) or leucocoria, eye pain, strabismus, or loss of vision (ocular larval migrans) (Bathrick, 1981). The most frequent cause of these diseases is Toxocara canis (dog) (Wolfrom et al., 1995) and Toxocara cati (cat) (Glickman and Magnava, 1993), but Baylisascaris procyonis in the raccoon is much more aggressive and therefore more pathogenic (Fox et al., 1988). Fatal or severe central nervous system disorders have been documented for mice, woodchucks, pigeons, domestic quail, turkeys, captive prairie dogs, and armadillos, and two human fatalities have been reported. Several other animal parasites have been associated with larval migrans-like syndromes. These include Ascaris suum (swine), Capillaria hepatica (rat), Angiostrongylus cantonensis (rat), Gnathostoma spinigerum (dogs and cats) (Bathrick, 1981), and Angiostrongylus costaricensis (cotton rats) (Levine, 1980). Human involvement has been reported with each of the above. The practices encountered in a properly managed animal facility are not conducive to the transmission of these parasites. Proper quarantine, surveillance, and treatment procedures drastically reduce the endoparasitic burden of laboratory animals. Routine sanitation eliminates most parasitic ova before they have undergone the embryonation necessary for infectivity. Education of personnel on standard hygiene practices further reduces the likelihood of zoonotic infection. Laboratory-housed nonhuman primates are presumed to be the most likely, although infrequent, source of parasitic infection for animal handlers (Orihel, 1970; Nasher, 1988).

Table III Zoonotic Helminth Parasites in the Laboratory Environment Disease

Etiology

Natural host(s)

Aberrant hosts

Cestodiasis

Hymenolepis nana

Rats, mice, hamsters, nonhuman primates

Humans

Strongyloidiasis

Strongyloides stercoralis, S. fulleborni

Nonhuman primates, dogs, cats, humans, Old World nonhuman primates

Humans

Ternidens infection Ancylostomiasis

Ternidens deminutus Ancy lostoma duodenale Necator americanus

Old World primates Humans Humans

Humans Nonhuman primates, pigs Nonhuman primates, pigs

Trichostrongylosis

Ruminants, pigs, dogs, rabbits, Old World nonhuman primates Old World primates

Humans

Oesophagostomiasis

Trichostrongylus colubriformis, T. axei Oesophagostomum spp.

Ascariasis

Ascaris lumbricoides

Old World primates

Humans

Enterobiasis

Enterobias vermicularis

Humans

Old world primates

Trichuriasis

Trichuris trichiura

Humans

Old world primates

Larval migrans (viscera)

Toxocara canis Toxocara cati Toxocara leonina Baylisascaris procyonis

Dogs and other canids Cats and other felids Dogs, cats, wild canids, felids Raccoons

Humans Humans Humans Humans and other animals

Larval migrans (cutaneous)

Ancylostoma caninum Ancylostoma braziliense Ancylostoma duodenale Uncinaria stenocephala Necator americanus

Dogs Dogs, Dogs, Dogs, Dogs,

Humans Humans Humans Humans Humans

cats cats cats cats

Humans

Comments Intermediate host is not essential to the life cycle of this cestode. Direct infection and internal autoinfection can also occur. Heavy infections result in abdominal distress, enteritis, anal pruritis, anorexia, and headache Oral and transcutaneous infections can occur in animals and humans. Heavy infections can produce dermatitis, verminous pneumonitis, enteritis. Internal autoinfection can occur Rare and asymptomatic Oral and transcutaneous routes of infection occur. Heavy infections produce transient respiratory signs during larval migration followed by anemia due to gastrointestinal blood loss Heavy infections produce diarrhea Heavy infections result in anemia. Encapsulated parasitic granulomas are usually an inocuous sequella to infection Infection occurs by ingestion of embryonated eggs only. Embryonation, requiring 2 or more weeks, ordinarily would not occur in laboratory. Heavy infections can produce severe respiratory and gastrointestinal tract disease Oral and inhalational infection can occur. Disease in humans characterized by perianal pruritis, irritability, and disturbed sleep Three-week embryonation makes laboratory infection highly unlikely. Heavy infection in humans results in intermittent abdominal pain, bloody stools, diarrhea, and occasionally rectal prolapse Chronic eosinophilic granulomatous lesions distributed throughout various organs. Should not be encountered in laboratory Infections in aberrant host produces granulomas in visceral organs with a predilection for the central nervous system Transcutaneous infection causes a parasitic dermatitis called "creeping eruption"

25. SELECTED Z

O

O

N

O

S

E

S

1

0

9

5

Fig. 6. Maculopapular dermatoses in humans associated with mite and flea bites. (a) Tropical rat mite; (b) flea (courtesy of American College of Laboratory Animal Medicine and Washington State University College of Veterinary Medicine); (c) cheyletiella mite.

Table IV Ectoparasites a,b Species Mites Obligate skin mites Sarcoptes scabiei subspecies Notoedres cati Nest-inhabiting parasites Ornithonyssus bacoti Ornithonyssus bursa Ornithonyssus sylviarum Dermanyssus gallinae Allodermanyssus sanguineus Ophionyssus natricis Haemogamasus pontiger Haemolaelaps casalis Eulaelaps stabularis Glycyphagus cadaverum Acaropsis docta Trixacarus caviae Facultative mites Cheyletiella spp. Dermatophagoides scheremtewskyi

Eutrombicula spp. Laelaps echidninus Ixodids (ticks) Rhipicephalus sanguineus Dermacentor variabilis Dermacentor andersoni Dermacentor occidentalis Ambylomma americanum Ixodes scapularis Ixodes spp. Ornithodorus spp. Argas persicus

Disease in humans

Animal host

Scabies Mange

Mammals Cats, dogs, rabbits

Dermatitis, murine typhus Dermatitis Dermatitis, encephalitis Dermatitis, encephalitis Dermatitis, rikettsialpox Dermatitis Dermatitis Dermatitis Dermatitis, tularemia Dermatitis, psittacosis Dermatitis, psittacosis Dermatitis

Rodents and other vertebrates, including birds Birds Birds Birds Rodents, particularly Mus musculus Reptiles Rodents, insectivores, straw bedding Birds, mammals, straw, hay Small mammals, straw bedding Birds Birds Guinea pigs

Dermatitis Dermatitis, urinary infections, pulmonary acariasis Human pest (chiggers), local pruritis

Cats, dogs, rabbits, bedding

Agent

WEE, c SLE d virus Rickettsia mooseri WEE, EEE, e SLE viruses

Rickettsia akari

Francisella tularensis Chlamydophila psittaci Chlamydophila psittaci

Feathers, animal feed, bird nests Chickens, occasional mammals obtained from natural habitat Potential Argentine hemorrhagic fever

Irritation, RMSF/tularemia, other diseases Irritation, RMSF/tularemia tick paralysis, other diseases Irritation, Colorado tick fever, Q fever, RMSF/other diseases Irritation, Colorado tick fever, RMSF/ tularemia Irritation, RMSF/tularemia Irritation, possible tularemia Lyme disease Irritation, relapsing fever Irritation, seldom bites humans, but can transmit anthrax, Q fever

Dogs Wild rodents, cottontail rabbits, dogs from endemic areas Small mammals, uncommon on dogs

Rickettsia rickettsii, Francisella tularensis See above

Small mammals, uncommon on dogs Wild rodents, dogs Dogs, wild rodents Dogs, cats, wild rodents Captive reptiles, wild animals, pigs

See above

Borrelia burgdonferi Borrelia recurrentis

Domestic fowl

Borrelia recurrentis

See above Ungrouped rhabdoviruses

continues

Table IV (Continued)

Species Fleas Ctenocephalides felis

Ctenocephalides canis (cat and dog fleas) Xenopsylla cheopis Nasopsyllus f asciatus

Leptopsylla segnis Echidnophaga gallinacea (sticktight flea) Pulex irritans

Animal host

Disease in humans

Dermatitis, vector of Hymenolepis diminuta, Dipylidium caninum Dermatitis, plague vector, Hymenolepis nana, H. diminuta Dermatitis, plague vector, Hymenolepis nana, H. diminuta murine typhus Hymenolepis diminuta, H. nana, murine typhus vector Potential plague vector Irritation

Dogs, cats

Mouse, rat, wild rodents

Yersinia pestis

Mouse, rat, wild rodents

Yersinia pestis

Rat Poultry Domestic animals (esp. pigs) and humans

Harbors salmonella

Found in laboratory animals that cause allergic dermatitis or from which zoonotic agents have been recovered in nature. /'Modified from Fox et al. (1984). c WEE, Western equine encephalitis. dSLE, St. Louis encephalitis. eEEE, Eastern equine encephalitis. IRMSF, Rocky Mountain spotted fever. a

Agent

1098

JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

However, a review cited only three cases of zoonotic helminth infections resulting from nonhuman primates. These animals had been kept as pets and not as laboratory animals. Thus, although helminth parasites should be recognized as potentially zoonotic in the laboratory environment, they represent a significantly smaller problem than that posed by possible viral and bacterial zoonosis.

IX.

ARTHROPOD INFESTATIONS

Health hazards to humans due to ectoparasite infestations from arthropods associated with laboratory animals are most often mild and limited to manifestations of allergic dermatitis. However, arthropods can serve as vectors to systemic illnesses such as rickettsial pox, tularemia, and Lyme disease. Those working with laboratory animals, particularly those species arriving directly from their natural habitat, should be familiar with the arthropods capable of transmitting these diseases. Mites probably pose the greatest health hazard, not only because they are the most common inhabitant in number and variety of species, but because they also readily transmit agents from almost every major group of pathogens: bacteria, chlamydia, rickettsia, viruses, protozoa, spirochetes, and helminths (Yunker, 1964). In addition, most of these mites are capable of producing severe allergic papular dermatitis in humans (Fox and Reed, 1978; Fox, 1982b) (Fig. 6). Control of mite infestation is primarily dependent on their habitats. Some, such as Sarcoptes sp. and Notoedres spp. are obligate parasites that require treatment of the host. Other mites, such as Ornithonyssus bacoti, which live most of the time off the animal, require treatment of the environment with appropriate insecticides (Markell et al., 1999). Ticks, with the exception of those in newly arrived dogs or wild animals brought into the laboratory, are rarely found in the well-managed animal facility. The brown dog tick, Rhipicephalus sanguineus, is an exception. It readily infests kennels and vivaria. Ticks, like mites, can transmit a variety of diseases, including Rocky Mountain spotted fever, tick-borne typhus, Lyme disease, and others (Table IV). Lyme borreliosis is a commonly reported tick-borne infection in Europe and North America. The illness is caused by a spirochete, Borrelia burgdorferi, which is transmitted during the blood feeding of ticks of the genus Ixodes. The larvae and nymphs feed readily on a wide range of hosts, including birds, and an abundance of reservoir hosts exists, usually small and medium-sized animals. Larger animals, such as deer, sheep, cows, or horses, must be present for the maintenance of the tick population since adult ticks only engorge successfully on larger animals. Transmission occurs through salivation during the feeding process on a host.

Control of ticks indoors is aimed primarily at the resting places of the unattached ticks and proper treatment of newly arrived animals, which are noted for harboring ticks. Fleas are notorious for their ability to transmit disease to humans, particularly plague and murine typhus. Three rodent fleas, Xenopsylla cheopis, Nasopsyllus fasciatus, and Leptopsylla segnis, have been found in a high percentage of urban dwellings in certain areas of the United States and are potential transmitters of disease in the laboratory. Apparently, X. cheopis in the past was readily established in animal facilities. At a Midwestern U.S. university, it inhabited rooms housing laboratory mice, where on two separate occasions fleas bit students (Yunker, 1964). Leptopsylla segnis, the mouse and rat flea, bites humans and is a vector for plague and typhus, serious diseases in humans. Leptopsylla segnis can also serve as an intermediate host for the rodent tapeworms Hymenolepis nana and H. diminuta, both of which can infect humans (Markell et al., 1999). The flea bite can be irritating and can cause allergic dermatitis. The cat flea, Ctenocephalides felis, is the most common flea in and around human dwellings in the United States. This flea is capable of experimentally transmitting plague and murine typhus, and therefore the potential exists for transmitting the disease to humans. Control of fleas consists of treatment of infested areas as well as the primary host; in the case of rodent fleas, the animal facility must be free of feral rodents and their entry to prevent introduction of these arthropods.

REFERENCES

Adams, S. R. (1995). Part B. Zoonoses, biohazards, and other health risks. In "Nonhuman Primates in BiomedicalResearch" (B. T. Bennett, C. R. Abee, and R. Henrickson, eds.), pp. 391-412. AcademicPress, San Diego. Adams, W. H., Emmons,R. W., and Brooks, J. E. (1970). The changing ecology of murine (endemic)typhus in southern California.Am. J. Trop. Med. Hyg. 19,311-318. Adams, J. R., Schmidtmann, E. T., and Azad, A. E (1990). Infection of colonized cat fleas, Ctenocephalidesfelis (Bouche), with a rickettsia-like microorganism. Am. J. Trop. Med. Hyg. 43, 400-409. Alexander, A. D. (1984). Leptospirosis in laboratory mice. Science 224, 1158. Allos, B. M., Lastovica, A. J., and Blaser, M. J. (1995). Atypical campylobacters and related microorganisms. In "Infections of the Gastrointestinal Tract" (M. J. Blaser, P. D. Smith, J. I. Ravdin, H. B. Greenberg, and R. L. Guerrant, eds.), pp. 849-865. Raven Press, New York. Ambrus, J. L., and Strandstrom, H. V. (1966). Susceptibilityof Old World monkeys to Yabavirus. Nature 211, 876. Ambrus, J. L., Strandstrom, H. V., and Kawinski,W. (1969). "Spontaneous" occurrence of a Yaba tumor in a monkey colony. Experimentia 25, 64-65. Anderson, L. C., Leary, S. L., and Manning, P. J. (1983). Rat-bite fever in animal research laboratory personnel. Lab. Anim. Sci. 33, 292. Andrei, G., and DeClerq, E. (1993). Molecular approaches for the treatment of hemorrhagic fever virus infections.Antiviral Res. 22, 45-75. Angus, K. N. (1983). Cryptosporidiosis in man, domestic animals, and birds: A review. J. R. Soc. Med. 76, 62-70.

25. SELECTED ZOONOSES Anonymous (1997). Zoonoses control. Lyssavirus infection in 3 fruit bats, Australia. Wkly. Epidemiol. Rec. 72, 194-196. Armstrong, J., and Hertzog, R. E. (1979). Giardiasis in apes and zoo attendants, Kansas City, Missouri. Vet. Public Health Notes 1, 7-8. Asher, M. S. (1989). Rickettsial diseases. In "Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections" (N. J. Schmidt and R. W. Emmons, eds.), pp. 1141-1164. American Public Health Association, Washington, D.C. Babudieri, B. (1959). Q fever: A zoonosis. Adv. Vet. Sci. 5, 82-182. Balayan, M. S. (1997). Epidemiology of hepatitis E virus infection. J. Viral Hepat. 4, 155-165. Barkin, R. M., Guckian, J. C., and Glosser, J. W. (1974). Infections by Leptospira ballum: A laboratory-associated case. South. Med. J. 67, 155-176. Barkley, W. E., and Richardson, J. H. (1984). The control of biohazards associated with the use of experimental animals. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 595-602. Academic Press, Orlando, Florida. Bathrick, M. E. (1981). Intraocular gnathostomiasis. Ophthalmology 99, 12931295. Bearcroft, W. G. C., and Jamieson, M. E (1958). An outbreak of subcutaneous tumors in rhesus monkeys. Nature 182, 195-196. Beaver, P. C., and Jung, R. C. (1985). "Animal Agents and Vectors of Human Disease," 5th ed. Lea and Febiger, Philadelphia. Benson, P. M., Malane, S. L., Banks, R., Hicks, C. B., and Hilliard, J. (1989). B virus (Herpesvirus simiae) and human infection. Arch. Dermatol. 125, 1247-1248. Berger, L., Volp, K., Mathews, S., Speare, R., and Timms, P. (1999). Chlamydia pneumoniae in a free-ranging giant barred frog (Mixophyes iteratus) from Australia. J. Clin. Microbiol. 37, 2378-2380. Bernard, K. W., Parham, G. L., Winkler, W. G., and Helmick, C. G. (1982). Q fever control measures: Recommendations for research facilities using sheep. Infect. Control 3, 461-465. Berridge, B. R., Fuller, J. D., de Azavedo, J. C., Low, D. E., Bercovier, H., and Frelier, P. E (1998). Development of specific nested oligonucleotide PCR primers for the Streptococcus iniae 16S-23S ribosomal DNA intergenic spacer. J. Clin. Microbiol. 36, 2778-2781. Besser, T. E., Gay, C. C., Gay, J. M., Hancock, D. D., Rice, D., Pritchett, L. C., and Erickson, E. D. (1997). Salmonellosis associated with S. typhimurium DT104 in the USA [letter; comment]. Vet. Rec. 140, 73. Bhatt, P. N., Jacoby, R. O., and Barthold, S. W. (1986). Contamination of transplantable murine tumors with lymphocytic choriomeningitis virus. Lab. Anim. Sci. 36, 136-139. Biggar, R. J., Woodall, J. P., Walter, P. D., and Haughie, G. E. (1975). Lymphocytic choriomeningitis outbreak associated with pet hamsters. J. Am. Vet. Med. Assoc. 232, 494-500. Blaser, M. J. (1985). Campylobacter species. In "Priniciples and Practices of Infectious Diseases" (G. L. Mandell, R. G. Douglas, and J. E. Bennett, eds.), p. 1221. Wiley and Sons, New York. Blaser, M. J., LaForce, E M., Wilson, N. A., and Wang, W. L. L. (1980). Reservoirs for human campylobacteriosis. J. Infect. Dis. 141, 665-669. Bowen, G. S., Calisher, C. H., Winkler, W. G., Kraus, A. L., Fowler, E. H., Garmon, D. W., Fraser, D. W., and Hinman, A. R. (1975). Laboratory studies of lymphocytic choriomeningitis virus outbreak in man and laboratory animals. Am. J. Epidemiol. 102, 233-240. Breman, J. G., Kalisa-Ruti, S., Steniowski, M. V., Zanotto, W., Gromyko, A. I., and Arita, E. (1980). Human monkeypox 1970-1979. Bull World Health Organ. 58, 165-182. Brooks, J. E. (1973). A review of commensal rodents and their control. Rev. Eviron. Control 3, 405-453. Burman, W. J., Cohn, D. L., Reves, R. R., and Wilson, M. L. (1995). Multifocal cellulitis and monoarticular arthritis as manifestations of H. cinaedi bacteremia. Clin. Infect. Dis. 20, 564-570.

1099 Carmichael, L. E., and Greene, C. E. (1998). Canine brucellosis. In "Infectious Diseases of the Dog and Cat" (C. E. Greene, ed.), pp. 248-257. Saunders, Philadelphia. Centers for Disease Control (CDC) (1987). Guidelines for the prevention of Herpesvirus simiae (B virus) infection in monkey handlers. MMWR 36, 680-689. Centers for Disease Control (CDC) (1989). Ebola virus infection in imported primatesmVirginia. MMWR 38, 831-832. Centers for Disease Control (CDC) (1990). Update: Ebola-related filovirus infection in nonhuman primates and interim guidelines for handling nonhuman primates during transit and quarantine. MMWR 39, 22-30. Centers for Disease Control (CDC) (1991). Update on adult immunization. Recommendations of the Immunization Practices Advisory Committee (ACIP). MMWR 40. Centers for Disease Control (CDC) (1992a). Anonymous survey for simian immunodeficiency virus (SIV) seropositivity in SIV-laboratory researchersm United States. MMWR 41, 814-815. Centers for Disease Control (CDC) (1992b). Seroconversion to simian immunodeficiency virus in two laboratory workers. MMWR 41, 678-681. Centers for Disease Control (1995). Outbreak of acute febrile illness and pulmonary hemorrhagemNicaragua. MMWR 44, 841-843. Centers for Disease Control and Prevention (CDCP) (1994). Laboratory management of agents associated with hantavirus pulmonary syndrome: Interim biosafety guidelines. MMWR 43, 1-18. Centers for Disease Control and Prevention (CDCP) (1996). Invasive infection due to Streptococcus iniae~Ontario, 1995-1996. MMWR 45, 650-653. Centers for Disease Control and Prevention (CDCP) (1997). Human monkeypoxmKasai Oriental, Democratic Republic of Congo, February 1996-October 1997. MMWR 46, 1168-1171. Centers for Disease Control and Prevention (CDCP) (1998). Fatal cercopithecine herpesvirus 1 (B virus) infection following a mucocutaneous exposure and interim recommendations for worker protection. MMWR 47, 1073-1076. Centers for Disease Control and Prevention (CDCP) (1999). Human rabies prevention~United States, 1999: Recommendations of the Advisory Committee on Immunization Practices. MMWR 48, 1-18. Centers for Disease Control and Prevention (CDCP) (2000). Fatal illnesses associated with a New World arenavirus~California, 1999-2000. MMWR 49, 709-711. Centers for Disease Control and Prevention-National Institutes of Health (CDCP-NIH) (1999). "Biosafety in Microbiological and Biomedical Laboratories," HHS Publ. (CDC) 93-8395, 4th ed. U.S. Government Printing Office, Washington, D.C. Chen, Z., Telfier, P., Gettie, A., Reed, P., Zhang, L., Ho, D. D., and Marx, P. A. (1996). Genetic characterization of new West African simian immunodeficiency virus SIVsm: Geographic clustering of household-derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse viruses from a single feral sooty mangabey troop. J. Virol. 70, 3617-3627. Chessman, B. S. (1972). Reactivation of toxoplasm oocyst production in the cat by infection with Isospora felis. Br. Vet. J. 128, 33-36. Childs, J. E., Glass, G. E., Korch, G. W., Ksiazek, T. G., and LeDuc, J. W. (1992). Lymphocytic choriomeningitis virus infection and house mouse (Mus musculus) distribution in urban Baltimore. Am. J. Trop. Med. Hyg. 47, 27-34. Childs, J. E., Colby, L., Krebs, J. W., Strine, T., Feller, M., Noah, D., Drenzek, C., Smith, J. S., and Rupprecht, C. E. (1997). Surveillance and spatiotemporal associations of rabies in rodents and lagomorphs in the United States, 1985-1994. J. Wildl. Dis. 33, 20-27. Chin, J., ed. (2000). "Control of Communicable Diseases Manual." American Public Health Association, Washington, D.C. Chiodini, R. J., and Sundberg, J. P. (1981). Salmonellosis in reptiles: A review. Am. J. Epidemiol. 113, 494.

1100

JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

Clayson, E. T., Innis, B. L., Myint, K. S., Narupiti, S., Vaughn, D. W., Giri, S., Ranabhat, P., and Shrestha, M. P. (1995). Detection of hepatitis E infections in swine among domestic swine in the Kathmandu Valley of Nepal. Am. J. Trop. Med. Hyg. 53, 228-232. Committee on Emerging Microbial Threats to Health (1992). Factors in emergence. In "Emerging Infections" (J. Lederberg, R. E. Shope, and S. C. Oaks Jr., eds.), pp. 34-112. National Academy Press, Washington, D. C. Committee on Occupational Health and Safety in Research Animal Facilities (1997). "Occupational Health and Safety in the Care and Use of Research Animals." Institute of Laboratory Animal Resources, National Research Council. National Academy Press, Washington, D. C. Committee on Urban Pest Management (1980). "Urban Pest Management." National Academy Press, Washington, D. C. Cotton, M. M., and Partridge, M. R. (1998). Infection with feline Chlamydia psittaci. Thorax 53, 75-76. Craven, R. B., and Barnes, A. M. (1991). Plague and tularemia in animal associated human infections. Infect. Dis. Clin. North Am. 1, 165-175. Dalgard, D. W., Hardy, S. L., Pearson, G. J., Pucak, G. J., Quander, Z., Zack, P. M., Peters, C. J., and Jahrling, P. B. (1992). Combined simian hemorrhagic fever and Ebola virus infection in cynomolgus monkeys. Lab. Anim. Sci. 42, 152-159. Davies, H. D., Sakuls, P., and Keystone, J. S. (1993). Creeping eruption. A review of clinical presentation and management of 60 cases presenting to a tropical disease unit. Arch. DermatoL 129, 588-591. Demers, R. Y., Thiermann, A., Demers, P., and Frank, R. (1983). Exposure to Leptospira icterohaemorrhagiae in inner-city and suburban children: A serologic comparison. J. Fam. Prac. 17, 1007-1011. Deming, M. S., Tauxe, R. V., Blake, B. A., Dixon, S. E., Fowler, B. S., Jones, T. S., Lockamy, E. A., Patton, C. M., and Sikes, R. O. (1987). Campylobacter enteritis at a university: Transmission from eating chicken and from cats. Am. J. Epidemiol. 126, 526-534. Dennis, D. T., and Hughes, J. M. (1997). Multidrug resistance in plague. N. Engl. J. Med. 337, 702-704. DiGiacomo, R. E, Harris, N. V., Huber, N. L., and Cooney, M. K. (1990). Animal exposures and antibodies to Toxoplasma gondii in a university population. Am. J. Epidemiol. 131, 729-733. Division of Tuberculosis Elimination. (2000). Core Curriculum on Tuberculosis: What the Clinician Should Know, 4th ed. Centers for Disease Control and Prevention, Atlanta. Dolan, M. J., Wong, M. T., Regnery, R. L., Jorgensen, J. H., Garcia, M., Peters, J., and Drehner, D. (1993). Syndrome of Rochalimaea henselae adenitis suggesting cat scratch disease. Ann. Intern. Med. 118, 331-336. Dubey, J. P. (1998). Advances in the life cycle of Toxoplasma gondii. Int. J. Parasitol. 28, 1019-1024. Dubey, J. P., Lappin, M. R., and Thulliez, P. (1995). Long-term antibody responses of cats fed Toxoplasma gondii tissue cysts. J. Parasitol. 81, 887893. Duma, R. J., Sonenshine, D. E., Bozeman, F. M., Veazey, J. M., Jr., Elisberg, B. L., Chadwick, D. P., Stocks, N. I., McGill, T. M., Miller, G. B., and MacCormack, J. N., Jr. (1981). Epidemic typhus in the United States associated with flying squirrels. J. Am. Med. Assoc. 245, 2318-2323. Dupont, H. L. (2000). Shigella species (bacillary dysentery). In "Practices and Principles in Infectious Diseases" (G. L. Mandell, J. E. Bennett, and R. Dolin, eds.), p. 2363. Churchill Livingston, Philadelphia. Dykes, A. C., Juranek, D. D., Lorenz, R. A., Sinclair, S., Jakubowski, W., and Davies, R. (1980). Municipal waterborne giardiasis: An epidemiologic investigation. Beavers implicated as a possible reservoir. Ann. Intern. Med. 92, 165-170. Dykewicz, C. A., Dato, V. M., Fisher-Hoch, S., Horwath, M. V., Perez-Oronoz, G., Ostroff, S. M., Gary, H., Jr., Schonberger, L. B., and McCormick, J. B. (1992). Lymphocytic choriomeningitis outbreak associated with nude mice in a research institute. J. Am. Med. Assoc. 267, 1349-1353. Edelglass, J. W., Douglass, M. C., Stiefler, R., and Tessler, M. (1982). Cuta-

neous larva migrans in northern climates. A dream vacation. J. Am. Acad. DermatoL 7, 353-358. Everett, K. D., Bush, R. M., and Andersen, A. (1999). Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov. each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of the organisms. Int. J. Syst. Bacteriol. 49, 415-440. Faine, S. (1991). Leptospirosis. In "Bacterial Infections of Humans" (A. S. Evans and P. S. Brachman, eds.), pp. 367-393. Plenum Medical Book Co., New York. Farhang-Azad, A., Traub, R., and Baqar, S. (1985). Transovarial transmission of murine typhus, rickettsiae in Xenopsylla cheopis fleas. Science 227, 543-545. Fayer, R., and Ungar, B. L. P. (1986). Cryptosporidium spp. and cryptosporidiosis. Microbiol. Rev. 50, 458. Fenner, E (1990). Poxviruses of laboratory animals. Lab. Anim. Sci. 40, 469480. Flowers, D. J. (1970). Human infection due to Mycobacterium marinum after a dolphin bite. J. Clin. Pediatr. 23, 475-477. Flynn, R. J. (1973). "Parasites of Laboratory Animals." Iowa State Univ. Press, Ames. Forbes, L. B., and Pantekoek, J. E (1988). Brucella canis isolates from Canadian dogs. Can. Vet. J. 29, 149. Formenty, P., Boesch, C., Wyers, M., Steiner, C., Donati, E, Dind, E, Walker, E, and LeGuenno, B. (1999a). Ebola virus outbreak among wild chimpanzees living in a rain forest of C6te d'Ivoire. J. Infect. Dis. 179, S120S126. Formenty, P., Hatz, C., Le Guenno, B., Stoll, A., Rogenmoser, P., and Widmer, A. (1999b). Human infection due to Ebola virus, subtype C6te d'Ivoire: Clinical and biologic presentation. J. Infect. Dis. 179, $48-$53. Fox, J. G. (1975). Transmissible drug resistance in Shigella and Salmonella isolated from pet monkeys and their owners. J. Med. Primatol. 4, 164. Fox, J. G. (1982a). Campylobacteriosis--a "new" disease in laboratory animals. Lab. Anim. Sci. 32, 625. Fox, J. G. (1982b). Outbreak of tropical rat mite dermatitis in laboratory personnel. Arch. Dermatol. 118, 676-678. Fox, J. G. (1998). "Biology and Diseases of the Ferret," 2nd ed. Williams and Wilkins, Baltimore. Fox, J. G. (1999). Man's worst friend: The rat. In "Infections of Leisure" (D. Schlossberg, ed), 2nd ed., pp. 249-279. ASM Press, Washington, D. C. Fox, J. G., and Lipman, N. S. (1991). Infections transmitted from large and small laboratory animals. In "Infectious Diseases of North America" (A. Weinberg and D. Weber, eds.), pp. 131-163. Saunders, Philadelphia. Fox, J. G., and Newcomer, C. E. (1990). Rodent-associated zoonoses and health hazards. In "Handbook of in Vivo Toxicity Testing" (D. C. Arnold, H. Grice, and D. Krewski, eds.), pp. 72-102. Academic Press, Orlando, Florida. Fox, J. G., and Reed, C. (1978). Cheyletiella infestation of cats and their owners. Arch. Dermatol. 117, 1233-1234. Fox, J. G., Newcomer, C. E., and Rozmiarek, H. (1984). Selected zoonoses and other health hazards. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 614-648. Academic Press, Orlando, Florida. Fox, A. S., Kazacos, K. R., Gould, N. S., Heydemann, P. T., Thomas, C., and Boyer, K. M. (1988). Fatal eosinophilic meningoencephalitis and visceral larva migrans caused by the raccoon ascarid Baylisascaris procyonis. N. Engl. J. Med. 312, 1619. Fox, J. G., Maxwell, K. O., Taylor, N. S., Runsick, C. D., Edmonds, P., and Brenner, D. J. (1989a). "Campylobacter upsaliensis" isolated from cats as identified by DNA relatedness and biochemical features. J. Clin. Microbiol. 27, 2376-2378. Fox, J. G., Taylor, N. S., Penner, J. L., Shames, B., Gurgis, R. V., and Thomson,

25. SELECTED Z

O

O

N

O

S

F. N. (1989b). Investigation of a zoonotically acquired Campylobacter jejuni enteritis with serotyping and restriction endonuclease DNA analysis. J. Clin. Microbiol. 27(11), 2423-2425. Fox, J. G., Perkins, S., Yan, L., Shen, Z., Attardo, L., and Pappo, J. (1996). Local immune response in Helicobacterpylori infected cats and identification of H. pylori in saliva, gastric fluid, and feces. Immunology 88, 400-406. Franz, D. R., Jahrling, P. B., Friedlander, A. M., McClain, D. J., Hoover, D. L., Bryne, W. R., Pavlin, J. A., Christopher, G. W., and Eitzen, E. M., Jr. (1997). Clinical recognition and management of patients exposed to biological warfare agents. J. Am. Med. Assoc. 278, 399-411. Gao, E, Bailes, E., Robertson, D. L., Chen, Y., Rodenburg, C. M., Michael, S. E, Cummins, L. B., Aurthur, L. O., Peeters, M., Shaw, G. M., Sharp, P. M., and Hahn, B. H. (1999). Origin of HIV-1 in the chimpanzee (Pan troglodytes troglodytes). Nature 397, 436-441. Gebhart, C. J., Fennell, C. L., Murtaugh, M. P., and Stamm, W. E. (1989). Campylobacter cinaedi is normal intestinal flora in hamsters. J. Clin. Microbiol. 27, 1692-1694. Geller, E. H. (1979). Health hazards for man. In "The Laboratory Rat" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), p. 402. Academic Press, New York. Gerrard, J. (1997). Fatal encephalitis and meningitis at the Gold Coast Hospital, 1980 to 1996. Commun. Dis. Intell. 21, 32-33. Gill, D. M., and Stone, D. M. (1992). The veterinarian's role in the AIDS crisis. J. Am. Vet. Med. Assoc. 201, 1683-1684. Glickman, L. T., and Magnava, J. E (1993). Zoonotic roundworm infections. Infect. Dis. Clin. North Am. 7, 717-732. Goh, S. H., Driedger, D., Gillett, S., Low, D. E., Hemmingsen, S. M., Amos, M., Chan, D., Lovgren, M., Willey, B. M., Shaw, C., and Smith, J. A. (1998). Streptococcus iniae, a human and animal pathogen: Specific identification by the chaperonin 60 gene identification method. J. Clin. Microbiol. 36, 2164- 2166. Haig, D. M., Mclnnes, C., Deane, D., Reid, H., and Mercer, A. (1997). The immune and inflammatory response to off virus. Comp. Immunol. Microbiol. Infect. Dis. 20, 197-204. Handt, L. K., Fox, J. G., Dewhirst, E E., Fraser, G. J., Paster, B. J., Yan, L., Rozimarek, H., Rufo, R., and Stalis, I. H. (1994). Helicobacter pylori isolated from the domestic cat: Public health implications. Infect. lmmun. 62, 23672374. Hanninen, M. L., Happonen, I., Saari, S., and Jalava, K. (1996). Culture and characteristics of Helicobacter bizzozeronii, a new canine gastric Helicobacter sp. Int. J. Syst. Bacteriol. 46, 160-166. Harmon, M. W., and Kendal, A. P. (1989). Influenza viruses. In "Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections" (N. J. Schmidt and R. W. Emmons, eds.), pp. 631-668. American Public Health Association, Washington, D.C. Hayami, M., Ido, E., and Miura, T. (1994). Survey of simian immunodeficiency virus among nonhuman primate populations. Curr. Top. Microbiol. Immunol. 188, 1-20. Heilmann, K. L., and Borchard, E (1991). Gastritis due to spiral shaped bacteria other than Helicobacterpylori: Clinical, histological, and ultrastructural findings. Gut 32, 137-140. Heneine, W., Switzer, W. M., Sandstrom, P., Brown, J., Vedapuri, S., Schable, C. A., Khan, A. S., Lerche, N. W., Schweizer, M., Neuman-Haeflin, D., Chapman, L. E., and Folks, T. M. (1998). Identification of a human population infected with simian foamy viruses. Nat. Med. 4, 391-392, 644645. Herwaldt, B. L., and Juranek, D. D. (1993). Laboratory acquired malaria, leishmaniasis, trypanosomiasis, and toxoplasmosis. Am. J. Trop. Med. Hyg. 48, 313-323. Hinman, A. R., Fraser, D. W., Douglas, R. D., Bowen, G. S., Krause, A. L., Winkler, W. G., and Rhodes, W. W. (1975). Outbreaks of lymphocytic choriomeningitis infection in medical center personnel. Am. J. Epidemiol. 101, 103-110.

E

S

1

1

0

1

Hjelle, B., Torrez-Martinez, N., Koster, E T., Jay, M., Ascher, M. S., Brown, T., Reynolds, P., Ettestad, P., Voorhees, R. E., Sharisky, J., Enscore, R. E., Sands, L., Mosely, D. G., Kioski, C., Bryan, R. T., and Sewell, C. M. (1996). Epidemiologic linkage of rodent and human hantavirus genomic sequences in case investigation of hantavirus pulmonary syndrome. J. Infect. Dis. 173, 781-786. Ho, A. C., and Rapuan, C. J. (1993). Pasteurella multocida keratitis and corneal laceration from a cat scratch. Ophthalmic Surg. 24, 346-348. Hollinger, R. B., and Glombicki, P. A. (1990). Hepatitis A virus. In "Principles and Practices of Infectious Diseases" (G. L. Mandell, D. R. Gordon, and J. E. Bennett, eds.), pp. 1383-1395. Churchill Livingstone, New York. Holmes, G. P., Hilliard, J. K., Klontz, K. C., Rupert, A. H., Schindler, C. M., Parrish, E., Griffin, D. G., Ward, G. S., Bernstein, N. D., Bean, T. W., Ball, M. R., Sr., Brady, J. A., Wilder, M. H., and Kaplan, J. E. (1990). Virus (Herpesvirus simiae) infection in humans: Epidemiologic investigation of a cluster. Ann. Intern. Med. 112, 833-839. Holmes, G. P., Chapman, L. E., Stewart, J. A., Straus, S. E., Hilliard, S. E., Davenport, D. S., and B Virus Working Group (1995). Guidelines for the prevention and treatment of B virus infections in exposed persons. Clin. Infect. Dis. 20, 421-439. Hombal, S. M., and Dincsoy, H. P. (1992). Pasteurella multocida endocarditis. Am. J. Clin. PathoL 98, 565-568. Hull, T. G. (1955). "Diseases Transmitted from Animals to Man," 4th edl Thomas Springfield, Illinois. Hyde, S. R., and Benirschke, K. (1997). Gestational psittacosis: Case report and literature review. Mod. Pathol. 10, 602-607. Ikeda, J. S., Hirsch, D. C., Jang, S. S., and Biberstein, E. L. (1986). Characteristics of Salmonella isolated from animals at a veterinary medical teaching hospital. Am. J. Vet. Res. 47, 232-235. Jaax, N. K., Davis, K. J., Geisbert, T. J., Vogel, P., Jaax, G. P., Topper, M., and Jahrling, P. J. (1996). Lethal experimental infection of rhesus monkeys with Ebola-Zaire (Mayinga) virus by the oral and conjunctival route of exposure. Arch. Pathol. Lab. Med. 120, 140-155. Jackson, L. A., Perkins, B. A., and Wenger, J. D. (1993). Cat scratch disease in the United States: An analysis of three national databases. Am. J. Public Health 83, 1707-1711. Jahrling, P. B. (1989). Arenaviruses and filoviruses. In "Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections" (N. J. Schmidt and R. W. Emmons, eds.), 6th ed., pp. 857-891. American Public Health Association, Washington, D.C. Jahrling, P. B., and Peters, D. J. (1992). Lymphocytic choriomeningitis virus, a neglected pathogen of man. Arch. Pathol. Lab. Med. 116, 486-488. Jay, M., Hjelle, B., Davis, R., Ascher, M., Baylies, H. N., Reilly, K., and Vugia, D. (1996). Occupational exposure leading to hantavirus pulmonary syndrome. Clin. Infect. Dis. 22, 841-844. Jezek, Z., and Fenner, E (1988). Human monkeypox. In "Monographs in Virology" (J. L. Melnick, ed.), 17, Karger, Basel. Jezek, Z., Arita, I., Szczeniowski, M., Paluka, K. M., Kalisa, R., and Nakano, J. H. (1985). Human tanapox in Zaire: Clinical and epidemiological observations on cases confirmed by laboratory studies. Bull. World Health Organ. 63, 1027-1035. Johnson, H. N. (1989). Rabies virus. In "Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections" (N. J. Schmidt and R. W. Emmons, eds.), 6th ed., pp. 893-923. American Public Health Association, Washington, D.C. Johnson, K. M. (1990). Lymphocytic choriomeningitis virus, lassa virus (Lassa Fever) and other arenaviruses. In "Principles and Practices of Infectious Diseases" (G. L. Mandell, D. R. Gordon, and J. E. Bennett, eds.), pp. 13291334. Churchill Livingstone, New York. Johnson, D. K., Morin, M. L., Bayne, K. B., and Wolfle, T. (1995). Laws, regulations, and policies. In "Nonhuman Primates in Biomedical Research" (B. T. Bennett, C. R. Abee, and R. Henrickson, eds.), pp. 15-31. Academic Press, San Diego.

1102

JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

Jones, J. W., Pether, J. V. S., Rainey, H. A., and Swinburn, C. R. (1993). Recurrent Mycobacterium bovis infection following a ferret bite [letter]. J. Infect. 26, 225-226. Jorgesen, D. M. (1997). Gestational psittacosis in a Montana sheep rancher. Emerg. Infect. Dis. 3, 191-194. Kabrane-Lazizi, Y., Fine, J. B., Elm, J., Glass, G. E., Higa, H., Diwan, A., Gibbs, C. J., Jr., Meng, X.-J., Emerson, S. U., and Purcell, R. H. (1999). Evidence for widespread infection of wild rats with hepatitis E virus in the United States. Am. J. Trop. Med. Hyg. 61, 331-335. Kalter, S. S., Milstein, C. H., Bocyk, L. H., and Cummins, L. B. (1978). Tuberculosis in nonhuman primates as a threat to humans. Dev. Biol. Stand. 41, 85-91. Kalter, S. S., Heberling, R. L., Cooke, A. W., Barry, J. D., Tian, P. Y., and Northam, W. J. (1997). Viral infections of nonhuman primates. Lab. Anim. Sci. 47, 461-467. Karp, B. E., Ball, N. E., Scott, C. R., and Walcoff, J. B. (1999). Rabies in two privately owned domestic rabbits. J. Am. Vet. Med. Assoc. 215,1824-1827. Kaufman, A. F., Boyce, J. M., and Martone, W. J. (1980). Trends in human plague in the United States. J. Infect. Dis. 141, 522. Kawamata, J., Yamanouchi, T., Dohmae, K., Miyamoto, H., Takahaski, M., Yamanishi, K., Kurata, T., and Lee, H. W. (1987). Control of laboratory acquired hemorrhagic fever with renal syndrome (HFRS) in Japan. Lab. Anim. Sci. 37, 431-436. Keifer, A., Lynch, G., and Conwill, D. (1980). Water-borne giardiasismCalifornia, Colorado, Pennsylvania, Oregon. MMWR 29, 121-123. Kemper, C. A., Lombard, C. M., Deresinki, S. C., and Tompkins, L. S. (1990). Visceral bacillary epithelioid angiomatosis: Possible manifestations of disseminated cat scratch disease in the immunocompromised host: A report of two cases. Am. J. Med. 89, 216-222. Khabbaz, R. E, Rowe, T., Murphey-Corb, M., Heneine, W. M., Schable, C. A., George, J. R., Pau, C. P., Parekh, B. S., Lairmore, M. D., Curran, J. W., Kaplan, J. E., Schochetman, G., and Folks, T. M. (1992). Simian immunodeficiency virus needlestick accident in a laboratory worker. Lancet 341), 271-273. Khabbaz, R. E, Heneine, W. M., George, J. R., Parekh, B. S., Rowe, T., Woods, T., Switzer, W. M., McClure, H. M., Murphey-Corb, M., and Folks, T. M. (1994). Brief report: Infection of a laboratory worker with simian immunodeficiency virus. N. Engl. J. Med. 330, 172-177. Kiehlbauch, J. A., Tauxe, R. V., Baker, C. N., and Wachsmuth, I. K. (1994). Helicobacter cinaedi-associated bacteremia and cellulitis in immunocompromised patients. Ann. Intern. Med. 121, 90-93. Kiehlbauch, J. A., Brenner, D. J., Cameron, D. N., Steigerwalt, A. G., Makowski, J. M., Baker, C. N., Patton, C. M., and Wachsmuth, I. K. (1995). Genotypic and phenotypic characterization of H. cinaedi and H. fennelliae strains isolated from humans and animals. J. Clin. Microbiol. 22, 29402947. Koehler, J. E., Glaser, C. A., and Tappero, J. W. (1994). Rochalimaea henselae infection: New zoonosis with the domestic cat as reservoir. J. Am. Med. Assoc. 271, 531-535. Kornegay, R. W., Giddens, W. E., Jr., Van Hoosier, J. L., Jr., and Morton, W. R. (1985). Subacute nonsuppurative hepatitis associated with hepatitis B virus infection in two cynomolgus monkeys. Lab. Anim. Sci. 35, 400-404. Kristensen, K. H., and Lautrop, H. (1962). A family epidemic caused by the whooping cough bacillus Bordetella bronchiseptica (Danish). Ugeskr. Laeger 124, 303-308. Ksiazek, T. G., West, C. P., Rollin, P. E., Jahrling, P. B., and Peters, C. J. (1999). ELISA for the detection of antibodies to Ebola viruses. J. Infect. Dis. 179, S192-S198. Kupper, J. L., Casey, H. W., and Johnson, D. K. (1970). Experimental Yaba and benign epidermal monkeypox in rhesus monkeys. Lab. Anim. Care 20, 979-988. Ladiges, W. C., DiGiacomo, R. E, and Yamaguchi, R. A. (1982). Prevalence of Toxoplasma gondii antibodies and oocytes in pound source cats. J. Am. Vet. Med. Assoc. 180, 1334-1335.

Lairmore, M. D., Kaplan, J. E., Daniel, M. D., Lerche, N. W., Nara, P. L., McClure, H. M., McVicar, J. W., McKinney, R. W., Hen&y, M., Cerone, P., Rayfield, M., Johnson, D. O., Purcell, R., Gibbs, J., Allan, J., Ribas, J. L., Klein, H. J., Jahrling, P. B., and Brown, B. (1989). Guidelines for the prevention of simian immunodeficiency virus infection in laboratory workers and animal handlers. J. Med. Primatol. 18, ~67-174. Langley, R. L., ed. (1999). Animal handlers. In "Occupational Medicine: State of the Art Reviews." Hanley and Belfus, Philadelphia. Langley, J. M., Marrie, T. H., and Covert, A. (1988). Poker player's pneumonia: An urban outbreak of Q fever following exposure to a parturient cat. N. EngL J. Med. 319, 354-356. Lankas, G. R., and Jensen, R. D. (1987). Evidence of hepatitis A infection in immature rhesus monkeys. Vet. Pathol. 24, 340-344. Lavelle, J. P., Landas, S., Mitros, F. A., and Conklin, J. L. (1994). Acute gastritis associated with spiral organisms from cats. Dig. Dis. Sci. 39, 744-750. LeBoit, P. E., Berger, T. G., Egbert, B. M., Yen, T. S. B., Stoler, M. H., Bonfiglio, T. A., Stauchen, J. A., English, C. K., and Wear, D. J. (1988). Epithelioid haemangioma-like vascular proliferations in AIDS: Manifestation of cat scratch disease bacillus infection? Lancet 1, 960-963. LeDuc, J. W. (1987). Epidemiology of Hantaan and related viruses. Lab. Anim. Sci. 37, 413. Lee, H. W. and Johnson, K. M. (1982). Laboratory acquired infections with Hantaan virus, the etiologic agent of Korean hemorrhagic fever. J. Infect. Dis. 146, 645-651. Le Guenno, B. (1997). Hemorrhagic fevers and ecological perturbations. Arch. Virol. 13, 191-199. Lehmann-Grube, F. (1982). Lymphocytic choriomeningitis virus. In "The Mouse in Biomedical Research" (H. J. Foster, J. D. Small, and J. G. Fox, eds.), II, pp. 231-266. Academic Press, New York. Leitman, T., Brooks, D., Moncada, J., Schachter, J., Dawson, D., and Dean, D. (1998). Chronic follicular conjunctivitis associated with Chlamydia psittaci or Chlamydia pneumoniae. Clin. Infect. Dis. 26, 1335 - 1340. Lemon, S. M., Binn, K. N., Marchwicki, R., Murphy, P. C., Ping, L. H., Jansen, R. W., Asher, L. V. S., Stapelton, J. T., Taylor, D. G., and LeDuc, J. W. (1990). In vivo replication and reversal to wild type of a neutralization resistant antigenic variant of hepatitis A virus. J. Infect. Dis. 161, 7-13. Levine, N. D. (1980). "Nematode Parasites of Domestic Animals and of Man." Minneapolis: Burgess Publishing Co. Levine, J. F., Levy, M. G., Walker, R. L., and Crittenden, S. (1988). Cryptosporidiosis in veterinary students. J. Am. Vet. Med. Assoc. 193,1413-1414. Lipman, N. S. (1996). Rat bite fevers. In "Current Therapy of Infectious Disease" (D. Schlossberg, ed.), pp. 451-455. Mosby-Yearbook, Philadelphia. Lorenz, H. J., Cornelie, J., Willems, H., and Baljer, G. (1998). PCR detection of Coxiella burnetii from different clinical specimens, especially bovine milk, on the basis of DNA preparation with silica matrix. Appl. Environ. Microbiol. 64, 4234-4237. Luzzi, G. A., Milne, L. W., and Waitkins, S. A. (1987). Rat-bite acquired leptospirosis. J. Infect. 15, 57-60. Macy, D. W. (1999). Plague. In "Infectious Diseases of the Dog and Cat" (C. E. Greene, ed.), 2nd ed. Saunders, Philadelphia. Maetz, H. M., Sellers, C. A., Bailey, W. C., and Hardy, G. E., Jr. (1976). Lymphocytic choriomeningitis from pet hamster exposure: A local public health experience. Am. J. Public Health 66, 1082-1085. Mansfield, K., and King, N. (1998). Viral diseases. In "Nonhuman Primates in Biomedical Research" (B. T. Bennett, C. R. Abee, and R. Henrickson, eds.), pp. 1-57. Academic Press, San Diego. Marini, R. E, Adkins, J. A., and Fox, J. G. (1989). Proven or potential zoonotic diseases of ferrets. J. Am. Vet. Med. Assoc. 195, 990. Markell, E. K., John, D. T., and Krotoski, W. A. (1999). "Medical Parasitology," 8th ed. Saunders, Philadelphia. Marrie, T. J. (1990). Epidemiology of Q fever. In "Q Fever: The Disease" (T. J. Marrie, ed.), pp. 49-70. CRC Press, Boca Raton, Florida. Marrie, T. J., Williams, J. C., Schlech, W. E, and Yates, L. (1986). Q fever pneumonia associated with exposure to wild rabbits. Lancet 1, 427-429.

25. SELECTED Z

O

O

N

O

S

McDade, J. E., and Fishbein, D. (1988). The rickettsiae. In "Laboratory Diagnosis of Infectious DiseasemPrinciples and Practice" (A. Balows, W. J. Hausler, Jr., M. Ohashi, and A. Turano, eds.), pp. 864-890. SpringerVedag, New York. McEvoy, M. B., Noah, N. D., and Pilsworth, R. (1987). Outbreak of fever caused by Streptobacillus moniliformis. Lancet 2, 1361-1363. Meng, X. J., Purcell, R. H., Halbur, P. G., Lehman, J. R., Webb, D. M., Tsareva, T. S., Haynes, J. S., Thacker, B. J., and Emerson, S. U. (1997). A novel virus in swine is closely related to the human hepatitis E virus. Proc. Natl. Acad. Sci. U.S.A. 94, 9860-9865. Meng, X. J., Halbur, P. G., Shapiro, M. S., Govindarajan, S., Bruna, J. D., Mushahwar, I. K., Purcell, R. H., and Emerson, S. U. (1998). Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. J. Virol. 72, 9714-9721. Mercer, A., Fleming, S., Robinson, A., Nettleton, P., and Reid, H. (1997). Molecular genetic analyses of parapoxviruses pathogenic for humans. Arch. Virol. Suppl. 13, 25-34. Miranda, M. E., Ksiazek, T. G., Retuya, T. J., Khan, A. S., Sanchez, A., Fulhorst, C. E, Rollin, P. E., Calaor, A. B., Manalo, D. L., Roces, M. C., Dayrit, M. M., and Peters, C. J. (1999). Epidemiology of Ebola (subtype Reston) in the Philippines, 1996. J. Infect. Dis. 179, S115-S119. Molba, K., Baggesen, D. L., Aarestrup, E M., Ebbesen, J. M., Engberg, J., Frydendahl, K., Gerner-Smidt, P., Petersen, A. M., and Wegener, H. C. (1999). An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype typhimurium DT104. N. Engl. J. Med. 341, 14201425. Monath, T. P. (1999). Ecology of Marburg and Ebola viruses: Speculations and directions for future research. J. Infect. Dis. 179, S127-S138. Montali, R. J., Ramsay, E. C., Stephensen, C. B., Wodey, M., Davis, J. A., and Holmes, K. V. (1989). A new transmissible viral hepatitis of marmosets and tamarins. J. Infect. Dis. 16t), 759-765. Moran, G. J., Talan, D. A., Mower, W., Newdow, M., Ong, S., Nakase, J. Y., Pinner, R. W., and Childs, J. E. (2000). Appropriateness of rabies postexposure prophylaxis treatment for animal exposures. J. Am. Med. Assoc. 284,10011007. Morita, C., Tsuchiya, K., Ueno, H., Muramatsu, Y., Kojimahara, A., Suzuki, H., Moriwaki, K., Jin, M. L., Wu, X. L., and Wang, E S. (1996). Seroepidemiological survey of lymphocytic choriomeningitis virus in wild house mice in China with particular reference to their subspecies. Microbiol. Immunol. 40, 313-315. Mufson, M. A. (1989). Parainfluenza viruses, mumps, and Newcastle disease virus. In "Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections" (N. J. Schmidt and R. W. Emmons, eds.), pp. 669-691. American Public Health Association, Washington, D.C. Mushatt, D. M., and Hyslop, N. E. (1991). Neurologic aspects of North American zoonoses. Infect. Dis. North Am. 5, 703-731. Nasher, A. K. (1988). Zoonotic parasitic infections of the Arabian sacred baboon Papio hamadryas arabicus, Thomas in Asir Province, Saudi Arabia. Ann. Parasitol. Hum. Comp. 63, 448-454. National Research Council (1997). "Occupational Health and Safety in the Care and Use of Research Animals," p. 154. National Academy Press, Washington, D. C. Nelson, J. D., Kusmiesz, H., Jackson, L. H., and Woodman, E. (1980). Treatment of Salmonella gastroenteritis with ampicillin, amoxicillin, and placebo. Pediatrics 65, 1125-1130. Neuman-Haeflin, D., Felps, U., Renne, R., and Schweizer, M. (1993). Foamy viruses. Intervirology 35, 196-207. Newcomer, C. E., Anver, M. R., Simmons, J. L., Wilcke, B. W., and Nace, G. W. (1982). Spontaneous and experimental infections of Xenopus laevis with Chlamydia psittaci. Lab. Anim. Sci. 32, 680-686. Ng, V. L., Boggs, J. M., York, M. K., Golden, J. A., Hollander, H., and Hadley, W. K. (1992). Recovery of Bordetella bronchiseptica from patients with AIDS. Clin. Infect. Dis. 15, 376-377. Nicklas, W., Kraft, V., and Meyer, B. (1993). Contamination of transplantable

E

S

1

1

0

3

tumors, cell lines, and monoclonal antibodies with rodent viruses. Lab. Anim. Sci. 43, 296-300. Niven, S. J. E (1961). Subcutaneous "growths" in monkeys with Yaba pox virus. J. Pathol. Bacteriol. 81, 1-14. Ordog, G. J. (1985). Rat bites: Fifty cases. Ann. Emerg. Med. 14, 126. Orihel, T. C. (1970). The helminth parasites of nonhuman primates and man. Lab. Anim. Care 20, 395-401. Orlicek, S. L., Welch, D. E, and Kuhls, T. L. (1993). Septicemia and meningitis caused by Helicobacter cinaedi in a neonate. J. Clin. Microbiol. 31, 569-571. Paegle, R. D., Tweari, R. P., Bernhard, W. N., and Peters, E. (1976). Microbial flora of the larynx, trachea, and large intestine of the rat after long-term inhalation of 100 percent oxygen. Anesthesiology 44, 287-290. Palmer, A. E. (1987). Herpesvirus simiae: Historical perspective. J. Med. PrimatoI. 16, 99-130. Parker, S. and Holliman, R. E. (1992). Toxoplasmosis and laboratory workers: A case control assessment of risk. Med. Lab. Sci. 49, 103-106. Pavia, A. T., and Tauxe, R. V. (1991). Salmonellosis: Nontyphoidal. In "Bacterial Infections of Humans. Epidemiology and Control" (A. S. Evans and P. S. Brachman, eds.), 2nd ed., pp. 573-592. Plenum Press, New York. Pier, G. B., and Madin, S. H. (1976). Streptococcus iniae sp. nov., a betahemolytic streptoccus isolated from an Amazon freshwater dolphin, Inia geoffrensis. Int. J. Syst. Bacteriol. 26, 545-553. Polt, S. S.; Dismukes, W. E., Flint, A., and Schaefer, J. (1982). Human brucellosis caused by Brucella canis: Clinical features and immune response. Ann. Intern. Med. 97, 717-719. Purcell, R. H., Hoofnagle, J. H., Ticehurst, J., and Gerin, J. L. (1989). Hepatitis viruses. In "Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections" (N. J. Schmidt and R. W. Emmons, eds.), pp. 957-1065. American Public Health Association, Washington, D.C. Ravdin, J. L. (1995). Amebiasis. Clin. Infect. Dis. 20, 1453-1466. Ravdin, J. I. (2000). Entamoeba histolytica. In "Principles and Practice of Infectious Diseases" (G. L. Mandell, J. E. Bennett, and R. Dolin, eds.), pp. 2798-2810. Churchill Livingstone, Philadelphia. Reese, N. C., Current, W. L., Ernst, J. V., and Barley, W. S. (1982). Cryptosporidiosis of man and calf: A case report and results of experimental infections in mice and rats. Am. J. Trop. Med. Hyg. 31, 226-229. Reimer, L. G. (1993). Q fever. Clin. Microbiol. Rev. 6, 193-198. Richter, C. P., Lehner, N. D. M., and Henrickson, R. V. (1984). Primates. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), p. 298. Academic Press, Orlando, Florida. Roberts, J. A., Lerche, N. W., Markowits, J. E., and Maul, D. H. (1988). Epizootic measles at the CRPRC. Lab. Anim. Sci. 38, 492. Rosner, W. W. (1987). Bubonic plague. J. Am. Med. Assoc. 191, 406-409. Rousseau, M. C., Saron, M. E, Brouqui, P., and Boureade, A. (1997). Lymphocytic choriomeningitis in France: Four case reports and a review of the literature. Eur. J. Epidemiol. 13, 817-823. Rupp, M. E. (1992). Streptobacillus moniliformis endocarditis: Case report and review. Clin. Infect. Dis. 14, 769-772. Russell, R. G., Sarmiento, J. I., Fox, J. G., and Panigrahi, P. (1990). Evidence of reinfection with multiple strains of C. jejuni and C. coli in Macaca nemestrina housed under hyperendemic conditions. Infect. Immun. 58, 2149. Ryabchikova, E. I., Kolesnikova, L. V., and Luchko, S. V. (1999). An analysis of features of pathogenesis in two animal models of Ebola virus infection. J. Infect. Dis. 179, S199-$202. Sanger, J. G., and Thiermann, A. B. (1988). Leptospirosis. J. Am. Vet. Med. Assoc. 193, 1250-1254. Schmaljohn, C., and Hjelle, B. (1997). Hantaviruses: A global disease problem. Emerg. Infect. Dis. 3, 95-104. Schriefer, M. E., Sacci, J. B., Jr., Dumler, J. S., Bullen, M. G., and Azad, A. E (1994). Identification of a novel rickettsial infection in a patient diagnosed with murine typhus. J. Clin. Microbiol. 32, 949-954. Schweizer, M., Falcone, V., Gange, J., Turek, R., and Neumann-Haefelin, D.

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JAMES G. FOX, CHRISTIAN E. NEWCOMER, AND HARRY ROZMIAREK

(1997). Simian foamy virus isolated from an accidentally infected human individual. J. Virol. 71, 4821-4824. Sens, M. A., Brown, E. W., Wilson, L. R., and Crocker, T. P. (1989). Fatal Streptobacillus moniliformis infection in a two month old infant. Am. J. Clin. Pathol. 91, 612-616. Serikawa, T., Iwaki, S., Mori, M., Muraguchi, T., and Yamada, J. (1989). Purification of Brucella canis cell wall antigen using immunosorbent columns and use of the antigen in enzyme-linked immunosorbent assay for specific diagnosis of canine brucellosis. J. Clin. Microbiol. 27, 837-842. Shaw, P. K., Brodsky, R. E., Lyman, D. O., Wood, B. T., Hibler, C. P., Healy, G. R., Macleod, K. I., Stahl, W., and Schultz, M. G. (1977). A community wide outbreak of giardiasis with evidence of transmission by a municipal water supply. Ann. Intern. Med. 87, 426-432. Shen, Z., Feng, Y., and Fox, J. G. (1999). Co-infection with enteric Helicobacter spp. and Campylobacter spp. in asymptomatic cats. Lab. Anim. Sci. 49, 434. Shevtsova, Z. V., Lapin, B. A., Doroshenko, N. V., Krilova, R. L., Korzaja, L. I., Lomovskaya, I. B., Dzhelieva, Z. N., Zairov, G. K., Stakhanova, V. M., Belova, E. G., and Sazehchenko, L. A. (1988). Spontaneous and experimental hepatitis A in Old World monkeys. J. Med. Primatol. 17, 177-194. Siegart, R. (1972). Marburg virus. Virol. Monogr. 11, 98. Slomka, M. J., Brown, D. W. G., and Clewley, J. P. (1993). Polymerase chain reaction for detection of Herpesvirus simiae (B virus) in clinical specimens. Arch. Virol. 131, 89-99. Smith, A. L., Singleton, G. R., Hansen, G. M., and Shellam, G. (1993). A serologic survey for viruses and Mycoplasma pulmonis among wild house mice (Mus domesticus) in southeastern Australia. J. Wildl. Dis. 29, 219-229. Smith, A. L., Black, D. H., and Ebede, R. (1998). Molecular evidence for distinct genotypes of monkey B virus (Herpesvirus simiae) which are related to the macaque host species. J. Virol. 72, 9224-9232. Smith, T. F. (1989). Chlamydia. In "Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections" (N. J. Schmidt and R. W. Emmons, eds.), pp. 1335-1340. American Public Health Association, Washington, D.C. Soulsby, E. J. L. (1969). "Helminths, Arthropods, and Protozoa of Domesticated Animals," 6th ed. Williams and Wilkins, Baltimore. Staat, M. A., Kruszon-Moran, D., McQuillan, G. M., and Kaslow, R. A. (1996). A population-based serologic survey of Helicobacter pylori infection in children and adolescents in the United States. J. Infect. Dis. 174, 11201123. Stevensen, C. B., Montali, R. J., Ramsay, E. C., and Holmes, K. V. (1990). Identification, using sera of exposed animals, of putative viral antigens in livers of primates with callitrichid hepatitis. J. Virol. 64, 6349-6354. Stevensen, C. B., Jacob, J. R., Montali, R. J., Holmes, K. V., Muchmore, E., Compans, R. W., Arms, E. D., Buchmeier, M. J., and Lanford, R. E. (1991). Isolation of an arenavirus from a marmoset with callitrichid hepatitis and its association with disease. J. Virol. 65, 3995-4000. Stevensen, C. B., Park, J. Y., and Blount, S. R. (1995). CDNA sequence analysis confirms that the etiologic agent of callitrichid hepatitis is lymphocytic choriomeningitis virus. J. Virol. 69, 1349-1352. Stoenner, H. G., and Maclean, D. (1958). Leptospirosis (ballum) contracted from Swiss albino mice. Arch. Intern. Med. 101, 706-710. Stolte, M., Wellens, E., Bethke, B., Ritter, M., and Eidt, H. (1994). Helicobacter heilmannii (formerly Gastrospirillum hominis) gastritis: An infection transmitted by animals? Scand. J. Gastroenterol. 29, 1061-1064. Storz, J. (1971). "Chlamydia and Chlamydia-Induced Diseases." Thomas, Springfield, Illinois. Talan, D. A., Citron, D. M., Abrahamian, E M., Moran, G. R., and Goldstein, E. J. C. (1999). Bacteriologic analysis of infected dog and cat bites. N. Engl. J. Med. 340, 85-92. Tauxe, R. V. (1999). Salmonella enteritidis and Salmonella typhimurium DT104: Successful subtypes in the modern world. In "Emerging Infections 3" (W. M. Scheld, W. A. Craig, and J. M. Hughes, eds.), pp. 37-52. ASM Press, Washington, D.C.

Taylor, J. E, Istre, G. R., McChesney, T. C., Satalowich, E T., Parker, R. L., and McFarland, L. M. (1991). Epidemiologic characteristics of human tularemia in the southwest-central states, 1981-1987. Am. J. Epidemiol. 133, 1032-1038. Teare, J. A., and Loomis, M. R. (1982). Epizootic of balantidiasis in lowland gorillas. J. Am. Vet. Med. Assoc. 181, 1345-1347. Teutsch, S. M., Juranek, D. D., Sulzer, A , Dubey, J. E, and Sikes, R. K. (1979). Epidemic toxoplasmosis associated with infected cats. N. Engl. J. Med. 300, 695-699. Thiermann, A. B. (1977). Incidence of leptospirosis in the Detroit rat population. Am. J. Trop. Med. Hyg. 26, 970-974. Tissot-Dupont, H., Torres, S., Nezri, M., and Raoult, D. (1999). Hyperendemic focus of Q fever related to sheep and wind. Am. J. Epidemiol. 150, 67-74. To, H., Sakai, R., Shirota, K., Kano, C., Abe, S., Sugimoto, T., Takehara, K., Morita, C., Takashima, I., Maruyama, T., Yamaguchi, T., Fukushi, H., and Hirai, K. (1998). Coxiellosis in domestic and wild birds from Japan. J. Wildl. Dis. 34, 310-316. Torten, M. (1979). Leptospirosis. In "CRC Handbook Series in Zoonoses" (J. H. Steele, ed.), 1, pp. 363-421. CRC Press, Cleveland. Tsai, T. E (1987). Hemorrhagic fever with renal syndrome: Mode of transmission to humans. Lab. Anim. Sci. 37, 428. Tsai, T. E, Bauer, S. E, Sasso, D. R., Whitfield, S. G., McCormick, J. B., Caraway, T. C., MacFarland, L., Bradford, H., and Kurata, T. (1985). Serological and virological evidence of a hantaan virus-related enzootic in the United States. J. Infect. Dis. 152, 126-136. Versalovic, J., and Fox, J. G. (1999). Helicobacter. In "Manual of Clinical Microbiology" (E" R. Murray, E. J. Baron, M. A. PfaUer, F. C. Tenover, and R. H. Yolken, eds.), 7th ed., pp. 727-738. ASM Press, Washington, D.C. Visvesvar, G. S., and Stehr-Green, J. K. (1990). Epidemiology of free-living amoebae infections. J. Protozool. 37, 25S-33S. Washburn, R. G. (2000). Streptobacillus moniliformis (rat-bite fever). In "Principles and Practice of Infectious Diseases" (G. L. Mandell, J. E. Bennett, and R. Dolin, eds.), p. 2422. Churchill Livingstone, Philadelphia. Weber, D. J., and Hansen, A. R. (1991). Infections resulting from animal bites. In "Infectious Disease Clinics of North America" (A. Weinber and D. Weber, eds.), Vol. 5, pp. 663-677. Saunders, Philadelphia. Webster, R. G. (1997). Influenza virus: Transmission between species and relevance to the emergence of the next human pandemic. Arch. Virol. 13, 105113. Weigler, B. J., Hird, D. W., Hilliard, J. K., Lerche, N. W., Roberts, J. A., and Scott, L. M. (1993). Epidemiology of cercopithecine herpesvirus 1 (B virus) infection and shedding in a large breeding cohort of rhesus macaques. J. Infect. Dis. 167, 257-267. Weigler, B. J., Scinicareillo, E, and Hilliard, J. (1995). Risk of venereal B virus (cercopithecine herpes virus 1) transmission on rhesus monkeys using molecular epidemiology. J. Infect. Dis. 171, 1139-1143. Weinstein, M. R., Litt, M., Kertesz, D. A., Wyper, E, Rose, D., Coulter, M., McGeer, A., Facklam, R., Ostach, C., Willey, B. M., Borczyk, A., and Low, D. E. (1997). Invasive infections due to a fish pathogen, Streptococcus iniae. N. Engl. J. Med. 337, 589-594. Weir, E. C., Bhatt, E N., Jacoby, R. O., Hilliard, J. K., and Morgenstern, S. (1993). Infrequent shedding and transmission of Herpesvirus simiae from seropositive macaques. Lab. Anita. Sci. 43, 541-544. Weisbroth, S. H. (1979). Bacterial and mycotic diseases. In "The Laboratory Rat" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), Vol. 1, pp. 194230. Academic Press, New York. Wells, D. L., Lipper, S. L., and Hilliard, J. (1989). Herpesvirus simiae contamination of primary rhesus monkey kidney cell cultures: CDC recommendations to minimize risks to laboratory personnel. Diagn. Microbiol. Infect. Dis. 12, 333-335. Wentworth, D. E., McGregor, M. W., Macklin, M. D., Neumann, V., and Hin-

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shaw, V. S. (1997). Transmission of swine influenza virus to humans after exposure to experimentally infected pigs. J. Infect. Dis. 175, 7-15. Wilkins, E. G. L., Millar, J. G. B., Cockcroft, P. M., and Okubadejo, O. A. (1988). Rat-bite fever in a gerbil breeder. J. Infect. 16, 177. Williams, S. G., Sacci, J. B., Jr., Schreifer, M. E., Andersen, E. M., Fujioka, K. K., Sorvillo, E J., Barr, A. R., and Azad, A. E (1992). Typhus and typhus-like rickettsiae associated with opossums and their fleas in Los Angeles County, California. J. Clin. Microbiol. 30, 1758-1762. Willy, E. M., Woodward, R. A., Thorton, V. B., Wolff, A. V., Flynn, B. MI, Heath, J. L., Villamarzo, Y. S., Smith, S., Bellini, W. J., and Rota, P. A. (1999). Management of a measles outbreak among Old World nonhuman primates. Lab. Anim. Sci. 49, 42-48. Wilson, M. L., Bretsky, P. M., Cooper, G. H., Jr., Egbertson, S. H., Van Kruningen, H. J., and Carter, M. L. (1997). Emergence of raccoon rabies in Connecticut, 1991-1994: Spatial and temporal characteristics of animal infection and human contact. Am. J. Trop. Med. Hyg. 54, 457-463. Wilson, C. A., Wong, S., Muller, J., Davidson, C. E., Rose, T. M., and Burd, P. (1998). Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells. J. Virol. 72, 3082-3087. Wolf, R. H., Gibson, S. V., Watson, E. A., and Baskin, G. B. (1988). Multidrug chemotherapy of tuberculosis in rhesus monkeys. Lab. Anim. Sci. 38, 2533. Wolfrom, E., Chene, G., Boisseau, H., Beylot, C., Geniaux, M., and Taieb, A. (1995). Chronic urticaria and Toxocara canis [letter]. Lancet 345, 196. Woodward, D. L., Khakhria, R., and Johnson, W. M. (1997). Human salmonellosis associated with exotic pets. J. Clin. Microbiol. 35, 2786-2790.

E

S

1

1

0

5

Woolfrey, B. E, and Moody, J. A. (1991). Human infections associated with Bordetella bronchiseptica. Clin. Microbiol. Rev. 4, 243-255. Wright, W. H. (1985). Laboratory-acquired toxoplasmosis. Am. J. Clin. Pathol. 28, 1. Wright, R., Johnson, D., Neumann, M., Ksiazek, T. G., Rollin, P., Keech, R. V., Bonthius, D. J., Hitchon, P., Grose, C. E, Bell, W. E, and Bale, J. E (1997). Congenital lymphocytic choriomeningitis virus infection syndrome: A disease that mimics congenital toxoplasmosis or cytomegalovirus infection. Pediatrics 100, E91-E96. Wyers, M., Formenty, P., Cherel, Y., Guigand, L., Fernandez, B., Boesch, C., and Le Guenno, B. (1999). Histopathological and immunohistochemical studies of lesions associated with Ebola virus in a naturally infected chimpanzee. J. Infect. Dis. 179, $54-$59. Yanase, T., Muramatsu, Y., Inouye, I., Okabayashi, T., Ueno, H., and Morita, C. (1998). Detection of Coxiella burnetii from dust in a barn housing dairy cattle. Microbiol. Immunol. 42, 51-53. Yunker, C. E. (1964). Infections of laboratory animals potentially dangerous to man: Ectoparasites and other arthropods, with emphasis on mites. Lab. Anim. Care 14, 455-465. Zangwill, K. M., Hamilton, D. H., Perkins, B. A., Regnery, R. L., Plikaytis, B. D., Hadler, J., Carter, M. L., and Wenger, J. D. (1993). Cat scratch disease in Connecticut: Epidemiology, risk factors, and evaluation of a new diagnostic test. N. Engl. J. Med. 329, 8-13.

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Chapter 2 6 Xenozoonoses: The Risk of Infection after Xenotransplantation M a r i a n G. M i c h a e l s

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Introduction .................................................

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Potential M e c h a n i s m s for C r o s s - S p e c i e s Infections . . . . . . . . . . . . . .

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D e c r e a s i n g Infections in S o u r c e A n i m a l s . . . . . . . . . . . . . . . . . . . . . .

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Potential Benefits of X e n o t r a n s p l a n t a t i o n and O t h e r Infectious Disease Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Issues after X e n o t r a n s p l a n t a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Immunologic and technical advances have led to a tremendous increase in the number of people benefiting from allotransplantation. Ironically, it is the success of the field that has led to investigations into the development of xenotransplantation. To a large part this has occurred because of the great scarcity of human organ and tissue donors. A risk for infection exists with the use of any biologic agent regardless of whether it is from a human or animal source. Accordingly, transmission of infections from donor organs, tissues, or cells has become well recognized as a cause of disease after allotransplantation (Ho and Dummer, 1990; Green and Michaels, 1997). As the human organ donor shortage continues to escalate and newer cellular therapies are explored, increasing attention has been given to the potential use of animal organs, tissues, or cells for human maladies through xenotransplantation. Similar to allotransplantation, issues reLABORATORY ANIMAL MEDICINE, 2nd edition

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garding transmission of infections from the graft to the human recipient arise with these procedures (Michaels and Simmons, 1994; Michaels, 1998; Chapman et al., 1995; Hammel et al., 1998). The potential for novel infections to emerge because of xenotransplantation, xenozoonoses, has led to public debate on whether the field should be permitted to progress. This chapter reviews issues of xenotransplantation as a vector for infection to humans. Our current awareness of donor-associated infections after allotransplantation and known zoonoses is used to help estimate the potential risk of xenotransplantation.

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Although the field of allotransplantation has advanced significantly over the years, infections remain a substantial cause Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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of morbidity and mortality. The major risk factor for severe infections is the use of nonspecific immunosuppression to prevent rejection of the new organ or tissue. This risk factor will still be present, perhaps to an even greater degree when attempting to cross species barriers through xenotransplantation. Allotransplantation teaches us that infectious microbes can be from the recipient's endogenous flora, the environment, or organisms harbored within the donated organ, tissues, or cells (Ho and Dummer, 1990; Green and Michaels, 1997). The first two sources, the environment and the recipient's endogenous flora, will be the same whether a person undergoes an allo- or xenotransplant. These types of infections can be serious and contributed to the deaths of five of six recipients of xenotransplants in two separate series from the early 1960s, using chimpanzee and baboon kidney transplants, respectively (Reemtsma et al., 1964; Starzl et al., 1964). Similarly, the first baboon-to-human liver xenotransplant recipient died from infection with Aspergillus, an environmental pathogen, after receiving aggressive immunosuppression (Starzl et al., 1993). A second recipient of a baboon liver xenotransplant also succumbed to infection, dying with multiorgan failure 26 days after transplantation largely because of sepsis from endogenous intra-abdominal bacteria (Starzl et al., 1994). These reports on human clinical trials of xenotransplantation identified infections that were caused by immunosuppression and surgical complications. The risks for infection and the types of infections were not significantly different from those that could occur after allotransplantation. However, it is the latter source of infections, the graft itself, that may lead to novel infections. The types of microorganisms, which are donor-associated after allotransplantation, are often predictable. In general, these infectious agents are maintained in a quiescent or intracellular state and are without outward signs of disease in the donor. In this fashion they are carried to the new host within the donor organ or the accompanying hematopoietic cells (Ho and Dummer, 1990; Green and Michaels, 1997; Michaels and Simmons, 1994; Michaels, 1998). Examples include blood-borne pathogens such as hepatitis B virus (HBV), hepatitis C virus (HCV), and retroviruses, along with some herpesviruses and parasites. Similar classes of organisms are of concern with animal organ transplantation and are worth examining more fully. Human herpesviruses--in particular, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV)--are important donor-associated infections after allotransplantation. Their transmission from donors was first suspected by epidemiologic evidence and later confirmed by using molecular techniques (Chou, 1986; Cen et al., 1991). Both HCMV and EBV cause more severe disease in naive hosts who undergo primary infection after transplantation (Rubin, 1990; Ho et al., 1985). In particular, seronegative recipients of organs from seropositive donors are at highest risk. However, even patients with previous immunity to HCMV or EBV can be reinfected with donor strains of these viruses (Chou, 1986; Cen et al., 1991; Rubin,

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1990). The fact that prior immunity does not offer complete protection from reinfection with different strains of the same virus suggests that it likewise will not afford complete protection from analogous animal viruses after xenotransplantation. Not all herpesviruses latent in the donor are transmissible by transplantation. For example, herpes simplex virus (HSV) and. varicella zoster virus (VZV) are latent in sensory ganglia and as such are not usually present in blood or in the transplanted graft. Consequently, they represent a very low risk of donor transmission and highlight the concept of relative risk for donor-associated infection based upon microbial tropism and individual properties of the microbe. Other viruses besides herpesviruses can be donor-transmitted. Blood-borne pathogens such as human immunodeficiency virus (HIV), HBV, and HCV have all been unintentionally transmitted after allotransplantation (Ho and Dummer, 1990; Green and Michaels, 1997; Pereira et al., 1991; Dummer et al., 1989; Simonds et al., 1992). Usually this happened in the period before the viruses were identified or screening tests were available (Pereira et al., 1991; Dummer et al., 1989). However, even in the era of universal screening, transmission occasionally occurs. Transmission of HIV from a single donor to all four recipients of his organs and three of four bone-graft recipients was reported from a patient who had negative HIV screening (Simonds et al., 1992). Retrospective analysis concluded that the donor must have recently been infected with HIV and not had time to mount a detectable antibody response prior to his own death, emphasizing the inherent limitations of all screening tests. Nonviral infections can be transmitted with human donor tissues. Parasites such as Toxoplasma gondii are transmissible if the donor has organisms within the transplanted graft. Toxoplasma gondii infection points out the relative risk of transmission based on the type of transplant performed. Although many types of organ recipients can develop toxoplasmosis from an infected graft, naive heart transplant recipients are at highest risk because of the protozoon's tropism for cardiac muscle (Wreghitt et al., 1989; Michaels et al., 1992; Mason et al., 1987; Mayes et al., 1995). Donor-associated infections are occasionally caused by acute infections such as bacteremia or viremia in the donor or from local infection of the graft, because symptoms were not apparent in the human donor. Prevention of disease from donor-transmitted microbes after allotransplantation relies largely on screening of donors. However, except in the case of living related donation, donor screening is limited by substantial time constraints. In addition, prophylaxis and surveillance of recipients who are at risk can help with decreasing disease from donor-transmitted infections (Ho and Dummer, 1990; Green and Michaels, 1997). As new agents are recognized to be donor-transmitted, new protocols for screening and surveillance must be developed. In a similar fashion, protocols for xenotransplant screening and surveillance may help in preventing and monitoring infections.

26. XENOZOONOSES: THE RISK OF INFECTION AFTER XENOTRANSPLANTATION

III.

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Potential M e c h a n i s m s for Cross-Species Infections

Transmission of an animal microbial agent to human recipients of xenotransplantation could occur by several mechanisms (Michaels and Simmons, 1994; Michaels, 1998). An organism could be infectious for both the source animal and the human recipient, such as Toxoplasma gondii, and transmissible in a similar fashion as in allotransplantation. Known zoonoses that are present in the transplanted tissue or accompanying cells gain easy access to the recipient environment. Animal viruses that are similar to analogous human viruses, even if not currently known to be zoonotic, also come into communication with human cells that they may not have had access to without transplantation and under these intimate circumstances might be capable of infecting humans. This has been postulated for animal herpesviruses such as cytomegalovirus (CMV) and EBV (Michaels and Simmons, 1994; Michaels, 1998; Michaels et aL, 1994). It is possible that some animal microbes would not be pathogenic in a healthy person but could cause disease after xenotransplantation because of the immunosuppressed environment. Another concern revolves around the potential for a crossover of genetic material between an animal virus and a human virus, leading to a more virulent recombinant organism. It is possible that some latent animal viruses present in the source animal tissue will be "species-specific" and therefore not transmissible to humans. However, it may be a problem if it can reactivate within the animal organ, leading to graft failure. This could happen because of the immunosuppressed environment to which the graft is exposed, compounded by the absence of specific immunity in the new human host. Finally, it is possible that some human viruses may be able to cause disease in the animal graft as well. The concept of species specificity deserves more thorough investigation. If true, it is possible that xenotransplantation carries less risk of donor-transmitted infections than allotransplantation. However, examples of transmission of viruses that were considered to be species-specific can be found where the consequences are severe, such as with the herpesvirus family. The alphaherpesvirus of macaques, Cercopithecine herpesvirus 1 (B virus), is well established as a virus that is capable of being more dangerous after crossing species lines. Although generally innocuous in the host species, it can cause fatal disease after inadvertent infection of a human (Artenstein et al., 1991; Holmes et al., 1995; Hilliard et al., 1989). Transmission is rare, demonstrating probable relative species specificity, but when it does transmit, disease is always serious. In addition, Cercopithecine herpesvirus was transmitted from a person to his spouse through direct contact with infectious secretions (Hilliard et al., 1989). The ability of a virus to be relatively harmless in one species but

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to cause more severe disease in another species and to have the potential for secondary transmission leads to concern for infections after xenotransplantation (Michaels and Simmons, 1994; Michaels, 1998; Chapman et al., 1995; Institute of Medicine, 1996; "Draft," 1996; Kennedy, 1996). Because Cercopithecine herpesvirus is an alphaherpesvirus that is latent in nerve endings and not in blood or organs, similar to HSV's behavior, it is anticipated to be of low risk for transmission via xenotransplantation; however, its disease severity makes any risk unacceptable. Baboons and pigs have been used as source animals for xenotransplantation. Although they do not harbor Cercopithecine herpesvirus 1, they do have analogous alphaherpesvirusesm simian agent 8 (SA8) and pseudorabies, respectively (Hilliard et al., 1989; Melby and Altman, 1976; Michaels et al., 1994). Thus far, active surveillance has not found transmission of SA8 to humans. Pseudorabies can cause fatal disease in sheep, dogs, and cattle but has not been proven to be infectious to nonhuman primates (Melby and Altman, 1976). However, an anecdotal report noted three immunocompetent humans with transient fever, weakness, and neurologic abnormalities to test positive for pseudorabies antibodies within 15 months of their clinical symptoms, suggesting transmission between swine and humans (Mravak et al., 1987). Further information on the mechanism of transmission was not available. As noted, members of the alphaherpesvirus family show that infections across species' lines can be dangerous, but these viruses are unlikely to be easily transmitted with xenotransplantation because of their site of latency. However, both baboons and swine harbor other herpesviruses that are likely to be in the cells or organs transplanted (Michaels et al., 1994; Edington et aL, 1988; Falk et aL, 1976). Because the analogous human viruses (HCMV and EBV) are recognized as important sources of donor-associated infections, the ability of these viruses to cross species lines is of practical concern for xenotransplantation. Transmission of CMV or EBV between disparate species has been suggested. The Towne strain of human CMV replicates in cultures of chimpanzee skin fibroblasts and baboon CMV replicates in human fibroblasts (Perot et al., 1992; Michaels et al., 1997a). In addition, neurologic disease in two humans has been attributed to primate CMV (Huang et al., 1978; Charamella et al., 1973; Martin et al., 1994, 1995). In the first case the Colburn CMV strain was reportedly isolated from a brain biopsy of an encephalopathic child and is homologous with an African green monkey CMV strain (GR2757) (Huang et al., 1978; Charamella et al., 1973). In a second case, an African green monkey-like CMV was repeatedly isolated from a woman diagnosed with chronic fatigue syndrome (Martin et al., 1994, 1995). Both cases suggest potential transmission of a simian CMV to humans, but neither provides evidence for how the transmission may have transpired. More direct implications for xenotransplantation are found with the isolation of baboon

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CMV from a blood specimen of a recipient of a baboon liver 1 month after transplantation but not subsequently (Michaels et al., 2001). Further evaluation and clinical correlation are ongoing; studies are investigating swine CMV's ability to be grown on human cell lines in vitro. Few studies on cross-species transmission of herpesviruses such as EBV are available, although it has long been known that human EBV is able to infect marmoset lymphocytes in the laboratory (Miller, 1921). Our studies have also found variable cross-reactivity of antibody tests directed against human EBV antigens, with antibodies found in baboons presumably against the analogous virus, Herpesvirus papio. Commercial tests for EBV viral capsid antigen found a high seropositivity rate in a baboon colony, whereas the majority of paired specimens were negative when tested for EBV nuclear antigen (EBNA). This finding may be due to differences in the amount of conservation of different sites of herpesviruses (Falk et al., 1976). The lack of available and easy-toperform techniques to differentiate human from animal viruses is a major problem with attempting to assess the true risk of xenozoonoses. Further study is also needed to investigate swine and baboon herpesviruses' abilities to adapt to the human environment in vivo and to recombine with other viruses found in the human. Human organs, blood, and other tissues have been inadvertent vehicles for transmitting retroviruses, in particular the lentivirus HIV. Retroviruses are often species-restricted, but similar to transmission of herpesviruses, cross-species transmission of retroviruses has occurred, sometimes with severe consequences (Benveniste et al., 1988; Khabbaz et al., 1992, 1994). For example, simian immunodeficiency virus (SIV) appears to be benign in its natural host, the African green monkey, but progresses to an AIDS-like disease when inoculated into macaques (Benveniste et al., 1988). Transmission is variable to other nonhuman primates; whereas inoculation of a specific SIV strain causes rapid disease and death in macaques, attempts to infect baboons with the same strain led to minimal or no disease and immunologic response (Miller, 1921). This virus - - S I V - - and HIV type 2 are genetically similar (Benveniste et al., 1988). Probable transmission to two humans who were occupationally exposed to SIV has been well documented (Khabbaz et al., 1992, 1994). One individual remained asymptomatic and gradually had loss of antibody against the virus over 2 years. The other person had virus isolated from peripheral blood cells, documenting ongoing active infection albeit without clinical symptoms (Khabbaz et al., 1994). Several strains of SIV of chimpanzees have been recently identified as the likely origin of HIV type 1, responsible for the human AIDS epidemic (Gao et al., 1999). Antibody against SIV has been found on only rare occasions in baboons; however, relatedness to HIV makes it an important virus for which to screen. Less is known about lentiviruses in pigs. Retroviruses other than lentiviruses may be transmissible with xenotransplantation, particularly if using nonhuman pri-

MARIAN G. MICHAELS

mates. Examples include simian T-lymphotropic virus (STLV), which is an oncogenic retrovirus found in many nonhuman primate populations. This virus has areas of sequence homology with regions of human T-lymphotropic virus type 1 (HTLV-1), which has been associated with leukemia (Homma et al., 1984). For this reason it is an important virus to identify and eliminate from a potential source animal, l~ven more prevalent in nonhuman primate colonies is the class of retroviruses termed foamy virus, named for the foamy cytopathic effect found to contaminate primary monkey cell lines in the virology laboratory. Surveillance of workers with occupational exposure to nonhuman primates has found several workers to be infected with foamy virus, although no disease has been noted (Schweizer et al., 1997; "Nonhuman primate spumavirus," 1997). The exact timing of infection could not be elicited but was thought to be at least 16-20 years in one case. Family members remained seronegative (Schweizer et al., 1997). Polymerase chain reaction (PCR) studies found DNA from foamy virus in two human recipients of baboon liver transplants (Allan et al., 1998). The virus was found in sites distant from the graft but was always in combination with baboon mitochondria in cells. The virus was not able to be isolated despite multiple cultures, and serologic studies were negative after transplantation. Accordingly, the interpretation of the finding of DNA from foamy virus is difficult; it may represent microchimerism rather than true infection of human cells. Despite the problems with interpretation in these two cases, the risk of infection appears to be high, and clinical relevance should be assessed. Exogenous retroviruses of swine are less well characterized than those of nonhuman primates. Some pig retroviruses reactivate after exposure to radiation and therefore may be at risk for reactivation under the influence of immunosuppressive drugs (Frazier, 1985). Although it may prove to be laborious, it would seem prudent to screen out source animals for xenotransplantation that harbor exogenous retroviruses. Endogenous retroviruses have been raised as concerns for xenotransplantation in particular, because they cannot be removed from source-animal populations by current rearing methods. These viruses have long been recognized, but only with the renewal of interest in clinical xenotransplantation have more in-depth studies of endogenous retroviruses begun to be conducted. All strains of pigs studied carry porcine endogenous retroviruses (PoERVs) within their genome (Patience et al., 1997; Le Tissuer et al., 1997; Martin et al., 1998; Wilson et al., 1998). Likewise, baboon endogenous virus (BaEV) is found in baboon genomes. In vitro studies show both PoERV and BaEV to be capable of infecting human cell lines (Patience et al., 1997; Le Tissuer et al., 1997; Martin et al., 1998; Wilson et al., 1998; Huang et al., 1989). A human who received baboon bone marrow for experimental treatment of HIV-1 had transient presence of BaEV detected at day 5 after transplantation but not subsequently (Michaels et al., 1997b). The finding was in conjunction with finding baboon mitochondria, again limiting in-

26. XENOZOONOSES: THE RISK OF INFECTION AFTER XENOTRANSPLANTATION

terpretation as to whether this represented chimerism rather than true infection. The largest review to date evaluated 160 people who had various pig tissue transplants up to 12 years earlier (Paradis et al., 1999). No patient was found to have detectable viremia. In addition, no persistent PoERV infection could be found; 23 patients had evidence of microchimerism up to 8 years after exposure to pig tissues. Other virus classes have been transmitted after allotransplantation and may likewise cause disease after xenotransplantation. For example, unrecognized acute viremia with adenovirus has caused graft failure after human liver transplantation (Varki et al., 1990). Because adenovirus can infect many animal species, similar concerns may exist. Alternatively, it is possible that xenotransplantation may cause less of a risk for this type of transmission of acute infectious agents, because animals can be reared under much more stringent conditions where surveillance for infections or unwellness could be detected. Hepatitis B and C viruses have been transmitted after allotransplantation and bring up another area where xenotransplantation has been suggested as a superior alternative to human grafts. Baboons appear to be resistant to infection with hepatitis B virus (Starzl et al., 1993, 1994; Michaels et al., 1996). Accordingly, livers from baboons were used in an attempt to transplant in two patients with end-stage hepatitis B virus liver disease (Starzl et al., 1993, 1994; Michaels et al., 1996). However, not all hepatitis viruses are species-specific; related strains of hepatitis E virus have been found in humans and swine, suggesting cross-species transmission (Meng et al., 1998). Numerous other viruses, some long recognized and others newly recognized, in animal populations can be added to the growing lists of potential xenozoonotic infections. Examples include encephalomyocarditis virus that has been found to infect multiple species, including humans, baboons, and swine (Hubbard et al., 1992; Kalter and Heberling, 1990), as well as reoviruses, circoviruses, and paramyxoviruses. Menangle virus is the agent in recently discovered zoonoses infecting pigs and humans in Australia and is believed to be harbored by flying foxes (Philbey et al., 1998; Halpin et al., 1999). It is imperative that animal sources are maintained under strict rearing methods and that issues of rodent, insect, or other animal infestation considered. Issues of animal husbandry and surveillance are considered in detail in the recently released Public Health Service guideline on infectious disease in xenotransplantation ("Public Health Services guideline," 2001). In addition to infection of a xenotransplant recipient with an animal virus, consideration must also be given to the possibility of recombination (Chou, 1989; Halliburton et al., 1977; Isfort et al., 1992). Mixed-strain isolates of human CMV can recombine in vitro with passage (Chou, 1989). Likewise, mouse studies have demonstrated that infection with two avirulent herpes simplex viruses can lead to recombinations that are lethal (Halliburton et al., 1977). It is even possible in some cases for different virus types to recombine if coinfection occurs, as shown

1111

by in vitro integration of reticuloendotheliosis virus (an avian retrovirus) into an avian herpesvirus (Marek's disease virus) (Remington et al., 1995). Consideration should also be given to whether it is possible for avian influenza virus to gain access to swine tissue v~ithin a xenotransplant recipient, leading to mixing of influenza viruses within a human host. Nonviral agents may also cause xenogeneic infections if present in tissues being transplanted, such as Toxoplasma cysts in the cardiac muscle of a transplanted heart, regardless of the donor species. Throughout the world, commercial herds of swine are commonly seropositive for T. gondii (Remington et al., 1995). Likewise T. gondii seroprevalence rates of 32% and 16% have been reported in captive and wild baboons, respectively (Michaels et al., 1994; McConnell et al., 1974). Parasites normally confined to the gastrointestinal tract could be a problem if extraintestinal infection occurs, as is possible for Entamoeba histolytica and some schistosoma species. Local epidemiologic considerations will influence the types of parasites considered. For example, Hepatocystis parasites were found in the blood and/or liver of 10% of baboons studied in South Africa but would be unexpected in the United States, whereas in Babesia species could be found in the latter country (McConnell et al., 1974; Persing, 1996). These parasites can be transmitted with a xenotransplant, as noted in an animal model in which a baboon developed severe anemia secondary to Hepatocystis kochi after an experimental cardiac xenotransplant from a macaque (Henderson, 1992). Consequently, source-animal populations should be raised in protected environments, where parasitic infestation is avoided ("Public Health Service guideline," 2001). Bacterial and fungal diseases, while less likely to be latent and therefore unrecognized in source animals, still require consideration. For example, Mycobacterium species can infect animals, including baboons and swine, that may not manifest clinical symptoms until disseminated end-stage disease (Cappucci, 1972). Often the source of tuberculosis in animal populations is from human caretakers; accordingly, serial tuberculin skin testing of human caregivers and source animals should be routine.

B.

Decreasing Infections in Source A n i m a l s

Concerns for xenozoonoses could be substantially diminished if source animals could be raised under strict germfree conditions. Small laboratory animals have been raised in gnotobiotic environments. Pigs have also been raised under these conditions to a lesser extent but have problems after several months of age because of their size and waste production. These environments also preclude the normal colonization of the gastrointestinal tract with normal microbial agents that help with the digestion of food. For this reason pigs raised in germfree conditions are less robust and may not be ideal organ sources (Michaels

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and Simmons, 1994; Michaels, 1998; Fishman, 1994). Consideration has therefore been given to raising source animals under controlled environments in which specific pathogens have been eliminated and the introduction of outside pathogens is prevented (Michaels and Simmons, 1994; Michaels, 1998; "Public Health Service guideline," 2001; Fishman, 1994; Ye et al., 1994). These specific pathogen-free (SPF) colonies are still logistically difficult for nonhuman primates because of their long time until sexual maturity, variable gestational period, single offspring per pregnancy, and dependent state during infancy. This is one of the reasons that nonhuman primates are considered to be less desirable a source animal. Despite these severe constraints, five retrovirus-free and herpesvirus B-free macaque colonies were developed for research purposes in the United States (Lerche et al., 1994). If nonhuman primates are to be used as a source of grafts as an alternative to allotransplantation, then this costly and time-consuming avenue must be explored. Vigilance must be strictly maintained against the accidental introduction of microbes to an established colony from human caretakers or outside animals (Lerche et al., 1994). In addition, concern remains for the possibility of xenozoonoses that were not initially screened out of the population. Prospective decisions about which microbial agents are going to screened out of a population must be developed and updated as new information about particular organisms becomes available. For example, when developing the protocol for screening the baboon to be used for a bone marrow transplant to attempt to reconstitute the immune system in a person with AIDS, microbes were classified into one of four groups: (1) absolute contraindications, (2) relative contraindications, (3) treatable microbes, or (4) unavoidable microbes (Huang et al., 1989). Absolute contraindications included microbial agents that were known to be zoonotic and dangerous to humans, even if they were not anticipated to be found in baboons that were born and raised in the United States. For example, SIV, STLV, filoviruses, T. gondii, Mycobacterium tuberculosis, and herpes B virus were put in this category, even though SIV and herpes B were not anticipated in baboon populations at all. Relative contraindications consisted of microbes that were hypothesized to be xenozoonotic but were unproven or with uncertain consequence. Examples included baboon herpesviruses and foamy virus. Treatable infections were microbes that could be identified and eradicated prior to bone marrow harvest such as Babesia species or gastrointestinal pathogens. The fourth category, unavoidable microbes, included BaEV and microbes that exist but are as yet unrecognized and thus clearly of indeterminate risk (Michaels et al., 1997). The categories were written with the most up-to-date information available but with the recognition that future protocols might move some of the infectious agents into different classes. For example, more recent information on baboon CMV would likely have moved that virus from the relative contraindication to the absolute contraindication classification.

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In addition to developing protocols for which organisms should be evaluated, it is important to determine which testing method will be used and to be prepared to change these methods as more sophisticated techniques are developed. As noted in a previous section, not all testing methods are equivalent. A serologic survey of baboons raised in the United States demonstrated great variability in identifying baboons with evidence of Herpesvirus papio, the EBV analog (Michaels et al., 1994). This highlights the need to develop more sensitive techniques specific to the agent being evaluated. To increase the sensitivity of screening, paired sera samples were sent to two laboratories that used different techniques to look for a wide variety of viruses; any positive finding was classified as a true positive (Michaels et al., 1994). Also recognizing that serologic surveys have the potential to miss an immunologic response to a recently encountered agent, select animals that tested negative initially were quarantined and retested over time. Swine can be reared more efficiently than nonhuman primates in controlled environments, which makes screening somewhat easier. However, prospective considerations for the types of screening are still necessary ("Public Health Service guideline," 2001; Fishman, 1994; Ye et al., 1994). One study evaluated 10 newborn piglets that were reared to be free of brucellosis, pseudorabies virus, atrophic rhinitis, and Mycoplasma hypopneumonia (Ye et al., 1994). The investigators cultured skin, urine, feces, and nasal swabs for bacteria and examined the tissues for fungi and parasites. Further testing included bacterial blood culture, as well as commercial serologic tests for antibody against human CMV, hepatitis B virus, hepatitis C virus, HIV, Treponema pallidum, and Toxoplasma gondii. Tests were performed serially and at necropsy. No pathogens were identified that the investigators considered as a risk for xenotransplantation. However, 2 of the 10 pigs had positive ELISA tests for HIV at one point in time. Further investigation revealed them to be negative, but this again emphasizes limitations that can exist with screening techniques.

Co Potential Benefits of Xenotransplantation and Other Infectious Disease Issues

It is possible that xenotransplantation may decrease the risk of some infections after transplantation. The rationale for using baboon livers in two patients with end-stage liver disease from chronic hepatitis B virus infection was based on the resistance of baboons to this virus (Starzl et al., 1993, 1994; Michaels et aL, 1996). Likewise the rationale of attempting to reconstitute the immune system of a patient with HIV through xenotransplantation was again based on the natural resistance of the baboon to HIV-1 (Michaels et al., 1997b). In both cases allotransplantation was known to result in reinfection of the human graft. In addition to this potential advantage, xenotransplanta-

26. XENOZOONOSES: THE RISK OF INFECTION AFTER XENOTRANSPLANTATION

tion may avoid many donor-transmitted infections by permitting source animals to be reared in controlled, SPF environments and surveyed against acute infections. Performing transplants as elective surgery rather than as emergency procedures would also decrease postoperative infectious complications.

D.

Issues after Xenotransplantation

SPF environments will help decrease the risk of xenozoonoses but will not eliminate it completely. For this reason, it is important for any recipient of xenogeneic tissue to undergo counseling about the potential risks and surveillance for new infections after xenotransplantation. In this fashion the true epidemiology and risks of xenotransplant infections will be recognized. Serial samples from the recipient and transplanted tissues should be collected for cultures and/or assays to look for agents that were known or suspected to be in the source animal, such as endogenous viruses. These recommendations have become part of the guidelines recommended by the U.S. Public Health Service (PHS) ("Public Health Service guideline," 2001). However, as noted, current techniques for screening may ultimately prove inadequate. Accordingly, archiving samples for future studies is important, and samples should be maintained for 50 years, as suggested by PHS ("Public Health Service guideline," 2001). Shared or centralized registries and repositories for archived specimens may help with evaluating potential infectious agents and are currently being evaluated as a pilot project (Michaels, 1998; Institute of Medicine, 1996; Kennedy, 1996; Halpin et al., 1999). In addition, the U.S. Department of Health and Human Services has formed a Secretary's Advisory Committee on Xenotransplantation to consider the complexities of xenotransplantation and to help with ongoing review of these procedures. All biologic agents have an inherent risk for transmitting infections, and our ability to recognize and prevent these infections is continually growing. Xenotransplantation has potential to offer life-saving tissues and grafts to a number of types of people who currently die because of the absence of available human donors. As the field grows, it is imperative that new techniques be developed to help identify and prevent novel infections.

REFERENCES

Allan, J. S., Broussard, S. R., Michaels, M. G., Starzl, T. E., Leighton, K. L., Whitehead, E. M., Comuzzie, A. G., Lanford, R. E., Leland, M. M., Switzer, W. M., and Heneine, W. (1998). Amplification of simian retroviral sequences from human recipients of baboon liver transplants. AIDS Res. Hum. Retroviruses 14, 821-824. Artenstein, A. W., Hicks, C. B., Goodwin, B. S., Jr., and Hilliard, J. K. (1991). Human infection with B virus following a needle stick injury. Rev. Infect. Dis. 13, 288-291.

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Benveniste, R. E., Morton, W. R., Clark, E. A., Tsai, C. C., Ochs, H. D., Ward, J. M., Kuller, L., Knott, W. B., Hill, R. W., and Gale, M. J. (1988). Inoculation of baboons and macaques with SIV/mne, a primate lentivirus closely related to HIV 2. Virology 2(6), 2091-2101. Cappucci, D. T. (1972). Tuberculosis from man to primates. Am. Rev. Respir. Dis. 106, 819-823. Cen, H., Breinig, M. C., Atchinson, R. W., Ho, M., and McKnight, J. L. C. (1991). Epstein-Barr virus transmission via the donor organs in solid organ transplantation: polymerase chain reaction and restriction fragment length polymorphism analysis of IR2, IR3, and IR4. Virology 65, 976. Chapman, L. E., Folks, T. M., Salomon, D. R., Patterson, A. E, Eggerman, T. E., and Noguchi, E D. (1995). Xenotransplantation and xenogeneic infections. N. Engl. J. Med. 333, 1498. Charamella, L. J., Reynolds, R. B., Ch'ien, L. T., and Alford, C. A., Jr. (1973). Biologic characterization of an unusual human cytomegalovirus isolated from the brain. ABST Vol. 373, Ann. Meet. Am. Soc. Med. 256. Chou, S. (1986). Acquisition of donor strains of cytomegalovirus by renaltransplant recipients. N. Engl. J. Med. 314, 1418. Chou, S. (1989). Reactivation and recombination of multiple cytomegalovirus strains from individual organ donors. J. Infect. Dis. 160, 11-15. Draft Public Health Service guideline on infectious disease issues in xenotransplantation. (1996). Fed. Regi. 61(September 23), 49920-49932. Dummer, J. S., Erb, S., Breinig, M., Ho, M., Rinaldo, C. R., Jr., Gupta, P., Ragni, M. V., Tzakis, A., Makowka, L., and Van Thiel, D. (1989). Infection with human immunodeficiency virus in the Pittsburgh transplant population. Transplantation 47, 134. Edington, N., Wrathall, A. E., and Done, J. T. (1988). Porcine cytomegalovirus (PCMV) in early gestation. Vet. Microbiol. 17, 117-128. Falk, L., Deinhardt, E, Nonoyama, M., Wolfe, L. G., and Bergholz, C. (1976). Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus. Int. J. Cancer 18, 798-807. Fishman, J. A. (1994). Miniature swine as organ donors for man: strategies for prevention of xenotransplant-associated infections. Xenotransplantation 1, 47-57. Frazier, M. E. (1985). Evidence for retrovirus in miniature swine with radiationinduced leukemia or metaplasia. Arch. Virol. 83, 83-97. Gao, E, Bailes, E., Robertson, D. L., Chen, Y., Rodenburg, C. M., Michael, S. E, Cummins, L. B., Arthur, L. O., Peeters, M., Shaw, G. M., Sharp, P. M., and Hahn, B. H. (1999). Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature 397, 436-441. Green, M., and Michaels, M. G. (1997). Infections in solid organ transplant recipients. In "Principles and Practice of Pediatric Infectious Diseases" (S. S. Long, L. Pickering, and C. Prober, eds.). Churchill Livingstone, New York. Halliburton, I. W., Randall, R. E., Killington, R. A., and Watson, D. H. (1977). Some properties of recombinants between type 1 and type 2 herpes simplex viruses. J. Gen. Virol. 36, 471-484. Halpin, K., Young, P. L., Field, H., and Mackenzie, J. S. (1999). Newly discovered viruses of flying foxes. Vet. Med. 68, 83-87. Hammel, J. M., Prentice, E., and Fox, I. J. (1998). Current status of xenotransplantation. Probl. Gen. Surg. 15, 189-201. Henderson, J. D., Jr. Diagnostic exercise: anemia in a baboon. (1992). Lab. Anim. Sci. 42, 514-515. Hilliard, J. K., Black, D., and Eberle, R. (1989). Simian alphaherpesviruses and their relation to the human herpes simplex viruses. Arch. Virol. 109, 83102. Ho, M., Miller, G., Atchinson, R. W., Breinig, M. K., Dummer, J. S., Andiman, W., Starzl, T. E., Eastman, R., Griffith, B. P., Hardesty, R. L., Bahnson, H. T., Hakala, T. R., and Rosenthal, J. T. (1985). Epstein-Barr virus infections and DNA hybridization studies in post-transplantation lymphoma and lymphoproliferative lesions: the role of primary infection. J. Infect. Dis. 152, 876. Ho, M., and Dummer, J. S. (1990). Risk factors and approaches to infections in transplant recipients. In "Principles and Practice of Infectious Diseases"

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(G. C. Mandell, G. R. Douglas, Jr., and J. E. Bennet eds), 2nd ed. Churchill Livingstone. Holmes, G. P., Chapman, L. E., Stewart, J. A., Hunt, R. D., O'Connell, M. J., Letvin, N. L., Daniel, M. D., Desrosiers, R. C., Yang, C. S., and Essex, M. (1995). Guidelines for the prevention and treatment of B-virus infections in exposed persons. Clin. Infect. Dis. 20, 421-439. Homma, T., Kanki, P. J., King, N. W., Strauss, S. E., Hilliard, J. K., and Davenport, D. S. (1984). Lymphoma in macaques: association with virus of HTLV family. Science 225, 716-718. Huang, E., Kilpatrick, B., Lakeman, A., and Alford, C. A. (1978). Genetic analysis of a cytomegalovirus-like agent isolated from human brain. J. Virol. 26, 718-723. Huang, L., Silberman, J., Rothschild, H., and Cohen, J. C. (1989). Replication of baboon endogenous virus in human cells. J. BioL Chem. 264, 88118814. Hubbard, G. B., Soike, K. F., Buffer, T. M., Carey, K. D., Davis, H., and Butcher, W. I. (1992). An encephalomyocarditis virus epizootic in a baboon colony. Lab. Anim. Sci. 42, 233-239. Institute of Medicine. (1996). "Xenotransplantation: Science, Ethics, and Public Policy." National Academy Press, Washington D.C. Isfort, R., Jones, D., Kost, R., Witter, R., and Kung, H. (1992). Retrovirus insertion into herpesvirus in vitro and in vivo. Proc. Natl. Acad. Sci. 89, 991995. Kalter, S. S., Heberling, R. L. (1990). Primate viral diseases in perspective. J. Med. Primatol. 19, 519-535. Kennedy, I. (1996). "Animal Tissue into Humans." Advisory Group on Ethics of Xenotransplantation, Dept. of Health, United Kingdom. Khabbaz, R. E, Rowe, T., Murphey-Corb, M., Heneine, W. M., Schable, C. A., George, J. R., Pau, C. P., Parekh, B. S., Lairmore, M. D., Curran, J. W., Kaplan, J. E., Schochetman, G., and Folks, T. M. (1992). Simian immunodeficiency virus needle stick accident in a laboratory worker. Lancet 340, 271-273. Khabbaz, R. F., Heneine, W., George, J. R., Parekh, B., Rowe, T., Woods, T., Switzer, W. M., McClure, H. M., Murphey-Corb, M., and Folks, T. M. (1994). Brief report. Infection of a laboratory worker with Simian immunodeficiency virus. N. Engl. J. Med. 330, 172-177. Lerche, N. W., Yee, J. L., and Jennings, M. B. (1994). Establishing specific retrovirus-free breeding colonies of macaques. Lab. Anim. Sci. 44, 217221. Le Tissuer, P., Stoye, J. P., Takeuchi, Y., Patience, C., and Weiss, R. A. (1997). Two sets of human-tropic pig retrovirus. Nature 389, 681-682. Martin, U., Kiessig, V., Blusch, J. H., Haverich, A., vonder Helm, K., Herden, T., and Steinhoff, G. (1998). Expression of pig endogenous retrovirus by primary porcine endothelial cellls and infection of human cells. Lancet 352, 692-694. Martin, W. J., Zeng, L. C., Ahmed, K., and Roy, M. (1994). Cytomegalovirusrelated sequence in an atypical cytopathic virus repeatedly isolated from a patient with chronic fatigue syndrome. Am. J. Pathol. 145, 440-451. Martin, W. J., Ahmed, K., Zeng, L. C., Olsen, J., Seward, J. G., and Seehrai, J. S. (1995). African green monkey origin of the atypical cytopathic "stealth virus" isolated from a patient with chronic fatigue syndrome. Clin. Diagn. ViroL 4, 93-103. Mason, J. C., Ordelheide, K. S., Grames, G. M., Thrasher, T. V., Harris, R. D., Bui, R. H., and Mackett, M. C. (1987). Toxoplasmosis in two renal transplant recipients from a single donor. Transplantation 44, 588. Mayes, J. T., O'Connor, A. R., Castellani, W., and Carey, W. (1995). Transmission of Toxoplasma gondii infection by liver transplantation. Clin. Infect. Dis. 21, 511. McConnell, E. E., Basson, P. A., De Vos, V., Myers, B. J., and Kuntz, R. E. (1974). A survey of diseases among 100 free-ranging baboons from the Kruger national park. Onderstepoort J. Vet. Res. 41, 97-167. Melby, E. C., and Altman, N. J., eds. (1976). "Handbook of Laboratory Animal Science," Vol. 3. CRC Press, Cleveland.

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Meng, X. J., Halbur, E G., Shapiro, M. S., Govindarajan, S., Bruna, J. D., Mushahwar, I. K., Purcell, R. H., and Emerson, S. U. (1998). Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. J. Virol. 72, 9714-9721. Michaels, M. G. (1998). Xenotransplant-associated infections. Lab. Anim. Sci. 48, 228-233. Michaels, M. G., and Simmons, R. L. (1994). Xenotransplant-associated zoonoses. Transplantation 57, 1. Michaels, M. G., Wald, E. T., Fricker, F. J., del Nido, P. J., and Armitage, J. (1992). Toxoplasmosis in pediatric recipients of heart transplants. Clin. Infect. Dis. 14, 847. Michaels, M. G., McMichael, J., Brasky, K., Kalter, S., Peters, R. L., Starzl, T. E., and Simmons, R. L. (1994). Screening donors for xenotransplantation: the potential for xenozoonoses. Transplantation 57, 1462. Michaels, M. G., Lanford, R., Demetris, A. J., Chavez, D., Brasky, K., Fung, J., and Starzl, T. E. (1996). Lack of susceptibility of baboons to infection with hepatitis B virus. Transplantation 61, 350-351. Michaels, M. G., Alcendor, D., St. George, K., Rinaldo, C. R., Jr., Ehrlich, G., and Becich Hayward, G. (1997a). Distinguishing baboon CMV from human CMV: importance for xenotransplantation. J. Infect. Dis. 176, 14761483. Michaels, M. G., Hilliard, J., Deeks, S., Gupta, P., Heneine, W., Pardi, D., Kaufmann, C., Rinaldo, C., St. George, K., Chapman, L., Folks, T., Colson, Y., Volberding, P., and Ildstat, S. (1997b). Baboon bone marrow xenotransplant in a patient with advanced HIV: a model for the evaluation of potential xenozoonoses. Proceedings of the Institute of Human Virology Annual Meeting, J. Acquired Immune Deficiency Syndromes and Human Retrovirology, Abstract 11, p. 3. Michaels, M. G., Jenkins, F. J., St. George, K., Nalesnik, M. A., Starzl, T. E., and Rinaldo, C. R. (2001). Detection of infectious baboon cytomegalovirus after baboon-to-human liver xenotransplantation. J. Virology 75, 28252828. Miller, G. (1990). Epstein-Barr virus: biology, pathogenesis, and medical aspects. In "Virology" (B. N. Fields and D. M. Knipe, eds.), p. 1921. Raven Press, New York. Mravak, S., Bienzle, U., Feldmeier, H., Hampl, H., and Habermehl, K. (1987). Pseudorabies in man. Lancet 1, 501. Nonhuman primate spumavirus infections among persons with occupational exposure--United States, 1996. (1997). J. Am. Med. Assoc. 277, 783-785. Paradis, K., Langford, G., Long, Z., Heneine, W., Sandstrom, P., Switzer, W. M., Chapman, L. E., Lockey, C., Onions, D., and Otto, E. (1999). Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science 285, 1236-1241. Patience, C., Takeuchi, Y., and Weiss, R. A. (1997). Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3, 282-286. Pereira, B. J. G., Milford, E. L., Kirkman, R. L., and Levey, A. S. (1991). Transmission of hepatitis C virus by organ transplantation. N. Engl. J. Med. 325, 454. Perot, K., Walker, C. M., and Spaete, R. R. (1992). Primary chimpanzee skin fibroblast cells are fully permissive for human cytomegalovirus replication. J. Gen. ViroL 73, 3281-3284. Persing, D. H. (1996). Nucleic acid-based pathogen discovery techniques. Potential application to xenozoonoses. Mol. Diagn. 1, 243. Philbey, A. W., Kirkland, P. D., Ross, A. D., Davis, R. J., Gleeson, A. B., Love, R. J., Daniels, P. W., Gould, A. R., and Hyatt, A. D. (1998). An apparently new virus (family Paramyxoviridae) infectious for pigs, humans, and fruit bats. Emerg. Infect. Dis. 4, 269-271. Public Health Service guideline on infectious disease issues in xenotransplantation. Reemtsma, K., McCracken, B. H., Schlegel, J. U., Pearl, M. A., Pearce, C. W., DeWitt, C. W., Smith, P. E., Hewitt, R. L., Flinner, R. L., and Creech, O. (1964). Renal heterotransplantation in man. Ann. Surg. 160, 384-410. Remington, J. S., McLeod, R., and Desmonts, G. (1995). Toxoplasmosis. In

26. XENOZOONOSES: THE RISK OF INFECTION AFTER XENOTRANSPLANTATION

"Infectious Diseases of the Fetus and Newborn Infant" (J. S. Remington and J. O. Klein, eds.), 4th ed., pp. 140-267. Philadelphia, Saunders. Rubin, R. H. (1990). Impact of cytomegalovirus infection on organ transplant recipients: Rev. Infect. Dis. 12(Suppl. 7), $754-66. Schweizer, M., Falcone, V., Gange, J., Turek, R., and Neumann-Haefelin, D. (1997). Simian foamy virus isolated from an accidentally infected human individual. J. Virol. 71, 4821-4824. Simonds, R. J., Holmberg, S. D., Hurwitz, R. L., Coleman, T. R., Bottenfield, S., Conley, L. J., Kohlenberg, S. H., Castro, K. G., Dahan, B. A., Schable, C. A., Rayfield, M. A., and Rogers, M. F. (1992). Transmission of human immunodeficiency virus type I from a seronegative organ and tissue donor. N. Engl. J. Med. 326, 726. Starzl, T. E., Marchioro, T. L., Peters, G. N., Kirkpatrick, C. H., Wilson, W. E. C., Porter, K. A., Rifldnd, D., Ogden, D. A., Hitchcock, C. R., and Waddel, W. R. (1964). Renal heterotransplantation from baboon to man: experience with six cases. Transplantation 2(6), 752-776. Starzl, T. E., Fung, J., Tzakis, A., Todo, S., Demetris, A. J., Marino, I. R., Doyle, H., Zeevi, A., Warty, V., Michaels, M., Kusne, S., Rudert, W. A., and

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Trucco, M. (1993). Baboon-to-human liver transplantation. Lancet 341, 65-71. Starzl, T. E., Tzakis, A., Fung, J. J., Todo, S., Demetris, A. J., Manez, R., Marino, I. R., Valdivia, L., and Murase, N. (1994). Prospects of clinical xenotransplantation. Transplant. Proc. 26(3), 1082-1088. Varki, N. M., Bhuta, S., Drake, T., and Porter, D. D. (1990). Adenovirus hepatitis in two successive liver transplants in a child. Arch. Pathol. Lab. Med. 114, 106. Wilson, C. A., Wong, S., Muller, J., Davidson, C. E., Rose, T. M., and Burd, P. (1998). Type C retrovirus released from primary peripheral blood mononuclear cells infects human cells. J. Virol. 72, 3082-3087. Wreghitt, T. G., Hakim, M., Gray, J. J., Balfour, A. H., Stovin, P. G., Stewart, S., Scott, J., English, T. A., and Wallwork, J. (1989). Toxoplasmosis in heart and heart and lung transplant recipients. J. Clin. Pathol. 42, 194. Ye, Y., Niekrasz, M., Kosanke, S., Welsh, R., Jordan, H. D., Fox, J. C., Edwards, W. C., Maxwell, C., and Cooper, D. K. C. (1994). The pig as a potential organ donor for man. Transplantation 57, 694-703.

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Chapter 2 7 Genetic Monitoring John J. Sharp, Evelyn E. Sargent, and Peter A. Schweitzer

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The Need for Genetically Defined Animals . . . . . . . . . . . . . . . . . . . . . . . . III. Sourcesand Monitoring of Genetic Variability . . . . . . . . . . . . . . . . . . . . . A. Genetic Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Incomplete Inbreeding and Residual Heterozygosity . . . . . . . . . . . . . C. Genetic Drift and Substrain Variation . . . . . . . . . . . . . . . . . . . . . . . . . D. Genetically Engineered Strains: Mutant Alleles . . . . . . . . . . . . . . . . . E. Mixed Genetic Backgrounds: Congenic Strains . . . . . . . . . . . . . . . . . IV. Colony Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Monitoring Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Physical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Immunological Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Biochemical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Chromosomes and Bones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. DNA-BasedMolecular Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Strain Performance Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

INTRODUCTION

The need for genetic monitoring of experimental laboratory animals has expanded and b e c o m e more complex as the technology to manipulate the m a m m a l i a n g e n o m e has advanced. Not only must the genetic background of strains be monitored but also specific genetic modifications, such as the presence of a transgene or targeted allele, must be monitored, often on a daily or weekly basis. The focus of this chapter will be on the genetic monitoring of mice, since it is the development of gene targeting in mice that has greatly added to the complexity of research tools available for biomedical research. LABORATORY ANIMAL MEDICINE, 2nd edition

1117 1118 1119 1119 ~120 1120 1120 1120 1121 1122 1122 1123 1123 1124 1124 1126 1126 1127

Research with rodents took a giant leap forward in the early part of 1900 with the rediscovery of Mendel's laws. Early studies focused primarily on the genetics of coat-color inheritance and cancer (Morse, 1978). The first inbred mouse strain, DBA, was described by C. C. Little in 1916 (Statts, 1996), and L. C. Strong subsequently began the development of additional inbred strains, or as he put it, "development of a better mouse," when such an effort was largely considered a waste of time (Strong, 1978). Strong's efforts led to the establishment of many of today's c o m m o n inbred strains. Subsequent to Strong's research, the inbred mouse strain b e c a m e the standardized laboratory model for m a n y scientific investigations. As more inbred strains were developed, a greater appreciation Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

1118

for the variability between the individual strains became recognized. This variability is manifested in many ways, including susceptibility to cancer, response to infectious agents, susceptibility to atherosclerosis, alcohol preference, etc. Such strain characteristics are evident whenever controlled breeding and selection take place (i.e., in dogs, cats, and horses) even though full inbreeding is not obtained. A fully inbred strain represents a unique combination of largely homozygous alleles that are responsible for the particular characteristics of that strain. It is this uniqueness that must be monitored in order to maintain the genetic purity of the inbred strain. Early on, only careful breeding practices and visible characteristics were available as tools to maintain genetic purity. However, it is possible for genetic contamination to occur (i.e., through accidental crossbreeding) without the observance of any visible characteristic to indicate that such an event took place. Genetic monitoring has evolved largely as biochemical and DNA-based molecular technology have developed. Selected biochemical and immunological markers were shown to be capable of representing a unique strain-distribution pattern for most inbred strains. With the advent of molecular technology such as Southern blotting and PCR (polymerase chain reaction), genetic monitoring has 9become more sophisticated, and there is a greater confidence level of genetic purity. In the first half of the twentieth century, scientists exchanged small numbers of animals with one another for their research projects. Today rodent-breeding centers supply research facilities around the world with ever increasing numbers of animal models. The number of new strains has increased dramatically in the last few years, particularly because of the development of gene targeting in the mouse (Sharp and Mobraaten, 1997). Both the total number and the variety of mice being used in biomedical research are rapidly expanding. For instance, the nonprofit Jackson Laboratory alone maintains 2500 different inbred, congenic, recombinant inbred, spontaneous mutant, transgenic, and targeted mutant mouse strains, a number that is growing daily. Commercial breeding centers carry many fewer strains, but they too are starting to distribute genetically engineered strains. It is vitally important that these centralized distribution centers monitor the genetic purity of the strains they distribute. A combination of proper colony-management practices and genetic monitoring must be employed to detect the occurrence of genetic deviants, thereby permitting corrective measures to be taken. Likewise, individual researchers must assume the responsibility for ensuring the genetic quality of the strains they maintain. They must be aware of the potential sources of genetic contamination and also practice proper colony management. Researchers need to understand the importance of genetic background and how it may affect the phenotype of a mutant strain. They must be vigilant for potential genetic errors or contamination both within their own colonies and in mice received from central breeding facilities. Experienced rodent researchers are

JOHN J. SHARP, EVELYN E. SARGENT, AND PETER A. SCHWEITZER

well aware of these issues, but there are many investigators today who are just starting to use genetically engineered strains and who have little awareness of the need for genetic monitoring or the consequences of the lack thereof. It is the purpose of this chapter to outline the potential sources of genetic variation in rodent (primarily mouse) colonies and to describe the current methods used to monitor or control this variation. These methodologies may be applied in both centralized breeding centers and individual research laboratories.

II.

THE NEED FOR GENETICALLY DEFINED ANIMALS

For our purposes "genetically defined" is an operational term that refers to the genetic homogeneity between individual animals of a breeding group 9If individual animals within a group are essentially identical with regard to their genomic structure (allelic variation), then they fit the definition of "genetically defined 9 Inbred strains and inbred strains that carry spontaneous or genetically engineered mutations (i.e., transgenes or targeted mutations) fit the definition of genetically defined. Outbred, random-bred, or hybrid strains (with the exception of F1 hybrids between two inbred strains), because of the variability between individuals within the group, are not genetically defined. Genetically engineered stocks that carry an explicitly defined mutation on a hybrid or mixed genetic background are in common use today. These strains are composed of both defined and undefined genetic elements. The argument over the use of genetically defined (inbred) versus genetically diverse (outbred) rodent strains has been carried on for years (e.g., Festing, 1999b; Festing and Wolff, 1995). Those who favor the use of outbred stocks argue that outbred stocks better match the genetically diverse human population and therefore provide more realistic models for human disease. However, the use of outbred stocks severely limits the range of experimentation available to the researcher and is especially limiting in the areas of gene discovery and gene function. Because of this limitation, genetically defined strains are best suited for most of today's rodent experimentation. If hybrid vigor or genetic diversity is felt to be necessary for a particular line of experimentation, then F1 animals, created by matings between two inbred strains, may be used (Silva et al., 1997). F1 animals provide both added diversity and animals of identical genetic background. Also, a mutant phenotype on an inbred background that does not mimic the human phenotype may very well do so on another background (Erickson, 1996). Taken as a whole, the inbred strains represent a huge amount of genetic diversity, a portion of which has been fixed within each individual strain. The extent of this diversity is largely underappreciated, but it provides an enormous reservoir for genomic research.

27. GENETIC MONITORING As a further consideration, the use of inbred strains (or strains carrying specific mutations on an inbred background) reduces experimental animal usage, a recommended goal of the National Institutes of Health and other national and international animal regulatory agencies (Festing, 1999a). Because of the genetic diversity among individuals, the use of an outbred stock requires large numbers of experimental animals in order to obtain statistically valid results. Inbred strains essentially eliminate experimental variability due to genetic factors, thus reducing animal usage. Many inbred strains were initially developed to study the role of genes in cancer (Strong, 1978). Studies of these strains led to the understanding that cancer is a multifactorial (multigenic) disease in which oncogenes, immune-function genes, tumorsuppressor genes, and other unidentified genes play a role in this disease. We now understand that much, if not most., human disease is multigenic. An inbred strain has a uniquecombination of homozygous genes that results in the particular characteristics of that strain (Biesiadecki et al., 1998; Crawley, 1996; Erickson, 1996; Festing, 2000). The variety of characteristics that differ between inbred strains is very broad. There are, for instance, strains that are susceptible to atherosclerosis and others that are resistant; strains that are uniquely susceptible to particular pathogens and others that are resistant; strains that are aggressive and others that are not, etc. To map, clone, and identify the genes affecting a particular trait (each is referred to as a quantitative trait locus, or QTL), it is necessary to carry out a mapping cross between the susceptible and resistant strains (e.g., Paigen, 1995). The homogeneity of inbred strains greatly facilitates the identification and mapping of genes that contribute to individual strain characteristics (Mu et al., 1999; Pitman et al., 1998). The rapid growth of genetically engineered rodent (particularly mouse) strains has led to a greater appreciation of the role of the genetic background on the expressed phenotype of a mutant allele (e.g., Homanics et al., 1999). Transgenic and targeted mutant strains of mice are often generated or initially studied on a mixed genetic background. In order to increase the range of experimentation and stabilize experimental results, these mutant alleles should be transferred to one or more inbred backgrounds. The observed phenotype of a mutant allele is often very different on different genetic backgrounds (Dietrich et al., 1993; Nielsen et al., 1995; Wakeland et al., 1997). Such variability again presents the opportunity to map, clone, and identify the modifying genes (Cormier et al., 1997; Gould et al., 1996). The chemical mutagen ENU (N-ethyl-N-methylnitrosourea) is used to generate mice carrying point mutations (Justice et al., 1999). Mutant alleles with a detectable phenotype may result if the mutation occurs within a gene or regulatory element. Such mutant strains are now being rapidly generated in order to provide strains for gene discovery. The majority of ENU mutagenesis programs are using inbred strains since they provide the

1119 most direct path to mapping and cloning the mutated genes or regulatory elements. In addition to inbred, genetically engineered, and chemically mutagenized mouse strains, there are other genetically defined strains that find great utility in today's research atmosphere. In particular, recombinant inbred and recombinant congenic strains are strains that provide great utility in QTL mapping.

III.

SOURCES AND M O N I T O R I N G OF GENETIC VARIABILITY

In this section we examine the sources of genetic variability that may occur within genetically defined strains of laboratory animals, particularly mice. In general, genetic variability may result from human error, genetic manipulation, or the accumulation and fixing of spontaneous mutations that occur within a colony (genetic drift). Attempts to control or limit this variability require a regular monitoring system and/or strict colonymanagement practices, depending on the potential source of the variability. These are discussed below.

A.

Genetic Contamination

Breeding errors possibly represent the most common and most disastrous cause of variation among genetically defined rodent colonies. For example, cage-labeling errors may result in the introduction of incorrect animals into a breeding pen. This is a particular problem if animal technicians do not understand strain and genetic nomenclature. The mistake may manifest itself by the appearance of an unexpected coat color, thus making the breeding error obvious and leading to removal of affected animals from the colony. However, if the two strains carry identical coat-color alleles, then the accidental contamination will probably go undetected. More subtle contamination may result from an undesirable mating between an offspring and a parent, which can occur with those strains of mice that become sexually mature around weaning age. Strict monitoring of breeding pairs and removal of young mice from breeding pens are necessary to prevent such an occurrence. These errors will have minimal effects when maintaining strains of inbred mice, but will have catastrophic effects on matings when two different inbred strains are mated to produce F1 hybrid mice. Regular genetic monitoring of colonies will detect such contamination, but the discovery of such errors may occur subsequent to the experimental use of the contaminated animals. Monitoring frequency is a decision that considers trade-offs between frequency and cost. Proper colony management is the most effective control measure against accidental contamination and includes thorough training of animal care technicians.

JOHN J. SHARP,EVELYNE. SARGENT,AND PETER A. SCHWEITZER

1120

B.

Incomplete Inbreeding and Residual Heterozygosity

Inbred strains are generated by brother-sister (full sib) matings and are largely isogenic. F20 has been the generation arbitrarily chosen to designate a strain "inbred"; however, at F20 the number of loci that remain unfixed is estimated to be 273 (Bailey, 1982). With additional inbreeding beyond F20, some of these alleles will become fixed and others lost as a result of the continual inbreeding process, and complete isogenicity is approached. If separate lines are created at F20, with continued inbreeding the number of loci differing between the two eventual substrains is estimated to be 117 (Bailey, 1982).

C.

Genetic Drift and Substrain Variation

All inbred strains suffer a low level of mutation that leads to genetic drift. The majority of mutations affect neither coding nor regulatory sequences, and therefore remain silent and are of little practical concern to the researcher. However, mutations that occur in coding or regulatory sequences may alter the physiology of a given strain. If such phenotypic deviants are not detected and removed from the colony, alteration of the strain characteristics is possible. Substrains separated from each other will independently diverge through the process of genetic drift. Thus, substrains from different suppliers will not be genetically identical. Some substrains differ from each other enough to reject skin grafts (Simpson et al., 1997). For these reasons, it is important to use a strain from a single source or supplier and when publishing, report the substrain designation (i.e., C57BL/6J, C57BL/6ByJ). Within a given location, minimizing the effects of genetic drift is most practically carried out by proper colony-management practices in which large colonies are generated by expansion from a single or small number of progenitors. Genetic drift is an inexorable process affecting all strains of mice and is essentially impossible to monitor, although molecular techniques do permit distinguishing even closely related substrains from each other (see Simpson et al., 1997, and Table III).

D.

Genetically Engineered Strains: Mutant Alleles

The ability to introduce specific, molecularly defined mutations into the mammalian genome requires that monitoring technology now include DNA-based molecular techniques such as Southern blotting and PCR for the purpose of monitoring specific mutant alleles. If the mutation is on an inbred background, then the background must be monitored as well. In cases where the mutation is on a mixed genetic background, monitoring is restricted to ensuring the presence or absence of the mutant allele. The cloning and sequencing of many sponta-

neous mutations now permit the molecular monitoring of these previously unknown molecular defects. Incorrect genotyping of a mutant allele can lead to colony heterogeneity and subsequent total loss of the desired allele from an entire colony. This is particularly problematic in cases where there is a selective advantage for loss of the desired allele, e.g., better breeding performance or longer reproductive life span. In fact, one of the cardinal indicators of potential genetic problems within a colony is an improvement in reproductive performance. Regular monitoring of new breeders introduced into these colonies will reduce the possibility that genotyping errors will spread rapidly through the colony.

E.

Mixed Genetic Backgrounds: Congenic Strains

Many initial studies with genetically engineered mice are carried out on mixed, or segregating, genetic backgrounds. For the reasons discussed above, the mutant allele will have its greatest utility on one or more inbred genetic backgrounds, and the development of congenic strains, generated by introgressing the mutation onto an inbred background through a series of backcrosses, is becoming commonplace. Complete backcrossing to the recipient strain background requires 10 to 12 backcross generations before the strain may be considered to be congenic, and it may take well over 3 years to complete. Genetic monitoring of the mutant allele is required at each backcross generation to identify carriers for the subsequent generation. The need to shorten the time to generate congenic strains, together with the development of PCR-amplified SSLP (simple sequence length polymorphism) markers (Dietrich et al., 1996), has led to protocols that reduce the time required to produce a congenic strain to 1.5-2 years. These "speed congenics" are now being created in as few as 5 backcross generations (Wakeland et al., 1997). The basis of the methodology lies in that fact that, statistically, each backcross generation reduces the residual heterozygosity by 50%, but individual mice lie in a normal distribution around this reduction (Fig. 1). Faster introgression of the desired mutant allele or transgene will occur if the best animal (the one with the least residual heterozygosity) is chosen for the breeder in the subsequent generation. About 20 carriers (preferably males) are screened with 100 or more SSLP markers (polymorphic between the donor and recipient backgrounds) at each backcross generation in order to choose the best breeder for the next generation. More than 3000 PCR reactions are required to generate a single congenic generated by this method, but the time reduction in generating the strain may balance the need for increased genetic monitoring. Genes closely linked to the mutation being introgressed during congenic strain generation are not reduced by 50% at each backcross generation. They are lost more slowly because the loss requires a crossover event between the linked gene and the

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N8

N9

N10

I

Nll

N12

Backcross Generation

Fig. 1. The data points represent the average percentage of host genome with increasing backcross generation. Individual animals will vary around this average. Using speed congenic protocols, the animal with the highest host genome (lowest residual heterozygosity) at each generation is picked to continue the backcrossing.

selected locus. These passenger genes may interact epistatically with the mutant gene, thereby affecting the observed phenotype. Allelic variation between the donor and recipient strain could result in a phenotype that is heavily influenced by either the donor or recipient alleles. In the case where epistatic interaction offers some advantage in viability or reproductivity, one allele may be selected over the other (Bailey, 1982). The size of the carryover DNA is statistically 40 cM (centimorgans) after 5 backcross generations and 20 cM after 10 (Fox and Witham, 1997). Assuming the number of genes in the mouse genome to be 75,000 and a total length of 1416 cM (autosomes + X chromosome), the number of carryover genes is about 2120 and 1060 after 5 and 10 backcross generations, respectively. In order to mimic an N 10 backcross when using marker-assisted protocols, this carryover piece should also be monitored. Breeders should be selected not only for enrichment of the recipient genome but also for a reduced size of the linked DNA.

IV.

COLONY M A N A G E M E N T

Probably the most common source of genetic variation within rodent colonies is human error. Good colony-management practices are vital to both reducing the occurrence of errors and to tracing and correcting an error once it has occurred. Good management includes maintaining an accurate record-keeping sys-

tem (pedigree charts), instituting a proper breeding system for each colony, and implementing a training program for animal technicians, who are the first line of defense in any genetic quality-control system. A proper breeding system is necessary to assure standardized genetic quality. In large breeding and research facilities, the pyramid breeding system incorporating a foundation stock (FS), pedigree expansion stock (PES), and production stock (PS) colony is the system of choice (Fox and Witham, 1997). The relative sizes of these colonies increase from FS to PS, thus the pyramid terminology. At the top of the pyramid is the foundation stock (FS) colony where all animals are pedigreed and inbred strains are maintained by strict brother-sister matings. Pedigree records have traditionally been maintained utilizing handwritten, bound pedigree books. Today there are several computer software packages that maintain pedigree records and are also capable of managing research projects (e.g., Locus Technology ; Progeny Software (Silver, 1993)). Within FS the pedigree system and small colony size permit the tracking of individual animals to a common ancestor within a few generations should it become necessary to purge the colony of deviants. The FS colony should be physically separated from other colonies to prevent major loss in cases of fire, disease, or natural disasters and to be able to maintain a strict health barrier. Offspring from FS breeders are used to supply the next layer in the pyramid, the PES colony.

1122

Strains in PES are also maintained as pedigreed pairs, and the offspring from PES matings provide breeders for the production stock (PS) colony. The PS matings are not pedigreed and are often set up as trio matings (two females and one male). Matings may be either sibling matings [brother-sister(s)] or random matings for strains that are at advanced generations of inbreeding. Offspring from random PS matings should never be used as breeders. Modified versions of this scheme may be utilized for small colonies with the provision that pedigree records should always be maintained. Measures should be taken within the animal rooms to protect against genetic contamination resulting from breeding error. Different-colored cards may be used for each strain, with the strain name and strain code printed on the card. Ideally, strains of identical coat color should not be housed in the same room. If they must be so housed, they should be separated by a strain of a different coat color. Capturing escapees will decrease the chance of random genetic contamination. Stray mice found in the room should be humanely euthanatized and never returned to a cage. The use of individually ventilated caging systems will further decrease random genetic contamination. Mice have been known to mate through conventional cage lids, and such access is not possible with ventilated caging. Implementation of the colony-management scheme requires a well-trained staff of animal care technicians. Technicians must be trained in basic genetics, proper colony-management practices, nomenclature, and record keeping. They must also be trained to recognize characteristic strain phenotypes and be able to detect deviants. A technician today is required to manage an increasing variety of new strains, and therefore, training must be an ongoing process. Properly trained animal care and research support personnel should be the first to identify phenotypic deviants, such as coat-color changes, behavioral changes, or a change in reproductive performance. Proper mechanisms for reporting of these events to genetic-monitoring personnel must also be in place. There are many resources describing characteristics of inbred strains, including online resources such as the Jackson Laboratory's Mouse Genome Informatics website, which contains Michael Festing's "Listing of Inbred Strains of Mice and Rats" (Festing, 2000). Cryopreservation of sperm or embryos is an effective method to protect against accidental loss of a strain due to genetic contamination, sudden infertility, a lethal mutation, disease, fire, or an environmental accident. The mutation rate of cryopreserved embryos is probably zero or close to zero, and thus genetic drift in a frozen strain is nonexistent (Bailey, 1977). Therefore, a strain maintained on the shelf for several years after the initial cryopreservation will have diverged from the frozen gametes or embryos, and the two should be considered sublines should the cryopreserved strain be recovered. As a quality-control measure, animals providing the gametes or embryos for cryopreservation, particularly those carrying mutant alleles, should be genotyped prior to their being used. Comprehensive record

JOHN J. SHARP, EVELYN E. SARGENT, AND PETER A. SCHWEITZER

keeping must be maintained for the cryopreservation process since many strains may be maintained in a single liquid nitrogen chamber.

V.

MONITORING METHODS

Today breeding and research facilities maintain inbred, congenic, recombinant inbred, transgenic, targeted mutant, spontaneous mutant, chemically induced mutant, and other genetically defined strains. There is a bewildering array of mouse strains that are now available to the researcher. Current estimates project there are over 435 inbred strains (not including substrains) and over 3000 targeted mutant strains, the numbers of which are rapidly increasing. Residual heterozygosity, genetic drift, subline divergence, human error, contamination, mutant alleles, passenger genes, and other sources of genetic variation all must be monitored or controlled. It is impossible to account for every gene in every rodent used in a research or breeding program, but various techniques are available that will assure strains are as homogeneous as is possible within the normal limits of technological and economic restraints. A genetic monitoring program will utilize many of the methods used for the past 10 to 20 years, but DNA-based molecular techniques are now playing an increasingly important role in the modern program. Molecular techniques not only permit the detection of subtle variations that were previously undetectable, but in many cases are more cost-effective than older methodologies. A genetic-monitoring program with DNA-based expertise is likely to find itself involved in activities that require whole genome characterization, such as the generation of marker-assisted congenic strains (speed congenics) or genemapping projects. A.

Physical Characteristics

Physical characteristics or behavior is often the first indication that an unplanned genetic event has occurred within a colony. It is necessary to be familiar with the individual strain characteristics in order to distinguish normal from deviant appearance. Coat color is the most obvious physical characteristic and should be the expected color for the strain (Table I). Other examples of normal strain-specific physical characteristics include a low frequency of bent tails and eyes of different sizes in PL/J mice, a low frequency of microphthalmia and hydrocephalus in C57BL/6 animals, and large body size in the MRL/MpJ and LG/J strains. Behavioral or neurological characteristics may also be useful in strain identification. For example, there is a 30% incidence of handling seizures within the NZB/B1NJ strain, young DBA mice are susceptible to audiogenic seizures, and the NOD strain is aggressive while mice of the C3H sub-

27. G E N E T I C M O N I T O R I N G

1123

Table I Coat Color Loci of Several CommonInbred Strains of Micea Strain A/J AKR/J B ALB/cJ SJL/J 129P3/J CBA/J CBA/CaJ C3H/HeJ C57BL/6J NZB/B lnJ DBA/1J DB A/2J

a

Tyrpl b

Tyr r

Myo5a d

p

Phenotypic coat color

a a + + AW + + + a a a a

Tyrp l b + Tyrp I b + + + + + + + Tyrp l b Tyrp l b

Tyr r Tyr c Tyrc Tyr ~ T y f -Ch/TyrC + + + + + + +

+ + + + + + + + + + Myo5a d Myo5a d

+ + + p p + + + + + + +

Albino Albino Albino Albino Light chinchilla or albino Agouti Agouti Agouti Nonagouti (black) Nonagouti (black) Dilute brown Dilute brown

aKey to loci symbols: a, nonagouti; A w, white-bellied agouti; Tyrpl b, brown; Tyr c, albino; Tyr c'ch, chinchilla; Myo5a d, dilute; p, pinkeye; +, wild-type. From Fox and Witham (1997).

strains are not (Festing, 2000). Such observations and familiarity with individual strains will often rule out what may be considered deviant characteristics, and animal handlers should be trained to recognize them.

B.

Immunological Techniques

Breeders and researchers have used tail skin grafting for many years to check strain purity. This procedure simultaneously detects changes in hundreds of histocompatibility (H) loci and thus is a extremely sensitive method to detect histocompatibility changes caused by substrain differentiation, contamination, residual heterozygosity, and mutation (Bailey, 1970a). This procedure requires little specialized equipment and therefore is available to most laboratories. In addition to detecting genetic differences, reciprocal circle grafting will determine if a mutation is a gain, a loss, or a gain-and-loss type (Bailey and Kohn, 1965). A major drawback to skin grafting for genetic monitoring is the lengthy time required to obtain results. Differences at the major histocompatibility complex (MHC) may reject in several days, but minor histocompatibility differences may take much longer. Thus, grafts should be observed for at least 100 days before conclusions are drawn about histocompatibility. Also, mice of some inbred strains are notorious for destroying each other's grafts (e.g., C57BL/10 and BALB/cByJ-Rb (8.12)5Bnr/J) and must be individually housed. Nonetheless, this technique is simple, checks hundreds of histocompatibility loci, and is still considered to be a useful tool for assessing strain histogenicity. In addition to skin grafting, many serological techniques have been developed for assessing genetic differences among strains, and these continue to be valuable tools for genetic monitoring programs. Many inbred strains of mice and rats differ at the MHC (H2 complex in mice and RT1 complex in rats). The MHC

haplotypes may be determined by a microcytotoxic assay (Shiroishi et aL, 1981), a technique that is particularly useful since it can be carried out using peripheral lymphocytes. This method can also be used to characterize lymphocytic (Cd) antigens and Thyl in mice (Boyse et al., 1968; Reif and Allen, 1966). Hemagglutination assays will also distinguish H2 haplotypes and other erythrocytic antigens (Snell and Cherry, 1972). Hemagglutination is a simple, specific, reliable assay using specific antisera, washed erythrocytes, and polyvinyl pyrrolidone (PVP) as the developing agent (Stimpfling, 1961). Such assays are scored macroscopically, and many animals can be typed simultaneously using several different antibodies. In rats, hemagglutination can also be used for typing erythrocytic antigens RT2, RT3, RT8, RT9, and RT11 (Gunther, 1990). The mixed lymphocyte reaction (MLR) has been used to examine major and minor histocompatibility differences in rodents (Klein, 1975). The MLR detects proliferation of lymphocytes (respondeB) stimulated by irradiated cells (stimulators) bearing histocompatibility differences. These assays typically require culturing responders and stimulators for 5 days, whereupon proliferation is assessed by pulsing the culture with tritiated thymidine. Although MLR is not widely used for routine genetic monitoring, it is considered to be the in vitro counterpart of skin grafting. Since the mutation rates for the H2 complex and H loci are considered to be high, estimated to be 2.25 x 10 -4 and 9.33 X 10 -4, respe&ively (Klein, 1975), it is wise to incorporate skin grafting, microcytotoxicity testing, hemagglutination, PCR, or MRL into a genetic-monitoring program.

C.

Biochemical Techniques

Assaying for various biochemical markers (variants of enzymes [isoenzymes] and proteins) is the most commonly used

1124

JOHN J. SHARP, EVELYN E. SARGENT, AND PETER A. SCHWEITZER

tool in a genetic quality-control program. Biochemical markers that are polymorphic between strains have been used to monitor inbred strains of mice (Roderick et al., 1981) and rats (Adams et al., 1990). They are technically easy to evaluate and are sensitive, cost-efficient, and reliable. Following isolation from a specific tissue, samples are run by electrophoresis on cellulose acetate plates, starch gels, or polyacrylamide gels, using a specified buffer, voltage, and time (Nomura et al., 1984; Adams eta/., 1990). Visualization of the bands follows staining, using either a general protein stain or a stain that is specific for the enzyme. A panel of biochemical markers can be selected to give a specific strain-distribution pattern (Table II). Usually, four or five biochemical markers plus coat color are sufficient to distinguish most common laboratory inbred strains. Ten biochemical markers will provide a unique profile for every independent inbred strain, enough information to identify a contamination and the likely source of the contaminant. However, such tests are less useful for discriminating among closely related substrains and congenic strains (Table II).

D.

C h r o m o s o m e s and Bones

Karyotype analysis can be done on rodents by using differential staining techniques to look at the G, Q, C, and R bands of the chromosome and can be a useful tool to identify inbred strains (Levan and Fredga, 1990; Miller et al., 1976), although it is not widely used. Karytotyping of these bands is usually carried out using bone marrow from a humanely euthanatized animal or by blood, tail, or ear culture techniques using tissue from live animals. After staining air-dried preparations of metaphase cells (Davisson and Akeson, 1987), photographs are taken and enlarged 3000 to 4000 times. The photographic images of chromosomes are cut out and arranged in order of decreasing size into groups of chromosome types according to the system established by the Committee on Standard Nomenclature for Mice (Mice, 1972; Evans, 1996) and the Committee for a Standardized Karyotype of Rattus norvegicus (Nomura et al., 1984). Since there is considerable size polymorphism among inbred strains, C (centromeric heterochromatin) banding may be used for distinguishing inbred strains (Akeson and Davisson, 1996). Osteometric traits have been used to study subline divergence (Bailey, 1970b), to identify inbred strains, and to detect mutations or contamination (Festing, 1972). Although vertebrae, the ulna, and the ilium have been used for measurements, most work has been done using the mandible (Festing and Lovell, 1980). Animals used for bone measurements must be of the same sex and age, and the soft tissues must be removed from the bones before analysis. Usually the right mandible, with the incisor removed, is used for measurements. The X and Y coordinates of 11 landmarks on the mesial surface are measured (Nomura et al., 1984), and the means of the measurements are statistically analyzed to discriminate strains (Hedrich, 1981).

E.

D N A - B a s e d M o l e c u l a r Techniques

Physical, immunological, and biochemical techniques have been the methods of choice for assessing genetic background quality in mouse and rat strains because of their ease, speed, and low expense. However, with the widespread sequencing of different alleles and the advent of relatively simple techniques for detecting sequence differences (e.g., PCR), DNA-based molecular assays are becoming more common as genetic qualitycontrol tools. Indeed, several of the serological techniques described above can be replaced by PCR-based assays. For example, 12 MHC haplotypes can be identified by two PCR reactions by amplifying the second intron of the H 2 - I Eb (Saha, 1996). DNA-based molecular techniques now provide an alternative to the standard alleles monitored in the immunological and biochemical methodologies described above. Southern blotting techniques (restriction fragment length polymorphism [RFLP] analysis) allowed the first linkage maps based on DNA polymorphisms to be constructed. Analysis by RFLP, using multilocus probes directed against microsatellite or viral sequences, has proven useful for monitoring both mouse and rat strains (e.g., Russell et al., 1993). Multilocus probes directed against common repeat sequences detect multiple loci in a single hybridization, enabling the simultaneous monitoring of many polymorphic loci. This approach works well for developing a molecular fingerprint of an inbred strain. However, these probes detect multiple related sequences whose map positions are rarely known, making the resulting complex mixture of alleles difficult to interpret. Using PCR to amplify SSLP markers provides a more definitive representation of genome constitution. PCR-amplified SSLP markers have became the standard markers used for genetic mapping in rodents and are now being utilized to characterize and monitor the genetic background of inbred strains of mice and rats (e.g., Simpson et al., 1997; Otsen et al., 1995). PCR-amplified SSLP markers detect length variation in dinucleotide repeat regions found in intergenic DNA as well as within genes (Dietrich et al., 1996). They are detected by amplifying genomic DNA using a pair of PCR primers specific for the unique sequences flanking the repeat region. These markers are highly polymorphic between inbred strains and can be used to distinguish closely related substrains. For example, previously indistinguishable substrains of the BALB/c, A, and C3H inbred strains can be differentiated using these markers (Table III). In a study of fifteen different 129 substrains and ten embryonic stem cells derived from them, SSLP markers provided detailed analysis leading to the classification of the substrains into three distinct lineages that agreed with historical information (Simpson et al., 1997). The study also resulted in strain nomenclature changes (Festing et al., 1999) and uncovered a previously unrecognized genetic contamination in one substrain. Because markers are numerous (over 6500 have been identified in the mouse) and have been

Table II Biochemical M a r k e r and M H C Strain-Distribution Patterns for Some C o m m o n l y Used Inbred Strains of Mice Locus Chromosome Strain

Akpl

Idhl

Pep3

Car2

G6pdl

Acads

Pgml

Gpil

Hbb

Esl

Grl

Apoal

Modl

Trf

Es3

EslO

H2

1

1

1

3

4

5

5

7

7

8

8

9

9

9

11

14

17

a

A/J a

b

a

b

b

b

a

a

a

d

b

a

b

a

b

c

a

A/HeJ a

b

a

b

b

b

a

a

a

d

b

a

b

a

b

c

a

a

AKR/J BALB/cJ

b b

b a

b a

a b

b c

b b

a a

a a

d d

b b

a a

a b

b a

b b

c a

b a

k d

SJL/J

b

b.

b

b

b

a

b

a

s

b

b

a

a

b

c

b

s

129P3/J

b

a

b

a

a

b

a

a

d

b

a

b

a

b

c

b

b

CBA/J CBA/CaJ

a b

b b

b b

b a

b b

a b

a b

b b

d d

b b

a a

b a

b b

a a

c c

b b

k k

b b

a a

b b

b b

b b

a a

b b

b b

d d

b b

a a

b b

a a

b b

c c

b b

k k b

C3H/HeJ a C3HeB/FeJ

a

C57BL/6J

a

a

a

a

a

a

a

b

s

a

a

a

b

b

a

a

NZB/BlnJ

b

a

c

a

b

b

b

a

d

b

a

a

b

b

c

b

d

DBA/1J

a

b

b

a

a

b

b

a

d

b

a

b

a

b

c

b

q

DBA/2J

a

b

b

b

b

b

b

a

d

b

a

b

a

b

c

b

d

a

The A and C 3 H substrains cannot be distinguished using biochemical markers or H2 determinations. Data from < h t t p : / / j a x m i c e . j a x . o r g / h t m l / h e a l t h / g e n e m a r k e r s a . s h t m l >

1126 Table III Inbred Mouse SubstrainsThat Can Be DistinguishedUsing SSLPAnalysisa

JOHN J. SHARP, EVELYN E. SARGENT, AND PETER A. SCHWEITZER

Transgenic strains should also be monitored for transgene copy number since the loss of copy number may severely affect the strain phenotype. In one case the reduction of transgene copy number (estimated from 25 to 18) in the transgenic strain B6SJL-TgN(SOD 1-G93A) 1Gur (Gurney et al., 1994), a model for amyotrophic lateral sclerosis, resulted in a 4-month delayed onset of the phenotype (unpublished results). Quantitative Southern blotting is suitable for this purpose, but control DNA of known copy number must be run in parallel. Newer techniques, such as real-time PCR, may replace Southern blotting in the future.

F.

apCR product size (bp) (authors,unpublisheddata, 1998). Shadedboxes indicate allelic differences.

genetically mapped, they are particularly useful for speed congenic protocols. In the near future, SNP (single nucleotide polymorphism) analysis is expected to replace SSLP analysis and will thus become a valuable addition to genetic-monitoring programs. Detection of SNP, expected to become much less expensive than SSLP analysis, will permit the monitoring of many more markers per animal than the current technology, and at less cost. The number of SNPs identified in the mouse is as yet relatively small, but is expected to grow rapidly. Monitoring genetically engineered strains also requires methods to detect specific mutant alleles rapidly and reliably. A PCR assay is best suited for monitoring transgenes and targeted mutant alleles because of its speed and the ease in developing new assays. The reliability of PCR is very high as long as the proper controls are run. False-positive results are controlled by simultaneously running known wild-type DNA. False-negative resuits will usually have little effect on an experiment or colony maintenance since that animal will be discarded. The only exception is if the animal is to be used as a littermate control, in which case it may be necessary to amplify an unrelated locus to determine if the negative result was due to the absence of sample DNA. For strains where the homozygous mutant is not viable or fertile, the strain is often maintained by matings between heterozygous parents. In this case it is necessary to be able to distinguish homozygotes, heterozygotes, and wild-type animals for experimental purposes, which may be problematic, especially if required on a routine basis. A PCR assay can usually be developed to distinguish the three genotypes for targeted mutant strains (Pekhletski and Hampson, 1996), but it may be necessary to develop a quantitative Southern blotting assay for strains carrying transgenes,

Strain P e r f o r m a n c e M o n i t o r i n g

Many individual loci in the mammalian genome are now able to be monitored, but genetic drift results in the accumulation of undetected spontaneous mutations that may impact on strain performance (phenotype). Monitoring strain performance has largely been ignored, but ideally it should be a component of a genetic-monitoring program since it is the only mechanism available to uncover performance variation due to the accumulation of unmonitored mutations. In addition, the phenotype of genetically engineered strains will be affected as congenic strains are generated, and characterizing this change may become the responsibility of a monitoring program. Performance monitoring may not be practical on a widespread basis, but centralized suppliers should consider such a function, if only for heavily used strains.

VI.

SUMMARY

Genetic monitoring has become more complex as the technology to manipulate the mammalian genome has become more sophisticated. The observation of physical, biochemical, and immunological characteristics is valuable for monitoring the inbred genetic background, but these characteristics are of limited use when monitoring the genetic defects found in today's genetically engineered strains. DNA-based molecular techniques, particularly PCR, provide the basis for monitoring the specific genetic alterations being produced in today's newer strains. These molecular techniques are also finding greater use in characterizing and monitoring genetic backgrounds, and they may be expected to extensively replace immunological and biochemical methodology in the future. The responsibility for monitoring genetically engineered animals has placed a need to incorporate the newest technology into genetic-monitoring programs, and this need will continue to exist as even newer technology becomes available.

27. GENETIC MONITORING

REFERENCES

Adams, M., Bender, K., Den Bieman, M., von Demling, O., Hedrich, H. J., et al. (1990). Laboratory protocols for detecting biochemical markers. In "Genetic Monitoring of Inbred Strains of Rats" (H. J. Hedrich, ed.). Gustav Fischer Vedag, Stuttgart. Akeson, E. C., and Davisson, M. T. (1996). Centromeric heterochromatin variants. In "Genetic Variants and Strains of the Laboratory Mouse" (M. E Lyon, S. Rastan, and S. D. M. Brown, eds.). Oxford Univ. Press, London. Bailey, D. W. (1970a). Four approaches to estimating number of histocompatibility loci. Transplant Proc. 2, 32-38. Bailey, D. W. (1970b). Rates of subline divergence in highly inbred strains of mice. J. Hered. 50, 26-30. Bailey, D. W. (1977). Genetic drift: The problem and its possible solution by frozen-embryo storage. Ciba Found. Symp., 291-303. Bailey, D. W. (1982). How pure are inbred strains of mice? Immunol. Today 3, 210-214. Bailey, D. W., and Kohn, H. I. (1965). Inherited histocompatibility changes in progeny of irradiated and unirradiated inbred mice. Genet. Res. 6, 330340. Biesiadecki, B. J., Brand, P. H., Metting, P. J., Koch, L. G., and Britton, S. L. (1998). Phenotypic variation in strength among eleven inbred strains of rats. Proc. Soc. Exp. Biol. Med. 219, 126-131. Boyse, E. A., Miyazawa, M., Aoki, T., and Old, L. J. (1968). Ly-A and Ly-B: Two systems of lymphocyte isoantigens in the mouse. Proc. R. Soc. Lond. B Biol. Sci. 170, 175-193. Cormier, R. T., Hong, K. H., Halberg, R. B., Hawkins, T. L., Richardson, P., et al. (1997). Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigenesis [see comments]. Nat. Genet. 17, 88-91. Crawley, J. N. (1996). Unusual behavioral phenotypes of inbred mouse strains. Trends Neurosci. 19, 181-182, 188-189. Davisson, M. T., and Akeson, E. C. (1987). An improved method for preparing G-banded chromosomes from mouse peripheral blood [published erratum appears in Cytogenet. Cell Genet. 48(3) (1988), 192]. Cytogenet. Cell Genet. 45, 70-74. Dietrich, W. E, Lander, E. S., Smith, J. S., Moser, A. R., Gould, K. A., et al. (1993). Genetic identification of Mom-1, a major modifier locus affecting Min-induced intestinal neoplasia in the mouse. Cell 75, 631-639. Dietrich, W. E, Miller, J., Steen, R., Merchant, M. A., Damron Boles, D., et al. (1996). A comprehensive genetic map of the mouse genome [see comments] [published erratum appears in Nature 381(6578), 172]. Nature 380, 149-152. Erickson, R. P. (1996). Mouse models of human genetic disease: Which mouse is more like a man? Bioessays 18, 993-998. Evans, E. P. (1996). Standard idiogram. In "Genetic Variants and Strains of the Laboratory Mouse" (M. E Lyon, S. Rastan, and S. D. M. Brown, eds.), Vol. 2, pp. 1446-1448. Oxford Univ. Press, Oxford. Festing, M. (1972). Mouse strain identification. Nature 238, 351-352. Festing, M. E (1999a). Reduction in animal use in the production and testing of biologicals [in process citation]. Dev. Biol. Stand. 101, 195-200. Festing, M. E (1999b). Warning: The use of heterogeneous mice may seriously damage your research [in process citation]. Neurobiol. Aging 20, 237-244, 245-246. Festing, M. E (2000). "Listing of Inbred Strains of Mice and Rats," Vol. 2000, Mouse Genome Database (MGD), Mouse Genome Informatics. Jackson Laboratory, Bar Harbor, Maine. Festing, M. E, and Lovell, D. P. (1980). Routine egentic monitoring of commercial and other mouse colonies in the U.K. using mandible shape: Five years of experience. In "Proceedings of the Seventh ICLAS Symposium" (A. Spiegel, S. Brichsen, and H. A. Solleveld, eds.), pp. 341-348. Fischer, Stuttgart.

1127 Festing, M. E, and Wolff, G. L. (1995). Re: Inbred strains of laboratory animals: Superior to outbred mice? [letter; comment]. J. Natl. Cancer Inst. 87, 1715-1716. Festing, M. E, Simpson, E. M., Davisson, M. T., and Mobraaten, L. E. (1999). Revised nomenclature for strain 129 mice. Mamm. Genome 10, 836. Fox, R. R., and Witham, B. A. (1997). "Handbook on Genetically Standardized JAX Mice," 5th ed. Jackson Laboratory, Bar Harbor, Maine. Gould, K. A., Luongo, C., Moser, A. R., McNeley, M. K., Borenstein, N., et al. (1996). Genetic evaluation of candidate genes for the Moml modifier of intestinal neoplasia in mice. Genetics 144, 1777-1785. Gunther, E. (1990). Laboratory methods to determine polymorphic loci and other variants. In "Genetic Monitoring of Inbred Strains of Rats" (H. J. Hedrich, ed.), pp. 79-99. Gustav Fischer Vedag, Stuttgart. Gurney, M. E., Pu, H., Chiu, A. Y., Dal Canto, M. C., Polchow, C. Y., et al. (1994). Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation [see comments] [published erratum appears in Science 269(5221), 149]. Science 264, 1772-1775. Hedrich, H. J. (1981). Genetic monitoring. In "The Mouse in Biomedical Research" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), Vol. 1, pp. 159-176. Academic Press, New York. Homanics, G. E., Quinlan, J. J., and Firestone, L. L. (1999). Pharmacologic and behavioral responses of inbred C57BL/6J and strain 129/SvJ mouse lines. Pharmacol. Biochem. Behav. 63, 21-26. Justice, M. J., Noveroske, J. K., Weber, J. S., Zheng, B., and Bradley, A. (1999). Mouse ENU mutagenesis [in process citation]. Hum. Mol. Genet. 8, 19551963. Klein, J. (1975). "Biology of the Mouse Histocompatibility-2 Complex. Principles of Immunogenetics Applied to a Single System." Springer-Verlag, New York. Levan, G., and Fredga, K. (1990). Cytogenetic markers. In "Genetic Monitoring of Inbred Strains of Rats" (H. J. Hedrich, ed.), pp. 42-58. Gustav Fischer Verlag, Stuttgart. Mice, C. o. S. G. N. f. (1972). Standard karyotype of the mouse, Mus musculus. J. Hered. 63, 69-71. Miller, D. A., Tantravahi, R., Dev, V. G., and Miller, O. J. (1976). Q- and C-band chromosome markers in inbred strains of Mus musculus. Genetics 84, 67-75. Morse, H. E., ed. (1978). "Origins of Inbred Mice." Academic Press, New York. Mu, J. L., Naggert, J. K., Svenson, K. L., Collin, G. B., Kim, J. H., et al. (1999). Quantitative trait loci analysis for the differences in susceptibility to atherosclerosis and diabetes between inbred mouse strains C57BL/6J and C57BLKS/J. J. Lipid Res. 40, 1328-1335. Nielsen, L. L., Gurnani, M., Catino, J. J., and Tyler, R. D. (1995). In wap-ras transgenic mice, tumor phenotype but not cyclophosphamide-sensitivity is affected by genetic background. Anticancer Res. 15, 385-392. Nomura, T., Esaki, K., and Tomita, T. (1984). "ICLAS Manual for Genetic Monitoring of Inbred Mice." Univ. of Tokyo Press, Tokyo. Otsen, M., Den Bieman, M., Winer, E. S., Jacob, H. J., Szpirer, J., et al. (1995). Use of simple sequence length polymorphisms for genetic characterization of rat inbred strains. Mamm. Genome 6, 595-601. Paigen, B. (1995). Genetics of responsiveness to high-fat and high-cholesterol diets in the mouse. Am. J. Clin. Nutr. 62, 458S-462S. Pekhletski, R., and Hampson, D. R. (1996). Differentiating allele combinations of a transgene using multiple primer PCR. Biotechniques 20, 956-958, 960. Pitman, W. A., Hunt, M. H., McFarland, C., and Paigen, B. (1998). Genetic analysis of the difference in diet-induced atherosclerosis between the inbred mouse strains SM/J and NZB/BINJ. Arterioscler. Thromb. Vasc. Biol. 18, 615-620. Reif, A. E., and Allen, J. M. (1966). Mouse thymic iso-antigens. Nature 209, 521-523. Roderick, T. H., Staats, J., and Womack, J. E. (1981). Strain distribution of polymorphic variants. In "Genetic Variants and Strains of the Laboratory Mouse" (M. H. Green, ed.), pp. 377-400. Gustav Fischer Verlag, Stuttgart.

1128 Russell, R. J., Festing, M. E, Deeny, A. A., and Peters, A. G. (1993). DNA fingerprinting for genetic monitoring of inbred laboratory rats and mice. Lab. Anim. Sci. 43, 460-465. Saha, B. K. (1996). Typing of murine major histocompatibility complex with a microsatellite in the class II Eb gene. J. Immunol. Methods 194, 77-83. Sharp, J., and Mobraaten, L. (1997). To save or not to save: The role of repositories in a period of rapidly expanding development of genetically engineered strains of mice. In "Transgenic Animals: Generation and Use" (L.-M. Houdebine, ed.), pp. 525-532. Harwood Academic Publishers, Switzerland. Shiroishi, T., Sagai, T., and Moriwaki, K. (1981). A simplified micro-method for cytotoxicity testing using a fiat-type titration plate for the detection of H-2 antigens. Microbiol. ImmunoL 25, 1327-1334. Silva, A. J., Simpson, E. M., Takahashi, J. S., Lipp, H.-P., Nakanishi, S., et al. (1997). Mutant mice and neuroscience: Recommendations concerning genetic background. Neuron 19, 755-759. Silver, L. M. (1993). Recordkeeping and database analysis of breeding colonies. Methods Enzymol. 225, 3-15.

JOHN J. SHARP, EVELYN E. SARGENT, AND PETER A. SCHWEITZER

Simpson, E. M., Linder, C. C., Sargent, E. E., Davisson, M. T., Mobraaten, L. E., and Sharp, J. J. (1997). Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16, 19-27. Snell, G. D., and Cherry, M. (1972). Loci determining cell surface alloantigens. In "RNA Viruses and Host Genome in Oncogenesis" (P. Emmelot and P. Bentvelzen, eds.), pp. 221-228. North-Holland, Amsterdam. Statts, J. (1996). The laboratory mouse. In "Biology of the Laboratory Mouse" (E. L. Green, ed.), pp. 1-9. Dover, New York. Stimpfiing, J. H. (1961). The use of PVP as a developing agent in mouse hemagglutination tests. Transplant. Bull. 27, 109-111. Strong, L. C. (1978). Inbred mice in science. In "Origins of Inbred Mice" (H. C. Morse, ed.), pp. 45-67. Academic Press, New York. Wakeland, E., Morel, L., Achey, K., Yui, M., and Longmate, J. (1997). Speed congenics: A classic technique in the fast lane (relatively speaking). Immunol. Today 18, 472-477.

Chapter 2 8 Transgenic and Knockout Mice Glenn M. Monastersky and James G. Geistfeld

I. II. III.

IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1129

Choice of Mouse Strains for Transgenic Programs

1130

..................

Production of Transgenic Mice: Animal Requirements . . . . . . . . . . . . . . . A. Donor Females . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Fertile Stud Males . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Recipient Females . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1131 1131 1133 1133

D. Sterile Stud Males . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Management of the Transgenic Mouse Colony . . . . . . . . . . . . . . . . . . . . .

1134 1134

A. B.

Health Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colony Breeding Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1134 1135

C. D.

Colony Record Keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preservation of Transgenic Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1138 1139

E.

Transgenic Mice and Intellectual Property Rights

1140

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

INTRODUCTION

For most of the twentieth century, increasing numbers of genetically defined laboratory mice have been described and incorporated into biological research. Initially, research using inbred strains was limited mostly to basic genetic studies in which biochemical or visual phenotypic expression patterns were observed. With the advent of molecular genetics in the 1960s, laboratory mice developed into much more critical research tools in which the genomic basis of disease and mutation could be examined at the level of individual genes. By the 1970s, the prospect of intentionally modifying the murine genome by the addition of new functional DNA was at hand (Jaenisch and Mintz, 1974; Jaenisch, 1976). By the early 1980s, the persistence of microinjected laboratory-derived DNA within the cells of liveLABORATORYANIMALMEDICINE,2nd edition

...............

1140

born mice (Gordon et al., 1980) and the functional expression of transgenes in mice (Brinster et al., 1981; Costantini and Lacy, 1981) were reported. Within a few years, major universities, medical schools, and a small number of pharmaceutical and biotechnology companies had created in-house transgenic mouse laboratories. Very quickly, it became apparent that the unique colony and data-management challenges of transgenic mouse operations required exceptional attention to technical detail. Expertise in transgenic colony management is critical because of the significant potential value of each transgenic lineage and the formidable housing and laboratory efforts required to develop each individual line. The mouse remains the primary choice for transgenic experimentation due to the relative ease of embryo and adult manipulation and the unparalleled depth of murine genetic knowledge. Today, transgenic mice are produced as models of human disease and toxicology, as models Copyright 2002, ElsevierScience (USA).All rights reserved. ISBN 0-12-263951-0

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GLENN M. MONASTERSKY AND JAMES G. GEISTFELD

for transgenic livestock protocols, and as in vivo systems in which mammalian (and nonmammalian) genetic expression may be investigated. The creation and production of transgenic mice entail an intensive sequence of procedures involving genetics, molecular biology, embryology, and animal science. Scientists design and construct DNA sequences that can be transferred into the mouse genome either randomly (i.e., via pronuclear microinjection, classical transgenic) or in a targeted fashion (i.e., via embryonic stem cell-mediated homologous integration, knockout transgenic techniques). If successful, the transferred DNA is integrated stably into one of the mouse chromosomes. If the transgene has been designed to produce a gain of function (i.e., classical transgenic), it may be expressed as mRNA, which encodes the transgenic protein. If the transgene-bearing mouse expresses a functional transgenic protein, a new phenotype (e.g., altered immune function or increased susceptibility to a disease process) may be apparent. In the majority of experiments, immediate breeding to homozygosity is desirable in order to assess the expression of the new phenotype through subsequent generations. Alternatively, if the inserted transgenic DNA has been designed to ablate or mutate the function of an endogenous gene (i.e., knockout transgenic), mRNA expression from the targeted locus will be either absent or modified. The resultant knockout mice may be of enormous potential value but must be bred to homozygosity (i.e., null genotype) in order to fully evaluate the phenotype. Many comprehensive reviews of laboratory mouse husbandry and transgenic methodologies already exist (Hogan et al., 1994; Pinkert, 1994; Monastersky, 1995). The purpose of this chapter is to offer practical discussionmand recommended solutionsm for many of the actual challenges faced by researchers and animal care professionals when working with transgenic mice.

II.

C H O I C E OF M O U S E S T R A I N S F O R T R A N S G E N I C PROGRAMS

The design of the DNA expression cassette determines the potential scientific value of a transgenic mouse. However, the most important factor in realizing the potential of a transgenic study involves the choice of mouse strain(s) used for the initial embryology and for the critical breeding strategies. The degree of expression and/or detection of a transgene may differ significantly when placed into different inbred and non-inbred genetic milieus. Individual laboratories frequently rely on the use of favorite strains based on many factors, including genotype, cost, availability, health history, and reproductive characteristics. The genotypes of the embryo donor mice frequently affect the expression pattern of the transgenic offspring and are especially

critical in knockout studies. Also, the investigator should review the frequencies of specific pathologies reported for well-known inbred and outbred strains. Exceptional strain-dependent tumor frequencies or metabolic deficiencies should be avoided for specific predicted transgenic phenotypes. The genotype of recipient pseudopregnant females, particularly with regard to coat-color genetics, also should be considered carefully. Because different strains exhibit different average litter sizes, this factor should also be considered in the choice of embryo transfer recipient strains. The following questions should be reviewed before proceeding with a transgenic mouse program. Is the genetic background critical to the experiment? If a defined genetic background is desired, an inbred strain with the appropriate genetics should be selected. Predictability of the effects of background genetics on the transgenic expression minimizes the variability in expression or phenotype between animals. If genetics is not apparently critical to the project, then hybrid animals with above-average vigor and breeding characteristics may be selected. Compared to inbred strains, many hybrid mouse strains, such as (C57BL/6 X SJL/J)F1, yield relatively larger numbers of oocytes in response to superovulation, and these embryos may also exhibit increased survival to implantation following microinjection. Hybrid animals also usually exhibit more reliable fertility characteristics and fairly large litter sizes. What are the behavioral characteristics of the parental strains ? Are they exceptionally aggressive? Do they frequently cannibalize their newborn litters? Are they attentive mothers? What are the reproductive characteristics of the potential parental strains ? What are the average litter sizes? Do the females respond well to superovulatory stimulation? Are gestation times usually predictable? What types of pathologies normally occur in the strains being considered? Could these pathologies mask the expected expression of the transgene? Could endogenous pathologies enhance, accentuate, or confound the predicted transgenic phenotype? Is coat color important or useful in the project ? What is the minimal acceptable health status for the project and within the facility ? The inbred strains most frequently used in transgenic research are C57BL/6, FVB/N, and 129/SvEv. Some characteristics of these strains are listed in Table I. The C57BL/6 probably has been the most widely used inbred strain for transgenic studies. The genetics and phenotypic variation of this mouse strain have been well characterized over several decades. The reproductive and maternal characteristics of this mouse are only fair to average, but the response of females to injected exogenous hormones is above average; relatively high yields of pronuclear embryos may be collected following superovulation and mating. For knockout mouse studies, C57BL/6 blastocysts have been used almost exclusively as recipients of genetically engi-

28. TRANSGENICAND KNOCKOUTMICE

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Table I

Mouse Strains CommonlyUsed in Transgenic Studies Strain

Litter size (average)

Ovulation response

C57BL/6

6

High

FVB/N

8

High

129/SvEv

8

Low

General comments Black coat; poor mothers--tendency to eat first litter; used for pronuclear microinjection and as blastocystrecipients for ES cells (knockouts; KO); mating strain for creation of inbred KO lines Albino coat; goodmothers; large pronuclei for microinjection Light-bellied, black agouti; good mothers; generationof ES cells; mating strain for creation of inbred KO lines

neered embryonic stem (ES) cells. These blastocysts reliably give rise to ES-cell chimeras, and the coat-color genetics usually facilitates analysis of the live-born mice. The FVB/N is another popular strain for pronuclear microinjection because many researchers agree that the pronuclei of the zygotes are somewhat larger and more distinctive than those collected from comparable strains (Taketo et al., 1991). These albino mice yield relatively large numbers of embryos and usually serve as reliable mothers. The 129/SvEv mouse frequently is used for breeding, because it has favorable reproductive traits and because many of the available ES cell lines are derived from 129/Sv substrains. The ES cell lines derived from 129/Sv mice are reputed to have a high incidence of germline transmission and are easily maintained in culture. Chimeric, knockout-positive mice produced from these cells may be mated to 129/SvEv inbred mice to increase and preserve the inbred background of the knockout line. Considerable genetic variation has been reported in the 129 substrains (Simpson et al., 1997). When the genetic background of the embryos is not considered a factor, microinjection procedures are usually performed with hybrid embryos because hybrids have better reproductive characteristics, high embryo survival rates (in vitro and in utero), and relatively high rates of transgene integration and retention. Popular hybrid mouse embryo donors include C57BL/6 X SJL, C57BL/6 X DBA/2, C57BL/6 X C3H, and C57BL/6 X CBA. The C57BL/6 X SJL (B6SJL F1) mouse is very widely used to generate embryos for pronuclear microinjection experiments (Brinster et al., 1985). For embryo transfer procedures, outbred Swiss albino stocks are very widely employed as recipients of microinjected embryos. Frequently, the ICR or Swiss-Webster stocks are used due to low cost and good maternal characteristics. The Swiss black mouse has become popular for the test mating of chimeras

to determine whether a knockout gene has been transmitted (i.e., production of agouti coat color). Advantages of using the Swiss black mouse include relatively large litter sizes and low cost.

llI.

PRODUCTION OF TRANSGENIC MICE: ANIMAL REQUIREMENTS

To produce transgenic mice, it is necessary to maintain four different types of colonies. The four different types of mice required may be generated in-house or more commonly, purchased commercially as needed to conserve valuable facility space: (A) embryo donor female mice (superovulated), (B) fertile stud male mice (proven breeders), (C) embryo transfer recipient female mice (pseudopregnant), and (D) sterile stud male mice (vasectomized).

A.

Donor Females

Sexually mature females are used to produce the zygotes donated for the creation of transgenic embryos. A transgenic research program requires a continuous supply of embryo donor females, which receive superovulatory hormones prior to breeding. As with all mouse colonies, predictable, well-controlled, familiar husbandry practices facilitate optimal performance. With embryo donation protocols, the control of the colony environment is even more critical because it can have a major impact on breeding and fertility and ultimately, on embryo yield. Obviously, the temperature, relative humidity, and properly timed light cycle of the mouse rooms are important. In addition, some easily avoided potential depressors of breeding success and embryo yield include the following: use of cold (i.e., 1~ to 10~ superovulatory hormone solutions unscheduled interruptions in the light or dark phases of the light cycle in the room use of females less than 48 hr after arrival on-site, especially after air transport cage-rack machinery vibrations or any type of repeated loud noises excessive or rough handling, especially after mating or during the dark phase bedding change within 24 hr after breeding housing of rats or nonrodent species within the room sacrifice of animals within the room continued presence of multiple males within the mating cage viral or bacterial infections (may be subclinical)

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GLENN M. MONASTERSKY AND JAMES G. GEISTFELD

Superovulation is performed to increase the number of mature oocytes ovulated and therefore the number of viable embryos collected. The administration of exogenous gonadotropins also serves to synchronize estrus and ovulation within a group of females. In addition, the number of female mice maintained for egg or blastocyst production is minimized compared to the use of naturally ovulated females (e.g., may be 2 - 3 times fewer females). The standard murine superovulatory regime involves the sequential intraperitoneal (IP) injection of two gonadotropins into young (i.e., 3- to 4-week-old) virgin females. The optimal age will vary by strain and must be determined for each laboratory. Several gonadotropic hormones, including luteinizing hormone (LH) and follicle-stimulating hormone (FSH), have been evaluated for their efficiency in eliciting increased ovarian follicle development and ovulation in mice to achieve superovulation and/or timed pregnancy. Almost all current protocols employ an initial IP injection of pregnant mare serum gonadotropin (PMSG) followed within 4 6 - 4 8 hr by an IP injection of human chorionic gonadotropin (hCG). The dose of each hormone administered per mouse usually ranges between 5.0 and 7.5 IU. Overdosage tends to elicit the production of increased numbers of unfertilized and abnormal oocytes. These hormones are supplied as a lyophilized powder and should be dissolved in either 0.9% NaC1 or phosphate-buffered saline to provide a final injection volume of around 0.10 to 0.15 ml. The dilution procedure will depend on the concentration of the hormone procured. Usually the stocks are diluted to 25-50 IU/ml and then subdivided into aliquots (1-2 ml), which are stored at - 2 0 ~ to - 8 0 ~ until thawed for use. A discard date (usually 6 12 months) should be included on the label of each frozen aliquot. Once thawed, remaining hormone must be discarded rather than refrozen for future use. In many laboratories, each new lot of PMSG and hCG is evaluated independently in a limited mouse biological assay before further use. This assay may evaluate the quantity and quality of eggs produced and their fertilization efficiency, as compared to those of control groups of

animals that have received appropriate injections of proven hormones. All IP injections should be performed rapidly and smoothly, injecting 0.10 to 0.15 ml of solution using a 22- to 26gauge sharp (i.e., used for fewer than 10 injections) needle to cause minimal stress to the animal. To maximize the effect of exogenous hormone injection, it is important to administer the second hormone (i.e., hCG) prior to the release of endogenous leutinizing hormone (LH). Endogenous LH secretion will occur at different times after ovarian follicle development has begun, depending on mouse strain and light-cycle timing. Secretion of LH and subsequent ovulation of the eggs usually begin within 48-56 hr after PMSG injection (actually, approximately 15-20 hr after the midpoint of the second dark period after PMSG injection). As previously mentioned, the donor females should be given several days to acclimate to the room and to adjust to the light-dark cycle (Van Ruiven et al., 1996). In most experiments for most strains, a 4 2 48 hr interval between the PMSG and hCG injections is usually optimal. For example, in a room with a 6 AM-6 PM light cycle, PMSG can be administered between 2 and 3 PM and the hCG would be administered 2 days later between 1 and 2 PM. Superovulated females should be mated (preferably 1:1) with proven stud males immediately following the hCG injections. Normal, motile male sperm will remain viable within the female reproductive tract for 1-2 days after deposition, and females should ovulate within 10-15 hr after hCG injection. Superovulation will synchronize estrus and ovulation and thereby permit the timed collection of predictable numbers of similarly staged pronuclear, 2-celled, 8-celled, and blastocyst-stage embryos (Table II). On the morning (i.e., between 6 and 9 AM) following hCG and pairing, successful mating may be validated by observing vaginal copulation plugs. Plug-positive and plugnegative females should be segregated at that time. Only positive-plugged females should be used for subsequent embryo collections. Negative females, optimally, should be isolated for 2 weeks to discount pregnancy before reuse in the program (Table III). At times, critically important females must be used

Table II Collection of MouseEmbryosfor Research: Timingof Developmenta Embryo stage

Time after fertilization and time of collectionb

Pronuclear-stage embryos 2-cell-stage emobryospost-hCG 8-ceU-stage embryos Compacted-morula-stageembryos Early-blastocyst-stageembryos Expanded-blastocyst-stageembryos

Morning and early afternoonof day 1; collected approximately20-22 hr post-hCG Morning of day 2; collected approximately42-44 hr post-hCG Day 3; collected approximately68-70 hr post-hCG Evening of day 3 Day 4 Late on day 4 and on day 5; collected approximately93-96 hr post-hCG ,,

Adapted fromMonastersky(1995). ~ day 1 is equivalentto plug day,i.e., the morningafter hCG injection and mating. Earlyblastocystsshouldenter the uterine cavity on day 4, and implantationinto the endometriumshouldoccur on day 5. a

28. TRANSGENIC AND KNOCKOUTMICE as embryo donors but are unresponsive to gonadotropin injections due to a specific transgenic phenotype, obesity, or old age. The use of an osmotic pump to deliver hormones to these females has been successful (Leveille and Armstrong, 1989). In dire situations, in vitro fertilization (IVF), gamete intrafallopian transfer (GIFT), or ovary transplantation may be attempted to save the lineage of a female founder mouse from extinction. As alluded to previously, gonadotropin overdosage, age, and mouse strain each may underlie an increase in the incidence of abnormal eggs. Typically, between 5 and 10% of FVB/N eggs are abnormal, whereas up to 25% of the eggs collected from a C57BL/6 superovulated female will be abnormal. The superovulated BALB/c produces many eggs, but a high percentage (sometimes 50% or more) are frequently unusable for manipulation. When the percentage of unusable oocytes exceeds 25%, the environment (e.g., stressors and health) and culture media should be evaluated for possible problems. Another potential influence on egg yield might be related to the specific litter number from which the ovulating female originated (Polites and Pinkert, 1994).

B.

Fertile Stud Males

Fertile stud males are mated to superovulated females to produce fertilized embryos for manipulation. Male mice reach sexual maturity by 2 months of age, and active studs should generally be replaced at 10-12 months of age due to usually decreasing reproductive performance. Males should be housed in individual cages to avoid fighting and stress. To increase breeding efficiency, males should be placed into the breeding cages at least 2 days prior to the introduction of females. Young males previously housed together should be isolated for 1-2 weeks prior to the introduction of females to mitigate the dominantmale effect, in which the dominant male will cause the suppression of testosterone synthesis (and spermatogenesis) in cagemates. Overly aggressive males that injure females should be removed from service immediately. The use of "proven-breeder" stud males is of immense value in maintaining acceptable rates of oocyte fertilization. To maximize the use of studs, it is convenient to designate distinct groups of proven males (e.g., A - C ) to be used on a rotation schedule. A reasonably successful plan would schedule monogamous overnight mating (2 PM tO 9 AM) within each male's individual (i.e., scent-marked) cage, followed by at least 72 hr of celibacy. Plugging efficiency should be recorded on the cage card for each male, and stringent criteria should be maintained to preserve proven-breeder status. Breeding observations should also include records of plug-positive females that yield unfertilized eggs. In these cases, the stud males probably are infertile and should be discarded or evaluated using test mating, vaginal lavage, and simple sperm-motility analysis. A useful

1133

rule of thumb for transgenic programs calculates that one proven-breeder stud male should be maintained for every seven pronuclear embryos or three blastocysts required daily.

C.

Recipient Females

Manipulated mouse embryos must be transferred surgically (embryo transfer [ET]) into recipient females in order to produce live-born offspring. These recipient females must support the gestation and postpartum development of the pups through weaning. Therefore, females must be chosen that are known within individual laboratories to exhibit satisfactory reproductive characteristics, mothering attributes, and lactation capabilities. In addition, the use of lean, hardy female animals will result in increased litter sizes and pregnancy rates because the ET surgery will be performed more rapidly and efficiently. The absolutely critical factor in achieving satisfactory results with the ET of manipulated embryos is the skill of the animal surgeon. Performance of each ET protocol with speed, accuracy, and minimal tissue insult and hemorrhage will translate into increased survival of the embryos and enhanced pregnancy data. As discussed in the following section, pseudopregnant mice are produced by mating with sterile males. The tactile and olfactory stimuli of mating will elicit pseudopregnancy in 50 to 80% of the females. Although no viable sperm are delivered at ejaculation, the brain, endocrine system, and reproductive tract epithelia of these females will be perfectly prepared to support pregnancy within the week following the sterile mating. For quality control, the selection of recipient females with different coatcolor genetics than that of the transferred embryos is suggested. Females usually are gang-caged (4-15 per cage, depending on cage size). In a nonsynchronized colony, at least 20% of the females are expected to be in estrus simultaneously. In many programs, females may be chosen randomly. In some programs, vaginal examinations for indications of estrus are performed. Superovulated females are not used since naturally mated females (i.e., nonsuperovulated) have a substantially higher rate of pregnancy after embryo transfer. The pseudopregnant female should be mated 1 day later than the developmental age of the transferred embroys. This asynchrony compensates for the delayed embryo development attributable to manipulation in vitro. Unused pseudopregnant females may be recycled back into the colony as soon as they resume estrous cycling within 8 12 days. The total number of females mated when producing pseudopregnant mice should be approximately 4 - 5 times the actual number needed for embryo transfers. Reduced pseudopregnancy frequencies may occur due to the same key environmental factors listed for embryo donor females in Section III,A. If a recipient female produces too many pups (e.g., more than 8-10) or dies, the pups can be cross-fostered onto a foster mother, which may be a female with a small, similarly aged

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GLENN M. MONASTERSKY AND JAMES G. GEISTFELD

litter. Alternatively, females may be specially bred to serve as foster mothers; these purpose-bred animals are mated 1 day after the ET-recipient females. Certain strains, for example, BALB/c or outbred Swiss, are favored by different investigators. To facilitate a successful transfer of newborn mice to a foster mother, no more than 8 pups should be transferred to a single mother. It is also recommended that an attempt be made to transfer existing cage odors to the new pups, which may be accomplished by moistening the pups lightly with water from the foster mother's water bottle and then placing them in a paper bag with bedding from the foster mother's cage. After the bag has been gently shaken for a few seconds, the foster mother is placed into the bag with the pups for about a half an hour. The behavior of the foster mother will predict the success of the litter transfer. If she begins to groom herself and the pups, she most likely will accept the new pups as her own. However, if she grabs the pups by the nape of the neck and begins to bury them in the bedding, she probably will reject fostering the pups. Transfer to another foster mother may be attempted at this time. It is important not to disturb or clean the foster cage for at least a week following transfer of the pups. Providing extra bedding or nesting material may also enhance the nurturing instincts of the foster female.

D.

Sterile Stud Males

The pseudopregnant female mice used as recipients for the embryo transfer of genetically modified embryos are prepared by mating with sterile males. Sterile breeding-age males (i.e., at least 6 weeks of age) are created by surgical vasectomy and mated to breeding-age females (i.e., usually 2 - 4 months of age). In order to permit postsurgical healing and to ensure sterility and mating performance prior to employment in actual experiments, these males should be test-mated about 2 weeks after surgery. Each vasectomized male should be bred with a cohort (e.g., 3 to 6) of healthy, breeding-age females. These females should exhibit vaginal plugs following mating yet must fail to become pregnant. An additional genetic safety measure is to select males that will produce offspring of a different coat color than that of the manipulated embryos. Males from any hybrid or outbred strain with good breeding characteristics may be selected for vasectomy. Because fertility is not an issue, these males can be mated polygamously (i.e., 1:2 or 1:3) on a daily basis if plugging frequencies are maintained. Performance generally will decline by 10-12 months of age, and new vasectomized males should be prepared on a regular schedule (e.g., 1 day per month) to provide replacements as needed. The number of vasectomized males available should equal approximately half the number of pseudopregnant females desired per week.

IV.

MANAGEMENT OF THE TRANSGENIC MOUSE COLONY

The effort involved in the creation and characterization of a transgenic line is significant. Transgenic animals frequently exhibit phenotypic and genotypic characteristics that challenge colony-management skills. Depending on the specific nature of the transgene construct, genetically engineered mice may exhibit immune incompetence, infection-promoting skin disorders, or a specific disease process or syndrome. Any of these types of transgene-mediated phenotypes may affect health, longevity, or reproductive performance. Therefore, the management of a transgenic facility is much more critical than the management of a conventional, nontransgenic facility. Four major support systems should be established before a transgenic line is created or imported into a facility: an established facility health program, including explicit methods for the importation of animals from other facilities explicit and well-documented breeding strategy standard operating procedures (SOPs) and guidelines a fastidious record-keeping system for reproductive and phenotypic data access to an internal or external embryo cryopreservation program These systems and procedures must be supported and managed by well-trained personnel.

A.

Health Profile

A high-quality transgenic mouse program should always use specific pathogen-free animals for production, for breeding, and for fostering. A reliable specific pathogen-free colony environment must be maintained by using high-quality animals, by carefully managing importation of new animals into the facility, and most important, by adhering to stringent animal care and husbandry SOPs. In addition, diligent health-monitoring protocols and smooth, rapid strategies for dealing with potential discoveries of pathogens within the facility must be in place and understood by all animal care personnel and researchers. The barrier .should be monitored on a regular schedule for all undesirable agents, using nontransgenic sentinel animals or immune-incompetent animals produced within the colony (Small, 1984). The health risks within a transgenic facility are more challenging than those within a nontransgenic program. Mice from a variety of sources are imported frequently, usually on a weekly basis. The health profiles of all imported animals must be reviewed carefully because "clean" animals from different

1135

28. TRANSGENIC AND KNOCKOUT MICE

vendors or laboratories will have differing criteria. Transgenic mice received from academic laboratories should absolutely be quarantined and evaluated before inclusion into a breeding program. Ideally, transgenic colonies should be maintained in defined barrier rooms and/or isolators with defined health profiles and dedicated personnel. Transfer of animals m and personnelmbetween colonies or barriers must be regulated stringently. Research animals that leave the colony for research purposes should not be returned to the colony. The production and breeding units should preferably be maintained within a barrier separated from the transgenic research animal housing, which may be managed as a conventional facility to make possible investigator access and use. Perhaps the most challenging aspect of transgenic animal management involves the long-term breeding strategies of important lineages. Maintaining health during continuous, possibly multiyear, breeding programs of critical animals presents a formidable task. When a pathogen enters this type of operation, it is extremely difficult to eradicate it without sacrificing all of the valuable animals and disinfecting the facility. Aseptic rederivation and embryo transfer procedures can be critical to save important lines after disease has been discovered (Geistfeld, 1991). Contamination of a colony

with a variety of viruses and bacteria, even in the absence of clinical disease, can impact the transgenic phenotype and experimental results (Lussier, 1988; National Research Council, 1991; Percy and Barthold, 1993; Waggie et al., 1994; Baker, 1998; Maggio-Price et al., 1998).

B. ColonyBreeding Strategies With all transgenic lines, only a single animal, the founder, is initially available for production. Therefore, it is imperative to perpetuate the line by breeding this founder as rapidly as possible. Obviously, colonies may be generated from male founders through polygamous and repeated mating within a relatively short time. Well-planned and reliably executed mouse-breeding strategies are critical to fully understanding and appreciating the expression pattern of a transgene. Table III reviews the time frames of various components of a transgenic mouse program. The strategies should be prepared by the researcher and discussed in detail with all participating animal care personnel. Expected phenotypic patterns and possible effects on health, behavior, or fertility should be reviewed. Other topics that

Table I I I Time Frames for Components of Transgenic Mouse Production and Breeding Programa Procedures

Time Frame

Goal

Creation and identification of transgenic mice (a) Microinjection of DNA into pronuclei or injection of ES cells into blastocysts (b) Birth of newborn pups (c) Tissue biopsy (d) Identification of founders

3 weeks

Obtain live-born litters

3 weeks 1-3 weeks 2-3 weeks

Weaning and biopsy; genomic DNA analysis Identification of transgenic founder animals Initiation of breeding for colony expansion, embryo collection, or rederivation

Breeding for colony expansion and generation of homozygotes (a) Mating pairs set up (b) Newborn transgenic animals delivered (c) Transgenic siblings identified (d) Sibling crosses set up (e) F1 X F1 litter(s)delivered (f) Identification of transgenic F2 animals

3 weeks 4 weeks 2 weeks 3 weeks 4 weeks 2 weeks

Newborn pups delivered Weaning and identification of F~ pups Transgenic F1 X F~ sibling crosses set up F1 x F1 litter(s) delivered Weaning and identification of transgenic F2 pups Initiation of outcross test matings for identification of putative homozygous animals Test litters born Identification of transgenic test pups; data evaluation Confident verification of homozygous animals

(g) Outcross test matings set up (h) Test animals born (i) Test matings initiated Breeding for embryo collection and transfer derivation (a) Mating pairs set up

3 weeks 4 weeks 4-16 weeks

(b) Embryo transfer derivation (c) Rederived pups born Breeding for rederivation surgery (hysterectomy) (a) Mating pairs set up (b) Rederived pups born

3 weeks 4 - 6 weeks

Embryo collection and initiation of cryopreservation or embryo transfer derivation Delivery of newborn rederived pups Serologic evaluation of rederived animals

3 weeks 4 - 6 weeks

Newborn pups delivered and placed with foster mothers Serologic evaluation of derived animals

a

Adapted from Monastersky and Geistfeld (1997).

1 week

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GLENN M. MONASTERSKY AND JAMES G. GEISTFELD

should be discussed include the importance of record keeping, potential sex-influenced expression and imprinting, and key early indicators of transgene-mediated pathology. The specific goals of the breeding program should be documented and reviewed by all relevant personnel. Priorities must be explicitly clear because the number of transgenic breeding animals almost always will be the key rate-limiting factor for the progress of the research program. Goals of the breeding program may include the following: stabilization of the transgene on a specific genetic background by backcrossing to an inbred strain breeding to transgenic homozygosity (especially for knockouts) utilization of sibling crosses to stabilize genetic and phenotypic expression expansion of the colony to provide animals for research production of embryos for cryopreservation surgical rederivation of the line When a founder is identified, it is important to expand the line as rapidly as possible by mating with wild-type mates. This procedure is more easily accomplished when the founder is a male because he can breed several females each week. The wild-type mates are usually from one of the parental strains of the founder. In most programs and for most lineages, it is advisable to mate to produce homozygous offspring as soon as possible (Table IV). For knockout heterozygotes, production of the homozygous (i.e., null) genotype is absolutely required. The evaluation of certain dramatic or dominant genotypes and Y chromosome-linked gene insertions may not require homozygous study. Homozygosity may elicit a phenotype much different from that observed in the founder (i.e., hemizygote). Once homozygous breeders are available, significant time and expense can be saved since all of their offspring will carry the transgene, obviating the need for test mating and genotyping. There is no evidence that homozygosity of a chromosomally integrated transgene is any more unstable or subject to mutation than a endogenous gene locus. It is usually wise to maintain a few backcross matings in case the homozygotes exhibit challenged reproduction due to phenotype and/or morbidity o r I i n the extreme casemexcessive mortality. Table IV

Predicted TransgeneZygosityof Offspringfrom Hemizygous(He) Transgenic(Tg) Mice Hemizygote(+/-) x non-Tg ( - / - )

Hemizygote(+/-) • Hemizygote(+/-)

0% Homozygous(+/+) 50% Hemizygous(+/- ) 50% Nontransgenic(-/-)

25% Homozygous(+/+) 50% Hemizygous(+/- ) 25% Nontransgenic( - / - )

1.

Knockout Mouse Breeding Strategies

With knockout mice, it is important to confirm germline transmission of the transgenic insertion as soon as possible after sexual maturity of the founder animal. Phenotypic evidence, most commonly a chimeric coat-color pattern, is not predictive of germline transmission. Polymerase chain reaction (PCR) amplification assay for knockout gene-specific sequence may be performed with ejaculated sperm from a male knockout founder (i.e., collected from postcoital vaginal lavage), if desired. Chimeric mice may occasionally be sterile hermaphrodites (Shomer et al., 1997). Because most ES cell lines have an XY karyotype, the sex ratio of chimeric mice is skewed toward males (Robertson et al., 1986). As a rule, any chimeric mouse with more than 50% agouti coat coloring should be test-bred to confirm transgene transmission. Chimeric males are usually crossed with nonagouti females, such as C57BL/6, 129/SvEv, or Black Swiss. Since the knockout phenotype can vary on different genetic backgrounds, it is wise to evaluate a mutation on at least one inbred and one outbred background. Because the ES cells modified by homologous recombination are heterozygous for the inserted DNA sequence, only 50% of the agouti offspring of a chimera X nonagouti mating will carry the mutation. Therefore, the genotypes of all offspring should be tested. As discussed previously, homozygous null knockout mice should be produced to assess the true effects of the lossof-gene function. 2.

Nontransgenic Strains for Transgenic Breeding Programs

The background strain can be very important to the expression of the transgene (e.g., Geflai, 1996). For example, gene expression and tumor formation in transgenic lines carrying human oncogenes can vary when the mice are backcrossed to different inbred or outbred strains (Pinkert, 1997). Initial generations of transgenic mice exhibit genetic characteristics similar to those of the embryo donor female. When the offspring of a founder are bred, the selection of a nontransgenic breeding strain may be critical to achieving a full appreciation of the value of the line. If the project research requires the placement of the transgene onto an inbred background, the offspring are usually mated with animals from the same inbred strain (or inbred component of a hybrid) as that of the founder or donor female. This strategy serves to standardize the genetic reliability of the line. The specific reproductive efficiency of the chosen inbred strain may also influence the selection process. Of course, breeders in an inbred transgenic foundation colony should be genetically monitored to assure the maintenance of genetic breeding fidelity (Whitmore et al., 1996). Various established breeding systems (Bennett and Vickery, 1970; Green, 1966) may be used for transgenic programs. Depending on goals and space, monogamous or a polygamous

28. TRANSGENICAND KNOCKOUTMICE

1137

2:1 harem mating system for males may be employed. When a breeding unit is being set up, the male should be placed in the cage 2 4 - 4 8 hr before entry of the females.

homozygosity. It is not uncommon to perform a minimum of 3 test matings to determine the gene transmittance frequency for mosaic or chimeric animals.

3.

4.

Genotype Analysis of Transgenic Mice

A transgenic founder always is hemizygous for the transgene (or, heterozygous for a knockout mutation). Although breeding to homozygosity is usually attempted for most transgenic lines within the first 6 months after a founder is born, homozygosity may not always be realized due to potential homozygous lethality or infertility resulting from certain transgenes. In all transgenic experiments, multiple lines expressing the same construct at different integration loci must be assessed to discount the impact of potential insertional mutations on expression patterns or embryonic survival. If homozygotes are not obtained after several litters of He X He matings within a single lineage have been examined, it may be assumed that the homozygous genotype somehow impairs embryonic or fetal development. In these cases, insertional mutation cannot be ignored as a possible factor, and studies for these lines must be restricted to analysis of He animals. Transgenic expressions related to endocrine or glandular (e.g., uterine) functions may also affect maternal transgenic female capabilities to support gestation. Regardless of which particular breeding strategies are selected for the study of a lineage, molecular genotype analysis must be performed for breeders, including putative homozygotes. Performance of this genetic quality-control program will provide great peace of mind because record-keeping errors and unplanned matings may occur in the best programs. Occasionally, especially with homozygous knockout mice, zygosity may be determined by phenotypic characteristics by observing either visual or biochemical markers of gene expression. The application of enzyme-linked immunosorbent assays (ELISAs) and Western protein blots are among the techniques used to detect biochemical phenotypic activities. It is also possible, with forethought, to employ a transgene construct with specific marker genes, such as the tyrosinase minigene (Overbeek, 1994). However, molecular analyses of DNA from a putative transgenic animal remain the most reliable methods of genotypic analysis. All breeders in a transgenic production or expansion program should be analyzed by either a Southern blot hybridization assay or by polymerase chain reaction (PCR) amplification. Either of these assays will confirm the presence of a transgene-specific nucleotide (i.e., DNA) sequence and with appropriate expertise and equipment, may be employed to detect zygosity for certain transgenes. The subsequent confirmation of zygosity by test mating is highly recommended. Test mating of a transgenic mouse must be performed fastidiously. The DNA analysis of weaned litters that contain fewer than 5 or 6 pups should be discouraged, and a minimum of 10 offspring from each transgenic animal should be evaluated in order to confirm

Special Circumstances

Each transgenic line may exhibit unique characteristics, which may create great difficulties in the analysis of expression and predicted pathology or with husbandry and breeding. The introduction of new genetic sequences into the chromosomes of an animal can also result in insertional mutations, which may be silent or may yield dramatic phenotypes. Also, the expression of certain reporter or fusion genes may produce tissue damage or interfere with reproduction (Pinkert et al., 1987; Yokoyama et al., 1993). Problems associated with the integration locus and/or knockout phenotype may include abnormal immune responses, altered life span, sex-influenced survival, and reduced litter size. The specific characteristics of an acquired line of transgenic mice must be understood upon receipt. It is important to discuss the line with the creator or vendor before using the animals in experiments or breeding programs. It is imperative that the idiosyncrasies of rare or expensive transgenic mice are familiar so that their care and breeding are performed expediently and efficiently. For example, the probable need for foster mothers, dietary supplementation, or an exceptionally sterile environment must be incorporated into the husbandry protocols. The altered immune competence and associated environmental requirements of certain lines may be critical, and the survival and/or experimental performance of these animals may require stringent barrier procedures and specially prepared husbandry supplies. Other characteristics that could impact the care and use of the animals would include highly aggressive behavior and a tendency for cannibalism. All animal care staff must be trained to detect the onset of specific phenotypic events such as tumor growth, hair loss, arthritis, and optic pathologies. Lifethreatening, transgene-mediated syndromes, such as diabetes, respiratory disorders, or intestinal blockage, must be expected and managed professionally. The welfare and comfort of the animals and the ultimate success of the research program depend on the training, attention, and performance of the daily animal care technicians. In animals created by pronuclear microinjection, it is possible for multiple copies of the transgene to integrate at more than one locus. These integrated transgenes will segregate independently and can yield a challenging genetic mosaicism and potentially confusing germline transmission patterns. It is possible, with time and effort, to establish true-breeding sublines for the independent insertion events, if desired (Wilkie et al., 1986). Between 10 and 30% of transgenic founders resulting from a single integration event may be mosaic due to integration of the transgene concatamers at some time after pronuclear

GLENN M. MONASTERSKY AND JAMES G. GEISTFELD

1138

fusion and DNA synthesis have occurred. These animals may exhibit a relatively reduced frequency of gene transmission to the F1 generation and an unreliable genotype of the germ cells formed during fetal development (Tinkle et al., 1994). Therefore, a founder (i.e., transgene-positive tail DNA) may produce one or more completely nontransgenic litters. 5.

Unique Husbandry Challenges

The general husbandry protocols for a transgenic line should follow the guidelines established for nontransgenic mice (Institute for Laboratory Animal Resources [ILAR], 1976; Foster et al., 1983; National Research Council, 1989). Extra care needs to be taken during the first year of existence of a colony to ensure survival until expansion breeding and embryo cryopreservation can be completed. For instance, water bottles, not automatic watering, should be used to prevent possible loss by drowning. If a particular line has difficulty eating pelleted feed, softened feed may have to be provided. Sufficient nesting material should be used to protect the pups from hypothermia, especially when a ventilated caging system is utilized. Specific transgenic colony husbandry concerns are reviewed in Monastersky (1995). C.

Colony Record Keeping

Transgenic animal production, genotype assessment, colony management, and phenotype observation generate an enormous amount of data that must be recorded properly and accurately so that they may be used to support the scientific conclusions of the research using the animals. The origin, breeding history, and individual characteristics of each transgene-bearing animal in a colony must be documented clearly and must be accessible. 1. Identification of Transgenic Animals and Lineages

It is important that each founder animal be given a unique descriptive identification code, which will be used to identify all of its offspring in subsequent generations. It is useful to use an abbreviation or acronym representing the transgene construct followed by the individual number of the founder (e.g., Glt-32001). Animals descended from the founder may be sequentially numbered so that their position within the family pedigree may be identified instantly (e.g., Glt-32-012 would be the twelfth transgenic animal identified within an F1 litter). The maintenance of a written or computer-based pedigree for each individual lineage is mandatory. The pedigree should note changes in breeding strains and the dates and types of deaths (e.g., 12/03/99-sac). This tool permits the easy correlation of inheritance and expression patterns, based on generation and sex, with phenotypic patterns and genotype analyses. It is recommended that each individual transgenic member of a lineage be physically marked, using subcutaneous transponders, ear tags,

or tattoos. All animal care staff must be aware that critical retrospective analysis of the expression pattern of a line may depend on accurate records of common events, including reproductive performance, litter sex ratios, life span, causes and dates of death, and the timing of phenotypic characteristics. Coatcolor patterns and imprinting phenomena must also be reported. For each lineage, the molecular biology information, including construct data and genotype analysis records, must be accessible whenever required. For each individual animal, recorded information should include the date of birth, sex, generation and identification number, line code, genotype, coat color, phenotypic expression data, genotype and phenotype sample information, backcrossing information, and the date and cause of death. Production information may include plugging data, number and size of litters, gestation times and parturition dates, lost and cannibalized litters, and the number of weaned pups produced. The use of well-maintained cage cards is mandatory. Cards should exhibit all normal mouse data in addition to transgenic lineage information and any treatment dates and the dates of any exceptional phenotypic events. Backcrossing and breeding notes must specify the sexes and genotypes of all animals and the ratio of males to females bred. The use of different-colored cage cards for each lineage is helpful. 2.

Nomenclature for Transgenic Lines

A universally accepted nomenclature system for transgenic mice would be of immense value. Although a few systems have been proposed, the widespread acceptance and use of a single nomenclature system across academia and industry remain to be accomplished. Critical items for a nomenclature system would include the designation of specific background strains or stocks used, the exact transgene construct, and any information regarding integration or insertion. Also, the laboratory of origin, the method of transgenesis, a complete backcrossing history, and the lineage designation constitute critical information. Health profiles and altered immune status may also be noted, if important. Obviously, including this formidable amount of information in a single designation is challenging, especially when an animal has a mixed background. One very important piece of information that many researchers tend to ignore is the "N" number. This is important both for lines that are continually backcrossed and for lines that have begun internal crossing. This information may be important, because certain lines, as they become genetically "fixed" (usually at N10 or N12), will behave differently than they did at lower N numbers. In 1992, the Institute for Laboratory Animal Resources (ILAR) printed a suggested transgenic nomenclature system. This system has been more widely accepted than any previous systems. In the ILAR system, the transgene symbol consists of four parts. The first part always consists of the letters Tg (i.e., transgene) plus one of three letters to designate the mode of DNA insertion (i.e., N, nonhomologous; R, retroviral vector; H,

28. TRANSGENICAND KNOCKOUTMICE

homologous recombination). The second part, which is not to exceed eight characters and should be included in parentheses, identifies the specific DNA insertion information. The insertion designation is determined by the creator and should identify the inserted sequence but not its location or phenotype. If the insertion uses sequences from a named gene, it is preferable that the designation contain the standard symbol (or a diminutive version) for the gene. Standard abbreviations (e.g., Im, insertional mutation; Rp, reporter sequence; Et, enhancer trap construct) may be used as part of the designation and should be placed at the end of the insert. New nomenclature designations may be checked through available resources, including ILAR or the Jackson Laboratory, to assure that inappropriate duplications or designations are avoided. The third part of the suggested nomenclature would be the laboratory-assigned number, which is the unique number assigned by the laboratory of origin to each confirmed stable insertion with germline transmittance. No two lines generated within a laboratory should have the same assigned number, even if the lines have the same insert integrated at different locations. The fourth and final part of the nomenclature designation would be the unique laboratory code, maintained by ILAR. It is recommended that each line of transgenic animals also be distinguished by the conventional designations for standard mouse strain or stock (preceding Tg symbol), if the information is not too complex. If these long and detailed nomenclature designations are used, especially when importing transgenic animals into a facility, the accuracy of the entire designation must be scrupulously recorded.

D.

Preservation of Transgenic Lines

The cryopreservation of preimplantation embryos collected from transgenic animals allows the long-term storage of valuable transgenic lines for an infinite period of time and the consquent ability to regenerate a colony of live animals in the future (Whittingham et al., 1977; Leibo et al., 1978; Weisburd, 1987; Nakagata, 1996). Cryopreserved embryos are protected from genetic mutation and drift, genetic contamination, and potential transgene rearrangement. Animals are unthreatened by disease, and most facility catastrophes and husbandry expenses are avoided (Reetz et al., 1988; Rouleau et al., 1992). Cryopreserved embryos may also be used for future support of patent claims, avoidance of international animal quarantine restrictions, and relatively cost-effective embryo transfer rederivation. Valuable lines that are not in current or imminent demand should be cryopreserved as soon as feasible, preferably as homozygotes to facilitate the future analysis and breeding of regenerated lines. Rodent embryos may be cryopreserved by slow-freezing methods, by fast-freezing methods, or by vitrification (Wood et al., 1987). Although 2-celled and 8-celled stages are the most commonly preserved, all stages of preimplantation embryos

1139 may be frozen successfully. The 8-celled mouse embryo exhibits favorable collection quantities, acceptable cryopreservation survival rates, and good postthaw recovery characteristics. The embryo can survive and produce a normal live-born mouse after ET, even if several of the totipotent blastomeres are damaged (Rulicke and Autenried, 1995). The cryopreservation of pronuclear-stage and and 1-celled embryos is more challenging because of the frequent damage to critical cytoskeletal structures and the more varied timing of these embryos at collection. Cryopreservation of blastocysts offers even more difficult challenges due to the dynamics of the fluid-filled blastocoele cavity. It is important to select normal-looking embryos with intact zona pellucidae for cryopreservation protocols so that future thawing and ET results may be enhanced. The major challenge to the survival of frozen embryos is the natural formation of intracellular ice crystals, which can damage the cell membrane and organelles. To avoid this catastrophe, embryos are usually frozen utilizing low molecular weight cryoprotectants (e.g., DMSO and glycerol), which permeate the individual blastomeres and modify the freezing points and fluid dynamics of the intracellular water. The cryoprotectants permit the controlled dehydration of the embryo by directly lowering the freezing point of the cell as the extracellular fluid is cooled down. The extracellular water is then induced to freeze rapidly (i.e., seeding) so that its vapor pressure is lower than the unfrozen intracellular water. This process concentrates the extracellular solutes, inducing a net flow of water out of the embryo to restore equilibrium across the cell membrane. Plastic vials or straws containing embryos in cooled, cryoprotectantsupplemented buffer solution are placed into programmed cryopreservation machines and frozen at a specific rate (e.g., 0.5~176 per min). As the embryonic cells lose water and shrink, the formation of intracellular ice is avoided. Embryos are usually frozen down to a target temperature of - 8 0 ~ and then stored in liquid nitrogen ( - 196~ When the frozen embryos are recovered from storage, the specific thawing procedure will depend on the initial freezing protocol and cryoprotectant choice. Slowly frozen embryos are usually thawed rapidly (e.g., > 1000~ per min). The intracellular cryoprotectant is toxic and must be removed from the embryonic cells as quickly as possible before the cells become metabolically active. To accomplish the rapid removal of the cryoprotectant agents, the thawed embryos are placed in a series of relatively large volumes of room-temperature buffer solution (e.g., M2 or DPBS). Expected survival and recovery rates for 8cell-stage embryos (e.g., cryopreserved by a slow-freeze process using DMSO) should range from 60 to 90%. Between 5 and 50% of the thawed embryos should become live-born following ET, with the skills of the embryology laboratory personnel determining the actual rates of success. The genotype of frozen transgenic embryos may be confirmed by performing a rapid PCR amplification for the transgene on a small aliquot of embryos immediately following thawing.

1140

GLENN M. MONASTERSKY AND JAMES G. GEISTFELD

The ability to freeze and recover viable, motile, fertilizationcapable mouse sperm is an enticing alternative to embryo freezing. Although common for the majority of mammalian species, mouse sperm cryopreservation has been exceptionally challenging. Nevertheless, this technique is rapidly developing (Songsasen and Leibo, 1998; Wakayama and Yanagimachi, 1998; Mobraaten, 1999).

The result of this array of intellectual property claims is that a given transgenic mouse is covered by multiple patents so that several licensing agreements must be negotiated before a line can be distributed freely.

REFERENCES E.

Transgenic Mice and Intellectual Property Rights

Transgenic technology has introduced a novel, and sometimes uncomfortable, complication into the animal research community. This new element involves intellectual property rights and the patenting of transgenic life-forms. Intellectual property rights may range from very broad coverage of a technique or process (e.g., recombinant DNA technology or pronuclear microinjection) or may protect the ownership of a specific gene sequence. Transgenic mouse patents may result in licensing or follow-through royalty agreements and have generated significant controversy in the research and laboratory animal communities. The reality of intellectual property rights must be confronted whenever transgenic and knockout mice are created, produced, or distributed. Obviously, the production, acquisition, and use of transgenic mice have become more complex and more costly as a result of patent protection for individual lineages, genes, and techniques. Transgenic mice may be covered by any or all of the following areas of intellectual property. (1) The first stage of the transgenic mouse creation process involves DNA cloning (Cohen and Boyer; assigned to Stanford University). Until 1998, any institution using recombinant DNA technology to construct transgene or knockout expression vectors had to obtain a license from Stanford. (2) The second stage of patent protection involves general creation techniques for genetically engineered animals. For example, the patents covering creation of embryonic stem cell knockout mice, "Techniques for Selection of Homologous Recombination Events m Gene Targeting Using Positive Negative Selection" (Capecchi; assigned to University of Utah) and the creation of pronuclear microinjection transgenic animals, "The Method of Genetic Transformation Resulting from Microinjection" (Wagner and Hoppe; assigned to DNX Corporation) are techniques that must be licensed. (3) The third patent stage covers specific creation techniques. For example, "Gene Targeting in Animal Cells Using Isogenic DNA Constructs" (Berns; Netherlands Cancer Institute) describes the creation of an inbred knockout mouse. (4) The fourth patent stage covers specific genetically mediated phenotypes. An example of this type of intellectual property would be the renowned "Oncomouse" patent: "Oncogene Bearing Rats and Mice" (Stewart and Leder; assigned to DuPont, Inc.). This patent applies to any transgenic or knockout model that is predisposed to genetically mediated tumor formation. (5) The fifth stage of patent protections covers the outright ownership of specific genes. An example of this property would be the "p53" patent (Donehower and Bradley; Baylor University).

Baker, D. G. (1998). Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin. Microbiol. Rev. (Vol. II), 231-266. Bennett, J. P., and Vickery, B. H. (1970). Rats and mice. In "Reproduction and Breeding Techniques for Laboratory Animals" (E. S. E. Hafez, ed.) Lea and Febiger, Philadelphia. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Senear, A. W., Warren, R., and Palmiter, R. D. (1981). Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27, 223-231. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., and Palmiter, R. D. (1985). Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. U.S.A. 82, 44384442. Costantini, E, and Lacy, E. (1981). Introduction of a rabbit B-globin gene into the mouse germ line. Nature 294, 92-94. Foster, H. L., Small, J. D., and Fox, J. G., eds. (1983). "The Mouse in Biomedical Research (Vol. III)mNormative Biology, Immunology, and Husbandry. Academic Press, San Diego. Geistfeld, J. G. (1991). Transgenic mouse colony management. Lab. Anim., 20, 21-29. Gerlai, R. (1996). Gene-targeting studies of mammalian behavior: Is it the mutation or the background genotype? Trends Neurosci. 19, 177-181. Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A., and Ruddle, E H. (1980). Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. U.S.A. 77, 7380-7384. Green, E. L. (1966). Breeding systems. In "Biology of the Laboratory Mouse" (E. L. Green, ed.). McGraw-Hill, New York. Hogan, B., Beddington, R., Costantini, E, and Lacy, E. (1994). "Manipulating the Mouse Embryo--A Laboratory Manual," 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, New York. Institute for Laboratory Animal Resources (ILAR) (1976). Long-term holding of laboratory rodents. In "ILAR News" 19(4), L1-L25. Institute for Laboratory Animal Resources (ILAR) (1992). Standardized nomenclature for transgenic animals. In "ILAR News" 34(4), 47-52. Jaenisch, R. (1976). Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. U.S.A. 73, 1260-1264. Jaenisch, R., and Mintz, B. (1974). Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc. Natl. Acad. Sci. U.S.A. 71, 1250-1254. Leibo, P., and Mazur, P. (1978). Methods for the preservation of mammalian embryos by freezing. In "Methods in Mammalian Reproduction" (J. C. Daniel, ed.), pp. 179-201. Academic Press, New York. Leveille, M. C., and Armstrong, D. T. (1989). Preimplantation embryo development and serum steroid levels in immature rats induced to ovulate or superovulate with pregnant mare's serum gonadotropin injection or folliclestimulating hormone infusions. Gamete Res. 23, 127-138. Lussier, G. (1988). Potential detrimental effects of rodent viral infections on long-term experiments. Vet. Res. Comm. 12, 199-217. Maggio-Price, L., Nicholson, K. L., Kline, K. M., Birkebak, T., Suzuki, I., Wil-

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son, D. L., Schauer, D., and Fink, E J. (1998). Diminished reproduction, failure to thrive, and altered immunologic function in a colony of T-cell receptor transgenic mice: Possible role of Citrobacter rodentium. Lab. Anim. Sci. 48(2), 145-155. Mobraaten, L. E. (1999). Cryopreservation in a transgenic program. Lab Anim. (January), pp. 15-18. Monastersky, G. M. (1995). Transgenic mouse strategies: Embryology and animal science. In "Strategies in Transgenic Animal Science" (G. M. Monastersky and J. M. Robl, eds.), pp. 3-36. ASM Press, Washington, D.C. Monastersky, G. M., and Geistfeld, J. G., (1997). The production and verification of transgenic mouse strains. Lab. Anim. (26), pp. 36-40. Nakagata, N. (1996). Use of cryopreservation techniques of embryos and spermatozoa for production of transgenic (TG) mice and for maintenance of TG mouse lines. Lab. Anim. Sci. 46, 236-238. National Research Council (1989). "Immunodeficient Rodents m A Guide to Their Immunobiology, Husbandry, and Use." National Academy Press, Washington, D.C. National Research Council (1991). "Infectious Diseases of Mice and Rats." National Academy Press, Washington, D.C. Overbeek, P. A. (1994). Factors affecting transgenic animal production. In "Transgenic Animal Technology, a Laboratory Handbook" (C. A. Pinkert, ed.), pp. 96-107. Academic Press, San Diego. Percy, D. H., and Barthold, S. W. (1993). "Pathology of Laboratory Rodents and Rabbits." Iowa State Univ. Press, Ames. Pinkert, C. A. (1997). The history and theory of transgenic animals. Lab. Anim. (26), pp. 29-34. Pinkert, C. A., ed. (1994). "Transgenic Animal TechnologymA Laboratory Handbook." Academic Press, New York. Pinkert, C. A., Ornitz, D. M., Brinster, R. L., and Brinster, R. D. (1987). An albumin enhancer located 10Kb upstream functions along with its promoter to direct efficient liver-specific expression in transgenic mice. Genes Dev. 1, 268-276. Polites, H. G., and Pinkert, C. A. (1994). DNA microinjection and transgenic animal production. In "Transgenic Animal TechnologymA Laboratory Handbook" (C. A. Pinkert, ed.), p. 23. Academic Press, New York. Reetz, I. C., Wullenweber-Schmidt, M., Kraft, V., and Hedrich, H. J. (1988). Rederivation of inbred strains of mice by means of embryo transfer. Lab. Anim. Sci. 38, 696-701. Robertson, E., Bradley, A., Kuehn, M., and Evans, M. (1986). Germ-line transmission of gene introduced into cultured pluripotential cells by retroviral vector. Nature 323, 445-448. Rguleau, A., Kovacs, P., Kunz, H. W., and Armstrong, D. T. (1992). Decontamination of rat embryos and transfer to SPF recipients for the production of a breeding colony. Theriogenology 37, 289. Rulicke, T., and Autenried, P. (1995). Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation. Lab. Anim. 29, 320-326.

1141 Shomer, N. H., Foltz, C. J., Li, K., and Fox, J. G. (1997). Diagnostic exercise: Infertility in two chimeric mice. Lab. Anim. Sci. 47(3), 321-323. Simpson, E. M., Linder, C. C., Sargent, E. E., Davisson, M. T., Mobraaten, L. E., and Sharp, J. J. (1997). Genetic variation among 129 substrains and its importance for "targeted mutagenesis" in mice. Nat. Genet. 16, 19-27. Small, J. D. (1984). Rodent and lagomorph health surveillance-quality assurance. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 709-723. Academic Press, San Diego. Songsasen, N., and Leibo, S. P. (1998). Live mice from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa. Lab. Anim. Sci. 48, 275-281. Taketo, M. Schroeder, A. C., Mobraaten, L. E., Gunning, K. B., Hanten, G., Fox, R. R., Roderick, T. H., Stewart, C. L., Lilly, E, Hansen, C. T., and Overbeek, P. A. (1991). FVB/N: An inbred mouse strain preferable for transgenic analyses. Proc. Natl. Acad. Sci. U.S.A. 88, 2065-2069. Tinkle, B. T., Bieberich, C. J., and Jay, G. (1994). Molecular approaches involved in mammalian gene transfer: Analysis of transgene integration. In "Transgenic Animal Technology--A Laboratory Handbook" (C. A. Pinkert, ed.), p. 230. Academic Press, San Diego. Van Ruiven, R., Meijer, G. W., van Zutphen, L. E M., and Ritkes-Hoitinga, J. (1996). Adaptation period of laboratory animals after transportation: A review. Scand. J. Lab. Anim. Sci. 23(4), 185-190. Waggie, K., Kagiyama, N., Allen, A., and Nomura, T., eds. (1994). "Manual of Microbiologic Monitoring of Laboratory Animals, 2nd ed. NIH Publ. 942498. National Institutes of Health, Bethesda, Maryland. Wakayama, T., and Yanagimachi, R. (1998). Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat. Biotech. 16, 639641. Weisburd, S. (1987). Beyond the cutting edge of cold. Sci. News 132, 129-144. Whitmore, S. P., Gilliam, A. E, Hendren, R. W., Lewis, S. E., Rao, G. N., and Whisnant, C. C. (1996). Genetic monitoring of inbred rodents from controlled production colonies through biochemical markers and skin grafting procedures. Lab. Anim. Sci. 46(2), 585-588. Whittingham, D. G., Lyon, M. E, and Glenistor, P. H. (1977). Re-establishment of breeding stocks of mutant and inbred strains of mice from embryos stored at - 196~ for prolonged periods. Genet. Res. 30, 287-299. Wilkie, T. M., Brinster, R. L., and Palmiter, R. D. (1986). Germline and somatic mosaicism in transgenic mice. Dev. Biol. 118, 9-18. Wood, M. J., Whittingham, D. G., and Rail, W. E (1987). The low temperature preservation of mouse oocytes and embryos. In "Mammalian DevelopmentmA Practical Approach" (M. Monk, ed.), pp. 255-280. IRL Press, Oxford. Yokoyama, T., Copeland, N. G., Jenkins, N. A., Montgomery, C. A., Elder, E E B., and Overbeek, P. A. (1993). Reversal of left-right asymmetry: A situs inversus mutation. Science 260, 679-682.

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Chapter 2 9 Factors That May Influence Animal Research Neil S. Lipman and Scott E. Perkins

I. II.

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Introduction .................................................

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Intrinsic Considerations

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Genetics ................................................

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Age ....................................................

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Sex ....................................................

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I m m u n e and Nutritional Status

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Circadian Rhythms

F.

Endocrine Factors

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Physical Factors ..........................................

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Chemical Factors .........................................

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Stressors ................................................

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I.

INTRODUCTION

Animal research involves the collection of data from carefully designed experiments. The validity of the research and the conclusions drawn from the data are influenced by many factors. Some of these factors may confound experimental results and therefore must be carefully considered and controlled. Otherwise, distortions may result that lead to false observations or conclusions. Although disease is recognized to complicate in vivo research, there is a wide variety of more subtle factors that may alter experimental findings. This chapter will provide an overview of the multitude of complicating factors that have been described in the literature. The reader should understand that there are likely additional factors yet to be recognized, as well as interactions among fac-

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tors that may also influence experimental outcomes. This chapter has been divided into two principal sections: intrinsic considerations are those inherent to the animal itself, such as its genotype; and extrinsic considerations are those external to the animal that may influence its response. In order to obtain reliable, meaningful results, an attempt should be made to control or standardize all known biological, environmental, and social factors when conducting experiments involving animals.

II.

INTRINSIC CONSIDERATIONS

Experimental animals vary among one another based on their genetic constitution, age, sex, health, and nutritional and

Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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immune status, as well as on other biological factors. Although animals with genetic uniformity are available and often used, other intrinsic factors must also be considered.

A.

Genetics

The genotype of an animal is an important consideration in designing an experimental protocol, as genetic differences clearly exist between species, breeds, and even strains. Both inbred and outbred animals are widely used in biomedical research, each having advantages and limitations. For outbred animals, the breeding colony from which they are obtained must be large enough to maintain heterogeneity, and management techniques must be employed to ensure genetic variability. Heterogeneity can be monitored by using computer models; analyzing biochemical, genetic, and immunologic markers; and examining physiologic variables (Groen and Lagerwerf, 1979; MacCleur et al., 1986; van Oorschot et al., 1992; WilliamsB langero, 1993). The development and availability of inbred strains permit researchers to address specific questions while generating reproducible and comparable data. However, genetic differences exist even when considering the same strain or even substrain, thus potentially altering experimental data (Heywood and Buist, 1983; Simpson et al., 1997). It is important to understand that the genetic integrity of an animal is not necessarily guaranteed by its nomenclature. Genetic divergence is common; thus, the genetic purity of inbred animals must be monitored and maintained (Festing, 1982; Hedrich, 1983; Threadgill et al., 1997). Genetic differences between individuals of the same inbred strain from the same colony may be the result of incomplete inbreeding, mismatings, inadvertent outcrossings with other strains, spontaneous mutations, chromosomal aberrations, or residual heterozygosity (Baily, 1982). H'Doubler et al. (1991) determined spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats to be genetically disparate despite derivation from the same parental Wistar stock. In animal studies using SHR rats, WKY rats have been used almost exclusively as controls due to their presumed homozygosity. However, skin graft experiments and analyses of immunologic and biochemical markers determined SHR and WKY rat strains to be heterogeneic, and some sources of WKY rats to be outbred stocks (H'Doubler et al., 1991). In addition, Kurtz et al. (1989) demonstrated genetic heterogeneity among WKY rats distributed from different commercial sources and even among a single breeding facility. The genetic variability may have been the result of the National Institutes of Health (NIH) distributing breeding stocks of WKY rats as early as the F 6 generation (Kurtz et al., 1987). Thus, rats designated WKY do not constitute an inbred strain. St. Lezin et al. (1992) demonstrated that SHR rats and their WKY controls share only approximately 50% of their DNA fingerprint bands in common, while the hy-

pertensive inbred Dahl salt-sensitive rats (SS/Jr strain) and their normotensive controls, Dahl salt-resistant rats (SR/Jr strain), share only approximately 80% of their DNA fingerprint bands in common. These findings provide evidence of extensive genetic polymorphism between spontaneously hypertensive rat strains and their corresponding normotensive controls and suggest that the continued comparison of these strains may have limited value in interpreting the results on the pathogenesis of hypertension. Today, commercial rodent vendors diligently control and monitor genetic integrity and carefully manage breeding practices for inbred and F1 hybrid colonies, using immunologic and biochemical markers, as well as evaluate polymorphic alleles, using molecular techniques (Cramer 1983; Groen 1977; Russell et al., 1993; Whitmore et al., 1996). When using animals generated by gene targeting, additional details must be considered. Choosing the best genetic strain or stock of mice when developing targeted mutant mice requires an extensive knowledge of their endogenous traits (Crawley et al., 1997). In transgenic animals, the number of copies and site of gene integration may not be known. Additionally, in "knockout" or "knockin" animals the integrated or deleted gene(s) may interact with other genes and extrinsic factors, producing unexpected results that may compromise the interpretation of the mutant phenotype. Threadgill et al. (1995) demonstrated that the phenotype of epidermal growth factor receptor (EGFR) knockout mice was dependent on the genetic background. Development of homozygous EGFR-deficient mutants resulted in peri-implantation death due to degeneration of the inner cell mass in CF-1 mice; midgestational death due to placental defects in 129/Sv mice; and early neonatal death in CD1, 129/Sv X C57BL/6, and 129/Sv x C57BL/6 X MF1 mice due to organ abnormalities (Threadgill et al., 1995; Sibilia et al., 1995). In prostaglandin E2 EP4 receptor-deficient mice on a 129 background, remodeling of the ductus arteriosus (DA) does not occur after birth, resulting in death of the pups. However, 5% of EP 4 ( - / - ) mice on a mixed genetic background survive, and with selective breeding of these mice, there is a 21% survival rate, suggesting that alleles at other loci can provide an alternative mechanism for DA closure (Nguyen et al., 1997). Spearow et al. (1999) detected large differences in susceptibility to juvenile male reproductive development by 17[3-estradiol (E2) in different strains of mice. For example, spermatid maturation was eliminated by low doses of E2 in C57BL/6 and C17/Jls mice, while there was little or no inhibition of spermatid maturation in CD-1 mice, even in response to 16 times the dose of E2. Crawley et al. (1997) provided an extensive overview of behavioral phenotypes of inbred mouse strains and the implications and recommendations for molecular studies based on these behavioral traits. Importantly, the understanding of behavioral phenotypes of the strain in which a mutation will be analyzed can avoid overinterpretation of the mutant phenotype (Crawley et al., 1997). As with inbred animals, comprehensive genetic monitoring, including monitoring

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the inserted gene and number of copies, should be performed to when evaluating defeat-induced learned submissiveness (Siegmaintain these unique resources and to verify the presence and fried et al., 1984). Therefore, species and strain selection of an zygosity of the genotype (National Research Council, 1996). animal may have profound effects on experimental results. Species and strains of animals may metabolize xenobiotics differently as a result of inherent quantitative and qualitative B. Age variations. Quantitative variations include differences in cytochrome P450 isoenzymes; cytochrome P450 concentrations; The age of an animal may affect research outcomes and has and competing isoenzyme reactions within the host. Qualitative variations generally involve differences in metabolic pathways. been shown to be a source of variability in rodent carcinogenicThey result from defective or absent enzymes or the ability of a ity studies (Hardisty, 1985; Haseman et al., 1989). Neonatal anspecies to conduct unique enzymatic reactions. Examples in- imals have immature body systems compared to those of adults, clude defects in mercapturic acid formation in guinea pigs; de- and depending on the genotype of the animal, the immunologic fects in glucuronidation in cats; deficient sulfate conjugation in competence of neonates is considerably more immature swine; the lack of N-acetyltransferase in dogs, preventing them (Williams and Weisburger, 1991). As an example, the total and from acetylating aromatic amines; and the singular ability of differential white blood cell count of dogs and cats less than dogs to biotransform 2,2 ',4,4 ',5,5 ' -hexachlorobiphenyl due to 6 months of age are higher than those of adults; the total leukoa unique cytochrome P450 isoenzyme present in their livers cyte, neutrophil, and lymphocyte counts in kittens are highly (Sipes and Gandolfi, 1991). Williams- Blangero et al. (1996) at- variable (Bounous et al., 1995); and neonatal dogs and cats tributed phenotypic variation in alanine transaminase (ALT) have lower blood pressures, stroke volumes, and peripheral vasactivity in chimpanzees to genetic factors. The authors provided cular resistance, and higher heart rates, cardiac output, plasma evidence that the genetic components causing variations in ALT volumes, and central venous pressures (Driscoll et al., 1979). levels can have significant effects on experimental parameters These differences reflect their immature sympathetic innervain hepatitis C research. Examples of intraspecies differences in- tion as compared to the more mature parasympathetic system. clude differences in rates of hexobarbital oxidation in Sprague- Other reported age differences include the demand for oxygen, Dawley and Wistar rats (Sipes and Gandolfi, 1991); differing respiratory rate, hepatic microsomal enzyme system developsensitivities to neurotoxicants such as diisopropyl fluorophos- ment, renal concentrating and diluting capability, and the abilphate (DFP) in Sprague-Dawley, Long-Evans, Fischer 344, and ity to thermoregulate (Hosgood, 1995). The ability to biotransWistar rats (Gordon and Watkinson, 1995); carcinogenic effects form xenobiotics is severely limited in fetuses and neonates of 7,12-dimethyl-benz[a] anthracene (DMBA) on different types due to the immaturity of their hepatic microsomal enzymes of mammary tissue in Wistar-Furth (WF) and Copenhagen (Sipes and Gandolfi, 1991). As an example, neonates are more (COP) rats (Kusunose et al., 1990); and differences in learning, sensitive to certain carcinogenic compounds (Williams and memory, and attention tasks among rat strains and even the same Weisburger, 1991). Conversely, enzyme activities decrease with strain obtained from different suppliers (Andrews, 1996; Craw- increasing age, which may lead to an increased toxicity of ley et al., 1997; Kacew et al., 1998). In addition, significant dif- xenobiotics that serve as their substrates. Older animals may ferences in acute cadmium intoxication have been demonstrated have decreased hepatic and renal blood flow, smaller livers, inin mice. Only C3H mice develop severe hepatocellular damage creased body fat, and decreased excretory capability (Sipes and and significantly lower metallothione induction at 6 hr post- Gandolfi, 1991). Han et al. (1998) demonstrated age-related cadmium injection compared to B ALB/c and DBA/2 strains changes in blood pressures of two strains of senescence(Hata et al., 1980). Strain differences are also observed with accelerated mice (SAM). The SAMP1 mice had a significantly chloroform toxicity in mice; the DBA/2J strain is sensitive, increased blood pressure with age, possibly due to progressive renal changes, and SAMP8 mice had a gradual decrease in while C57BL/6J is resistant (Hill et al., 1975; Vessel et al., 1976). Chloroform toxicity in DBA/2J mice is due to faster re- blood pressure after 5 - 7 months of age. All of these age-related nal conversion of chloroform to phosgene, a reactive intermedi- biochemical and physiologic factors may alter the response to ate, compared to that in C57BL/6J mice. Inbred strains of mice xenobiotics and confound experimental results. also differ with respect to the frequency, type, and age at which neoplasia develops (Fox and Witham, 1997) and also in sensoC. Sex rimotor gating as measured by differences in prepulse inhibition of auditory and tactile startle responses (Paylor and Crawley, The gender of the animal may also influence experimental 1997). A/J and C57BL/6J mice demonstrate differences in anxiety-related behavioral phenotypes and responses to di- outcome. Marked differences in pharmacologic and toxicologic azepam, methyl-[3-carboline 3-carboxylate, and benzodiazepine responses to a variety of xenobiotics have been demonstrated (Mathis et al., 1994, 1995). Additionally, DBA/2 mice have a in male and female rats (Sipes and Gandolfi, 1991). Female greater endogenous analgesic response than C57BL/6 mice rats have a reduced capacity to biotransform certain chemicals,

NEIL S. LIPMANAND SCOTT E. PERKINS

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including hexobarbital and parathion. Thus, they sleep significantly longer than males when given an equivalent dose of hexobarbital, and parathion is approximately twice as toxic to female rats than male rats (Sipes and Gandolfi, 1991). Genderspecific forms of cytochrome P450 have been discovered in rats (Sipes and Gandolfi, 1991). The renal conversion of chloroform to phosgene occurs 10 times faster in male mice than in female mice, making male mice susceptible and female mice resistant to chloroform toxicity (Sipes and Gandolfi, 1991; Vessel et al., 1976). Additional examples of sex-related differences are observed when cognitive testing rats and examining the development of DMBA-induced mammary tumors in Wistar-Furth rats, as the incidence of tumors is 100% in females and only 19% in males (Andrews, 1996). However, in Copenhagen rats, the neoplastic response to DMBA-induced mammary tumors is not sex-dependent (Kusunose et al., 1990).

D.

Immune and Nutritional Status

The immune and nutritional status of an experimental animal may have profound effects on the experimental outcome, depending on the nature of the research. The immune system has many functions, including homeostasis of leukocyte differentiation and maturation, immunoglobulin production, and immune surveillance, providing the host a defense system against microbes, neoplastic cells, and environmental agents (Dean and Murray, 1991). Resistance to infectious agents may be dependent on the type of immune response that the host generates. For example, CBA/J mice are highly resistant to Leishmania major, as they develop a predominantly cell-mediated or THl-type response; whereas BALB/c mice are not resistant, as they develop a predominantly humoral or TH2-type response (McEachron et al., 1995). The response to murine leukemia virus (E-55 +) is different in B ALB.K mice and in B 10.BR mice, which express the same H-2 haplotype but differ in genetic background. One hundred percent of BALB.K mice develop leukemia and generate a predominantly TH2 response to infection, while B 10.BR mice generate a predominantly TH1 response and do not develop the final leukemic phase (McEachron et al., 1995). In contrast, it is the TH2 response that confers resistance to helminth infections in BALB.K mice but the TH1 response in B 10.BR mice (McEachron et al., 1995). Immunologic dysfunction, including hypersensitivity and allergy, autoimmunity, and immunodeficiency may influence experimental outcome. There is a wide variety of agents that alter immune function, including chemicals, drugs, food additives, metals, and microbes (Dean and Murray, 1991). The increased sensitivity to chemical carcinogens in neonatal animals may be related to immunologic mechanisms. Penn (1988) demonstrated an alteration in the rate and extent of tumor development in humans with different types of immunodeficiencies.

An animal's nutritional status is dependent on the type(s) of food provided, method and amount of feeding, appetite, and age (Sipes and Gandolfi, 1991). Various dietary conditions, such as mineral, vitamin, and protein deficiencies; lipid composition; and the composition of the diet, alter the biotransformation of xenobiotics (Sipes and Gandolfi, 1991). For example, vitamins C, E, and B complex are involved in cytochrome P450 regulation, and their deficiency reduces the rate of xenobiotic biotransformation (Sipes and Gandolfi, 1991). E.

Circadian Rhythms

Circadian rhythms are endogenous rhythms of physiologic functions and are partially influenced by the time of day, intensity of light, and the suprachiasmatic nuclei (Hastings, 1970; Scheving et al., 1983). Many behavioral, biochemical, and physiologic parameters display rhythmic minima and maxima daily, occurring at specific times of the day and night (Hastings, 1970; Mock et al., 1978). These include blood counts; blood coagulation times; concentrations of CD-3 molecules on the surface of T cells; T-cell response to mitogens; plasma steroid concentrations; body temperature; thermal preference; sensitivity to audiogenic seizure induction; susceptibility to endotoxin and pneumococcus exposure; infectivity pattern following murine leukemia virus inoculation; drug metabolism and toxicity, including barbiturate sleep times and response to ethanol administration; susceptibility to neoplasia; DNA synthesis; protein synthesis; and release of urinary chemosignals that accelerate puberty (Alder and Friedman, 1968; Berger, 1980, 1981; Bowman et al., 1970; Cayen et al., 1972; Chedid and Nair, 1972; Deimling and Schnell, 1980; Drickamer, 1982; Feigin et al., 1969; Gordon et al., 1998; Halberg et al., 1973; Haus et al., 1983; Izquierdo and Gibbs, 1972; Jori et al., 1971; LeBouton and Handler, 1971; Levi et al., 1991; McEachron et al., 1995; Mitropoulos et al., 1972; Nair and Casper, 1969; Nash and Llanos, 1971; Radzialowski and Bousquet, 1968; Ramaley, 1972; Romero, 1976; Scheving and Pauly, 1967; Scheving et al., 1968, 1978; Tavadia et al., 1972; Wada and Asakura, 1970; Wongwiwat et al., 1972; Zbiesieni, 1980). Caution should be exercised when comparing data that have been collected at different times. F.

Endocrine Factors

The endocrine system is one of the principal control systems of the body, producing hormones that regulate both cellular and metabolic functions (Williams and Weisburger, 1991). The interrelationship of the endocrine and nervous systems is essential to integrate a variety of factors that are important in an animal's response to general arousal, physiologic processes, and aversive stimuli (Clark et al., 1997).

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29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH

Sex hormones are important determinants of cytochrome P450 enzyme activity (Sipes and Gandolfi, 1991). Testosterone administered to female rats increases their ability to biotransform xenobiotics. The opposite effect is observed when male rats are castrated (Sipes and Gandolfi, 1991). Certain chemicals have been shown to disrupt the endocrine system, leading to cancer induction in specific target organs, such as the adrenals, uterus, or thyroid (Williams and Weisburger, 1991). Gonadectomy, hypophysectomy, or adrenalectomy alters hormonal level(s), potentially resulting in a variety of biological changes (Williams and Weisburger, 1991). Neonatal gonadectomy of CE and DBA strains of mice leads to a high incidence of estrogen-secreting adrenal tumors (Fekete et al., 1941; Murthy et al., 1970). Lipman et al. (1993) postulated that hypersecretion of trophic pituitary hormones in gonadectomized ferrets may result in stimulation of the adrenal gland, producing adrenal gland tumors. Induced ovulators, restricted from normal reproductive activity, may develop hormonal alterations and subsequent biological responses. Prolactin-secreting pituitary adenomas may develop in nulliparous rabbits because of exposure of pituitary acidophils to high concentrations of plasma estrogens secreted by ovarian follicles (Lipman et al., 1994). This hypothesis is supported by the development of prolactin-secreting tumors in rats following prolonged administration of natural or synthetic estrogens (Ito, 1976). Interspecies variations at the hypothalamic-pituitary axis appear to have an important bearing on the differential activities of estrogens and antiestrogens (Hart, 1990). Toxicity of estrogens is species-dependent. Carnivores are more susceptible than rodents (Hart, 1990). Female ferrets may develop estrogen-induced bone marrow suppression due to prolonged estrus or an estrogen-secreting ovarian remnant resulting in pancytopenia and death (Bernard et al., 1983; Sherrill and Gorham, 1985).

Ill.

EXTRINSIC CONSIDERATIONS

A.

Physical Factors

The provision of a stable environment for the conduct of animal research is essential to ensure the integrity of both the animals and the results obtained. The environment to which the animals are exposed must be considered from two perspectives, the m a c r o e n v i r o n m e n t of the room in which the animals are housed, and the cage m i c r o e n v i r o n m e n t with which the animals have direct and prolonged contact. Dependent on the species housed and the caging system utilized, macro- and microenvi-

ronmental conditions may be vastly dissimilar. The principal factors influencing the microenvironment include the macroenvironment, the caging system, and the animals. Extensive effort is devoted to maintaining the temperature, relative humidity, and air quality within the macroenvironment. Guidelines (the "Guide") have been established for these parameters to ensure the well-being of the animals, as well as to provide a stable environment (National Research Council, 1996). Alterations and fluctuations of environmental conditions are well recognized to influence results in a variety of disciplines. For example, the effect of environment on mouse behavior was recently examined in a variety of inbred strains and a targeted mutant mouse line. Despite using animals of the exact genotype, the same experimental setup and procedures, and the same caging, and controlling for the effects of shipment, differences in results were observed and attributed to environmental influences, including differences in air handling and humidity (Crabbe et al., 1999).

1. Temperature The thermoneutral zone (TNZ) of an animal is the temperature range in which homeotherms exert minimal energy to maintain core body temperature. When an animal is exposed to temperatures above or below the TNZ, both behavioral and physiologic adjustments are made to ensure homeostasis, including postural adjustments, huddling, piloerection, peripheral vascular dilatation or constriction, alterations in the respiratory rate and pattern, and food consumption. If alterations in ambient temperature persist or the adaptive adjustments are inadequate, changes in the animal's basal metabolic rate result (Clough, 1982; Newton, 1978). However, the TNZ is not necessarily identical to the temperature range providing optimal development, comfort, reactivity, and adaptability, or for particular species such as the rat, even to the animal's thermal preference (Gordon et al., 1991; Weihe, 1965). Gwosdow and Besch (1985) have proposed that the temperatures to which rats are acclimated can alter their TNZ and set-point temperatures. Importantly, metabolic adaptation can occur within minutes of temperature change (Hart, 1963). Alterations in ambient temperature have greater impact on small mammals, such as rodents, because of their large surface area-body weight ratios. The confounding influence of environmental temperature on research has been recognized for over 50 years. However, most reports reflect the effects of temperature extremes; few describe more subtle changes expected to occur in modern animal holding facilities. In mice and rabbits, both the onset and severity of hypoglycemia and associated seizures were observed to differ with ambient temperature when the biological effects of insulin were assayed (Chen, 1943; Johlin, 1944). The influence of temperature on studies of drug-induced toxicity is well recognized. The toxicity of sympathomimetic amines such as amphetamine

NElL S. LIPMAN AND SCOTT E. PERKINS

1148

increases with a rise in ambient temperature, while the toxicity of others, e.g., ephedrine, decreases (Chance, 1957). Malberg and Seiden (1998) demonstrated that an environmental temperature change of only 2 ~C, from 20 ~ to 22 ~C, resulted in a drop in core body temperature in 3,4-methylenedioxymethamphetamine (MDMA)-treated rats. Not only is the temperature at which a study is conducted important, but the thermal history of the animals has also been demonstrated to play an important role. Mice exposed to cold (4 ~C) for 7 days prior to evaluation of the [3-agonist isoproterenol at 24~ demonstrated enhanced toxicity (up to 10,000 times) when compared to mice maintained at 24~ for the entire test period (Balazs et al., 1962). Weihe (1973) has suggested that differences in drug toxicity attributed to high cage-population density may actually have resulted from difficulty in thermoregulation. Reduction in temperature, as well as its elevation, is also of importance. Hypothermia resulting from housing rats in isolation has been attributed to a decrease in systemic clearance of antipyrine, a marker compound for hepatic oxidative metabolism and for the estimation of total body water (Brunner et al., 1994). Three drug toxicity curves have been described with respect to environmental temperature: (1) A V- or U-shaped curve with minimal toxicity observed at or near the TNZ, with increasing toxicity observed above or below thermal neutrality, characterizes many centrally acting drugs affecting the thermoregulatory system; (2) a linear relationship between toxicity and increasing temperature, as exemplified by sympathomimetic amines, which stimulate heat production; and (3) a skewed relationship in which no change in toxicity is obtained until the TNZ is exceeded, after which a linear relationship is observed (Fuhrman and Fuhrman, 1961). Effects of temperature are not limited to drug toxicity. Macroenvironmental temperature elevation in a rat production colony for 2 days at 31.6~ resulted in death of 33% of the animals. Twenty-five percent of the surviving males, 4 - 6 6 days of age at time of exposure, were sterile as a result of bilateral testicular atrophy (Pucak et al., 1977). Rabbits whose dams were exposed to elevated temperatures (33~ 14 days prepartum responded differently to cold exposure, endotoxin administration, and noradrenalin infusion, than those whose dams were maintained in a stable environment (Cooper et al., 1980). Reported effects on lactation include reduced latency to first milk ejection and reduction in the quantity of milk produced by directly impacting the activity of the mammary alveolae in rats (Jans and Woodside, 1987; Yagil et al., 1976). Exposure to elevated environmental temperatures for extended periods during rearing can influence morphological features, including tail, ear, paw, and salivary gland size in rodents (Caputa and Demicka, 1995; Clough, 1982). Reductions in growth rate, litter size, and neonate viability, which may extend into subsequent generations, have been reported in rodents exposed to low temperatures (Barnett, 1965, 1973).

2.

Relative Humidity (RH)

Both high and low RH have the potential to affect research. Elevation in RH directly impacts the ability of animals to thermoregulate, as evaporative heat loss is essential for core body temperature control in homeotherms. Rats consume approximately 5% more food when maintained at the same temperature but at 35 % as compared to 75 % RH (Weihe et al., 1961). These findings may be of relevance to studies in which test agents or chemicals are administered in the food or in which the amount of food consumed is critical. Mice are more active at low RH (Clough, 1982). Low humidity has also been shown to delay sexual maturity (Drickamer, 1990a). Environmental moisture impacts the viability and transmission of infectious microorganisms. Transmission of Sendai virus is enhanced by high RH (60-70% as compared to 40-45%), whereas transmission of influenza virus is reduced, as the virus survives best at 17-25% RH (Hemmes et al., 1960; Schulman and Kilbourne, 1962; van der Veen, et al., 1972). In general, the viability of microorganisms appears to be lowest when RH is 50% (Anderson and Cox, 1967). RH has a dramatic impact on the generation of intracage ammonia, especially in static rodent isolator cages (Corning and Lipman, 1991; Gamble and Clough, 1976). In these cages, microenvironmental RH may be as much as 38% higher than macroenvii'onmental RH. RH has implications for skin-absorption studies, particularly when animals devoid of fur are used. Variation in RH alters absorption of the topically applied substances by altering evaporative rates, the animals' peripheral circulation, and the material's viscosity (Clough, 1982). Variation would also be expected when test substances are administered as aerosols. Ringtail in rats, which in part is genetically determined and influenced by both caging and nutrition, is characterized by annular constrictions on the tail skin, which may lead to tail sloughing. The incidence of ringtail increases when the RH falls below 40% and the environmental temperature falls outside the animal's TNZ (Flynn, 1959; Njaa et al., 1957). The syndrome is observed most frequently in young animals. Ringtail has also been observed in mice and M y s t r o m y s albicaudatus (Nelson, 1960; Stuhlman and Wagner, 1971). 3. Air Exchange and Composition

Small laboratory animals breathe considerable quantities of air. It is estimated that a mouse breathes approximately 35 liters of air per day under normal conditions (Clough, 1982). The composition of the air is important not only to the animal, but also to the researcher because of the potential implications for research. The quality of air to which an animal is exposed is dependent on a multitude of factors, including but not limited to geographic location, particularly with respect to proximity to

29. FACTORS THAT

MAY INFLUENCEANIMAL RESEARCH

heavy industry and urban populations; the scope of its treatment by the HVAC (heating, ventilation, air conditioning) system; and the location of air intake(s), especially in consideration to exhaust locations of the building and to automobile traffic. Air may contain particulates and/or volatile substances that may be injurious to the respiratory system, skin, and mucous membranes, or may be absorbed and cause systemic effects. Microenvironmental pollutants, principally the metabolic waste gases ammonia and carbon dioxide, may be of major importance. In addition, cage components, feed, and bedding may potentially off-gas undesirable substances (Perkins and Lipman, 1995; Vessel et al., 1976; Wade et al., 1968). The quality of microenvironmental air is influenced by the caging system employed, the strain or stock of animal housed, bedding utilized, and housing density, as well as macroenvironmental conditions (Choi et al., 1994; Corning and Lipman, 1991; Hasenau et al., 1993; Perkins and Lipman, 1995). Although of concern for all laboratory-housed species, poor microenvironmental air quality is of particular concern for rodents maintained in isolator caging. Static isolator caging systems used to house rodents significantly impede air exchange, leading to accumulation of gaseous pollutants, notably ammonia (NH3) and carbon dioxide (CO2) (Corning and Lipman, 1991; Gamble and Clough, 1976; Keller et al., 1989; Murakami, 1971; Serrano, 1971; Simmons et al., 1968). Considerable intercage variability in microenvironmental conditions is frequently observed when housing animals of the same strain and biomass in identical caging (Corning and Lipman, 1991). Ammonia is formed by urease-producing bacteria or bedding containing heat-labile urealytic and urease-activating enzymes, which convert urea in urine and feces into ammonia. Microenvironmental NH 3 may reach 350 ppm within 7 days when housing the maximum number of mice prescribed in the "Guide" (Perkins and Lipman, 1996). This concentration exceeds, by as much as 14-fold, limits of 25 ppm established as an 8 hr timeweighted average (TWA) by the American Conference of Governmental Industrial Hygienists (ACGIH) for human exposure in the workplace (ACGIH, 1995). Physiologic alterations and interference with research may occur at NH 3 concentrations that can be observed in static isolator cages. Ammonia is a potent respiratory irritant that can induce morphologic changes, including reduction in the number of cilia of the respiratory epithelium, hyperplasia of epithelial cells, and formation of glandular crypts in respiratory and olfactory epithelium (Broderson et al., 1976; Gamble and Clough, 1976). In addition, exposure of M y c o p l a s m a p u l m o n i s - i n f e c t e d rats to NH3 concentrations observed in isolator cages (2-250 ppm) enhances M. p u l m o n i s isolation and severity of infection (Broderson et al., 1976; Schoeb et al., 1982). Pneumocystosis and a variety of immunosuppressive effects have also been attributed to elevations in NH3 (Gordon et al., 1980; Targowski et al., 1984; Walzer et al., 1989). It has also been suggested that NH 3 inhibits select com-

1149

ponents of the hepatic microsomal enzyme (HME) system and is a contributing factor in the development of corneal opacities in a variety of inbred and F1 hybrid mouse strains (Van Winkle and Balk, 1986; Vessel et al., 1976). Concentrations of CO2 may also be significantly elevated in static isolator cages (Corning and Lipman, 1991; Huerkamp and Lehner, 1994; Serrano, 1971). Concentrations up to 4000 ppm higher than those observed in the macroenvironment, when housing the maximum biomass permissible in the "Guide," have been reported (Perkins and Lipman, 1995). However, maximum reported concentrations do not exceed ACGIH exposure limits of 5000 ppm established as an 8 hr TWA (ACGIH, 1995). As a result, minimal concern has been raised with its elevation. However, CO2 is a respiratory and cardiovascular stimulant and has the potential to act as an asphyxiant by displacing oxygen. Physiologic alterations that could influence research investigations are clearly possible. The potential of other microenvironmental contaminants must also be considered. Bolon et al. (1991) questioned the presence of an uncharacterized pollutant in isolator cages while evaluating the inhalational effects of methyl bromide on F344 rats. Although elevated ammonia concentrations were contributory to the nasal lesions induced by methyl bromide, the olfactory sensory-cell loss observed could not be attributed to either agent. In another study, acetic acid (0.86 ppm) was determined to have been off-gassed from corncob bedding in isolator cages, although no adverse effects are known to occur at the concentrations observed (Perkins and Lipman, 1995). Volatile chemicals used for research purposes, such as gas anesthetics, and agents used for sanitization and environmental control (e.g., disinfectants and pesticides), have the potential to influence the air the animals breathe. The effects of aromatic hydrocarbons released from cedar and pine bedding on induction of hepatic microsomal enzymes have been well documented (Vessel et al., 1976; Wade et al., 1968). Similarly, pesticides used intentionally or introduced inadvertently and a variety of room-deodorizer constituents may similarly alter hepatic microsomal enzyme function (Cinti et al., 1976; Conney and Burns, 1972; Hodgson et al., 1980; Jori et al., 1969; Robacker et al., 1981). Modern pesticides frequently contain organophosphates or carbamates that may alter mammalian cholinergic transmission, even at low levels of exposure. Pesticides, frequently applied as a spray in an oil base or an aqueous carrier, are likely to result in concentrations in ambient air that increase the risk of direct animal contact (Pakes et al., 1984). Pesticides are known to induce immune cell dysfunction, act as tumor promoters, be toxicants, and act as antiandrogens (Kelce and Earl Grey, 1999; Rought et al., t999). Odorants, such as citrus fragrance used in cleaning products, have been observed to restore stress-induced immunosuppression, decrease locomotor activity during open field testing, and reduce total immobility time in a forced swimming test (Komori et al., 1995a,b).

1150

Because of the high volatility of odorants or other volatile chemicals, sufficient amounts of their vapor at concentrations sufficient to affect the animals may drift into animal holding rooms from corridors or storage sites (Lindsey et al., 1978). Clough (1982) has raised concerns regarding the possible effects that electrically charged airborne molecules generated by the treatment of air by the HVAC system may have on research. Air-conditioned buildings with metal duct distribution systems have a negative ion concentration that is typically 5% of the outdoor value. Rats exposed to a positively charged atmosphere do not do as well in select behavioral tests (Clough, 1982).

4.

Noise

The impact of intense noise of frequencies detectable by humans on both the physiology and behavior of laboratory animals has been recognized for many decades (Peterson, 1980). However, the effects of noise of lower intensity and ultrasound have received considerably less attention. Noise can induce auditory effects, principally destruction of auditory structures and degradation of hearing. Of considerable importance are the nonauditory effects attributed to exposure to sounds of particular intensities and frequencies. The hearing range of species used in the laboratory overlaps only partially with that of humans. Rodents, cats, dogs, and small primates can detect ultrasounds (> 20 kHz) that are outside a human's hearing range. While the human ear is most sensitive to sounds at 2 kHz, many rodents have peak auditory sensitivities in the range of 30 to 60 kHz (Bell, 1974; Heffner and Heffner, 1980). Ultrasounds are used for communication in rodents, small primates, and possibly cats (Bell et al., 1972; Brown, 1976; Milligan et al., 1993; Sales and Pye, 1974). Ultrasound communication is important to a number of intraspecies interactions in rodents, including those be' tween dam and pup, in establishing dominance hierarchies, and during mating (Hofer and Shair, 1978; Sales and Pye, 1974; Sales and Smith, 1980; White and Barfield, 1987). Low-frequency sounds (< 20 kHz) have been demonstrated to alter water consumption; blood pressure; blood corticosteroid, glucose, and insulin concentrations; reproductive performance; body weight; eosinophil counts; immune responsiveness; tumor resistance; histology of the pituitary gland; and learning ability, as well as to induce adrenal and cardiac hypertrophy and hypertension in rodents (Anthony, 1962; Anthony and Harclerode, 1959; Armario et al., 1985; Buckley and Smookler, 1970; Fay, 1988; Fink and Iturrian, 1970; Geber, 1970; Jensen and Rasmussen, 1970; Lockett, 1970; Morseth et al., 1985; Wolstenholme and O'Connor, 1967; Zondek and Tamari, 1967). Noise can induce effects that remain long after it is removed. Barlow (1972) observed that pups born to mice stressed by sound during pregnancy had reduced learning ability. Ultrasound has been observed to reduce fertility and productivity, cause diuresis and increased urinary sodium secretion, in-

NEIL S. LIPMANAND SCOTT E. PERKINS duce audiogenic seizures, reduce locomotor activity, and destroy auditory structures in rodents (Fink and Iturrian, 1970; Lockett, 1970; Peterson, 1968; Pye, 1973; Sales, 1991; Zondek and Tamari, 1967). Structural damage to the auditory system can occur even though subjective (conditioned) responses to sound stimuli remain normal (Catlin, 1986). The direct application of ultrasound to the body wall of the rat, at power similar to that used for human echography, resulted in a significant decrease of fetuses detectable at embryonic day 15 (Bologne et al., 1983). Ultrasound has been shown to alter behavior in cattle, horses, poultry, sheep, and swine (Algers, 1984). Sound has also been demonstrated to entrain circadian rhythms in several species (Menaker and Eskin, 1966; Mrosovsky, 1988; Richter, 1968). Ultrasonic irradiation (> 1 MHz) has been shown to interfere with prenatal development in both the mouse and hamster (Shoji et al., 1975; Weinland, 1963). As this frequency is above the hearing range of any species, the mechanism is presumed to be nonauditory. The ability of sound to induce audiogenic seizures, a model for human epilepsy, has been well studied. Audiogenic seizure activity has been studied principally in mice and rats, although a variety of other species are susceptible (Pierson and Liebmann, 1992; Clough, 1982). Genetically susceptible strains of mice include the AKR, BALB/c, CBA, C57, and DBA/2, although weanlings of any murine strain can be made susceptible if they are audio-conditioned between 15 and 21 days of age, when they are most sensitive (Clough, 1982). Some mouse strains remain susceptible to seizure activity for several months after conditioning (Clough, 1982). Both Sprague-Dawley and Wistar rats can be made epileptogenic by noise exposure as neonates, although the severity of seizures and the age dependence of maximum severity differ among stocks and epilepsyprone substrains (Pierson and Liebmann, 1992). Anthony (1962) recommended that noise levels in animal facilities not exceed 85 dB, a level equivalent to the current 8 hr time-weighted average established for human occupational exposure to noise (ACGIH, 1995). The noise generated by the daily activity of animal facility personnel, by the use of equipment routinely employed to meet animal husbandry and research needs, as well as by particular species such as rabbits and dogs, may be of sufficient intensity to result in behavioral and physiologic alterations. Milligan et al. (1993) examined soundpressure levels in animal holding rooms at five institutions housing a variety of species at both low (0.01-12.5 kHz) and high (12.5-70 kHz) frequencies for periods up to 24 hr. The authors concluded that high sound levels were most frequently generated by human activity at frequencies within the audible range of animals, but often outside the human audible range. Another study undertaken to determine the presence and source of ultrasound in laboratories and animal holding facilities demonstrated that 24 of 39 sources monitored, including cage washers, oscilloscopes, and video display terminals, emitted ultrasonic sound, as did running water taps and rotating glass

29. FACTORSTHATMAYINFLUENCEANIMALRESEARCH

stoppers (Sales et al., 1988). Of particular concern was the finding that some equipment produced only ultrasound that was inaudible to humans. Concerns regarding the activation of fire alarm annunciators and the effect on animals of the noise generated by them led to the development of an annunciator that produces noise below the auditory frequency threshold of mice and rats (Clough and Fasham, 1975). The increased use of ventilated caging systems and laminar flow and biological safety cabinets within animal holding rooms heightens concerns regarding noise generation in both the macroenvironment, where it may impact personnel, and the microenvironment, where it may affect animals (Perkins and Lipman, 1996). Construction noise ranging between 70 and 100 dB at 50 to 2000 Hz has been demonstrated to alter changes in 3,-aminobutyric acid (GABA) release and uptake in rat amygdaloid and hippocampal slices (Fernandes and File, 1993).

5.

Light

The impact of light, including its periodicity, intensity, and wavelength, on the reproduction, behavior, and physiology of mammalian species is well documented. Light is an important synchronizer of circadian ~rhythm. In the absence of light, the cycle length of diurnal rhythms deviates more than an hour from the usual 24 hr period (Weihe, 1976). The time during which the nadir or peak of a specific parameter occurs shifts with an alteration of the light-dark (L: D) cycle. The synchronizing effect of light on the 24 hr clock and the amplitude of the cycle are closely associated with its intensity. The direction of amplitude shift frequently depends on whether the species is nocturnal or diurnal. Aschoff (1960) demonstrated that the nocturnal hamster's wheel-running activity decreased as light intensity increased. Intensity of lighting also affects reproductive physiology. The incidence of anestrus increases significantly when hamsters are exposed to light at an intensity less than 15 lux, and fecundity rates are highest when rats are housed at 250 lux as compared to 6 other ambient light intensities (Weihe et al., 1969). Additionally, light intensity has been reported to affect the mean vaginal opening time, ovarian and uterine weights, estrous cycle length, preweaning mortality, and defecation rates in rodents (Donnelly and Saibaba, 1993; Hautzinger and Piacsek, 1973; Porter et al., 1963; Weihe et al., 1969; Williams, 1971). Phototoxic retinopathy occurs in humans and a variety of animal species. It is most commonly reported in rodents, especially albino rat stocks and strains. Light intensity well below that which causes thermal epidermal burns leads to retinal damage. In addition to light intensity and albinism, other variables, including photoperiod duration, body temperature, nocturnality, the light level under which the animals were raised, age, hormone status, and time of day during light exposure, all affect the extent of photoreceptor damage (Duncan and O'Steen, 1985;

1151

Lanum, 1978; Semple-Rowland and Dawson, 1987a,b; Weihe, 1976). Continuous illumination as low as 110 lux for 7 to 10 days can damage photoreceptor cells in rats (Noell and Albrecht, 1971). Numerous studies indicate that retinal degeneration may occur at illumination levels observed in animal holding rooms (Bellhorn, 1980; ILAR, 1978; LaVail, 1976; O'Steen and Anderson, 1972; O'Steen et aL, 1973; Shear et al., 1973). Current recommendations for illumination at the cage level are light levels between 130 and 325 lux to prevent photoreceptor degeneration in sensitive species, unless animals were raised at extremely low (6 lux) light levels, which may require lower than the recommended light levels (National Research Council, 1996; Semple-Rowland and Dawson, 1987a,b). The location of cages on a rack is important, as light intensity decreases with the square of the distance from its source. The intracage light intensity may differ by as much as 80-fold in transparent plastic cages from the top to the bottom of a rack, while differences up to 20-fold have been observed within a single cage (Weihe et al., 1969). Greenman et al. (1982) concluded that differences in retinal morphology observed in BALB/c mice used in a chronic toxicity study were due to cage position. The caging system utilized, as well as the shelf, rack, and room positions in which the cages are located, should be rotated to reduce complications induced by light (Weihe et al., 1969). Photoperiodicity is an important stimulant and regulator of reproduction. Complex neuroendocrine pathways initiated at the retinal photoreceptors result in the release of hypothalamic hormones, including the gonadotropins (Brainard et al., 1997). Many, but not all, commonly used laboratory species remain highly sensitive to changes in photoperiod. The decline in the reproductive performance of hamsters when the light phase is shortened, accompanied by a regression of the size and activity of their gonads, is well established (Nelson and Zucker, 1987). The marked increases in body weight associated with increased testicular size, elevations in plasma testosterone, and spermatogenesis in the male squirrel monkey are also photoperioddependent (Baldwin, 1968; DuMond and Hutchinson, 1967; Mendoza et al., 1978). When species are maintained in the laboratory under constant photoperiod, many of these seasonal reproductive changes disappear; however, seasonal fluctuations in fecundity, sex ratios of litters, body weight at weaning, and age of sexual maturation are observed in rodents (Drickamer, 1977, 1984, 1990b; Lee and McClintock, 1986). The optimal photoperiod is unknown for most species. A 12L: 12D cycle is used for most species; however, longer photoperiods (14L) are used by some laboratories for rodent breeding (Mulder, 1971). Estrous cycle length has been shown to increase in SpragueDawley rats from a 4- to - 5-day cycle when photoperiod is increased from 12L to 16L (Hoffman, 1973). Photoperiod has also been observed to affect body weight gain and feed intake in livestock (Tucker et al., 1984). Alternating light-dark cycles weekly has been used to induce mild chronic stress in Brown

1152

Norway rats, resulting in a decrease in the cellular immune response while the spontaneous tumor incidence remain unchanged (Kort and Weijma, 1982; Kort et al., 1986). Contamination with as little as 0.2 lux light exposure during the dark phase of the light cycle can reset the circadian rhythm in rodents (Brainard et al., 1983; Minneman et al., 1974). Dauchy et al. (1997) determined that light contamination (0.2 lux) during the dark phase of the light cycle altered growth, lipid uptake, and metabolism of the transplantable Morris hepatoma in rats by inhibiting melatonin secretion. The function of time-controlled lighting systems should be carefully monitored. Although continuous dark cycles are highly unlikely to go undetected, the provision of continuous illumination for varying periods may not be noticed unless monitored. Continuous lighting has an overstimulating effect on reproduction, leading to cessation of cycling, permanent vaginal cornification, and development of excess ovarian follicles in rodents (Hoffmann, 1973; Weihe, 1976). Constant light also enhances the growth and metabolism of tumors (Dauchy et al., 1997). Although few laboratory species, except cats and many nonhuman primate species, have color vision, the wavelength and hence, color of light, have been shown to alter both behavioral and physiologic parameters in many species. Light wavelength, including the color generated by fluorescent lights, has been shown to alter voluntary wheel-running activity, the time of vaginal opening, reproductive organ weights, body weight, submandibular gland development, sexual maturity, and the development of dental caries in various rodent species (Hautzinger and Piacsek, 1973; Saltarelli and Coppola, 1979; Sharon et al., 1971; Spalding et al., 1969a,b; Wurtman, 1975). 6.

Radiation

Electromagnetic and ionizing are the two principal forms of radiation. Electromagnetic radiation consists of oscillating electric and magnetic fields composed of different wavelengths or frequencies. Ultraviolet, visible, and infrared light, and microwaves and radio waves are forms of electromagnetic radiation (ER). Electromagnetic radiation can alter biological responses and thus possibly influence the actions of drugs and the outcomes of disease (Izmerov, 1985; Wilkening and Sutton, 1990). An increased level of ultraviolet B (UVB) radiation in the environment due to ozone depletion has increased the risks of ocular damage, immunosuppression, immune modulation, and cancer (Goettsch et al., 1994; Longstreth et al., 1998; Selgrade et al., 1997). In animal studies, UVB-related immunosuppression has had a negative effect on the outcome of some infectious diseases and cancers (Goettsch et al., 1994; Longstreth et al., 1998). Infrared light diminishes diabetes induced by alloxan in rats. Hapke (1983) proposed that infrared radiation alters blood glucose regulation and is antidiabetogenic. Exposure of laboratory animals to electromagnetic fields has

NEIL S. LIPMAN AND SCOTT E. PERKINS

demonstrated a variety of biOlogical effects, including lymphomas, pulmonary sarcomas, hepatomas, and mammary and skin tumors. However, other animal studies have found no carcinogenic effects (Harris et al., 1998; Repacholi, 1997; Repacholi et al., 1997). Although further studies are required, human exposure to high field strength and low-level, extremely lowfrequency electric and magnetic fields, and chronic exposure to static magnetic fields have been associated with biological effects such as pediatric leukemias and other cancers, adverse pregnancy outcomes, and changes in eye structure and function (Repacholi, 1998; Repacholi and Greenebaum, 1999). Exposure to sinusoidal, bipolar oscillating magnetic fields has been reported to cause malformations in chicken embryos (Bryan and Gildersleeve, 1988). Ionizing radiation is the result of rays and particles producing enough energy to release free electrons from atoms, leaving the atom electrically charged or ionized. Gamma rays, X rays, and atomic particles (a and [3) are types of ionizing radiation. They can cause harmful biological effects, such as damage of DNA resulting in genetic, teratogenic, and somatic effects, including death; and increases in the incidence of neoplasms (Burkart et al., 1999; Report to the Congress, 1981; U.S. Regulatory Commission, 1981, 1987). 7.

Caging and Housing-Related Issues

Modern caging is generally manufactured from stainless steel or synthetic polymers. These materials differ dramatically in their thermal conductivity. Metabolic rate, evaporative water loss, and colon temperatures are altered when rats are housed in cages with floors made from metal or plastic for periods as short as 60 min, as were the animals' responses to MDMA, a psychoactive drug (Gordon and Fogelson, 1994). Cage design is also recognized to influence animal health. Pododermatitis can occur in rats housed for extended periods on wire caging (Anver and Cohen, 1979). Wire caging has also been associated with an increased incidence and severity of urologic syndrome in AKR mice (Everitt et al., 1988). The provision of a complex cage environment for rodents, in lieu of housing in standard shoe-box cages, enhances cell proliferation and improves response in behavioral tests following implantation of intracerebral grafts and lesions of the hippocampus and cortex (Galani et al., 1997; Kelche et al., 1988; Kempermann et al., 1998). However, cage enrichment, especially when providing nest boxes or modifications that allow spatial and visual separation, may result in the loss of stable dominance hierarchies, leading to increased aggression and neuroendocrine alterations dependent on the individual animal's social position (Haemisch and Gartner, 1994, 1997; Haemsich et al., 1994). Cage enrichment has been shown to decrease immune function, reducing resistance following challenge with Babesia microti in CFLP mice (Barnard et al., 1996). The influence of cage size has also been reported. The febrile

29. FACTORS THAT MAY INFLUENCEANIMAL RESEARCH

response to lipopolysaccharide (LPS) is less when hamsters are housed in cages providing 200 cm 2 of floor space as compared to cages providing 1815 cm 2. The author theorized that the diminished response was a result of the stress from housing in a small cage, as glucocorticoids and other stress hormones alter the response to LPS (Kuhnen, 1997, 1998). In examining play activity in young rats, Siegel and Jensen (1986) determined that animals housed in smaller cages exhibited greater social play as defined by pinning behavior. When housed in groups of 3 in shoe-box caging providing 32.2, 64.5, 96.8, or 129 cm 2 per mouse, C57BL/6 mice provided the least floor area consumed or wasted more water and responded more vigorously to a T-cell mitogen than mice provided greater space (Fullwood et al., 1998). However, an increase in aggressive behavior as well as adrenal gland weight and plasma glucocorticoid concentration was observed as progressively more space was provided. Provision of greater floor space to male mice alters the animals' dominance rank (Poole and Morgan, 1976). Cage size was not observed to significantly influence the behavior or physiology of dogs and rhesus monkeys (Hite et al., 1977; Line et aL, 1989). The use of ventilated caging for rodent housing may potentially alter research findings. Although intracage ventilation improves microenvironmental conditions, excessive intracage ventilation, especially when air is supplied at the level of the cage, may lead to chilling and dehydration, with neonates and hairless mutants being particularly sensitive. Intracage air velocities as high as 100 linear feet per minute (lfpm) have been measured in a commonly used caging system (Tu et al., 1997). As 20~ air moving at 60 lfpm has a cooling effect of 7~ exposure of animals to a ventilated cage may alter behavioral and physiologic responses (Weihe, 1971). Ventilated caging systems may cause pheromone dilution and alter breeding. A negative synergistic effect has been reported between ventilated cages and the use of automatic watering systems, resulting in increased mouse pup mortality (Huerkamp et al., 1994). The generation of noise and vibration by these systems is an additional consideration that may influence experimental findings (Lipman, 1999). 8.

Miscellaneous

The effect of vibration on animals has been subject to minimal evaluation. However, occupational exposure of humans to vibration has been associated with a variety of physiologic and biochemical alterations (Tzvetkov et al., 1992). Changes in lipid metabolism, electrolyte and trace element concentration, acid-base balance, and reproductive function are altered in animals exposed to vibration (Shenaeva, 1990; Tzvetkov, 1993). With the increasing use of ventilated caging systems worldwide, many of which contain blower systems mounted directly on the rack, the possibility of continuous long-term exposure to low levels of vibration should be considered. Sanz et al. (1988)

1153

attributed anomalies detected while evaluating the molecular mechanism of toxic substances to meteorological (storms), geological (earthquakes), and astronomical (lunar phase) events.

B. 1.

Chemical Factors

Xenobiotics (Other than Pharmaceuticals)

Xenobiotics are any chemicals or compounds that are foreign to a biological system. Exposure to xenobiotics may occur via the air, water, diet, bedding, caging, and/or equipment, or may be intentionally introduced pharmacologic agents as part of the routine conditioning or experimental procedure. The effect or toxicity of a xenobiotic is based on its dose and disposition. Absorption, distribution, biotransformation, and excretion all affect the disposition of a xenobiotic (Sipes and Gandolfi, 1991). In addition, host barriers, i.e., the skin, lungs, and alimentary tract, and the physical and chemical composition of a xenobiotic also affect its toxicity. A xenobiotic or its metabolites may cause physiologic alterations in the animal and thus affect the outcome of the experiment by altering immune function, and by acting as a mutagen and/or a teratogen (Dean and Murray, 1991; Sipes and Gandolfi, 1991). Examples include aflatoxins; heavy metals such as lead, mercury, and cadmium; organochlorine insecticides; and commonly administered anesthetic agents (Degraeve, 1981; Elis and DiPaolo, 1967; Gerber et al., 1980; Ito and Ingalls, 1981). a.

Biotransformation

Compounds are generally altered chemically by enzymatic activity before being excreted. This process yields metabolites that are more hydrophilic and thus more readily eliminated. Xenobiotic biotransformation is affected by the physiochemical properties of the compound as well as by its protein-binding ability, the dose, and route of administration, Biotransformation is also affected by a variety of host factors including species, strain, age, sex, time of exposure, state of its biotransforming enzymes, nutritional and disease status, and environmental factors (Sipes and Gandolfi, 1991). For example, cadmium accumulation from diet, water, and ambient air is greater in the tissues of hypertension-sensitive Dahl rats than of hypertensionresistant rat strains (Ohanian and Iwai, 1980). b.

Diet

The diet is usually the principal source of xenobiotic compound exposure (Torronen et al., 1994). Ideally, laboratory animal diets should not contain compounds that can alter experimental response. However, many animal diets contain natural and synthetic chemical compounds that may have significant effects on physiologic processes and thus alter the experimental

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NElL S. LIPMAN AND SCOTT E. PERKINS

outcome, especially in pharmacologic and toxicologic studies had decreased blood levels of cholesterol and phospholipid. The (Schecter et al., 1996; Torronen et al., 1994). Diets may contain differences were attributed to the level and composition of the inducers, suppressors, activators, inhibitors, and substrates that fiber fraction of the cereal-based diet (Rutten and de Groot, influence P450 levels (Torronen et al., 1994; Yang et al., 1992). 1992). Additionally, transgenic mice expressing a human breast Feed contaminants include chlorinated hydrocarbons, organo- cancer oncogene, c-neu, fed a diet containing fiber from nonphosphates, polychlorinated biphenyls, heavy metals, aflatox- purified cereal ingredients, had delayed development of mamins, nitrates, nitrosamines, and estrogenic compounds (Edwards mary cancer (Rao et al., 1997). The amount of dietary fat may et al., 1979; Newberne and McConnell, 1980; Silverman and influence the biotransformation and response of mice to barbiAdams, 1983). Many contaminants are found naturally in plant turates. The differences in response result from hepatic fat acmaterials or are agricultural residues. Diets may also be con- cumulation and a redistribution of the lipophilic barbiturate into taminated during storage or formulation. Examples include adipose tissue (Hapke, 1983). In chimpanzees, certain diets inaflatoxin contamination of corn, wheat, and other cereals during crease the urinary excretion of isoflavonoids, phytoestrogens, storage; the presence of phytoestrogens in dietary constituents; daidzein, and enterolactone, which may be partially responsible and contamination of diets with estrogenic compounds during for resistance to the development of estrogen-induced cancers formulation (Hadlow et al., 1955; Wogan, 1968; Wright and (Musey et al., 1995). Additional human and animal studies sugSeibold, 1958). Thigpen et al. (1987a) developed a standardized gest that dietary phytoestrogens help prevent cancer, heart dismouse bioassay for detecting estrogenic activity in rodent diets. ease, and osteoporosis through a variety of mechanisms (Kurzer Female mice, weaned at 15 days, receiving diet containing di- and Xu, 1997). Variations in the quantity or availability of essential vitamins ethylstilbestrol (DES) or negative control diet were sacrificed at 22 days, and uterine weights and uterine:body weight (U:BW) and minerals may alter drug-metabolizing systems, affect memratios were evaluated. Mice receiving the DES diet had sig- brane integrity, or predispose to the effects of carcinogens nificant increases in both uterine weights and U:BW ratios, (Newberne and McConnell, 1980). Deficiencies in calcium, thus providing a standardized bioassay for evaluating rodent di- copper, iron, magnesium, and zinc have been shown to decrease ets. Using this bioassay, Thigpen et al. (1987b) evaluated com- cytochrome P450 enzyme levels and redox reactions (Sipes and mercially available rodent diets and determined two diets to Gandolfi, 1991). Hypervitaminosis A has been associated with have estrogenic activity. However, additional studies concluded teratogenic effects in rabbits, including fetal resorptions, aborthat fats and carbohydrates in the food caused increases in uter- tions, and stillbirths (DiGiacomo et al., 1992). Miller et al. ine weights and in U :BW ratios, thus partially accounting for (1997) demonstrated that the amount of dietary iron intake in the estrogen-like uterine growth-promoting activity (Thigpen common marmosets can affect liver iron content and health. Diet al., 1987c). Variations in the constituents and formulation ets high in iron, 350-500 ppm, can lead to hepatic hemosideroof the diet result in a wide variety, type, and concentration of sis with subsequent effects, including death. Excessive caloric intake may have a serious impact, as caloric chemical contaminants as well as nutrient quality (Newberne and McConnell, 1980; Wise, 1982). Variations in dietary con- restriction has been documented to be beneficial to the host for stituents may alter the toxicity of chemical contaminants and resisting effects of aging, degeneration and infectious diseases, potentially affect the animal's response to specific drugs or neoplasia, and the toxicity of chemical agents (Anderson et al., 1985; Masoro, 1992; Newberne and McConnell, 1980; Pickerchemicals (Sipes and Gandolfi, 1991). The protein and fat content may have profound effects on ing and Pickering, 1984; Ross and Bras, 1973). Restriction of physiologic processes and on the toxicity of certain xenobiotics. dietary energy maintains most physiologic systems in a youthOlovson (1986) demonstrated dramatically increased breeding ful state, retarding a broad spectrum of disease processes, and efficiency, decreased mortality, and increased body-weight gains delays death due to neoplasms (Masaro, 1992). Further, in aniin both male and female cats when dietary fat content was in- mals and humans, dietary restriction has a marked effect on the creased from 15 to 27% in a conventional cat-breeding colony. endocrine system and the cell-cycling rate in various organs In inbred rats, both diet and strain strongly influence the num- (Williams and Weisburger, 1991). Laboratory diets may be contaminated by carcinogenic niber, size, and hemoglobin content of red blood cells (Hackbarth et al., 1983). Rats maintained on a low-protein diet have de- trosamines (Edwards et al., 1979; Walker et al., 1979) and nicreased lethality and hepatotoxicity produced by dimethylni- trates (Newberne and McConnell, 1980), which can be controsamine, while rats fed diets with a high polyunsaturated fat verted to nitrosamines in the gastrointestinal tract. In rodents, content have decreased concentrations of cytochrome P450 exposure to heavy metals, such as lead and cadmium, has been isoenzymes (Sipes and Gandolfi, 1991). In rats, aflatoxin B1- shown to suppress disease resistance (Cook et al., 1975; induced hepatic neoplasms were more significant when corn oil Hemphill et al., 1971); suppress the effects of endotoxin (Cook was the source of dietary fat compared to beef fat as the source et al., 1974); cause immunosuppression (Blakley et al., 1980; (Newberne et al., 1979). In comparing a cereal-based and a Koller, 1979; Loose et al., 1978); and influence reproductive purified diet, rats, mice, and hamsters fed the cereal-based diet performance (Degraeve, 1981; Gerber et al., 1980), and may be

29.

FACTORSTHATMAY INFLUENCEANIMAL RESEARCH

carcinogenic (Goyer, 1991). The type and quantity of feed provided to rats may cause differences in central nervous system responsiveness and function (Kacew et al., 1998). In cats, increased dietary cysteine promotes higher methionine, homocysteine, glutathione, and oxidized glutathione concentrations in blood (Fettman et al., 1999). Aged female beagle dogs fed a diet with n-6 to n-3 fatty acid ratio of 1:4 had increased total lymphocyte and CD4 § T-cell counts and a decrease in CD4 § to CD8 + ratio after vaccination with a keyhole limpet hemocyanin suspension (Hall et al., 1999). Finally, food deprivation can have a dramatic effect on the outcome of toxicology studies, as deprivation can induce hepatic microsomal enzymes and reduce the concentration of cofactors and conjugating agents (Sipes and Gandolfi, 1991). Good Laboratory Practice (GLP) Regulations published by the Food and Drug Administration require compounds be excluded that could act synergistically or antagonistically with test compounds, or that could be expected to produce a result similar to that of the test compound (McSheehy, 1983). c.

Water

Animal drinking water is generally supplied from a local potable water source that meets standards applied to human consumption. Depending on geographic location; area geology; the use of surface or well water; proximity to industrial, agricultural, or urban centers; and the type of water treatment used, the water consumed is subject to considerable variation. Drinking water may be contaminated by pesticides, heavy metals, radionuclides, and other compounds that may produce biological effects (Cantor, 1997; Surbeck, 1995). In Europe, a variety of pharmaceuticals, including antibiotics, analgesics, antiseptics, beta-blockers, and cholesterol-lowering drugs, were detected in the drinking-water supply (Garcia, 1998). There is epidemiologic evidence of an association between drinking-water contaminants, including pesticides, arsenic, volatile organics, asbestiform fibers, and radionuclides, and the formation of one or more types of cancer in humans (Cantor, 1997). Nitrogen fertilizers and pesticides have been used worldwide since the 1960s, and pesticide contamination of drinkingwater supplies has increased over the past decade (Morales et al., 1993; Shapiro, 1980; Taets et al., 1998). In Spain, contamination of drinking water by nitrates has been associated with a significant increase in bladder cancer in humans (Morales et al., 1993). Raszyk et al. (1995) demonstrated increased mutagenicity, via the Ames test, in pesticide and polychlorinated biphenyl (PCB)-contaminated drinking water collected on swine farms, establishing that mutagens present in drinking water may jeopardize animal health. Kligerman et al. (1993) demonstrated cytogenetic damage in splenocytes of male F344 rats and female B6C3F1 mice following exposure to simulated California groundwater contaminated with a mixture of pesticides and the fertilizer ammonia nitrate. Herbicides have also

1155

been shown to cause chromosomal damage to Chinese hamster ovary (CHO) cells in vitro (Taets et al., 1998). Heavy metals, such as lead, copper, cadmium, nickel, and silver, may contaminate drinking water; and lead contamination in drinking water has been associated with adverse pregnancy outcomes in humans including cardiovascular defects and stillbirths (Aschengrau et al., 1993). Ronis et al. (1998a,b) evaluated chronic lead exposure in pregnant SD rats. Pregnant rats exposed at embryonic day 5 to lead acetate, 0.05-0.45% (w/v), in drinking water had a dose-responsive decrease in neonatal birth weights and crown-to-rump lengths and a subsequent delay in sexual maturity. The disruptions in reproductive physiology were accompanied by a significant decrease in neonatal sex steroids and suppression of sex hormones during puberty. These results suggest the reproductive axis of the rat is sensitive to lead during development. Zheng et al. (1996) exposed weanling male SD rats to lead acetate in drinking water, 0, 50, or 250 ktg/ml, and demonstrated a dose-responsive decrease in production of choroid plexus transthyretin, a major cerebrospinal fluid (CSF) protein manufactured by the choroid plexus that is responsible for the transport of thyroid hormones to the developing brain. Water treatment can be used to minimize microbial contamination; however, many forms of treatment result in physiologic alterations that may affect experimental data (Fidler, 1977; Hall et al., 1980; Hermann et al., 1982; Homberger et al., 1993). Chlorine may be introduced into the water supply as a component of water treatment. Chlorine may cause alterations in the immune response (Exon et al., 1987; Hermann et al., 1982), and chlorination by-products may be carcinogenic and mutagenic (Koivusalo and Vartiainen, 1997; Morris et al., 1992; Pilotto, 1995). Trihalomethanes are formed as a result of interactions between chlorine or bromine with methane groups from natural organic materials. Chloroform, a trihalomethane, is found in relatively high concentrations and has demonstrated biological impact, including cytotoxicity, increased DNA synthesis, and carcinogenesis (Lee et al., 1998; Lipsky et al., 1993; Vessel et al., 1976). For GLP studies, the quality of the water must be analyzed and documented to ensure that the water does not contain chemical pollutants (McSheehy, 1983). The processing and delivery system of water in a lab animal facility may affect water constituents. Hall et al. (1980) evaluated the effects of acidified drinking water on select biological phenomena of normal and immunosuppressed male mice. Depending on the pH of the water and the acid (hydrochloric vs. sulfuric) used for acidification, there may be a decrease in weight gain, water consumption, and number of bacteria species isolated from the terminal ileum, with more pronounced changes noted in immunosuppressed mice. The authors concluded that the acidification of drinking water was not innocuous and it should be evaluated as an environmental variable. Lohmiller and Lipman (1998) documented increases in silicon concentration and formation of silicon crystals from autoclaving glass water bottles. The increase in silicon and variations

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NEIL S. LIPMAN AND SCOTT E. PERKINS

from bottle to bottle in silicon concentration could cause alterations in experimental variables. Renal lesions have been induced in guinea pigs following the experimental administration of silica-containing compounds (Dobbie and Smith, 1982). Kennedy and Beal (1991) evaluated rubber water bottle stoppers for mineral content and mineral-leaching ability into both deionized and acidified-deionized drinking water. Minerals were present in all three types of stoppers evaluated. Acidifieddeionized drinking water typically leaches more minerals from the stoppers. The authors concluded that certain types of stoppers may be more suitable for particular nutritional and toxicologic studies. 2.

Pharmaceuticals

Pharmaceutical agents are administered to laboratory animals for a host of reasons. For example, pharmaceuticals are administered to induce and maintain anesthesia, to relieve pain, to prevent or treat microbial disease, or to activate an inducible promoter that turns on or off specific genes. In general, their administration is necessary but may be ancillary to the primary experimental goal. Pharmaceuticals may result in physiologic changes, distinct from those expected from their principal mechanism of action, or they may alter the metabolism of other chemicals and therefore alter experimental results. Importantly, effects induced by pharmaceutical agents may frequently be dose- and species-dependent. a.

Anesthetics, Tranquilizers, and Analgesics

The induction and maintenance of general anesthesia lead to significant physiologic alterations, principally of the cardiovascular, pulmonary, and nervous systems. Anesthetics, tranquilizers, and analgesic combinations that minimize physiologic disturbance of the system under study are usually selected. This choice is most critical when experimentation is conducted while the animal is under anesthesia, as most significant cardiopulmonary alterations return to normal following recovery. However, some anesthetics may induce physiologic and behavioral changes distinct from their cardiopulmonary effects, which may persist after the animal has awakened from anesthesia. Anesthetics may be directly toxic. Commonly used anesthetics such as avertin, xylazine, and the combination ketamine and xylazine have been shown to induce tissue injury. When administered at clinically relevant doses, and depending on the species and route of administration, examples of tissue injury include pulmonary parenchymal damage, muscle necrosis, peritonitis, corneal calcium deposition, and keratoconjunctivitis sicca (Celly et al., 1999; Gaertner et al., 1987; Guillet et al., 1988; Kufoy et al., 1989; Smiler et al., 1990; Zeller et al., 1998). Lesions associated with the use of avertin in mice are speculated to have resulted from improper anesthetic prepara-

tion or the concentration administered rather than from any inherent toxicity of the anesthetic (Papaioannou and Fox, 1993; Weiss and Zimmerman, 1999). Methoxyflurane is nephrotoxic in F344 rats, causing a dose-related diabetes insipidus syndrome (Clifford, 1984; Mazze et al., 1973). Reactive intermediates formed during reductive metabolism of halothane and isoflurane under hypoxic conditions lead to hepatic toxicity (Eger et al., 1987; Harper et al., 1982). Anesthetics may enhance or inhibit the toxicity of other agents. Barbiturates and xylazine induce hepatic cytochrome P450-metabolizing enzymes that may influence the metabolism of other chemicals (Nossaman et al., 1990). Enflurane, halothane, and methoxyflurane have been shown to inhibit cytochrome P450-dependent type I substrates (Rice and Fish, 1987). The authors speculate that methoxyflurane, because of its high lipid solubility, would have long-lasting effects. The effects of anesthetics on the immune system are well recognized. Halothane reduces the responsiveness of lymphocytes to mitogensi decreases their chemotactic, phagocytic, and transforming capabilities, as well as their ability to synthesize RNA and protein; and inhibits cell-mediated cytotoxicity, neutrophil and monocyte chemotaxis, and neutrophil phagocytosis (Bruce, 1972, 1975; Cullen, 1974; Cullen et al., 1972, 1976; Mougdil, 1986). Decreased neutrophil and monocyte chemotaxis has been associated with administration of isoflurane and methoxyflurane (Mougdil, 1986). A variety of anesthetics and opioid analgesics, including avertin, ether, fentanyl, halothane, isoflurane, ketamine-xylazine, morphine, sufentanil, and sevoflurane, reduce the cytotoxic activity of natural killer (NK) cells, a lymphocyte subtype involved in nonspecific immune response to tumors, viruses, and select bacteria in the postoperative period (Beilin et al., 1989; Markovic and Murasko, 1990, 1991, 1993; Markovic et al., 1993). Hyporesponsiveness of NK cells lasts for at least 11 days after anesthesia (Markovic et al., 1993). Markovic and Murasko (1993) hypothesized that the anesthetic effects on NK cell activity results from the induction of CD8 § cells that suppress stimulation of NK cells by interferon. Following anesthesia, NK cells fail to respond to interferon or poly I:C, an inducer of endogenous interferon synthesis, as they do normally (Markovic and Murasko, 1990, 1991; Markovic et al., 1993). Anesthetic-induced NK cell depression strongly accelerated progression of spontaneous lung metastasis produced by the 3LL Lewis lung carcinoma and B16 melanoma (Katzav et al., 1986; Shapiro et al., 1981). The administration of local anesthetics inhibits lymphocyte capping; depresses phagocytosis in neutrophils; reduces both the number and function of CD4 § and CD 19 § cells; alters lymphocyte secretion of interferon, tumor necrosis factor, interleukin (IL)-I, and soluble IL-2 receptor following stimulation by a variety of mitogens; and increases plasma endothelin-like immunoreactivity (Brand et al., 1998; Corsi et al., 1995; Kutza et al., 1997; Sato et al., 1996; Shirakami et al., 1995). Local anesthetics, including procaine, lidocaine, butacaine, tetracaine,

29.

FACTORSTHAT MAYINFLUENCEANIMALRESEARCH

and dibucaine, have been shown to enhance the toxicity of the bleomycin derivative, peplomycin (Mizuno and Ishida, 1982). The effects of anesthetics on cardiovascular function are well recognized. At concentrations used clinically, most volatile anesthetics depress the contractile force of the heart. The mechanisms underlying the negative inotropic effects of these agents, although partially involving the effect of Ca 2§ on the myofibrillar apparatus, are not completely understood (Bosnjak, 1991). Volatile anesthetics, including halothane, isoflurane, and sevoflurane, have an inhibitory effect upon both vascular and tracheal smooth muscle, leading to both vascular and airway dilatation (Kai et al., 1998; Mercier and Denjean, 1996; Zhang and List, 1996). These agents attenuate and prevent airway smooth muscle constriction when exposed to allergen and leukotriene D4 (Tudoric et al., 1995). These effects are mediated by influencing Ca 2§ sensitivity (Kaiet al., 1998; Zhang and List, 1996). Effects have been described following the use of select anesthetic agents, which can be of critical importance to experimental outcome although they may be clinically inapparent. The application of the topical anesthetic benzocaine is associated with methemoglobinemia in a variety of species (Davis et al., 1993). A 2 sec burst of anesthetic spray or direct application of 56 mg of benzocaine increased methemoglobin concentrations sufficiently to substantially alter cardiovascular and pulmonary function (Davis et al., 1993; Lagutchik et al., 1992). The widely used a2-adrenergic agonist xylazine lowers basal plasma insulin concentrations and abolishes the rise in insulin following glucose administration, resulting in elevations in fasting glucose and glucose intolerance in multiple species (Brockman, 1981; Goldfine and Arieff, 1979; Hsu, 1988; Koppel et al., 1982). Select anesthetics have been shown to exert effects on the neuroendocrine system. Reported alterations include both increased and decreased cortisol and catecholamine secretion; increased concentrations of serum growth hormone, thyroxine, antidiuretic hormone, and renin; and decreased secretion of aldosterone and testosterone (Bardin and Peterson, 1967; Fariss et al., 1969; Oyama, 1973; Pettinger et al., 1975). Anesthetics may also influence behavior. The analgesic buprenorphine was associated with pica behavior in the rat. Rats administered this agent ingested hardwood bedding on which they were maintained (Clark et al., 1997b). b.

Euthanasia Agents

There are limited studies examining the effects of euthanasia technique on experimental results. Butler et al. (1990) observed differences in prostacyclin production in aortic tissue and response of aortic and colonic smooth muscle to acetylcholine when rabbits and rats were sacrificed using methoxyflurane, carbon dioxide, and pentobarbital. Cervical dislocation with or without methoxyflurane or pentobarbital anesthesia, and CO2 and halothane overdose were observed to alter both mitogen-

1157

induced lymphoproliferation and the induction of alloantigenspecific cytolytic T lymphocytes in mice (Howard et al., 1990). Decapitation has been associated with dramatic increases in plasma catecholamine concentrations in rats, presumably caused by environmentally induced changes in sympathoadrenal medullary activity (Popper et al., 1977). Carbon dioxide inhalation, dependent on concentration, has varying effects on brain excitability, causes acidosis, and decreases the cerebral concentrations of both sodium and potassium (Granholm and Siesjo, 1969; Pincus, 1969). It is also reported to cause tissue petechiation, particularly in the lungs. The euthanasia agent T-61 causes intravascular hemolysis, which interferes with serum hexosaminidase measurements; artifactual damage of the pulmonary parenchyma, characterized by congestion, edema, and endothelial necrosis; and endothelial swelling of the renal glomerular tufts (Doughty and Stuart, 1995; Port et al., 1978; Prien et al., 1988). The pulmonary architecture is extremely sensitive to effects of a variety of euthanasia techniques (Feldman and Gupta, 1976). Most euthanasia techniques are unsuitable to maintain the integrity of enzymatically labile neurochemicals; therefore, microwave irradiation is utilized by neuroscientists to fix brain neurochemicals and metabolites in vivo while maintaining the brain's anatomic integrity (Stavinoha, 1993). c.

Antimicrobials

The potential of antibiotics to influence physiologic response is well founded. As they are commonly administered to animals used in biomedical research, it is imperative to recognize the nature and scope of their potential effects. Antibiotics may be toxic to a variety of laboratory animal species. Guinea pigs, hamsters, gerbils, and rabbits may develop fatal enterotoxemias, as a variety of antibiotics alter the bacterial microflora in the gut, permitting colonization and proliferation of either toxin-producing Clostridium difficile or C. spiroforme, dependent on the animal species, the route of administration, and the dose (Borriello, 1995; DeLong and Manning, 1994; Frisk, 1987). Although a host of antibiotics may induce the disease, penicillin and clindamycin are most commonly associated. Dihydrostreptomycin toxicity occurs in the gerbil (Wightman et al., 1980). Procaine, used in some penicillin formulations, is toxic to guinea pigs, mice, and rabbits (Galloway, 1968). The adverse effects of the aminoglycosides on renal function and hearing, as well as the potential of fluoroquinolones to damage cartilage, are well recognized by clinicians (Krasula and Pernet, 1991; Riviere and Spoo, 1995). Strain differences in the sensitivity to aminoglycoside (tobramycin) nephrotoxicity have been reported in Fischer 344 and Sprague-Dawley rats (Reinhard et al., 1991). Toxicity also fluctuates temporally in association with diurnal cycles (Linet al., 1996). Cardiovascular dysfunction has been associated with the administration of particular classes of antibiotics. Aminoglycosides produce negative inotropic effects in both cardiac and

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NEIL S. LIPMAN AND SCOTT E. PERKINS

arterial muscle (Adams, 1976). Additionally, they alter the positive inotropism of other agents, including the catecholamines and calcium (Adams, 1976). These effects result from the interference with calcium-dependent membrane phenomena and inhibition of ganglionic neurotransmission (Adams et al., 1976). Lincomycin at high doses disrupts myocardial conductance and is arrhythmogenic in dogs (Daubeck et al., 1974). Aminoglycosides also inhibit synaptic transmission at the somatic myoneural junction (Adams, 1973). The clinical significance of neurotransmission blockade relates to the potential for respiratory arrest when antibiotics are administered concurrently with anesthetics or neuromuscular blocking agents. Antibiotics can also influence immune responsiveness. The tetracyclines have been extensively evaluated. They reduce delayed-type hypersensitivity (DTH) responses; inhibit mitogeninduced lymphocyte proliferation and NK cell activity; and alter granulocyte adherence, migration, and phagocytic ability (Belsheim et al., 1983; Goh and Ferrante, 1984; Ingham et al., 1991; Potts et al., 1983a,b; Thong and Ferrante, 1979, 1980). Similarly, the aminoglycosides have been shown to selectively suppress lymphocyte response to mitogens, depress chemotaxis, and inhibit the microbiocidal activity of phagocytes (Hauser and Remington, 1982; Metcalf and Wilson, 1987). Ciprofloxacin also causes immune perturbation (Jimenez-Valera et al., 1995). When administered to mice prior to immunization with sheep red blood cells, the IgG response is suppressed; however, the IgM response remains unchanged. When the antibiotic is administered 3 days after immunization, the IgM response is enhanced. These authors also demonstrated that ciprofloxacin suppressed the DTH response, inhibited mitogen stimulation of lymphocytes, caused leukopenia, and increased the number of granulocyte-macrophage colony-forming cells in the bone marrow. Both trimethoprim-sulfamethoxazole and chloramphenicol have been shown to depress the anamnestic antibody response following immunization (Hauser and Remington, 1982). Antibiotics may influence the pharmacokinetics and metabolism of other agents. Chloramphenicol inhibits hepatic microsomal enzymes. The concurrent administration of chloramphenicol and ketamine-xylazine or pentobarbital prolongs the duration of anesthesia (Adams and Dixit, 1970; Nossaman et al., 1990). The fiuoroquinolones compete with GABA receptors and therefore may interfere with studies involving the central nervous system (Green and Halliwell, 1997). Bacitracin, gentamicin, and nystatin alter cecocolonic motility and increase fecal excretion of dry matter and water in rats, and amoxicillinclavulanate alters intestinal motility in humans (Caron et al., 1991; Cherbut et al., 1991). Therefore, the concurrent administration of antibiotics with test compounds should be carefully considered. d.

Miscellaneous

There are countless other pharmaceuticals and biologics that would be expected to alter physiologic responses and there-

fore complicate the research utilization of the animals receiving them. Careful consideration must be given to the use and selection of any agent administered. Parasiticides and multivalent vaccines, both frequently administered to laboratory animals, are provided as examples. The anthelmintic levamisole has a variety of immune-potentiating effects, including stimulating cell-mediated immune responses, enhancing the rate of T-lymphocyte differentiation, and increasing the activity of effector lymphocytes (Brunner and Muscoplat, 1980). Ivermectin, commonly used both as an anthelmintic and acaricide, is toxic to certain strains of young mice as well as some genetically altered mice (Schinkel et al., 1994; Skopets et al., 1996). The lack of drug-transporting P-glucoproteins and other developmentally sensitive features of the blood-brain barrier are factors in its toxicity. Ivermectin also has immunostimulatory properties associated with the altered function of T-helper cells (Blakley and Rousseaux, 1991). Vaccination of dogs with modified live vaccines may be associated with thrombopathia and may alter immune function. Vaccination with mixed vaccines, routinely used for disease prevention in puppies and adult dogs, altered DTH responses for at least 2 months postvaccination (Miyamoto et al., 1992). In addition, vaccination of dogs reversed immune suppression in puppies resulting from halothane anesthesia (Taura et al., 1995). 3. Pheromones Pheromone signals are an important form of communication in many species and are involved in social and sexual behaviors in mice and rats (McClintock, 1998; Monahan and Maxson, 1998; Mucignat-Caretta et al., 1998; Vagell and McGinnis, 1998). In mammals, pheromones are detected by the vomeronasal organ, an olfactory sensory structure in the brain (Matsunami and Buck, 1997). The endocrine system of vertebrates and higher invertebrates has been shown to produce a variety of cyclic nucleotides, peptides, fatty acids, prostaglandins, and sterols with endocrine-altering effects (Brown, 1998). Male reproductive behavior in Syrian hamsters is dependent on pheromones from the female and presence of gonadal steroid hormones (Romeo et al., 1998). In mice, pheromones may have an inhibitory influence on action-potential generation and on cAMP levels in receptor cells of the vomeronasal organ, where olfactory receptor cells are thought to detect pheromone-like molecules important for reproductive physiology (Guo et al., 1997; Moss et al., 1998; Zhou and Moss, 1997). Animal odors from conspecifics or a differing species may lead to behavioral or physiologic alterations. Odors emanating from ferrets housed in an adjacent animal holding room have been observed to negatively impact the breeding performance of hamsters (G. E. Schneider, personal communication, 1988). Cat odor resulted in an increased release and decreased uptake of GAB A in hippocampal and cortical brain slices derived from exposed rats as compared to those derived from rats exposed to a neutral odor (File et al., 1993). When stressed, rats can release

29. FACTORSTHAT MAYINFLUENCEANIMALRESEARCH

odors (alarm chemosignals) from their body surface into the air as well as into their urine. These odors act as inhibitory or stimulatory activity signals on conspecifics (Mackay-Sim and Laing, 1981a,b). It was erroneously believed that normal blood (rat) exerted similar effects; however, it has been subsequently determined that the effects exerted by blood are dependent on the stress to which the rats were subject prior to blood collection and not simply due to a factor present in normal rat blood (Mackay-Sim and Laing, 1981a). BALB/c mice exposed for 24 hr to odors from donor mice that were foot-shocked had suppressed cellular and humoral immune function (Cocke et al., 1993). Odors from unfamiliar male mice cause primary developmental defects displayed by embryos developing in a deteriorated uterine environment (Chung et al., 1997).

C.

Microbial Agents

Microbes frequently confound research findings. As a result, there is a tremendous effort to produce, distribute, maintain, and use animals free of microbial pathogens. The effects that microbes may exert on biomedical research are multifaceted. Clinical disease, with its attendant morbidity and mortality, can devastate a research project, especially when disease strikes well into a study of prolonged course. More commonly however, the effects are more insidious. Microbes may induce histologic or biochemical alterations that increase the difficulty of interpreting results, or of greater concern, may lead to their misinterpretation. Diseases causing hepatic injury or renal damage may alter the biotransformation and excretion of experimentally administered compounds (Sipes and Gandolfi, 1991). Subtle immunomodulatory effects and the subsequent impact of microbial pathogens may go unrecognized. The routine use of genetically altered animal models, many of which have phenotypes that are influenced by the microbial flora with which they are associated, requires an improved understanding of the interaction between flora and host to fully understand and exploit these models. As the scope of this section is limited, the reader is referred to more extensive reviews on the impact of infectious agents on research by Bhatt et al. (1986), Institute of Laboratory Animal Resources (ILAR) (1991), and Baker (1998). The agents included in this section have the potential to cause pathologic lesions in a variety of organs. As descriptions of these can be found elsewhere in this book, they are not included in the discussion below. I.

Viruses a.

M o u s e Hepatitis Virus ( M H V )

Mouse hepatis virus is considered to be the most important and common viral pathogen of mice. The virus is distributed worldwide, is highly contagious, contaminates transplantable

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tumors and cell lines, and has repeatedly demonstrated its ability to alter host physiology and impact research. Having the potential to induce both immune stimulation and suppression, MHV may significantly alter the immune system. The effects of MHV in immunocompetent mice, following natural or experimental infection, include inducing involution and apoptosis in the thymus (Cray et al., 1993; Godfraind et al., 1995; Lee et al., 1994); affecting the number and tumoricidal activity of macrophages (Bo0rman et al., 1982); decreasing the proliferative responses of lymphocytes to mitogens (Cray et al., 1993; de Souza et al., 1991; Even et aL, 1995; Kryzstyniak and Dupuy, 1983); triggering polyclonal immunoglobulin production (Lardans et al., 1996); stimulating or depressing antibody response following immunization, dependent on on the timing of the immunization with respect to infection (Lahmy and Virelizier, 1981; Leray et al., 1982; Virelizier et aL, 1976); altering mucosal immune responsiveness (Casebolt et al., 1987); reducing the number of lactate dehydrogenase-elevating virus (LDV)permissive macrophages, delaying plasma lactate dehydrogenase elevation after infection (Dillberger et al., 1987; Even et al., 1995); altering allograft rejection (Cray et al., 1993); depressing phagocytic activity (Gledhill et al., 1965; Williams and DiLuzzio, 1980); inducing macrophage procoagulant activity (Levy et al., 1981); increasing production of interferon (IFN), IL-1, IL-2, IL- 6, IL-12, tumor necrosis factor (TNF), afetoprotein, and antiretinal antibodies (Coutelier et al., 1995; Even et al., 1995; Hooks et al., 1993; Mallucci, 1964; Pearce et al., 1994; Piazza et al., 1965; Schindler et al., 1982); depressing production of IL-2 and IL-4 (de Souza et al., 1991); altering the behavior of ascitic tumors and expression of cell surface markers on T cells (Fox et al., 1977; Nelson, 1959); activating NK cells (Schindler et al., 1982; Tardieu et al., 1980); inducing anemia, leukopenia, and thrombocytopenia (Hunstein et al., 1969; Namiki et al., 1977; Piazza et al., 1965); altering the course or susceptibility of mice to Sendai virus, pneumonia virus of mice (PVM), K virus, and leukoviruses as well as E p e r y t h r o z o o n coccoides, Salmonella typhimurium, and Schistosoma m a n s o n i (Braunsteiner and Friend, 1954; Carrano et al., 1984; Fallon et al., 1991; Gledhill, 1961; Gledhill et al., 1965; Lavelle and Bang, 1973; Manaker, 1961; Nelson, 1952a,b; Niven et al., 1952; Tisdale, 1963; Warren et al., 1969); altering hepatic fermokinetics (Tiensiwakul and Husain, 1979; Vacha et al., 1994); inducing immune-mediated demyelinization (Houtman and Fleming, 1996); reducing the incidence of diabetes in NOD mice (Wilberz et al., 1991); and altering hepatic regeneration, the number of hepatic sinusoidal fenestrae, and the proliferative activity of the bowel (Carthew, 1981; Barthold et al., 1982; Steffan et al., 1995). Mouse hepatitis virus also modulates a variety of enzyme systems. Hepatic isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, and aspartate transaminase are all markedly increased during infection, while cytochrome P450 microsomal enzymes, including those induced by phenobarbital, NADPH oxidase, aniline hydroxylase, and succinate hydrogenase, are significantly decreased

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NElL S. LIPMAN AND SCOTT E. PERKINS

(Budillon et al., 1972, 1973; Cacciatore and Antoniello, 1971; Paradisi et al., 1972; Ruebner and Hirano, 1965). Observations have shown that MHV also induces a number of important changes in immunocompromised mouse strains. Importantly, the extent and severity of lesions induced are considerably more severe in immunosuppressed animals (Huang et al., 1996; Ishida et al., 1978; Sebesteny and Hill, 1974; Ward et al., 1977). Mouse hepatitis virus also affects the immune system, causing spontaneous differentiation of lymphocytes bearing T-cell markers (Scheid et al., 1975; Tamura et al., 1978); enhances both the IgM and IgG antibody responses to sheep erythrocytes (Tamura et al., 1978; Tamura and Fujiwara, 1979); increases the number and phagocytic activity of macrophages (Tamura et al., 1980); enhances NK cell activity (Tamura et al., 1981); leads to xenograft rejection (Akimaru et al., 1981; Kyriazis et al., 1979); results in hepatosplenic myelopoiesis (Ishida et al., 1978); induces production of IL-1, IL-2, IL-6, and tumor necrosis factor (TNF) (Pearce et al., 1994); inhibits hepatic regeneration following partial hepatectomy (Carthew, 1981); and leads to the increase of a variety of hepatic enzymes and bilirubin (Huang et al., 1996). b.

Sendai Virus

Infection with Sendai virus (SV), an important pathogen of rats and mice, not only affects the respiratory system for which it has tropism, but also alters both humoral and cell-mediated immunity. Sendai virus depresses pulmonary bacterial clearance by altering pulmonary macrophage function (Degre and Glasgow, 1968; Degre and Solberg, 1971). The effects of SV on pulmonary macrophages include altering phagocytosis; inhibiting of phagosome-lysosome fusion; decreasing lysosomal enzymes; altering Fc and non-Fc receptor-mediated attachment; and altering the ability to degrade ingested bacteria (Jakab, 1981; Jakab and Warr, 1981). The effects of SV on the immune system are well characterized. The virus inhibits the response of T lymphocytes to mitogens (Garlinghouse and Van Hoosier, 1982; Roberts, 1982; Wainberg and Israel, 1980); causes a lifelong increase in cytotoxic T-cell precursors (Doherty et al., 1994); increases NK cell cytolytic activity and both the IgM and IgG splenic primary plaque-forming cell responses to sheep erythrocytes (Brownstein and Weir; 1987; Clark et al., 1979); stimulates TNF and interferon (INF) expression (Milone and Fitzgerald-Bocarsly, 1998; Payvandi et al., 1998; Uhl et al., 1998); enhances rejection of skin isografts (Streilein et al., 1981); reduces the severity of adjuvant-induced arthritis (Garlinghouse and Van Hoosier, 1978); inhibits the development of leukemia following inoculation of mice with Friend leukemia virus (Wheelock, 1966); and decreases the tumorigenicity of transplantable tumors (Matsuya et al., 1978; Takeyama et al., 1979). The effects of SV on immunity have been reported to be lifelong and include an increased prevalence of spontaneous autoimmune diseases (Kay, 1978, 1979; Kay et al., 1979). Prior or concurrent infection with SV alters the neoplastic response

of the respiratory system to carcinogens (Hall et al., 1985; Nettesheim et al., 1974, 1981; Parker, 1980; Peck et al., 1983), and the virus has also been shown to alter wound healing and interfere with early embryonic development and fetal growth (Kenyon, 1983; Lavilla-Apelo et al., 1992). c.

Lactate D e h y d r o g e n a s e - E l e v a t i n g

Virus (LDV)

Infection with LDV results in a lifelong viremia, which is the principal reason that LDV is one of the most common contaminants of transplantable tumors, infectious inocula, and biological materials originating from or passaged in mice. Elevation of one isozyme of lactate dehydrogenase, resulting from reduced clearance, is the hallmark of the disease, although other serum enzymes may also be elevated (Brinton, 1982). Carbon particle and asparaginase clearance is also reduced (Mahy et al., 1965; Notkins and Scheele, 1964; Riley et al., 1970). The immune system is also adversely affected by LDV. Infection with LDV results in elevation of serum gamma globulin concentrations, impairs the antigen-presenting capacity of macrophages, and alters the humoral immune response, dependent on chronicity of infection (Isakov et al., 1982a,b,c; Mergenhagen et aL, 1967; Michaelides and Simms, 1980; Notkins et al., 1966; Riley et al., 1975); causes polyclonal lymphocyte activation (Michaelides and Simms, 1980); delays allograft rejection (Howard et al., 1969); inhibits contact sensitivity to 2,4-dinitro fiuorbenzene (DNFB) (Hayashi et al., 1991); prevents experimental allergic encephalomyelitis (Inada and Mims, 1986); reduces autoimmune disease, adhesion molecule expression, and antinuclear antibody formation in NZB x NZWF1 mice (Hayashi et al., 1993; Kameyama and Hayashi, 1994; Oldstone and Dixon, 1972); depresses graft versus host disease (Notkins, 1971); stimulates NK cell activity and the production of IFN (Koi et al., 1981; Nicklas et al., 1988); alters macrophage superoxide anion production (Hayashi et al., 1992a,b); inhibits binding of asparaginase to monocytes (Mori et al., 1992); suppresses streptozotocin-induced insulitis (Hayashi et al., 1994); and potentiates the severity of Eperythrozoon coccoides, Plasm o d i u m yoelii, and Listeria m o n o c y t o g e n e s infections (Bonventre et al., 1980; Fitzmaurice et al., 1974; Henderson et al., 1978; Riley, 1964). Lactate dehydrogenase-elevating virus also enhances or suppresses the growth of transplantable and inducible tumors. Tumors transplanted shortly after infection demonstrate enhanced growth because of cellular immune suppression (Bailey et al., 1965; Michaelides and Schlesinger, 1974; Riley et al., 1978). Bittner agent-induced mammary tumors and the development of pulmonary adenomas following treatment with urethane are both suppressed by LDV (Riley, 1966; Theiss et al., 1980). d.

Lymphocytic Choriomeningitis Virus ( L C M )

Lymphocytic choriomeningitis virus is an important zoonotic pathogen and a common contaminant of transplantable tumors,

29. FACTORSTHATMAYINFLUENCEANIMALRESEARCH cell lines, and biological materials. The virus induces a persistent infection of T cells, resulting in lifelong viremia and viral shedding. The effects of LCM on research, following natural and experimental infection, include developing severe cellular and humoral immune suppression (Bro-Jorgensen and Volkert, 1974; Bro-Jorgensen et al., 1975; Guttler et al., 1975; LehmannGrube et al., 1972; Mims and Wainwright, 1968; Thomsen et al., 1982; Wu-Hsieh et al., 1988); enhancing NK cell activity, interferon production, macrophage function, and virus-specific cytotoxic T-cell proliferation (Blanden and Mims, 1973; Pfau et al., 1982; Ronco et al., 1981; Welsh, 1978; Welsh and Doe, 1980; Zinkernagel and Doherty, 1975, 1979); increasing endothelial adhesion molecules in serum (Christensen et al., 1995; Marker et al., 1995); altering cytokine gene expression (Colle et al., 1993); delaying skin allograft and tumor rejection (Guttier et al., 1975; Lehmann-Grube et al., 1972); increasing susceptibility of mice to ectromelia virus, E. coccoides, bacterial endotoxin, and irradiation (Barlow, 1964; Bro-Jorgensen and Volkert, 1972; Hotchin, 1962; Mims and Wainwright, 1968; Seamer et al., 1961); inhibiting the tumorigenic potential of polyoma virus, the Bittner agent, and Rauscher leukemia virus (Hotchin, 1962; Padnos and Molomut, 1973; Yuon and Barski, 1966); inactivating experimental hepatitis B infection (Guidotti et al., 1996); preventing the development of insulin-dependent diabetes mellitus in the BB rat and NOD mouse (Dyrberg et al., 1988; Oldstone, 1990; Schwimmbeck et al., 1988); and altering murine behavior, synaptic plasticity, and cognitive functions (de la Torre et al., 1996; Gold et al., 1994). Mouse Parvovirus and Rat Parvovirus Type 1 (MPV-1; RPV-1)

Mouse parvovirus and rat parvovirus type 1 have been identified as viruses that infect the mouse and rat, respectively. Although recognized serologically for several years, MPV-1 was first isolated after infecting and interfering with cultures of CD8 +, CD4 +, and y8 T-cell clones used for studying lymphocyte activation and immunoregulatory mechanisms (McKisic et al., 1993). In addition to causing lyric infection and inhibiting proliferation of T cells, MPV-1 was shown to inhibit T-cell proliferation following exposure to IL-2 and antigen; depress the proliferative response of spleen and lymph node from antigen-primed mice; and accelerate tumor allograft rejection, not by directly infecting the graft, but by inducing a "bystander help" effect (McKisic et al., 1993, 1996). RPV-1 is not as well characterized. It may suppress the development of lymphoid tumors (Jacoby and Ball-Goodrich, 1995). f.

Kilham Rat Virus (KRV) and H-1 Virus

Kilham rat virus contaminates transplantable tumors and cell lines. The virus alters in vitro responses of lymphocytes to mitogens and allogeneic lymphoid cells (Campbell et al., 1977); affects cytotoxic T-cell activity (Darrigrand et al., 1984); and

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induces interferon production (Kilham et al., 1968). Kilham rat virus also suppresses the development of leukemia induced by the Moloney murine leukemia virus (Bergs, 1969); induces diabetes mellitus in the diabetes-resistant BB/Wor strain (Brown et al., 1993); alters lipid metabolism (Schuster et aL, 1991); alters leukocyte adhesion to aortic endothelium (Gabaldon et al., 1992); and may reduce the incidence of Yersinia-associated arthritis (Gripenberg-Lerche and Toivanen, 1993, 1994). H-1 virus inhibits tumor induction by DMBA and adenovirus in hamsters (Toolan and Ledinko, 1968; Toolan et al., 1982); causes hepatic necrosis in rats exposed to other pathogens and hepatotoxic chemicals (Kilham and Margolis, 1970; Ruffolo et al., 1966); and may reduce the incidence of arthritis associated with Yersinia (Gripenberg-Lerche and Toivanen, 1993, 1994). As both viruses replicate in and are cytolytic to dividing cells, they also have the potential to alter fetal development or to be teratogenic. g.

Minute Virus o f Mice (MVM)

Minute virus of mice is a common contaminant of transplantable tumors, cell lines, and virus stocks. Evidence that MVM may interfere with research is based principally on studies of the immunosuppressive variant, MVM(i), although experimental inoculation of the prototype strain, MVM(p), inhibits the growth of intraperitoneally administered Ehrlich ascites tumor cells (Guetta et al., 1986). It is unclear whether MVM(i) occurs as a natural pathogen of mice; however, MVM(i) is immunosuppressive (Segovia et al., 1995); cytolytic to T-cell clones (Tattersall and Cotmore, 1986); inhibits cytotoxic T lymphocytes (Bonnard et al., 1976; Herbermann et al., 1977); suppresses the response of T cells to mitogens (McMaster et al., 1981); and suppresses T helper-B cell responses (Engers et al., 1981). h.

Sialodacryoadenitis Virus (SDA)

Sialodacryoadenitis virus may potentially interfere with research involving the lacrimal and salivary glands, the respiratory system, the eyes, or fetal and neonatal development. Effects associated with SDA infection include alteration of estrous cycles (Utsumi et al., 1991); embryonic and neonatal mortality (Utsumi et al., 1991); impairment of olfaction and chemoreception for up to 2 weeks postexposure (Bihun and Percy, 1995); alteration of growth rates in young rats (Utsumi et al., 1980); reduction in food consumption and weight gain (Nunoya et al., 1977); depletion of epidermal growth factor in salivary glands (Percy et al., 1988); and reduction of IL-1 production by alveolar macrophages (Boschert et al., 1988). 2.

Bacteria

a.

Citrobacter rodentium

The etiologic agent of transmissible murine colonic hyperplasia significantly alters the cytokinetics of the colonic mucosal

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NElL S. LIPMAN AND SCOTT E. PERKINS

epithelium (Barthold, 1979). Both the susceptibility to colonic neoplasia and the latent period for induction are increased in mice infected with C. rodentium and exposed to the carcinogen, 1,2-dimethylhydrazine (Barthold and Beck, 1980; Barthold and Jonas, 1977). The hyperplastic colonic lesions may also be misinterpreted as preneoplastic, as they resemble focal atypia. Citrobacter rodentium may also cause immunologic alterations, including inhibition of antigen-specific cytotoxic T-cell activity (Maggio-Price et al., 1998). b.

Clostridium piliforme

Frequently a subclinical infection, Clostridium piliforme causes overt Tyzzer's disease when animals are subject to experimental manipulations, such as whole body X-irradiation (Taffs, 1974; Takagaki et al., 1966); treatment with cortisone or adrenocorticotropic hormone (ACTH) (Takagaki et al., 1966; Yamada et al., 1969); feeding with a high-protein diet (Maejima et al., 1965); exposure to carbon tetrachloride (Takenaka and Fujiwara, 1975); and intraperitoneal passage of ascitic tumors (Craigie, 1966). Tyzzer's disease increases hepatic transaminases and alters the pharmacokinetics of trimethoprim and warfarin (Fries and Ladefoged, 1979; Naiki et al., 1965). c.

Helicobacter spp.

The importance of this expanding genus on research is becoming well recognized. Helicobacter hepaticus has considerable potential to influence research studies, as the bacterium is associated with progressive hepatitis, proliferative typhlitis, and/or colitis in immunocompetent and immunodeficient mouse strains (Foltz et al., 1998; Li et al., 1998; Ward et al., 1994a,b); causes hepatocellular and hepatocholangiolar adenomas and carcinomas in A/JCr and B6C3F1 mice (Fox et al., 1996a; Hailey et al., 1998; Ward et al., 1994a,b); elevates hepatic transaminase levels in serum (Fox et al., 1996b); and serves as an immunologic target promoting the development of inflammatory bowel disease in gene-targeted mice and in an adoptively transferred severe combined immunodeficient (SCID) mouse model (Cahill et al., 1997; Kullberg et al., 1998). The proneoplastic effects of H. hepaticus on the liver are mediated by increasing the levels of select cytochrome P450 isoforms involved in carcinogen bioactivation (Chomarat et al., 1997; Sipowicz et al., 1997); enhancing glutathione S-transferase activities while decreasing the activity of glutathione peroxidase and the content of glutathione (Chomarat et al., 1997; Sipowicz et al., 1997); increasing the production of superoxide (Chomarat et al., 1997); altering cytokinetics (Nyska et al., 1997); stimulating cyclin D expression, accelerating the development and progression of hepatic tumors in carcinogen-treated mice (Diwan et al., 1997); up-regulating a variety of growth factors, cell cycle proteins, and transcription factors (Ramljak et al., 1998); and producing a cytotoxin (Taylor et al., 1995). A1-

though not as well characterized, H. bilis induces similar lesions in immunocompromised mice and rats, but no association with hepatic neoplasia has been reported (Franklin et al., 1998; Haines et al., 1998; Shomer et al., 1997). d.

Mycoplasma pulmonis

Naturally occurring mycoplasmosis of rodents is an insidious disease that may significantly impact research in a variety of disciplines, especially long-term studies, as the organism may disseminate widely in the host. Mycoplasma pulmonis infection is likely to impact studies of the respiratory tract, as it alters mucociliary clearance (Cassell et al., 1981); ciliary function (Irvani and van As, 1972; Westerberg et al., 1972); cell kinetics (Wells, 1970); and immune function, as it alters both the number and subpopulation distribution of lymphocytes in the lung (Cole et al., 1975; Davis et al., 1982; Naot et al., 1979a,b). Mycoplasma pulmonis has been demonstrated to enhance the pulmonary response to carcinogens (Schreiber et al., 1972). Infection of the genital tract alters fecundity, as M. pulmonis affects embryo implantation, spermatozoan motility, and fertilization; skeletal development and ossification are also altered (Cassell, 1982; Fraser and Taylor-Robinson, 1977; Goeth and Appel, 1974; Lal et al., 1980; Leader et al., 1970). The effects of M. pulmonis on the immune system include increasing NK cell activity (Lai et al., 1987); suppressing humoral antibody response (Aguila et al., 1988b); stimulating the production of mitogenic substances for B and T cells (Proust et al., 1985; Ross et al., 1992); enhancing production of TNF, IL-1, IL-6, and interferon (Faulkner et al., 1995); and reducing the incidence and severity of collagen and adjuvant-induced arthritides (Taurog et al., 1984). Subclinical mycoplasmosis exacerbates vitamin A and E deficiency (Lindsey et al., 1986). e.

Pasteurella multocida

Pasteurella multocida causes a variety of clinical syndromes in rabbits, all of which can seriously impact research. More subtle effects of pasteurellosis have also been described. Nasal instillation of P. m u l t o c i d a - f r e e rabbits with a suspension of the organism resulted in the expression of the adhesion molecule VCAM-1 (Richardson et al., 1997a). Richardson et al. (1997b) also demonstrated the ability of P. multocida to enhance atherosclerosis, as naturally and experimentally infected rabbits fed a high-lipid diet had increased VCAM-1 expression as well as enhanced aortic sudanophilia. f

Pseudomonas aeruginosa

A common commensal in many species as well as a ubiquitous environmental contaminant, Pseudomonas aeruginosa is of major importance to immunocompromised subjects and studies employing the use of indwelling catheters and other percuta-

29. FACTORSTHATMAYINFLUENCEANIMALRESEARCH

neously implanted devices. Effects associated with infection include high mortality following administration of immunosuppressive drugs (Hazlett et al., 1977; Millican, 1963; Pierson et al., 1976; Rosen and Berk, 1977, Schook et al., 1977; Urano and Maejima, 1978); premature death after exposure to lethal radiation, cytomegalovirus infection, or cold stress (Flynn 1963a,b,c; Halkett et al., 1968; Hamilton and Overall, 1978; Hammond et al., 1954, 1955; Hightower et al., 1966; Vincent et al., 1955); increased fibrosis following airway infection (Mclntosh et al., 1992); depressed contact sensitivity to oxazolone (Campa et al., 1975, 1976, 1977); altered pulmonary epithelium fluid transport (Pittet et al., 1996); stimulation of the release of immunosuppressive factors from macrophages (Marshall et al., 1993); thymic atrophy and apoptosis (Wang et al., 1994); proliferation of immature splenic T cells in nude mice (Dixon and Misfeldt, 1994); T-cell dependent immune suppression (Haslov et al., 1992); enhanced cardiac automaticity and depression from hypoxia (Kwiatkowska-Patzer et al., 1993); impaired wound healing (Heggers et al., 1992); and altered behavioral and clinical pathologic parameters following experimental wound infection (Bradfield et al., 1992). 3.

Fungi

Infections caused by fungi, with the exception of Pneumocystis carinii in immunocompromised hosts, are relatively uncommon in laboratory-bred animals. Pneumocystis carinii alters alveolar capillary permeability (Yoneda and Waltzer, 1980); induces activating and inhibitory innate cellular immune-response mechanisms (Warschkau et al., 1998); increases levels of TNF, IL-1, IL-6, IL-8, and arachidonic acid and its metabolites (Castro et al., 1993; Chen et al., 1992, 1993; Kolls et al., 1993; Lipschik et al., 1996; Pottratz et aL, 1998); induces expression of the adhesion molecule ICAM-1 and inhibits cyclin-dependent kinase activity and fibrinogen expression in pulmonary epithelium (Limper et al., 1998; Simpson-Haidaris et al., 1998; Yu and Limper, 1997); alters pulmonary GTP-binding proteins and the amount and type of surfactant produced (Kernbaum et al., 1983; Oz and Hughes, 1997; Sheehan et al., 1986); and modifies the uptake of intratracheally administered compounds (Mordelet-Dambrine et al., 1992). 4.

Parasites

Parasites can confound the experimental use of animals in a variety of ways, including inducing an immune response, altering immune responsiveness, competing with the host for nutrients, consuming body fluids, altering cytokinetics, stimulating or depressing tissue growth, causing mechanical obstructions, altering host physiology and biochemistry, and affecting behavior. They may also transmit or enhance the pathogenicity of other infectious agents. In addition, anthelmintic and acaricide administration may significantly alter the biology of the host.

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Some examples of the effects of parasites on experimental results are well known. The carcinogenicity of saccharin was questioned, as the rats used to evaluate the compound were infected with Trichosomoides crassicauda, which causes hyperplasia or papillomatosis of the urinary bladder (Homburger, 1978). Gongylonema neoplasticum is associated with esophageal tumors in the rat. However, the effects of parasites are likely to be more subtle. Pinworms are commonly found in rodents and can alter a variety of biological processes. Oxyuriasis is reported to reduce the development of adjuvant-induced arthritis (Pearson and Taylor, 1975); induce splenic T- and B-cell proliferation and occasional germinal center formation (Beattie et al., 1981); increase the antibody response to sheep erythrocytes (Sato et al., 1995); retard growth (Wagner, 1988); impede colonic water and electrolyte absorption (Lubcke et al., 1992); accelerate the development of the hepatic monooxygenase system (Mohn and Philipp, 1981); be associated with the development of lymphomas in nude mice (Baird et al., 1982); and cause a significant reduction of activity in behavioral studies (McNair and Timmons, 1977). Fur mites, also commonly found in rodents, may significantly alter the histologic features of the skin, activate the immune system, alter animal behavior, and inhibit reproduction. Specifically, mites cause secondary amyloidosis (Galton, 1963; Weisbroth, 1982); alter the production of both IL- 2 and IL-4 and the immunoglobulin isotype profile in serum (Jungmann et al., 1996a,b); cause lymphocytopenia and granulocytosis (Jungmann et al., 1996a,b); and reduce the contact sensitivity reaction to oxazolone (Laltoo and Kind, 1979). The response to mites in mice is strain-specific. The inbred mouse strain NC develops an exaggerated clinical and immunologic response to infestation with Myocoptes musculinus (Morita et al., 1999). The NC strain develops severe pruritis, skin lesions, and elevated serum IgE levels as compared to similarly infested BALB/c and C57BL/6 strains. Encephalitozoon cuniculi infects a broad host range; however, infections are uncommon in most species bred for the laboratory with the exception of the rabbit. Parasite-induced lesions in the brain and kidney may make interpretation of experimental evaluation of these organs difficult. Encephalitozoon cuniculi may also alter immune responsiveness. The protozoan is reported to increase NK cell activity (Niederkorn et al., 1983; Niederkorn, 1985); reduce cellular and humoral responses to a variety of immunogens (Cox, 1977; Cox and Gallichio, 1978; Didier and Shadduck, 1988; Niederkorn et al., 1981; Waller et al., 1978); and alter host responsiveness to transplantable tumors (Arison et al., 1966; Meiser et al., 1971; Petrie, 1966). D.

Stressors

Stress is the effect produced by external or internal factors that induce an alteration in an animal's biological equilibrium

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(National Research Council, 1992). The alterations that result, dependent on the severity, nature, and duration of the stressor, may significantly impact experimental findings. The response to stress is influenced by a variety of factors not limited to host sex, age, and genetics. Stress results in stimulation of the hypothalamic-pituitary axis, resulting in release of corticosteroids from the adrenal cortex and catecholamines from the adrenal medulla. Opioids, released centrally and peripherally, also mediate stress effects (Madden et al., 1977; Plotnikoff et al., 1985; Wybran, 1985). Stress elicits changes in numerous immunologic processes. A variety of murine tumor-host model systems have been used to demonstrate the effects of stress on tumor development (Fitzmaurice, 1988; Riley, 1975; Sklar and Anisman, 1979; Visintainer et al., 1982). Stress-induced alterations in tumor model systems include tumor rejection inhibition, decreased tumor survival, and altered tumor latency. Riley (1975) demonstrated that the incidence of mammary tumors in C3H/He mice carrying the Bittner agent could be altered from 7 to 92% at 400 days of age by increasing the degree of environmental stress to which the mice were subject. Stress may be a direct result of experimental manipulation or may be caused by environmental or psychosocial factors. Many stressors are discussed in this chapter. Handling, acute and chronic restraint, social interaction with humans, and activity of personnel in animal holding rooms are additional factors that have been reported to result in physiologic and/or behavioral changes, that, dependent on the design and nature of the study, could influence results (Long et al., 1991; Misslin, 1982; Nerem et al., 1980; Saibaba et al., 1996; Tuli et al., 1995a,b). One stress to which almost all animals are subject is that which occurs during transport between institutions. Numerous studies have documented the impact of transportation in a variety of laboratory species. Alterations attributable to shipment include elevation of plasma glucocorticoid concentrations, neutrophilia, lymphopenia, hyperglycemia, changes in serum biochemical indices, reduced splenic NK cell activity, depressed humoral and cell-mediated responses to sheep red blood cells, thymic atrophy, depressed food consumption, weight loss, depressed reproductive performance, behavioral alterations, increased susceptibility to disease, and decreased latency and incidence of tumors (Aguila et al., 1988a; Bean-Knudsen and Wagner, 1987; Dymsza et al., 1963; Hayssen, 1998; Landi et al., 1982; Peters and Kelly, 1977; Riley, 1975; Toth and January, 1990; Tuli et al., 1995b; Wallace, 1976). In general, altered immunologic parameters return to baseline within 48 hr after arrival, although corticosterone levels remained elevated in mice when measured at 48 hr (Aguila et al., 1988; Landi et al., 1982; Toth and January, 1990). Behavioral and reproductive function may require greater acclimatization before returning to normal. A 96 hr acclimatization period was insufficient for mouse behavior to return to normal after transport (Tuli

NElL S. LIPMAN AND SCOTT E. PERKINS

et al., 1995b). Reproductive performance was depressed in transatlantic-shipped deer mice for 110 days after arrival (Hayssen, 1998). Alterations attributed to transportation are detectable even when animals were shipped by environmentally controlled ground transportation (Aguila et al., 1988; Toth and January, 1990). When comparing air to ground transport, investigators found either no differences or greater physiologic change induced by air transport (Aguila et al., 1988; Landi et al., 1981; Toth and January, 1990). Transportation-induced changes could be detected following transport for as little as 4 hr (Toth and January, 1990). Interinstitutional shipment of animals is not the only source of transport stress. Drozdowicz et al. (1990) examined the effects of in-house transport in mice and found that moving animals on a cart for 12 min led to an increase in plasma corticosterone, a decrease in circulating white blood cells and lymphocytes, and reduction in thymic weight. Corticosterone, WBC, and lymphocyte counts returned to normal within 4 hr of transport; however, the circadian release of corticosterone remained abnormal for 1 day. Social interaction is an important experimental consideration. An isolation stress p h e n o m e n o n is well recognized in rodents. Housing animals in isolation influences a variety of behavioral and physiologic parameters, including creatinine clearance; urine flow rates; fractional reabsorption of sodium; antipyrine clearance; plasma glucose, triglyceride, and corticosterone levels; the incidence of spontaneous tumors; the prevalence of degenerative joint disease; humoral and cell-mediated immune response; body weight; behavioral reactivity; ethanol consumption, drug toxicity, and susceptibility to challenge with infectious agents; and the response of tumor allografts to chemotherapy (Benton and Brain, 1981; Brown and Grunberg, 1995, 1996; Brunner et al., 1994; Capitanio and Lerche, 1998; Consolo et al., 1965; Dairman and Balazs, 1970; Everitt et al., 1988; Haseman et al., 1994; Hilakivi-Clarke and Dickson, 1995; Jessop and Bayer, 1989; Karp et al., 1993; Kerr et al., 1997; Meisel et al., 1990; Nyska et al., 1998; Parker and Radow, 1974; Perez et al., 1997; Rabin and Salvin, 1987; Rao and Lindsey, 1988; Shanks et al., 1994; Steplewski et al., 1987; Vadiei et al., 1990; Vargas-Rivera et al., 1990; Weinreich et al., 1996). Temporary maternal separation may cause sufficient stress and nutritional deprivation to delay development. For example, neonatal rat pups separated from their dam for 6 hr a day resulted in delayed developmental landmarks, including righting ability, negative geotaxis, and eye opening. Body and brain weights were also decreased in comparison to those of controls (Vitarella et al., 1998). The effects of housing density and group size have been long recognized and must be distinguished from each other. Housing density reflects the number of animals housed per unit space. Group size can be increased without altering housing density by increasing cage space proportionally as the population is increased. When the same floor space per animal is provided, dif-

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH

ferences in behavioral and physiologic parameters are more easily detected when comparing housing in isolation to that in groups, as differences between group sizes are difficult to distinguish (Bell et al., 1972; Davis, 1978). Increased housing density has been associated with a variety of biological effects, including declines in reproductive performance and increases in aggressive behavior (Christian and LeMunyan, 1958; Davis, 1978; Welch and Welch, 1969). In general, increasing housing density has an immunosuppressive effect. Increases in plasma corticosterone, reduction in the number of peripheral granulocytes and lymphocytes, and reductions in superoxide production and neutrophil phagocytic activity have been reported with increasing housing density (Peng et al., 1989; Tsukamoto et al., 1994). It has also been shown to increase the susceptibility of mice to coxsackievirus and Plasmodium berghei infection, enhance the diabetogenic response of streptozotocin, decrease antibody synthesis, depress homograft rejection, enhance the development of autoimmune disease, and alter the size and constituents of various regions of the brain (Johnson et al., 1963; Mazelis et al., 1987; Plaut et al., 1969; Solomon, 1969; Vessey, 1964; Welch et al., 1974). However, select studies on housing density have reported immune enhancement that may have resuited from differences in gender, strain, and housing condition (Joasoo and McKenzie, 1976; MacManus et al., 1971).

REFERENCES

Adams, H. R. (1973). Neuromuscular blocking effect of aminoglycoside antibiotics in nonhuman primates. J. Am. Vet. Med. Assoc. 163, 613-616. Adams, H. R. (1976). Antibiotic-induced alterations of cardiovascular reactivity. Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 1148-1150. Adams, H. R., and Dixit, B. N. (1970). Prolongation of pentobarbital anesthesia by chloramphenicol in dogs and cats. J. Am. Vet. Med. Assoc. 156, 902905. Adams, H. R. Teske, R. H., and Mercer, H. D. (1976). Anesthetic-antibiotic interrdationships. J. Am. Vet. Med. Assoc. 168, 409-412. Aguila, H. N., Pakes, S. P., Lai, W. C., and Lu, Y. S. (1988a). The effect of transportation stress on splenic natural killer cell activity in C57BL/6J mice. Lab. Anim. Sci. 38, 148-151. Aguila, H. N., Lai, W. C., Lu, Y. S., and Pakes, S. P. (1988b). Experimental Mycoplasma pulmonis infection of rats suppresses humoral but not cellular immune response. Lab. Anim. Sci. 38, 138-142. Akimaru, K., Stuhlmiller, G. M., and Seigler, H. E (1981). Influence of mouse hepatitis virus on the growth of human melanoma in the peritoneal cavity of the athymic mouse. J. Surg. Oncol. 17, 327-339. Alder, R., and Friedman, S. B. (1968). Plasma cortisone response to environmental stimulation: Effects of duration of stimulation and the 24-hour adrenocortical rhythm. Neuroendocrinology 3, 378-386. Algers, B. (1984). A note on behavioural responses of farm animals to ultrasound. Appl. Anim. Behav. Sci. 12, 387-391. American Conference of Governmental Industrial Hygienists (1995). "19951996 Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices." ACGIH, Cincinnati.

1165 Anderson, J. D., and Cox, C. S. (1967). Microbial survival. In "Airborne Microbes" (P. H. Gregory and J. L. Monteith, eds.), 17th Symposium of the Society of General Microbiology, pp. 203-226. Cambridge Univ. Press, Cambridge. Anderson, D. J., Watson, A. L., and Yunis, E. J. (1985). Environmental and genetic factors that influence immunity and longevity in mice. Basic Life Sci. 35, 231-240. Andrews, J. S. (1996). Possible confounding influence of strain, age, and gender on cognitive performance in rats. Brain Res. Cogn. Brain Res. 3, 251267. Anthony, A. (1962). Criteria for acoustics in animal housing. Lab. Anim. Care. 13, 340-347. Anthony, A., and Harclerode, J. E. (1959). Noise stress in laboratory rodents. II. Effects of chronic noise exposures on sexual performance and reproductive function of guinea pigs. J. Acoust. Soc. Am. 31, 1437. Anver, M. R., and Cohen, B. J. (1979). Lesions associated with aging. In "The Laboratory Rat" (H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), pp. 377-399. Academic Press, New York. Arison, R. N., Cassaro, J. A., and Pruss, M. P. (1966). Studies on murine ascitesproducing agent and its effect on tumor development. Cancer Res. 26, 1915-1920. Armario, A., Castellanos, J. M., and Balasch, J. (1985). Chronic noise stress and insulin secretion in male rats. Physiol. Behav. 34, 359-361. Aschengrau, A., Zierler, S., and Cohen, A. 1993. Quality of community drinking water and the occurrence of late adverse pregnancy outcomes. Arch. Environ. Health 48, 105-113. Aschoff, J. (1960). Exogenous and endogenous components in circadian rhythms. Cold Spring Harbor Symp. Quant. Biol. 25, 11-28. Bailey, J. M., Clough, J., and Lohaus, A. (1965). Influence of LDH viruses on growth of Erhlich ascites tumor in mice. Proc. Soc. Exp. Biol. Med. 119, 1200-1204. Baily, D. W. (1982). How pure are inbred strains of mice? Immunol. Today 3, 210-214. Baird, S. M., Beattie, G. M., Lannom, R. A., Lipsick, J. S., Jensen, E C., and Kaplan, N. O. (1982). Induction of lymphoma in antigenically stimulated athymic mice. Cancer Res. 42, 198-206. Baker, D. G. (1998). Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin. Microbiol. Rev. 11, 231-266. Balazs, T., Murphy, J. B., and Grice, H. C. (1962). The influence of environmental changes on the cardiotoxicity of isoprenaline in rats. J. Pharm. Pharmacol. 14, 750-755. Baldwin, J. D. (1968). The social behavior of the adult male squirrel monkey in a seminatural environment. Folia Primatol. 9, 281-314. Bardin, C. W., and Peterson, R. E. (1967). Studies of androgen production by the rat: Testosterone and androstenedione content of blood. Endocrinology 80, 38-44. Barlow, J. L. (1964). Hyperreactivity to endotoxin in mice infected with lymphocytic choriomeningitis virus. In "Bacterial Endotoxins" (M. Landy and W. Braun, eds.), pp. 448-454. Quinn and Bodin, Rahway, New Jersey. Barlow, S. M. (1972). Teratogenic effects of restraint, cold, and audiogenic stress in mice and rats. Ph.D. thesis, University of London. Barnard, C. J., Behnke, J. M., and Sewell, J. (1996). Environmental enrichment, immunocompetence, and resistance to Babesia microti in male mice. Physiol. Behav. 60, 1223-1231. Barnett, S. A. (1965). Adaptation of mice to cold. Biol. Rev. 40, 5-51. Barnett, S. A. (1973). Maternal processes in the cold-adaptation of mice. Biol. Rev. 48, 477-508. Barthold, S. W. (1979). Autoradiographic cytokinetics of colonic mucosal hyperplasia in mice. Cancer Res. 39, 24-29. Barthold, S. W., and Beck, D. (1980). Modification of early dimethylhydrazine carcinogenesis by colonic mucosal hyperplasia. Cancer Res. 40, 44514455.

1166 Barthold, S. W., and Jonas, A. M. (1977). Morphogenesis of early 1,2dimethylhydrazine-induced lesions and latent period reduction of colon carcinogenesis in mice by a variant of Citrobacterfreundii. Cancer Res. 37, 4352-4360. Barthold, S. W., Smith, A. L., Lord, P. E S., Bhatt, P. N., Jacoby, R. O., and Main, A. J. (1982). Epizootic coronaviral typhlocolitis in suckling mice. Lab. Anim. Sci. 32, 376-383. Bean-Knudsen, D. E., and Wagner, J. E. (1987). Effect of shipping stress on clinicopathologic indicators in F344/N rats. Am. J. Vet. Res. 48, 306-308. Beattie, G. M., Baird, S. M., Lipsick, J. S., Lannom, R. A., and Kaplan, N. O. (1981). Induction of T- and B-lymphocyte responses in antigenically stimulated athymic mice. Cancer Res. 41, 2322-2327. Beilin, B., Martin, E C., Shavit, Y., Gale, R. P., and Liebeskind, J. C. (1989). Suppression of natural killer cell activity by high-dose narcotic anesthesia in rats. Brain Behav. Immun. 3, 129-137. Bell, R. W. (1974). Ultrasounds in small rodents: Arousal produced and arousal producing. Dev. Psychobiol. 7, 39-42. Bell, R. W., Nitschke, W., and Zachman, T. A. (1972). Ultrasounds in three inbred strains of young mice. Behav. Biol. 7, 805-814. Bellhorn, R. W. (1980). Lighting in the animal environment. Lab. Anim. Sci. 30, 440-450. Belsheim, J., Blomqvist, C., Lofberg, J., and Gnarpe, H. (1983). Tetracycline influence on leukocyte functions. Acta Otorhinolaryngol. Belg. 37, 635-648. Benton, D., and Brain, P. E (1981). Behavioral and adrenocortical reactivity in female mice following individual or group housing. Dev. Psychobiol. 14, 101-107. Berger, J. (1980). Seasonal influences on circadian rhythms in the blood picture of the laboratory mice. I. Leucocytes and erythrocytes. II. Lymphocytes, eosinophils, and segmented neutrophils. Z. Versuchstierkd. 22, 122-134. Berger, J. (1981). Seasonal variations in reticulocyte counts in blood of laboratory mice and rats. Short communication. Z. Versuchstierkd. 23, 8-12. Bergs, V. V. (1969). Rat virus-mediated suppression of leukemia induction by Moloney virus in rats. Cancer Res. 28, 1669-1672. Bernard, S. L., Leathers, C. W., Brobst, D. E, and Gorham, J. R. (1983). Estrogen-induced bone marrow depression in ferrets. Am. J. Vet. Res. 44, 657-661. Bhatt, E N., Jacoby, R. O., Morse, H. C., III, and New, A. E., eds. (1986). "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research." Academic Press, Orlando, Florida. Bihun, C. G., and Percy, D. H. (1995). Morphologic changes in the nasal cavity associated with sialodacryoadenitis virus infection in the Wistar rat. Vet. Pathol. 32, 1-10. Blakley, B. R., and Rousseaux, C. G. (1991). Effect of ivermectin on the immune response in mice. Am. J. Vet. Res. 52, 593-595. Blakley, B. R., Sisodia, C. S., and Mukkur, T. K. (1980). The effect of methylmercury, tetraethyl lead, and sodium arsenite on the humoral immune response in mice. Toxicol. Appl. Pharmacol. 52, 245-254. Blanden, R. V., and Mims, C. A. (1973). Macrophage activation in mice infected with ectromelia or lymphocytic choriomeningitis viruses. Aust. J. Exp. Biol. Med. Sci. 51, 393-398. Bologne, R., Demoulin, A., Schaaps, J. E, Hustin, J., and Lambotte, R. (1983). Influence of ultrasonics on the fecundity of female rats. C.R. Seances Soc. Biol. Fil. 177, 381-387. Bolon, B., Bonnefoi, M. S., Roberts, K. C., Marshall, M. W., and Morgan, K. T. (1991). Toxic interactions in the rat nose: Pollutants from soiled bedding and methyl bromide. Toxicol. Pathol. 19, 571-579. Bonnard, B. D., Manders, E. K., Campbell, D. A., Herbermann, R. B., and Collins, M. J. (1976). Immunosuppressive activity of a subline of the mouse El-4 lymphoma. Evidence for minute virus of mice causing the inhibition. J. Exp. Med. 143, 187-205. Bonventre, E F., Hubel, H. C., Michael, J. G., and Nickol, A. D. (1980). Impaired resistance to bacterial infection after tumor implant is traced to lactic dehydrogenase virus. Infect. Immun. 30, 316-319.

NEIL S. LIPMAN AND SCOTT E. PERKINS Boorman, G. A., Luster, M. I., Dean, J. H., Cambell, M. L., Lauer, L. A., Talley, E A., Wilson, R. E., and Collins, M. J. (1982). Peritoneal and macrophage alterations caused by naturally occurring mouse hepatitis virus. Am. J. Pathol. 106, 110-117. Borriello, S. P. (1995). Clostridial disease of the gut. Clin. Infect. Dis. 20(Suppl. 2), $242-$250. Boschert, K. R., Schoeb, T. R., Chandler, D. B., and Dillehay, D. L. (1988). Inhibition of phagocytosis and interleukin- 1 production in pulmonary macrophages from rats with sialodacryoadenitis virus infection. J. Leukoc. Biol. 44, 87-92. Bosnjak, Z. J. (1991). Cardiac effects of anesthetics. In "Mechanisms of Anesthetic Action in Skeletal, Cardiac, and Smooth Muscle" (T. J. J. Blanck and D. M. Wheeler, eds.), pp. 91-96. Plenum Press, New York. Bounous, D., Bordreaux, M. K., and Hoskins, J. D. (1995). Hematology of normal dogs and cats and responses to disease. In "Veterinary Pediatrics, Dogs and Cats from Birth to Six Months" (J. D. Hoskins, ed.), pp. 337-353. Saunders, Philadelphia. Bowman, R. E., Wold, R. C., and Sackett, G. P. (1970). Circadian rhythms of plasma 17-hydrocorticosteroids in the infant monkey. Proc. Soc. Exp. Biol. Med. 133, 342-344. Bradfield, J. E, Schachtman, T. R., McLaughlin, R. M., and Steffen, E. K. (1992). Behavioral and physiologic effects of inapparent wound infection in rats. Lab. Anim. Sci. 42, 572-578. Brainard, G. C., Richardson, B. A., King, T. S., Matthews, S. A., and Reiter, R. J. (1983). The suppression of pineal melatonin content and N-acetyltransferase activity by different light irradiances in the Syrian hamsters: A doseresponse relationship. Endocrinology 113, 293-296. Brainard, G. C., Rollag, M. D., and Hanifin, J. P. (1997). Photic regulation of melatonin in humans: Ocular and neural signal transduction. J. Biol. Rhythms 12, 537-546. Brand, J. M., Kirchner, H., Poppe, C., and Schmucker, P. (1998). Cytokine release and changes in mononuclear cells in peripheral blood under the influence of general anesthesia. Anaesthesist 47, 379-386. Braunsteiner, H., and Friend, C. (1954). Viral hepatitis associated with transplantable mouse leukemias. I. Acute hepatic manifestations following treatment with urethane or methylformamide. J. Exp. Med. 100, 665-677. Brinton, M. A. (1982). Lactate dehydrogenase-elevating virus. In "The Mouse in Biomedical Research, Vol. II: Diseases" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), pp. 193-208. Academic Press, New York. Brockman, R. P. (1981). Effect of xylazine on plasma glucose, glucagon, and insulin concentrations in sheep. Res. Vet. Sci. 30, 383-384. Broderson, J. R., Lindsey, J. R., and Crawford, J. E. (1976). The role of environmental ammonia in respiratory mycoplasmosis of rats. Am. J. Pathol. 85, 115-127. Bro-Jorgensen, K., and Volkert, M. (1972). Haemopoietic defects in mice infected with lymphocytic choriomeningitis virus. I. The enhanced x-ray sensitivity of the virus infected mice. Acta Pathol. Microbiol. Scand. 80, 845-852. Bro-Jorgensen, K., and Volkert, M. (1974). Defects in the immune system of mice infected with lymphocytic choriomeningitis virus. Infect. Immun. 9, 605-614. Bro-Jorgensen, K., Guttler, F., Jorgensen, P. N., and Volkert, M. (1975). T lymphocyte function as the principal target of lymphocytic choriomeningitis virus-induced immunosuppression. Infect. Immun. 11, 622-629. Brown, A. M. (1976). Minireview: Ultrasound and communication in rodents. Comp. Biochem. Physiol. 53, 313-317. Brown, J. W. (1998). Metabolic and membrane-altering toxins, molecular differentiation factors, and pheromones in the evolution and operation of endocrine-signaling systems. Horm. Metab. Res. 30, 66-69. Brown, D. W., Welsh, R. M., and Like, A. A. (1993). Infection of peripancreatic lymph nodes but not islets precedes Kilham rat virus-induced diabetes in BB/Wor rats. J. Virol. 67, 5873-5878. Brown, K. J., and Grunberg, N. E. (1995). Effects of housing on male and fe-

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH

male rats: Crowding stresses male but calms females. Physiol. Behav. 58, 1085-1089. Brown, K. J., and Grunberg, N. E. (1996). Effects of environmental conditions on food consumption in female and male rats. Physiol. Behav. 60, 293297. Brownstein, D. G., and Weir, E. C. (1987). Immunostimulation in mice infected with Sendai virus. Am. J. Vet. Res. 48, 1692-1696. Bruce, D. L. (1972). Halothane inhibition of phytohemagglutinin-induced transformation of lymphocytes. Anesthesiology 36, 201-205. Bruce, D. L. (1975). Halothane inhibition of RNA and protein synthesis of PHA-treated human lymphocytes. Anesthesiology 42, 11-14. Brunner, C. J., and Muscoplat, C. C. (1980). Immunomodulatory effects of levamisole J. Am. Vet. Med. Assoc. 176, 1159-1162. Brunner, L. J., Dipiro, J. T., and Feldman, S. (1994). Metabolic cage isolation reduces antipyrine clearance in rats. J. Pharm. Pharmacol. 46, 581-584. Bryan, T. E., and Gildersleeve, R. P. (1988). Effects of nonionizing radiation on birds. Comp. Biochem. Physiol. A 89, 511-530. Buckley, J. P., and Smookler, H. H. (1970). Cardiovascular and biochemical effects of chronic intermittent neurogenic stimulation. In "Physiological Effects of Noise" (B. L. Welch and A. S. Welch, eds.), pp. 75-84. Plenum Press, New York. Budillon, G., Carella, M., and Coltorti, M. (1972). Phenobarbital liver microsomal induction in MHV-3 viral hepatitis of the mouse. Experientia 28, 1011-1012. Budillon, G., Carella, M., DeMarco, E, and Mazzacca, G. (1973). Effect of phenobarbital on MHV-3 viral hepatitis of the mouse. Pathol. Microbiol. 39, 461-466. Burkart, W., Jung, T., and Frasch, G. (1999). Damage pattern as a function of radiation quality and other factors. C.R. Acad. Sci. III. 322, 89-101. Butler, M. M., Griffey, S. M., Clubb, E J., Jr., Gerrity, L. W., and Campbell, W. B. (1990). The effect of euthanasia technique on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility. Lab. Anim. Sci. 40, 277-283. Cacciatore, L., and Antoniello, S. (1971). Arginase activity of mouse serum and liver tissue in some conditions of experimental liver damage. Enzymologia 41, 112-120. Cahill, R. J., Foltz, C. J., Fox, J. G., Dangler, C. A., Powrie, E, and Schauer, D. B. (1997). Inflammatory bowel disease: An immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus. Infect. Immun. 65, 3126-3131. Campa, M., Garizelli, C., and Falcone, G. (1975). Depression of contact sensitivity and enhancement of aritibody response in Pseudomonas aeruginosa-infected mice. Infect. Immun. 12, 1252-1257. Campa, M., Garizelli, C., Ferrannini, E., and Falcone, G. (1976). Evidence for suppressor cell activity associated with depression of contact sensitivity in Pseudomonas aeruginosa-infected mice. Clin. Exp. Immunol. 26, 355362. Campa, M., Ferrannini, E., Colizzi, V., and Garzelli, G. (1977). Distribution of 51 Cr-labeled lymph node cells in Pseudomonas aeruginosa-infected mice. J. Nucl. Med. Allied Sci. 21, 37-42. Campbell, D. A., Staal, S. P., Manders, E. K., Bonnard, G. D., Oldman, R. K., Salzman, L. A., and Herbermann, R. B. (1977). Inhibition of in vitro lymphoproliferative responses by in vivo passaged rat 13762 mammary adenocarcinoma cells. II. Evidence that Kilham rat virus is responsible for the inhibitory effect. Cell. lmmunol. 33, 378-391. Cantor, K. P. (1997). Drinking water and cancer. Cancer Causes Control 8, 292-308. Capitanio, J. P., and Lerche, N. W. (1998). Social separation, housing relocation, and survival in simian AIDS: A retrospective analysis. Psychosom. Med. 60, 235-244. Caputa, M., and Demicka, A. (1995). Warm rearing modifies temperature regulation in rats. J. Physiol. PharmacoL 46, 195-203. Caron, E, Ducrotte, P., Lerebours, E., Colin, R., Humbert, G., and Denis, P.

1167 (1991). Effects of amoxicillin-clavulanate combination on the motility of the small intestine in human beings. Antimicrob. Agents Chemother. 35, 1085-1088. Carrano, V., Barthold, S. W., Beck, D. L., and Smith, A. L. (1984). Alteration of viral respiratory infections of mice by prior infection with mouse hepatitis virus. Lab. Anim. Sci. 34, 573-576. Carthew, P. (1981). Inhibition of the mitotic response in regenerating mouse liver during viral hepatitis. Infect. Immun. 33, 641-642. Casebolt, D. B., Spalding, D. M., Schoeb, T. R., and Lindsey, J. R. (1987). Suppression of immune response induction in Peyer's patch lymphoid cells from mice infected with mouse hepatitis virus. Cell. Immunol. 1t)9, 97103. Cassell, G. H. (1982). The pathogenic potential of mycoplasmas: Mycoplasma pulmonis as a model. Rev. Infect. Dis. 4, S 18-$34. Cassell, G. H., Lindsey, J. R., Davis, J. K., Davidson, M. K., Brown, M. B., and Mayo, J. G. (1981). Detection of natural Mycoplasma pulmonis infection in rats and mice by an enzyme-linked immunosorbent assay. Lab. Anim. Sci. 31, 676-682. Castro, M., Morgenthaler, T. I., Hoffman, O. A., Standing, J. E., Rohrbach, M. S., and Limper, A. H. (1993). Pneumocystis carinii induces the release of arachidonic acid and its metabolites from alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 9, 73-81. Catlin, E I. (1986). Noise-induced hearing loss. Am. J. Otol. 7, 141-149. Cayen, M. N., Givner, M. L., and Kraml, M. (1972). Effect of diurnal rhythm and food withdrawal on serum lipid levels in the rat. Experientia 28, 502503. Celly, C. S., Atwal, O. S., McDonell, W. N., and Black, W. D. (1999). Histopathologic alterations induced in the lungs of sheep by use of alpha2adrenergic receptor agonists. Am. J. Vet. Res. 60, 154-161. Chance, M. R. A. (1957). The contribution of environment to uniformity. Collected Papers Lab. Anim. Bur. 6, 59-73. Chedid, A., and Nair, V. (1972). Diurnal rhythm in endoplasmic reticulum of rat liver. Electron microscopic study. Science 175, 176-179. Chen, K. K., Anderson, R. C., Steldt, E A., and Mills, C. A. (1943). Environmental temperature and drug action in mice. J. Pharmacol. Exp. Ther. 79, 127-132. Chen, W., Havell, E. A., Moldawer, L. L., Mclntyre, K. W., Chizzonite, R. A., and Harmsen, A. G. (1992). Interleukin 1: An important mediator of host resistance against Pneumocystis carinii. J. Exp. Med. 176, 713-718. Chen, W., Havell, E. A., Gigliotti, E, and Harmsen, A. G. (1993). Interleukin-6 production in a murine model of Pneumocyctis carinii pneumonia: Relation to resistance and inflammatory response. Infect. lmmun. 61, 97-102. Cherbut, C., Ferre, J. P., Corpet, D. E., Ruckebusch, Y., and Delori-Laval, J. (1991). Alterations of intestinal microflora by antibiotics. Effects on fecal excretion, transit time, and colonic motility in rats. Digest Dis. Sci. 36, 1729-1734. Choi, G. C., McQuinn, J. S., and Jennings, B. L., Hassett, D. J., and Michaels, S. E. (1994). Effect of population size on humidity and ammonia levels in individually ventilated microisolation rodent caging. Contemp. Top. Lab. Anim. Sci. 33, 77- 81. Chomarat, P., Sipowicz, M. A., Diwan, B. A., Fornwald, L. W., Awasthi, Y. C., Anver, M. R., Rice, J. M., Anderson, L. M., and Wild, C. P. (1997). Distinct time courses of increase in cytochromes P450, 1A2, 2A5, and glutathione S-transferases during the progressive hepatitis associated with Helicobacter hepaticus. Carcinogenesis 18, 2179-2190. Christensen, J. P., Johansen, J., Marker, O., and Thomsen, A. R. (1995). Circulating intercellular adhesion molecule- 1 (ICAM- 1) as an early and sensitive marker for virus-induced T cell activation. Clin. Exp. Immunol. 1tl2, 268273. Christian, J. J., and LeMunyan, C. D. (1958). Adverse effects of crowding on lactation and reproduction of mice and two generations of their progeny. Endocrinology 63, 517-529. Chung, H. J., Reyes, A. B., Watanabe, K., Tomogane, H., and Wakasugi, N.

1168 (1997). Embryonic abnormality caused by male pheromonal effect in pregnancy block in mice. Biol. Reprod. 57, 312-319. Cinti, D. L., Lemelin, M. A., and Christian, J. (1976). Induction of liver microsomal mixed-function oxidases by volatile hydrocarbons. Biochem. Pharmacol. 25, 100-103. Clark, E. A., Russell, P. H., Egghart, M., and Horton, M. A. (1979). Characteristics and genetic control of NK-cell-mediated cytotoxicity activated by naturally acquired infection in the mouse. Int. J. Cancer 24, 688-699. Clark, J. D., Rager, D. R., and Calpin, J. P. (1997a). Animal well-being. III. An overview of assessment. Lab. Anim. Sci. 47, 580-585. Clark, J. A., Jr., Myers, P. H., Goelz, M. E, Thigpen, J. E., and Forsythe, D. B. (1997b). Pica behavior associated with buprenorphine administration in the rat. Lab. Anim. Sci. 47, 300-303. Clifford, D. H. (1984). Preanesthesia, anesthesia, analgesia, and euthanasia. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 527-562. Academic Press, New York. Clough, G. (1982). Environmental effects on animals used in biomedical research. Biol. Rev. 57, 487-523. Clough, G., and Fasham, J. A. L. (1975). A "silent" fire alarm. Lab. Anim. 9, 193-196. Cocke, R., Moynihan, J. A., Cohen, N., Grota, L. J., and Ader, R- (1993). Exposure to conspecific alarm chemosignals alters immune responses in BALB/c mice. Brain Behav. Immun. 7, 36-46. Cole, B. C., Golightly-Rowland, L., and Ward, J. R. (1975). Arthritis in mice induced by Mycoplasma pulmonis: Humoral antibody and lymphocyte responses of CBA mice. Infect. lmmun. 12, 1083-1092. Colle, J. H., Saron, M. F., and Truffa-Bachi, P. (1993). Altered cytokine genes expression by conA-activated spleen cells from mice infected by lymphocytic choriomeningitis virus. Immunol. Lett. 35, 247-253. Conney, A. H., and Burns, J. J. (1972). Metabolic interactions among environmental chemicals and drugs. Science 178, 576-586. Consolo, S., Garrattini, S., and Valzelli, L. (1965). Sensitivity of aggressive mice to centrally acting drugs. J. Pharm. PharmacoL 17, 594-595. Cook, J. A., Marconi, E. A., and Di Luzio, N. R. (1974). Lead, cadmium, endotoxin interaction: Effect on mortality and hepatic function. ToxicoL Appl. Pharmacol. 28, 292-302. Cook, J. A., Hoffman, E. O., and Di Luzio, N. R. (1975). Influence of lead and cadmium on the susceptibility of rats to bacterial challenge. Proc. Soc. Exp. Biol. Med. 15tl, 741-747 Cooper, K. E., Ferguson, A. V., and Veale, W. L. (1980). Modification of thermoregulatory responses in rabbits reared at elevated environmental temperatures. J. Physiol. (Lond.) 3tl3, 165-172. Corning, B. E, and Lipman, N. S. (1991). A comparison of rodent caging system based on microenvironmental parameters. Lab: Anim. Sci. 41, 498503. Corsi, M., Mariconti, P., Calvillo, L., Falchi, M., Tiengo, M., and Ferrero, M. E. (1995). Influence of inhalational, neuroleptic, and local anaesthesia on lymphocyte subset distribution. Int. J. Tissue React. 17, 211-217. Coutelier, J. P., Van Broeck, J., and Wolf, S. E (1995). Interleukin-12 gene expression after viral infection in the mouse. J. Virol. 69, 1955-1958. Cox, J. C. (1977). Altered immune responsiveness associated with Encephalitozoon cuniculi infection in rabbits. Infect. Immun. 15, 392-395. Cox, J. C., and Gallichio, H. A. (1978). Serological and histological studies on adult rabbits with recent, naturally acquired encephalitozoonosis. Res. Vet. Sci. 24, 260-261. Crabbe, J. C., Wahlsten, D., and Dudek, B. C. (1999). Genetics of mouse behavior: Interactions with laboratory environment. Science 284, 16701672. Craigie, J. (1966). Bacillus piliformis (Tyzzer) and Tyzzer's disease of the laboratory mouse. II. Mouse pathogenicity of B. piliformis grown in embryonated eggs. Proc. R. Soc. Lond. 165, 61-78. Cramer, D. V. (1983). Genetic monitoring techniques in rats. ILAR News 26, 15-19.

NEIL S. LIPMAN AND SCOTT E. PERKINS Crawley, J. N., Belknap, J. K., Collins, A., Crabbe, J. C., Frankel, W., Henderson, N., Hitzeman, R. J., Maxson, S. C., Miner, L. L., Silva, A. J., Wehner, J. M., Wynshaw-Boris, A., and Paylor, R. (1997). Behavioral phenotypes of inbred mouse strains: Implications and recommendations for molecular studies. Psychopharmacology 132, 107-124. Cray, C., Mateo, M. O., and Altman, N. H. (1993). In vitro and long-term in vivo immune dysfunction after infection of BALB/c mice with mouse hepatitis virus strain A59. Lab. Anim. Sci. 43,169-174. Cullen, B. E (1974). The effects of halothane and nitrous oxide on phagocytosis and human leukocyte metabolism. Anesth. Analg. 53, 531-536. Cullen, B. E, Sample, W.F., and Chretien, P. B. (1972). The effect of halothane on phytohemagglutinin-induced transformation of human lymphocytes in vitro. Anesthesiology 36, 206-212. Cullen, B. E, Duncan, P. G., and Ray-Keil, L. (1976). Inhibition of cellmediated cytotoxicity by halothane and nitrous oxide. Anesthesiology 44, 386-390. Dairman, W., and Balazs, T. (1970). Comparison of liver microsome enzyme systems and barbiturate sleep times in rats caged individually or communally. Biochem. PharmacoL 19, 951-955. Darrigrand, A. A., Singh, S. B., and Lang, C. M. (1984). Effects of Kilham rat virus on natural killer cell-mediated cytotoxicity in Brown Norway and Wistar Furth rats. Am. J. Vet. Res. 45, 200-202. Daubeck, J. L., Daughety, M. J., and Petty, C. (1974). Lincomycin-induced cardiac arrest: A case report and laboratory investigation. Anesth. Analg. 53, 563 -567. Dauchy, R. T., Sauer, L. A., Blask, D. E., and Vaughan, G. M. (1997). Light contamination during the dark phase in "photoperiodically controlled" animal rooms: Effect on tumor growth and metabolism in rats. Lab. Anim. Sci. 47, 511-518. Davis, D. E. (1978). Social behavior in a laboratory environment. In "Laboratory Animal Housing," pp. 44-63. National Academy of Sciences, Washington, D.C. Davis, J. K., Thorp, R. B., Maddox, P. A., Brown, M. B., and Cassell, G. H. (1982). Murine respiratory mycoplasmosis in F344 and LEW rats: Evolution of lesions and lung lymphoid cell populations. Infect. Immun. 36, 720729. Davis, J. A., Greenfield, R. E., and Brewer, T. G. (1993). Benzocaine-induced methemoglobinemia attributed to topical application of the anesthetic in several laboratory animal species. Am. J. Vet. Res. 54, 1322-1326. Dean, J. H., and Murray, M. J. (1991). Toxic responses of the immune system. In "Casarett and Doull's Toxicology, the Basic Science of Poisons" (M. O. Amdur, J. Doull, and C. D. Klassen, eds.), pp. 282-333. Pergamon Press, New York. Degraeve, N. (1981). Carcinogenic, teratogenic, and mutagenic effects of cadmium. Mutat. Res. 86, 115-135. Degre, M., and Glasgow, L. A. (1968). Synergistic effect in viral bacterial infection. I. Combined infection of the respiratory tract in mice with parainfluenza virus and Hemophilus influenza. J. Infect. Dis. 118, 449-462. Degre, M., and Solberg, L. A. (1971). Synergistic effect in viral bacterial infection. III. Histopathologic changes in the trachea of mice following viral and bacterial infection. Acta PathoL Microbiol. Scand. 79, 129-136. Deimling, M. J., and Schnell, R. C. (1980). Circadian rhythms in the biological response and disposition of ethanol in the mouse. J. Pharmacol. Exp. Ther. 213, 1-8. de La Torre, J. C., Mallory, M., Brot, M., Gold, L., Koob, G., Oldstone, M. B., and Masliah, E. (1996). Viral persistence in neurons alters synaptic plasticity and cognitive functions without destruction of brain cells. Virology 220, 508-515. DeLong, D., and Manning, P. J. (1994). Bacterial diseases. In "The Biology of the Laboratory Rabbit" (P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds.), pp. 129-170. Academic Press, Orlando, Florida. de Souza, M. S., Smith, A. L., and Bottomly, K. (1991). Infection of BALB/ cByJ mice with the JHM strain of mouse hepatitis virus alters in vitro

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH splenic T cell proliferation and cytokine production. Lab. Anim. Sci. 41, 99-105. Didier, E. S., and Shadduck, J. A. (1988). Modulated immune responsiveness associated with experimental Encephalitozoon cuniculi infection in BALB/c mice. Lab. Anim. Sci. 38, 680-684. DiGiacomo, R. F., Deeb, B. Ji, and Anderson, R. J. (1992). Hypervitaminosis A and reproductive disorders in rats. Lab. Anim. Sci. 42, 250-254. Dillberger, J. E., Monroy, P., and Altman, N. H. (1987). Delayed increase in plasma lactic dehydrogenase activity in mouse hepatitis virus-infected mice subsequently infected with lactic dehydrogenase virus. Lab. Anim. Sci. 37, 792-794. Diwan, B. A., Ward, J. M., Ramljak, D., and Anderson, L. M. (1997). Promotion by Helicobacter hepaticus-induced hepatitis of hepatic tumors initiated by N-nitrosodimethylamine in male A/JCr mice. Toxicol. Pathol. 25, 597-605. Dixon, D. M., and Misfeldt, M. L. (1994). Proliferation of immature T cells within the splenocytes of athymic mice by Pseudomonas exotoxin A. Cell Immunol. 158, 71-82. Dobbie, J. W., and Smith, M. J. B. (1982). Silicate nephrotoxicity in the experimental animal: The missing factor in analgesic nephropathy. Scott. Med. J. 27, 10-16. Doherty, P. C., Hou, S., and Tripp, R. A. (1994). CD8 § T-cell memory to viruses. Curr. Opin. Immunol. 6, 545-552. Donnelly, H., and Saibaba, P. (1993). Light intensity and the oestrous cycle in albino and normally pigmented mice. Lab. Anim. 27, 385-390. Doughty, M. J., and Stuart, D. (1995). Quantification of the hemolysis associated with use of T-61 as a euthanasia agent in rabbitsma comparison with euthanyl (pentobarbital sodium) and the impact on serum hexosaminidase measurements. Can. J. Physiol. Pharmacol. 73, 1274-1280. Drickamer, L. C. (1977). Seasonal variation in litter size, body weight, and sexual maturation in juvenile female house mice (Mus muculus). Lab. Anita. 11, 159-162. Drickamer, L. C. (1982). Acceleration and delay of sexual maturation in female mice via chemosignals: Circadian rhythm effects. Biol. Reprod. 27, 596601. Drickamer, L. C. (1984). Seasonal variations in acceleration and delay of sexual maturation in female mice by urinary chemosignals. J. Reprod. Fertil. 72, 55-58. Drickamer, L. C. (1990a). Environmental factors and age of puberty in female house mice. Dev. Psychobiol. 23, 63-73. Drickamer, L. C. (1990b). Seasonal variation in fertility, fecundity, and litter sex ratios in laboratory and wild stocks of house mice. Lab. Anim. Sci. 40, 284-288. Driscoll, D. J., Gillette, P. C., Lewis, R. M., Hartley, C. J., and Schwartz, A. (1979). Comparative hemodynamic effects of isoproterenol, dopamine, and dobutamine in the newborn dog. Pediatr. Res. 13, 1006-1009. Drozdowicz, C. K., Bowman, T. A., Webb, M. L., and Lang, C. M. (1990). Effect of in-house transport on murine plasma corticosterone concentration and blood lymphocyte populations. Am. J. Vet. Res. 51, 1841-1846. DuMond, E V., and Hutchinson, T. C. (1967). Squirrel monkey reproduction: The fatted male phenomenon and seasonal spermatogenesis. Science 158, 1467-1470. Duncan, T. E., and O' Steen, W. K. (1985). The diurnal susceptibility of rat retinal photoreceptors to light-induced damage. Exp. Eye Res. 41, 497-507. Dymsza, H. A., Miller, S. A., Maloney, J. E, Goff, U., Wolf, M. H., and Fine, D. H. (1963). Equilibration of the laboratory rat following exposure to shipping stresses. Lab. Anim. Care 13, 60-65. Dyrberg, T., Schwimmbeck, P. L., and Oldstone, M. B. A. (1988). Inhibition of diabetes in BB rats by virus infection. J. Clin. Invest. 81, 928-931. Edwards, G. S., Fox, J. G., Policastro, P., Goff, U., Wolf, M. H., and Fine, D. H. (1979). Volatile nitrosamine contamination of laboratory animal diets (letter). Cancer Res. 39, 1857-1858. Eger, E. I., II, Johnson, B. H., Strum, D. P., and Ferrell, L. D. (1987). Studies of

1169 the toxicity of 1-653, halothane, and isoflurane in enzyme-induced, hypoxic rats. Anesth. Analg. 66, 1227-1229. Elis, J., and DiPaolo, J. A. (1967). Aflatoxin B 1. Induction of malformations. Arch. Pathol. 83, 53-57. Engers, H. D., Louis, J. A., Zubler, R. H., and Hirt, B. (1981). Inhibition of T cell mediated functions by MVM(i), a parvovirus closely related to minute virus of mice. J. Immunol. 127, 2280-2285. Even, C., Rowland, R. R., and Plagemann, P. G. (1995). Mouse hepatitis virus infection of mice causes long-term depletion of lactate dehydrogenaseelevating virus-permissive macrophages and T lymphocyte alterations. Virus Res. 39, 355-364. Everitt, J. I., Ross, P. W., and Davis T. W. (1988). Urologic syndrome associated with wire caging in AKR mice. Lab. Anim. Sci. 38, 609-611. Exon, J. H., Koller, L. D., O'Reilly, C. A., and Bercz, J. P. (1987). Immunotoxicologic evaluation of chlorine-based drinking water disinfectants, sodium hypochlorite, and monochloramine. Toxicology 44, 257-269. Fallon, M. T., Benjamin, W. H., Jr., Schoeb, T. R., and Briles, D. E. (1991). Mouse hepatitis virus strain UAB infection enhances resistance to Salmonella typhimurium in mice by inducing suppression of bacterial growth. Infect. Immun. 59, 852-856. Fariss, B. L., Hurley, T. J., Hane, S., and Forsham, P. H. (1969). Reduction of testicular testosterone in rats by ether anesthesia. Endocrinology 84, 940942. Faulkner, C. B., Simecka, J. W., Davidson, M. K., Davis, J. K., Schoeb, T. R., Lindsey, J. R., and Everson, M. P. (1995). Gene expression and production of tumor necrosis factor alpha, intedeukin 1, interleukin 6, and gamma interferon in C3H/HeN and C57BL/6N mice in acute Mycoplasma pulmonis disease. Infect. Immun. 63, 4084-4090. Fay, R. (1988). Hearing in vertebrates: A psychophysics data book. Hill Fay Associates, Winnetka, Illinois. Feigin, R. D., San Joaquin, V. H., Haymond, M. W., and Wyatt, R. G. (1969). Daily periodicity of susceptibility of mice to pneumococcal infection. Nature 224, 379-380. Fekete, E., Wooley, G., and Little, C. C. (1941). Histological changes following ovariectomy in mice. I. DBA high tumor strain. J. Exp. Med. 74, 1-7. Feldman, D. B., and Gupta, B. N. (1976). Histopathologic changes in laboratory animals resulting from various methods of euthanasia. Lab. Anim. Sci. 26, 218-221. Fernandes, C., and File, S. E. (1993). Beware the builders: Construction noise changes [ 14C]GABA release and uptake from amygdaloid and hippocampal slices in the rat. Neuropharmacology 32, 1333-1336. Festing, M. E W. (1982). Genetic contamination of laboratory animal colonies: An increasingly serious problem. ILAR News 25, 6-10. Fettman, M. J., Valerius, K. D., Ogilvie, G. K., Bedwell, C. L., Richardson, K. L., Walton, J. A., and Hamar, D. W. (1999). Effects of dietary cysteine on blood sulfur amino acid, glutathione, and malondialdehyde concentrations in cats. Am. J. Vet. Res. 60, 328-333. Fidler, I. J. (1977). Depression of macrophages in mice drinking hyperchlorinated water. Nature 270, 735-736. File, S. E., Zangrossi, H., Jr., and Andrews, N. (1993). Novel environment and cat odor change GABA and 5-HT release and uptake in the rat. Pharmacol. Biochem. Behav. 45, 931-934. Fink, G. B., and Iturrian, W. B. (1970). Influence of age, auditory conditioning, and environmental noise on sound induced seizures and seizure threshold in mice. In "Physiological Effects of Noise" (B. L. Welch and A. S. Welch, eds.), pp. 211-226. Plenum Press, New York. Fitzmaurice, M. A. (1988). Physiological relationships among stress, viruses, and cancer in experimental animals. Int. J. Neurosci. 39, 307-324. Fitzmaurice, M. A., Riley, V., and Santisteban, G. A. (1974). Biological synergism between the LDH-virus and Eperythrozoon coccoides: Studies on the mechanism. Pathol. Biol. 20, 743-750. Flynn, R. J. (1959). Studies on the etiology of ringtail of rats. Proc. Anim. Care Panel 9, 155-160.

1170 Flynn, R. J. (1963a). Pseudomonas aeruginosa infection and its effects on biological and medical research. Lab. Anim. Care 13, 1-6. Flynn, R. J. (1963b). Pseudomonas aeruginosa infection and radio-biological research at the Argonne National Laboratory: Effects, diagnosis, epizootiology, and control. Lab. Anim. Care 13, 25-35. Flynn, R. J. (1963c). The diagnosis of Pseudomonas aeruginosa infection in mice. Lab. Anim. Care 13, 126-130. Foltz, C. J., Fox, J. G., Cahill, R., Murphy, J. C., Yan, L., Shames, B., and Schauer, D. B. (1998). Spontaneous inflammatory bowel disease in multiple mutant mouse lines: Association with colonization by Helicobacter hepaticus. Helicobacter 3, 69-78. Fox, R. R., and Witham, B. A., eds. (1997). Strain selection. In "Handbook on Genetically Standardized JAX Mice," pp. 18-20. The Jackson Laboratory, Bar Harbor, Maine. Fox, J. G., Murphy, J. C., and Igras, V. E. (1977). Adverse effects of mouse hepatitis virus on ascites myeloma passage in the BALB/c mouse. Lab. Anim. Sci. 27, 173-179. Fox, J. G., Li, X., Yan, L., Cahill, R. J., Hurley, R., Lewis, R., and Murphy, J. C. (1996a). Chronic proliferative hepatitis in A/JCr mice associated with persistent Helicobacter hepaticus infection: A model of helicobacter-induced carcinogenesis. Infect. Immun. 64, 1548-1558. Fox, J. G., Yan, L., Shames, B., Campbell, J., Murphy, J. C., and Li, X. (1996b). Persistent hepatitis and enterocolitis in germfree mice infected with Helicobacter hepaticus. Infect. Immun. 64, 3673-3681. Franklin, C. L., Riley, L. K., Livingston, R. S., Beckwith, C. S., BeschWilliford, C. L., and Hook, R. R. (1998). Enterohepatic lesions in SCID mice infected with Helicobacter bilis. Lab. Anim. Sci. 48, 334-339. Fraser, L. R., and Taylor-Robinson, D. (1977). The effect of Mycoplasma pulmonis on fertilization and preimplantation development in vitro of mouse eggs. Fertil. Steril. 28, 488-498. Fries, A. S., and Ladefoged, O. (1979). The influence of Bacillus piliformis (Tyzzer) infections on the reliability of pharmacokinetic experiments in mice. Lab. Anim. 13, 257-261. Frisk, C. S. (1987). Bacterial and mycotic diseases. In "Laboratory Hamsters" (G. L. Van Hoosier, Jr. and C. W. McPherson, eds.), pp. 111-133. Academic Press, Orlando, Florida. Fuhrman, G. J., and Fuhrman, F. A. (1961). Effects of temperature on the action of drugs. Annu. Rev. Pharmacol. 1, 65-78. Fullwood, S., Hicks, T. A., Brown, J. C., Norman, R. L., and McGlone, J. J. (1998). Floor space needs for laboratory mice: C57BL/6 males in solidbottom cages with bedding. ILAR J. 39, 29-36. Gabaldon, M., Capdevila, C., and Zuniga, A. (1992). Effect of spontaneous pathology and thrombin on leukocyte adhesion to rat aortic endothelium. Atherosclerosis 93, 217-228. Gaertner, D. J., Boschert, K. R., and Schoeb, T. R. (1987). Muscle necrosis in Syrian hamsters resulting from intramuscular injections of ketamine and xylazine. Lab. Anim. Sci. 37, 80-83. Galani, R., Jarrard, L. E., Will, B. E., and Kelche, C. (1997). Effects of postoperative housing conditions on functional recovery in rats with lesions of the hippocampus, subiculum, or entorhinal cortex. Neurobiol. Learn. Mem. 67, 43-56. Galloway, J. H. (1968). Antibiotic toxicity in white mice. Lab. Anim. Care 18, 421-425. Galton, M. (1963). Myobic mange in the mouse leading to skin ulceration and amyloidosis. Am. J. Pathol. 43, 855-865. Gamble, M. R., and Clough, G. (1976). Ammonia build-up in animal boxes and its effect on rat tracheal epithelium. Lab. Anim. 10, 93-104. Garcia, T. (1998). Pharmaceutical drugs appearing in Europe's drinking water. In "Enviro Link News Service," <www.envirolink.org>. Garlinghouse, L. E., Jr., and Van Hoosier, G. L. (1978). Studies on adjuvantinduced arthritis, tumor transplantability, and serologic response to bovine serum albumin in Sendai virus infected rats. Am. J. Vet. Res. 39, 297-300. Garlinghouse, L. E., Jr., and Van Hoosier, G. L., Jr. (1982). The suppression of

NElL S. LIPMAN AND SCOTT E. PERKINS lymphocytic mitogenesis in Sendai virus infected rats. Am. Soc. Microbiol. Abstr. Annu. Meet., 258. Geber, W. E (1970). Cardiovascular and teratogenic effects of chronic intermittent noise stress. In "Physiological Effects of Noise" (B. L. Welch and S. A. Welch, eds.), pp. 85-90. Plenum Press, New York. Gerber, S. B., Leonard, A., and Jacquet, P. (1980). Toxicity, mutagenicity, and teratogenicity of lead. Mutat. Res. 76, 115-141. Gledhill, A. W. (1961). Enhancement of the pathogenicity of mouse hepatitis virus (MHV- 1) by prior infection of mice with certain leukemia agents. Br. J. Cancer 15, 531-538. Gledhill, A. W., Bilbey, D. L. J., and Niven, J. S. E (1965). Effect of certain murine pathogens on phagocytic activity. Br. J. Exp. Pathol. 46, 433-442. Godfraind, C., Holmes, K. V., and Coutelier, J. P. (1995). Thymus involution induced by mouse hepatitis virus A59 in BALB/c mice. J. Virol. 69, 65416547. Goeth, H., and Appel, K. R. (1974). Experimentelle untersuchungen uber die beeintrachtigung der fertilitat durch mycoplasmeninfektionen des genitaltraktes. Zentralbl. Bakteriol. 228, 282-289. Goettsch, W., Garssen, J., De Gruijl, E R., and Van Loveren, H. (1994). Effects of UV-B on the resistance against infectious diseases. Toxicol. Lett. 72, 359-363. Goh, D. H., and Ferrante, A. (1984). In vitro inhibition of natural killer cell activity by doxycycline. Int. J. Immunopharmacol. 6, 51-54. Gold, L. H., Brot, M. D., Polis, I., Schroeder, R., Tishon, A., de la Torre, J. C., Oldstone, M. B., and Koob, G. E (1994). Behavioral effects of persistent lymphocytic choriomeningitis virus infection in mice. Behav. Neural. Biol. 62, 100-109. Goldfine, I. D., and Arieff, A. I. (1979). Rapid inhibition of basal and glucosestimulated insulin release by xylazine. Endocrinology 105, 920-922. Gordon, C. J., and Fogelson, L. (1994). Metabolic and thermoregulatory responses of the rat maintained in acrylic or wire-screen cages: Implications for pharmacological studies. Physiol. Behav. 56, 73-79. Gordon, C. J., and Watkinson, W. P. (1995). Strain differences in the laboratory rat: Impact on the autonomic, behavioral, and biochemical response to cholinesterase inhibition. J. Toxicol. Environ. Health 45, 59-73. Gordon, A. H., Hart, P. D., and Young, M. R. (1980). Ammonia inhibits phagosome-lysosome fusion in macrophages. Nature 286, 79-80. Gordon, C. J., Lee, K. L., Chen, T. L., Killough, P., and Ali, J. S. (1991). Dynamics of behavioral thermoregulation in the rat. Am. J. Physiol. 261, R705-R711. Gordon, C. J., Becker, P., and Ali, J. S. (1998). Behavioral thermoregulatory responses of single- and group-housed mice. Physiol. Behav. 65, 255-262. Goyer, R. A. (1991). Transplacental transfer of cadmium and fetal effects. Fundam. Appl. Toxicol. 16, 22-23. Granholm, L., and Siesjo, B. K. (1969). The effect of combined respiratory and nonrespiratory alkalosis on energy metabolites and acid-base parameters in the rat brain. Acta Physiol. Scand. 81, 307-314. Green, M. A., and Halliwell, R. E (1997). Selective antagonism of the GABA(A) receptor by ciprofloxacin and biphenylacetic acid. Br. J. Pharmacol. 122, 584-590. Greenman, D. L., Bryant, P., Kodell, R. L., and Sheldon, W. (1982). Influence of cage shelf level on retinal atrophy in mice. Lab. Anim. Sci. 32, 353-356. Gripenberg-Lerche, C., and Toivanen, P. (1993). Yersinia associated arthritis in SHR rats: Effect of the microbial status of the host. Ann. Rheum. Dis. 52, 223 -228. Gripenberg-Lerche, C., and Toivanen, P. (1994). Variability in the induction of experimental arthritis: Yersinia associated arthritis in Lewis rats. Scand. J. Rheumatol. 23, 124-127. Groen, A. (1977). Identification and genetic monitoring of mouse inbred strains using biochemical polymorphisms. Lab. Anim. 11, 209-214. Groen, A., and Lagerwerf, A. J. (1979). Genetic heterogeneity and genetic monitoring of mouse outbred stocks. Lab. Anim. 13, 81-85. Guetta, E., Graziani, Y., and Tal, J. (1986). Suppression of Ehrlich ascites

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH tumors in mice by minute virus of mice. J. Natl. Cancer Inst. 76, 11771180. Guidotti, L. G., Borrow, P., Hobbs, M. V., Matzke, B., Gresser, I., Oldstone, M. B., and Chisari, E V. (1996). Viral cross talk: Intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver. Proc. Natl. Acad. Sci. 93, 4589-4594. Guillet, R., Wyatt, J., Baggs, R. B., and Kellogg, C. K. (1988). Anestheticinduced corneal lesions in developmentally sensitive rats. Invest. Ophthalmol. Vis. Sci. 29, 949-954. Guo, J., Zhou, A., and Moss R. L. (1997). Urine and urine-derived compounds induce c-fos mRNA expression in accessory olfactory bulb. Neuroreport 8, 1679-1683. Guttler, E, Bro-Jorgensen, K., and Jorgensen, P. N. (1975). Transient impaired cell-mediated tumor immunity after acute infection with lymphocytic choriomeningitis virus. Scand. J. Immunol. 4, 327-336. Gwosdow, A. R., and Besch, E. L. (1985). Effect of thermal history on the rat's response to varying environmental temperature. J. Appl. Physiol. 59, 413419. Hackbarth, H., Burow, K., and Schimansky, G. (1983). Strain differences in inbred rats: Influence of strain and diet on haematological traits. Lab. Anim. 17, 7-12. Hadlow, W. J., Grimes, E. E, and Jay, G. E. (1955). Stilbesterol-contaminated feed and reproductive disturbances in mice. Science 122, 643-644. Haemisch, A., and Gartner, K. (1994). The cage design affects intermale aggression in small groups of male laboratory mice: Strain specific consequences on social organization and endocrine activations in two inbred strains (DBA/2J and CBA/J). J. Exp. Anim. Sci. 36, 101-116. Haemisch, A., Voss, T., and Gartner, K. (1994). Effects of environmental enrichment on aggressive behavior, dominance hierarchies, and endocrine states in male DBA/2J mice. Physiol. Behav. 56, 1041-1048. Hailey, J. R., Haseman, J. K., Bucher, J. R., Radovsky, A. E., Malarky, D. E., Miller, R. T., Nyska, A., and Maronpot, R. R. (1998). Impact of Helicobacter hepaticus infection in B6C3F1 mice from twelve National Toxicology Program two-year carcinogenesis studies. Toxicol. Pathol. 26, 602611. Haines, D. C., Gorelick, P. L., Battles, J. K., Pike, K. M., Anderson, R. J., Fox, J. G., Taylor, N. S., Shen, Z., Dewhirst, F. E., Anver, M. R., and Ward, J. M. (1998). Inflammatory large bowel disease in immunodeficient rats naturally and experimentally infected with Helicobacter bilis. Vet. Pathol. 35, 202208. Halberg, E, Haus, E., Cardoso, S. S., Scheving, L. E., Kuhl, J. E, Shiotsuka, R., Rosene, G., Pauly, J. E., Runge, W., Spalding, J. E, Lee, J. K., and Good, R. A. (1973). Toward a chronotherapy of neoplasia tolerance of treatment depends upon host rhythm. Experientia 29, 909-934. Halkett, J. A. E., Davis, A. J., and Natsios, G. A. (1968). The effect of cold stress and Pseudomonas aeruginosa gavage on the survival of three-week-old Swiss mice. Lab. Anim. Care 18, 94-96. Hall, J. E., White, W. J., and Lang, C. M. (1980). Acidification of drinking water: Its effects on selected biologic phenomena in male mice. Lab. Anim. Sci. 30, 643-651. Hall, W. C., Lubet, R. A., Henry, C. J., and Collins, M. J., Jr. (1985). Sendai virus disease processes and research complications. In "Complications of Viral and Mycoplasmal Infections in Rodents to Toxicology Research and Testing" (T. E. Hamm, Jr., ed.), pp. 25-52. Hemisphere Press, Washington, D.C. Hall, J. A., Wander, R. C., Gradin, J. L., Shi-Hua, D., and Jewell, D. E. (1999). Effect of dietary n-6 to n-3 fatty acid ratio on complete blood and total white blood cell counts, and T-cell subpopulations in aged dogs. Am. J. Vet. Res. 60, 319-327. Hamilton, J. R., and Overall, J. C., Jr. (1978). Synergistic infection with murine cytomegalovirus and Pseudomonas aeruginosa in mice. J. Infect. Dis. 137, 775-782. Hammond, C. W., Tompkins, M., and Miller, C. P. (1954). Studies on the sus-

1171 ceptibility to infection following ionizing radiation. I. The time of onset and duration of the endogenous bacteremias in mice. J. Exp. Med. 99, 405410. Hammond, C. W., Ruml, D., Cooper, D. B., and Miller, C. P. (1955). Studies on susceptibility to infection following ionizing radiation. III. Susceptibility of the intestinal tract to oral inoculation with Pseudomonas aeruginosa. J. Exp. Med. 102, 403-411. Han, J., Hosokawa, M., Umezawa, M., Yagi, H., Matsushita, T., Higuchi, K., and Takeda, T. (1998). Age-related changes in blood pressure in the senescence-accelerated mouse (SAM): Aged SAMP1 mice manifest hypertensive vascular disease. Lab. Anim. Sci. 48, 256-263. Hapke, H. J. (1983). The effects of environment and environmental monitoring on pharmacology studies. In "The Importance of Laboratory Animal Genetics, Health, and the Environment in Biomedical Research" (E. C. Melby Jr. and M. W. Balk, eds.), p. 207. Academic Press, Orlando, Florida. Hardisty, J. E (1985). Factors influencing laboratory animal spontaneous tumor profiles. Toxicol. PathoL 13, 95-104. Harper, M. H., Collins, P., Johnson, B., Eger, E. I., II, and Biava, C. (1982). Hepatic injury following halothane, enflurane, and isoflurane anesthesia in rats. Anesthesiology 56, 14-17. Harris, A. W., Basten, A., Gebski, V., Noonan, D., Finnie, J., Bath, M. L., Bangay, M. J., and Repacholi, M. H. (1998). A test of lymphoma induction by long-term exposure of E mu-Pim 1 transgenic mice to 50 Hz magnetic fields. Radiat. Res. 149, 300-307. Hart, J. E. (1990). Endocrine pathology of estrogens: Species differences. Pharmacol. Ther. 47, 203-218. Hart, J. S. (1963). Physiological responses to cold in non-hibernating homeotherms. In "Temperature: Its Measurement and Control in Science and Industry" (C. M. Herzfeld, ed.), Vol. 3, Part 3. Van Nostrand Reinhold, New York. Haseman, J. K., Huff, J. E., Rao, G. N., and Eustis, S. L. (1989). Sources of variability in rodent carcinogenicity studies. Fundam. Appl. Toxicol. 12, 793804. Haseman, J. K., Bourbina, J., and Eustis, S. L. (1994). Effect of individual housing and other experimental design factors on tumor incidence in B6C3F1. Fundam. Appl. Toxicol. 23, 44-52. Hasenau, J. J., Baggs, R. B., and Kraus, A. L. (1993). Microenvironments in microisolation cages using BALB/c and CD-1 Mice. Contemp. Top. Lab. Anim. Sci. 32, 11-16. Haslov, K., Fomsgaard, A., Takayama, K., Fomsgaard, J. S., Ibsen, P., Fauntleroy, M. B., Stashak, P. W., Taylor, C. E., and Baker, P. J. (1992). Immunosuppressive effects induced by the polysaccharide moiety of some bacterial lipopolysaccharides. Immunology 186, 378-393. Hastings, J. W. (1970). The biology of circadian rhythms from man to microorganisms. N. Engl. J. Med. 282, 435-441. Hata, A., Tsunoo, H., Nakajima, H., Shintaku, K., and Kimura, K. (1980). Acute cadmium intoxication in inbred mice: A study on strain differences. Chem. Biol. Interact. 32, 29-39. Haus, E., Lakatua, D., Swoyer, J., and Sackett-Lundeen, L. (1983). Chronobiology in hematology and immunology. Am. J. Anat. 168, 467-517. Hauser, W. E., Jr., and Remington, J. S. (1982). Effect of antibiotics on the immune response. Am. J. Med. 72, 711-716. Hautzinger, G. M., and Piacsek, B. E. (1973). Influence of duration, intensity, and spectrum of light exposure on sexual maturation of female rats. Fed. Proc. 32, 213. Hayashi, T., Koike, Y., Hasegawa, T., Tsurudome, M., Ozaki, M., Yamamoto, H., and Onodera, T. (1991). Inhibition of contact sensitivity by interferon in mice infected with lactic dehydrogenase virus. J. Comp. Pathol. 104, 357366. Hayashi, T., Ozaki, M., Ami, Y., Onodera, T., and Yamamoto, H. (1992a). Increased superoxide anion release by peritoneal macrophages in mice with a chronic infection of lactic dehydrogenase virus. J. Comp. Pathol. 106, 93-98.

1172 Hayashi, T., Ozaki, M., Onodera, T., Ami, Y., and Yamamoto, H. (1992b). Macrophage function in the acute phase of lactic dehydrogenase virus infection of mice: Suppression of superoxide anion production in normal mouse peritoneal macrophages by interferon-alpha in vitro. J. Comp. PathoL 106, 183-193. Hayashi, T., Noguchi, Y., and Kameyama, Y. (1993). Suppression of development of anti-nuclear antibody and glomerulonephritis in NZB X NZWF1 mice by persistent infection with lactic dehydrogenase virus: Possible involvement of superoxide anion as a progressive effector. Int. J. Exp. Pathol. 74, 553-560. Hayashi, T., Hashimoto, S., and Kawashima, A. (1994). Effect of infection by lactic dehydrogenase virus on expression of intercellular adhesion molecule- 1 on vascular endothelial cells of pancreatic islets in streptozotocininduced insulitis of CD-1 mice. Int. J. Exp. Pathol. 75, 211-217. Hayssen, V. (1998). Effect of transatlantic transport on reproduction of agouti and nonagouti deer mice, Peromyscus maniculatus. Lab. Anim. 32, 55-64. Hazlett, L. D., Rosen, D. D., and Berk, R. S. (1977). Pseudomonas eye infections in cyclophosphamide-treated mice. Invest. Ophthalmol. Vis. Sci. 16, 649-652. H'Doubler, P. B., Jr., Petersen, M., Shek, W., Auchincloss, H., Abbott, W. M., and Orkin, R. W. (1991). Spontaneously hypertensive and Wistar Kyoto rats are genetically disparate. Lab. Anim. Sci. 41, 471-473. Hedrich, H. J. (1983). Overview of the state of the art genetic monitoring. In "The Importance of Laboratory Animal Genetics, Health, and the Environment in Biomedical Research" (E. C. Melby, Jr. and M. W. Balk, eds.), p. 75. Academic Press, Orlando, Florida. Heffner, R., and Heffner, H. (1980). Hearing in the elephant (Elephas maximus). Science 208, 318-320. Heggers, J. P., Haydon, S., Ko, E, Hayward, P. G., Carp, S., and Robson, M. C. (1992). Pseudomonas aeruginosa exotoxin A: Its role in retardation of wound healing: The 1992 Lindberg award. J. Burn Care Rehabil. 13, 512518. Hemmes, J. H., Winkler, K. C., and Kool, S. M. (1960). Virus survival as a seasonal factor in influenza and poliomyelitis. Nature 188, 430-431. Hemphill, E E., Kaeberle, M. L., and Buck, W. B. (1971). Lead suppression of mouse resistance to Salmonella typhimurium. Science 172, 1031-1032. Henderson, D. C., Tosta, C. E., and Wedderburn, N. (1978). Exacerbation of murine malaria by concurrent infection with lactic dehydrogenase-elevating virus. Clin. Exp. Immunol. 33, 357-359. Herbermann, R. B., Nunn, M. E., Holden, H. T., Staal, S., and Djeu, J. Y. (1977). Augmentation of natural cytotoxic activity of mouse lymphoid cells against syngeneic and allogeneic target cells. Int. J. Cancer 19, 555-564. Hermann, L. M., White, W. J., and Lang, C. M. (1982). Prolonged exposure to acid, chlorine, or tetracycline in drinking water: Effects on delayed-type hypersensitivity, hemagglutination titers, and reticuloendothelial clearance rates in mice. Lab. Anim. Sci. 32, 603-608. Heywood, R., and Buist, D. P. (1983). The effects of health and health monitoring on toxicology studies. In "The Importance of Laboratory Animal Genetics, Health, and the Environment in Biomedical Research" (E. C. Melby, Jr. and M. W. Balk, eds.), p. 25. Academic Press, Orlando, Florida. Hightower, D., Uhrig, H. S., and Davis, J. I. (1966). Pseudomonas aeruginosa infection in rats used in radiobiology research. Lab. Anim. Care 16, 85-93. Hilakivi-Clarke, L., and Dickson, R. B. (1995). Stress influence on development of hepatocellular tumors in transgenic mice overexpressing TGF alpha. Acta Oncol. 34, 907-1012. Hill, R. N., Clemens, T. L., Liu, D. K., Vessell, E. S., and Johnson, W. D. (1975). Genetic control of chloroform toxicity in mice. Science 190, 159-161. Hite, M., Hanson, H. M., Bohidar, N. R., Conti, P. A., and Mattis, P. A. (1977). Effect of cage size on patterns of activity and health of beagle dogs. Lab. Anim. Sci. 27, 60-64. Hodgson, E., Kulkarni, A. P., Fabacher, D. L., and Robacker, K. M. (1980). Induction of hepatic drug metabolizing enzymes in mammals by pesticides: A review. J. Environ. Sci. Health 15, 723-754.

NElL S. LIPMAN AND SCOTT E. PERKINS Hofer, M. A., and Shair, H. (1978). Ultrasonic vocalization during social interaction and isolation in 2-week-old rats. Dev. Psychobiol. 11, 495-504. Hoffman, J. C. (1973). The influence of photoperiods on reproductive functions in female mammals. In "Endocrinology, Vol. II, Handbook of Physiology, Section 7," pp. 57-77. American Physiological Society, Washington, D.C. Homberger, E R., Pataki, Z., and Thomann, P. E. (1993). Control of Pseudomonas aeruginosa infection in mice by chlorine treatment of drinking water. Lab. Anim. Sci. 43, 635-637. Homburger, E (1978). Saccharin and cancer. N. Engl. J. Med. 297, 560-561. Hooks, J. J., Percopo, C., Wang, Y., and Detrick, B. (1993). Retina and retinal pigment epithelial cell autoantibodies are produced during murine coronavirus retinopathy. J. Immunol. 151, 3381-3389. Hosgood, G. (1995). Anesthesia and surgical considerations. In "Veterinary Pediatrics, Dogs and Cats from Birth to Six Months" (J. D. Hoskins, ed.), pp. 562-575. Saunders, Philadelphia. Hotchin, J. (1962). The biology of lymphocytic choriomeningitis virus infection: Virus-induced immune disease. Cold Spring Harbor Symp. Quant. Biol. 27, 479-499. Houtman, J. J., and Fleming, J. O. (1996). Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM. J. Neurovirol. 2, 101-110. Howard, R. J., Notkins, A. L., and Mergenhagen, S. E. (1969). Inhibition of cellular immune reactions in mice infected with lactic dehydrogenase virus. Nature 211, 873-874. Howard, H. L., McLaughlin-Taylor, E., and Hill, R. L. (1990). The effect of mouse euthanasia technique on subsequent lymphocyte proliferation and cell mediated lympholysis assays. Lab. Anim. Sci. 40, 510-514. Hsu, W. H. (1988). Yohimbine increases plasma insulin concentrations and reverses xylazine-induced hypoinsulinemia in dogs. Am. J. Vet. Res. 49, 242-244. Huang, D. S., Emancipator, S. N., Fletcher, D. R., Lamm, M. E., and Mazanec, M. B. (1996). Hepatic pathology resulting from mouse hepatitis virus infection in severe combined immunodeficiency mice. Lab. Anim. Sci. 46, 167-173. Huerkamp, M. J., and Lehner, N. D. M. (1994). Comparative effects of forcedair, individual cage ventilation or an absorbent bedding additive on mouse isolator cage microenvironment. Contemp. Top. Lab. Anim. Sci. 33, 69-72. Huerkamp, M. J., Dillehay, D. L., and Lehner, N. D. M. (1994). Effect of intracage ventilation and automatic watering on outbred mouse reproductive performance and weanling growth. Contemp. Top. Lab. Anim. Sci. 33, 58-62. Hunstein, W., Perings, E., Eggeling, B., and Uhl, N. (1969). Panmyelophthisis and viral hepatitis. Experimental study with the MHV-3 mouse hepatitis virus. Acta Haematol. 42, 336-346. Inada, T., and Mims, C. A. (1986). Infection of mice with lactic dehydrogenase virus prevents development of experimental allergic encephalomyelitis. J. Neuroimmunol. 11, 53-56. Ingham, E., Turnbull, L., and Keamey, J. N. (1991). The effects of minocycline and tetracycline on the mitotic response of human peripheral bloodlymphocytes. J. Antimicrob. Chemother. 27, 607-617. Institute of Laboratory Animal Resources (ILAR) (1978). "Guide for the Care and Use of Laboratory Animals." National Academy of Sciences, Washington, D.C. Institute of Laboratory Animal Resources (ILAR) (1991). "Infectious Diseases of Mice and Rats." National Academy Press, Washington, D.C. Irvani, J., and van As, A. (1972). Mucus transport in the tracheobronchial tree of normal and bronchitic rats. J. Pathol. 106, 81-93. Isakov, N., Feldman, M., and Segal, S. (1982a). Acute infection of mice with lactic dehydrogenase virus (LDV) impairs the antigen-presenting capacity of their macrophages. Cell. Immunol. 66, 317-332. Isakov, N., Feldman, M., and Segal, S. (1982b). Lactic dehydrogenase virus (LDV) impairs the antigen-presenting capacity of macrophages yet fails to affect their phagocytic activity. Immunobiology 162, 15 - 27.

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH

Isakov, N., Feldman, M., and Segal, S. (1982c). The mechanism of modulation of humoral immune responses after infection of mice with lactic dehydrogenase virus. J. Immunol. 128, 969-975. Ishida, T., Tamura, T., Ueda, K., and Fujiwara, K. (1978). Hepatosplenic myelosis in the nude mouse naturally infected with mouse hepatitis virus. Jpn. J. Vet. Sci. 40, 739-743. Ito, A. (1976). Animal model of human disease: Pituitary tumors.Am. J. Pathol. 83, 423-426. Ito, T., and Ingalls, T. H. ( 1981). Sodium pentobarbital-induced mutations in the hamster. Arch. Environ. Health 36, 316-320. Izmerov, N. E (1985). Current problems of nonionizing radiation. Scand. J. Work Environ. Health 11, 223-227. Izquierdo, J. N., and Gibbs, S. J. (1972). Circadian rhythms of DNA synthesis and mitotic activity in hamster cheek pouch epithelium. Exp. Cell Res. 71, 402-408. Jacoby, R. O., and Ball-Goodrich, L. J. (1995). Parvovirus infections of mice and rats. Semin. ViroL 6, 329-333. Jakab, G. J. (1981). Interactions between Sendai virus and bacterial pathogens in the murine lung: A review. Lab. Anim. Sci. 31, 170-177. Jakab, G. J., and Warr, G. A. (1981). Immune enhanced phagocytic dysfunction in pulmonary macrophages infected with parainfluenza 1 (Sendai) virus. Am. Rev. Respir. Dis. 124, 575-581. Jans, J. E., and Woodside, B. (1987). Effects of litter age, litter size, and ambient temperature on the milk ejection reflex in lactating rats. Dev. Psychobiol. 20, 333-344. Jensen, M. M., and Rasmussen, A. E, Jr. (1970). Audiogenic stress and suscep9tibility to infection. In "Physiological Effects of Noise" (B. L. Welch and A. S. Welch, eds.). Plenum Press, New York. Jessop, J. J., and Bayer, B. M. (1989). Time-dependent effects of isolation on lymphocyte and adrenocortical activity. J. Neuroimmunol. 23, 143-147. Jimenez-Valera, M., Sampedro, A., Moreno, E., and Ruiz-Bravo, A. (1995). Modification of immune response in mice by ciprofloxacin. Antimicrob. Agents Chemother. 39, 150-154. Joasoo, A., and McKenzie, J. M. (1976). Stress and immune response in rats. Int. Arch. Allergy Appl. Immunol. 5tl, 659-663. Johlin, J. M. (1944). Influence of atmospheric temperature upon reaction of rabbits to insulin. Proc. Soc. Exp. Biol. Med. 55, 122-124. Johnson, T., Lavender, J. E, Hultin, E., and Rasmussen, A. E, Jr. (1963). The influence of avoidance-learning stress on resistance to coxsackie B virus in mice. J. Immunol. 91, 569-579. Jori, A., Bianchetti, A., and Prestini, E E. (1969). Effect of essential oils on drug metabolism. Biochem. Pharmacol. 18, 2081-2085. Jori, A., Di Salle, E., and Santini, V. (1971). Daily rhythmic variation and liver drug metabolism in rats. Biochem. Pharmacol. 29, 2965-2969. Jungmann, E, Guenet, J. L., Cazenave, E A., Coutinho, A., and Huerre, M. (1996a). Murine acariasis. I. Pathological and clinical evidence suggesting cutaneous allergy and wasting syndrome in BALB/c mouse. Res. Immunol. 147, 27-38. Jungmann, E, Freitas, A., Bandeira, A., Nobrega, A., Coutinho, A., Marcos, M. A., and Minoprio, E (1996b). Murine acariasis. II. Immunological dysfunction and evidence for chronic activation of Th-2 lymphocytes. Scand. J. Immunol. 43, 604-612. Kacew, S., Dixit, R., and Ruben, Z. (1998). Diet and rat strain as factors in nervous system function and influence of confounders. Biomed. Environ. Sci. 11, 203-217. Kai, T., Bremerich, D. H., Jones, K. A., and Warner, D. O. (1998). Drugspecific effects of volatile anesthetics on Ca 2+ sensitization in airway smooth muscle. Anesth. Analg. 87, 425-429. Kameyama, Y., and Hayashi, T. (1994). Suppression of development of glomerulonephritis in NZB • NZWF1 miceby persistent infection with lactic dehydrogenase virus: Relations between intercellular adhesion molecule-1 expression on endothelial cells and leucocyte accumulation in glomeruli. Int. J. Exp. Pathol. 75, 295-304.

1173 Karp, J. D., Moynihan, J. A., and Ader, R. (1993). Effects of differential housing on the primary and secondary antibody responses of male C57BL/6 and BALB/c mice. Brain Behav. Immun. 7, 326-333. Katzav, S., Shapiro, J., Segal, S., and Feldman, M. (1986). General anesthesia during excision of a mouse tumor accelerates postsurgical growth of metastases by suppression of natural killer cell activity. Isr. J. Med. Sci. 22, 339345. Kay, M. M. B. (1978). Long term subclinical effects of parainfluenza (Sendai) infection on immune cells of aging mice. Proc. Soc. Exp. Biol. Med. 158, 326-331. Kay, M. M. B. (1979). Parainfluenza infection of aged mice results in autoimmune disease. Clin. Immunol. Immunopathol. 12, 301-315. Kay, M. M. B., Mendoza, J., Hausman, S., and Dorsey, B. (1979). Age related changes in the immune system of mice of eight medium and long lived strains and hybrids. II. Short- and long-term effects of natural infection with parainfluenza type 1 virus (Sendai). Mech. Ageing Devel. 11, 347362. Kelce, W. R., and Earl Grey, I. (1999). Environmental antiandrogens: In vitro and in vivo screening mechanisms. Lab. Anim. 28, 26-32. Kelche, C., Dalrymple-Alford, J. C., and Will, B. (1988). Housing conditions modulate the effects of intracerebral grafts in rats with brain lesions. Behav. Brain Res. 28, 287-295. Keller, L. S. E, White, W. J., Snider, M. T., and Lang, M. C. (1989). An evaluation of intracage ventilation in three animal caging systems. Lab. Anim. Sci. 39, 237-242. Kempermann, G., Brandon, E. E, and Gage, E H. (1998). Environmental stimulation of 129/SvJ mice causes increased cell proliferation and neurogenesis in the adult dentate gyrus. Curr. Biol. 8, 939-942. Kennedy, B. W., and Beal, T. S. (1991). Minerals leached into drinking water from rubber stoppers. Lab. Anim. Sci. 41, 233-236. Kenyon, A. J. (1983). Delayed wound healing in mice associated with viral alteration of macrophages. Am. J. Vet. Res. 44, 652-656. Kernbaum, S., Masliah, J., Alcindor, L. G., Bouton, C., and Christol, D. (1983). Phospholipase activities of bronchoalveolar lavage fluid in rat Pneumocystis carinii pneumonia. Br. J. Exp. Pathol. 64, 75-80. Kerr, L. R., Grimm, M. S., Silva, W. A., Weinberg, J., and Emerman, J. T. (1997). Effects of social housing condition on the response of the Shionogi mouse mammary carcinoma (SC115) to chemotherapy. Cancer Res. 57, 1124-1128. Kilham, L., and Margolis, G. (1970). Pathogenicity of minute virus of mice (MVM) for rats, mice, and hamsters. Proc. Soc. Exp. Biol. Med. 133,14471551. Kilham, L., Buckler, C. E., Ferm, V. H., and Baron, S. (1968). Production of interferon during rat virus infection. Proc. Soc. Exp. Biol. Med. 129, 274278. Kligerman, A. D., Chapin, R. E., Erexson, G. L., Germolec, D. R., Kwanyuen, E, and Yang, R. S. (1993). Analyses of cytogenetic damage in rodents following exposure to simulated groundwater contaminated with pesticides and a fertilizer. Mutat. Res. 3tl0, 125-134. Koi, M., Saito, M., Ebina, T., and Ishida, N. (1981). Lactate dehydrogenaseelevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice. Microbiol. Immunol. 25, 565-574. Koivusalo, M., and Vartiainen, T. (1997). Drinking water chlorination byproducts and cancer. Rev. Environ. Health 12, 81-90. Koller, L. D. (1979). Effects of environmental contaminants on the immune system. Adv. Vet. Sci. Comp. Med. 23, 267-295. Kolls, J. K., Beck, J. M., Nelson, S., Summer, W. R., and Shellito, J. (1993). Alveolar macrophage release tumor necrosis factor during murine Pneumocyctis carinii pneumonia. Am. J. Respir. Cell Mol. Biol. 8, 370-376. Komori, T., Fujiwara, R., Tanida, M., Nomura, J., and Yokoyama, M. M. (1995a). Effects of citrus fragrance on immune function and depressive states. Neuroimmunomodulation 2, 174-180.

1174 Komori, T., Fujiwara, R., Tanida, M., and Nomura, J. (1995b). Potential antidepressant effects of lemon odor in rats. Eur. Neuropsychopharmacol. 5, 477-480. Koppel, J., Kuchar, S., Mozes, S., Petrusova, K., and Jasenovec, A. (1982). Changes in blood sugar after administration of xylazine and adrenergic blockers in the rat. Vet. Med. (Praha) 27, 113-118. Kort, W. J., and Weijma, J. M. (1982). Effect of chronic light-dark shift stress on the immune response of the rat. Physiol. Behav. 29, 1083-1087. Kort, W. J., Zondervan, P. E., Hulsman, L. O., Weijma, I. M., and Westbroek, D. L. (1986). Light-dark-shift stress, with special reference to spontaneous tumor incidence in female BN rats. J. Natl. Cancer Inst. 76, 439-446. Krasula, R. W., and Pernet, A. G. (1991). Comparison of organ-specific toxicity of temafloxacin in animals and humans. Am. J. Med. 91, 38-41. Kryzstyniak, K., and Dupuy, J. M. (1983). Immunodepression of lymphocyte response in mouse hepatitis virus 3 infection. Biomed. Pharmacother. 37, 68-74. Kufoy, E. A., Pakalnis, V. A., Parks, C. D., Wells, A., Yang, C. H., and Fox, A. (1989). Keratoconjunctivitis sicca with associated secondary uveitis elicited in rats after systemic xylazine/ketamine anesthesia. Exp. Eye Res. 49, 861-871. Kuhnen, G. (1997). The effect of cage size and environmental enrichment on the generation of fever in golden hamster. Ann. N.Y. Acad. Sci. 813, 398400. Kuhnen, G. (1998). Reduction of fever by housing in small cages. Lab. Anim. 32, 42-45. Kullberg, M. C., Ward, J. M., Gorelick, P. L., Caspar, P., Hieny, S., Cheever, A., Jankovic, D., and Sher, A. (1998). Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin- 10 (IL- 10)-deficient mice through an ILl2- and gamma interferon-dependent mechanism. Infect. Immun. 66, 51575166. Kurtz, T. W., and Morris, R. C., Jr. (1987). Biological variability in WistarKyoto rats. Implications for research with the spontaneously hypertensive rat. Hypertension 10, 127-131. Kurtz, T. W., Montano, M., Chan, L., and Kabra, P. (1989). Molecular evidence of genetic heterogeneity in Wistar-Kyoto rats: Implications for research with the spontaneously hypertensive rats. Hypertension 13, 188-192. Kurzer, M. S., and Xu, X. (1997). Dietary phytoestrogens. Annu. Rev. Nutr. 17, 353-381. Kusunose, N., Shoji, T., Tsubara, A., Yamamoto, M., and Morii, S. (1990). Strain difference in neoplastic response to DMBA powder dusted onto mammary tissues between Wistar/Furth and Copenhagen strains of rats. Jpn. J. Exp. Med. 60, 291-297. Kutza, J., Gratz, I., Afshar, M., and Murasko, D. M. (1997). The effects of general anesthesia and surgery on basal and interferon stimulated natural killer cell activity of humans. Anesth. Analg. 85, 918-923. Kwiatkowska-Patzer, B., Patzer, J. A., and Heller, L. J. (1993). Pseudomonas aeruginosa exotoxin A enhances automaticity and potentiates hypoxic depression of isolated rat hearts. Proc. Soc. Exp. Biol. Med. 202, 377383. Kyriazis, A. P., DiPersio, L., Michaei, J. G., and Pesce, A. J. (1979). Influence of the mouse hepatitis virus (MHV) infection on the growth of human tumors in the athymic mouse. Int. J. Cancer 23, 402-409. Lagutchik, M. S., Mundie, T. G., and Martin, D. G. (1992). Methemoglobinemia induced by a benzocaine-based topically administered anesthetic in eight sheep. J. Am. Vet. Med. Assoc. 201, 1407-1410. Lahmy, C., and Virelizier, J. L. (1981). Prostaglandins as probable mediators of the suppression of antibody production by mouse hepatitis virus infection. Ann. Immunol. 132, 101-105. Lai, W. C., Pakes, S. P., Lu, Y. S., and Brayton, C. F. (1987). Mycoplasma pulmonis infection augments natural killer cell activity in mice. Lab. Anim. Sci. 37, 299-303. Lal, J. G., Lary, J. M., and Conover, D. L. (1980). Embryotoxicity of Mycoplasma pulmonis infection in rats. Paper presented at the annual session

NEIL S. LIPMAN AND SCOTT E. PERKINS of the American Association for Laboratory Animal Science, Indianapolis, Indiana, October 5-10. (Abstract No. 84). Laltoo, H., and Kind, L. S. (1979). Reduction in contact sensitivity reactions to oxazolone in mite-infested mice. Infect. Immun. 26, 30-35. Landi, M. S., Kreider, J. W., Lang. C. M., and Bullock, L. P. (1982). Effects of shipping on the immune function in mice. Am. J. Vet. Res. 43, 1654-1657. Lanum, J. (1978). The damaging effects of light on the retina. Empirical findings, theoretical and practical implications. Surv. Ophthalmol. 22, 221249. Lardans, V., Godfraind, C., van der Logt, J. T., Heesen, W. A., Gonzalez, M. D., and Coutelier, J. P. (1996). Polyclonal B lymphocyte activation induced by mouse hepatitis virus A59 infection. J. Gen. Virol. 77, 1005-1009. La Vail, M. M. (1976). Survival of some photoceptor cells in albino rats following long-term exposure to continuous light. Invest. Ophthalmol. 15, 64-72. Lavelle, G. C., and Bang, E B. (1973). Differential growth of MHV (PRI) and MHV (C3H) in genetically resistant C3H mice rendered susceptible by eperythrozoon infection. Arch. Gesamte Virusforsch. 41, 175-184. Lavilla-Apelo, C., Kida, H., and Kanagawa, H. (1992). The effect of experimental infection of mouse preimplantation embryos with paramyxovirus Sendai. J. Vet. Med. Sci. 54, 335-340. Leader, R. W., Leader, I., and Witschi, E. (1970). Genital mycoplasmosis in rats treated with testosterone propionate to produce constant estrus. J. Am. Vet. Med. Assoc. 157, 1923-1925. LeBouton, A. V., and Handler, S. D. (1971). Persistent circadian rhythmicity of protein synthesis in liver of starved rats. Experientia 27, 1031-1032. Lee, T. M., and McClintock, M. K. (1986). Female rats in a laboratory display seasonal variation in fecundity. J. Reprod. Fertil. 77, 51-59. Lee, S. K., Youn, H. Y., Hasegawa, A., Nakayama, H., and Goto, N. (1994). Apoptotic changes in the thymus of mice infected with mouse hepatitis virus, MHV-2. J. Vet. Med. Sci. 56, 879-882. Lee, J. E, Liao, P. M., Tseng, D. H., and Wen, P. T. (1998). Behavior of organic polymers in drinking water purification. Chemosphere 37, 1045-1061. Lehmann-Grube, E, Niemeyer, I., and Lohler, J. (1972). Lymphocytic choriomeningitis of the mouse. IV. Depression of the allograft reaction. Med. Microbiol. Immunol. 158, 16-25. Leray, D., Dupuy, C., and Dupuy, J. M. (1982). Immunopathology of mouse hepatitis virus type 3 infection. IV. MHV3-induced immunodepression. Clin. Immunol. Immunopathol. 23, 539-547. Levi, E, Canon, C., Dipalma, M., Florentin, I., and Misste, J. L. (1991). When should the immune clock be reset? Ann. N.Y. Acad. Sci. 618, 312-329. Levy, G. A., Leibowitz, J. L., and Edington, T. S. (1981). Induction of monocyte procoagulant activity by murine hepatitis virus type 3 parallels disease susceptibility in mice. J. Exp. Med. 154, 1150-1163. Li, X., Fox, J. G., Whary, M. T., Yan, L., Shames, B., and Zhao, Z. (1998). SCID/NCr mice naturally infected with Helicobacter hepaticus develop progressive hepatitis, proliferative typhlitis, and colitis. Infect. Immun. 66, 5477-5484. Limper, A. H., Edens, M., Anders, R. A., and Leof, E. B. (1998). Pneumocystis carinii inhibits cyclin-dependent kinase activity in lung epithelial cells. J. Clin. Invest. 101, 1148-1155. Lin, L., Grenier, L., LeBrun, M., Bergeron, M. G., Thibault, L., Labrecque, G., and Beauchamp, D. (1996). Day-night treatment difference of tobramycin serum and intrarenal drug distribution and nephrotoxicity in rats: Effects of fasting. Chronobiol. Int. 13, 113-121. Lindsey, J. R., Conner, M. W., and Baker, H. J. (1978). Physical, chemical, and microbial factors affecting biologic response. In "Symposium on Laboratory Animal Housing" (Institute for Laboratory Animal Resources), pp. 3743. National Academy of Sciences-National Resources Council, Washington, D.C. Lindsey, J. R., Davidson, M. K., Schoeb, T. R., and Cassell, G. H. (1986). Murine mycoplasmal infections. In "Complications of Viral and Mycoplasmal Infections in Rodents to Toxicology Research and Testing" (T. E. Hamm, Jr., ed.), pp. 91-121. Hemisphere Press, Washington, D.C.

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH Line, S. W., Morgan, K. N., Markowitz, H., and Strong, S. (1989). Influence of cage size on heart rate and behavior in rhesus monkeys. Am. J. Vet. Res. 50, 1523-1526. Lipman, N. S. (1999). Isolator rodent caging systems (state of the art): A critical review. Contemp. Top. Lab. Anim. Sci. 38, 9-17. Lipman, N. S., Marini, R. P., Murphy, J. C., Zhibo, Z., and Fox, J. G. (1993). Estradiol-17 beta-secreting adrenocortical tumor in a ferret. J. Am. Vet. Med. Assoc. 11, 1522-1525. Lipman, N. S., Zhi-Bo, Z., Andrutis, K. A., Hurley, R. J., Fox, J. G., and White, H. J. (1994). Prolactin-secreting pituitary adenomas with mammary dysplasia in New Zealand white rabbits. Lab. Anim. Sci. 44, 114-120. Lipschik, G. Y., Treml, J. E, and Moore, S. D. (1996). Pneumocystis carinii glycoprotein A stimulates intedeukin-8 production and inflammatory cell activation in alveolar macrophages and cultured monocytes. J. Eukaryot. Microbiol. 43, 16S-17S. Lipsky, M. M., Skinner, M., and O'ConneU, C. (1993). Effects of chloroform and bromodichloromethane on DNA synthesis in male F344 rat kidney. Environ. Health Perspect. 101 (Suppl. 5), 249-252. Lockett, M. E (1970). Effects of sound on endocrine function and electrolyte excretion. In "Physiological Effects of Noise" (B. L. Welch and A. S. Welch, eds.), pp. 21-42. Plenum Press, New York. Lohmiller, J. J., and Lipman, N. S. (1998). Silicon crystals in water of autoclaved glass bottles. Contemp. Top. Lab. Anim. Sci. 37, 62-65. Long, N. C., Morimoto, A., Nakamori, T., and Murakami, N. (1991). The effect of physical restraint on IL-lbeta- and LPS-induced fever. Physiol. Behav. 50, 625-628. Longstreth, J., de Gruijl, E R., Kripke, M. L., Abseck, S., Arnold, E, Slaper, H. I., Velders, G., Takizawa, Y., and Van Der Leun, J. C. (1998). Health risks. J. Photochem. Photobiol. B. 46, 20-39. Loose, L. D., Silkworth, J. B., and Simpson, D. W. (1978). Influence of cadmium on the phagocytic and microbicidal activity of murine peritoneal macrophages, pulmonary alveolar macrophages, and polymorphonuclear neutrophils. Infect. Immun. 22, 378-381. Lubcke, R., Hutcheson, E A., and Barbezat, G. O. (1992). Impaired intestinal electrolyte transport in rats infested with the common parasite Syphacia muris. Dig. Dis. Sci. 37, 60-64. MacCleur, J. W., VandeBerg, J. L., Read, B., and Ryder, O. A. (1986). Pedigree analysis by computer simulation. Zoo Biol. 5, 147-160. Mackay-Sim, A., and Laing, D. G. (1981a). Rats' response to blood and body odors of stressed and non-stressed conspecifics. Physiol. Behav. 27, 503510. Mackay-Sim, A., and Laing, D. G. (198 lb). The sources of odors from stressed rats. Physiol. Behav. 27, 511-513. MacManus, J. P., Whitfield, J. E, and Youdale, T. (1971). Stimulation by epinephrine of adenyl cyclase activity, cyclic AMP formation, DNA synthesis, and cell proliferation in populations of rat thymic lymphocytes. J. Cell. Comp. Physiol. 77, 103-106. Madden, J., Akil, H., Patrick, R. L., Barchas, J. D. (1977). Stress-induced parallel changes in central opioid levels and pain responsiveness in the rat. Nature 265, 358-360. Maejima, K., Fujiwara, K., Takagaki, Y., Naiki, M., Kurashina, H., and Tajima, Y. (1965). Dietetic effects on experimental Tyzzer's disease of mice. Jpn. J. Exp. Med. 35, 1-10. Maggio-Price, L., Nicholson, K. L., Kline, K. M., Birkebak, T., Suzuki, I., Wilson, D. L., Schauer, D., and Fink, P. J. (1998). Diminished reproduction, failure to thrive, and altered immunologic function in a colony of T-cell receptor transgenic mice: Possible role of Citrobacter rodentium. Lab. Anim. Sci. 48, 145-155. Mahy, B. W. J., Rowson, K. E. K., Parr, C. W., and Salaman, H. H. (1965). Studies on the mechanisms of action of Riley virus. I. Action of substances affecting the reticuloendothelial system on plasma enzyme levels in mice. J. Exp. Med. 122, 967-981. Malberg, J. E., and Seiden, L. S. (1998). Small changes in ambient temperature

1175 cause large changes in 3,4-methylenedioxymethamphetamine (MDMA)induced serotonin neurotoxicity and core body temperature in the rat. J. Neurosci. 18, 5086-5094. Mallucci, L. (1964). Mouse hepatitis virus and interferon production in normal and regenerating liver. Arch. Gesamte Virusforsch. 15, 91-97. Manaker, R. A., Piczak, C. W., Miller, A. A., and Stanton, M. E (1961). A hepatitis virus complicating studies with mouse leukemia. J. Natl. Cancer Inst. 27, 29-45. Marker, O., Scheynius, A., Christensen, J. P., and Thomsen, A. R. (1995). Virusactivated T cells regulate expression of adhesion molecules on endothelial cells in sites of infection. J. Neuroimmunol. 62, 35-42. Markovic, S. N., and Murasko, D. M. (1990). Anesthesia inhibits poly I:C induced stimulation of natural killer cell cytotoxicity in mice. Clin. Immunol. Immunopathol. 56, 202-209. Markovic, S. N., and Murasko, D. M. (1991). Inhibition of induction of natural killer activity in mice by general anesthesia (avertin): Role of interferon. Clin. Immunol. ImmunopathoL 60, 181-189. Markovic, S. N., and Murasko, D. M. (1993). Anesthesia inhibits interferoninduced natural killer cell cytotoxicity via induction of CD8 § suppressor cells. Cell. Immunol. 151, 474-480. Markovic, S. N., Knight, P. R., and Murasko, D. M. (1993). Inhibition of interferon stimulation of natural killer cell activity in mice anesthetized with halothane or isoflurane. Anesthesiology 78, 700-706. Marshall, J. C., Ribeiro, M. B., Chu, P. T., Rotstein, O. D., and Sheiner, P. A. (1993). Portal endotoxemia stimulates the release of an immunosuppressive factor from alveolar and splenic macrophages. J. Surg. Res. 55, 14-20. Masoro, E. J. (1992). Aging and proliferative homeostasis: Modulation by food restriction in rodents. Lab. Anim. Sci. 42, 132-137. Mathis, C., Paul, S. M., and Crawley, J. N. (1994). Characterization of benzodiazepine-sensitive behaviors in the A/J and C57BL/6J inbred strains of mice. Behav. Genet. 24, 171-180. Mathis, C., Neumann, P. E., Gershenfeld, H., Paul, S. M., and Crawley, J. N. (1995). Genetic analysis of anxiety-related behaviors and responses to benzodiazephine-related drugs in AXB and BXA recombinant inbred mouse strains. Behav. Genet. 25, 557-568. Matsunami, H., and Buck, L. B. (1997). A multigene family encoding a diverse array of putative pheromone receptors in mammals. Cell 90, 775-784. Matsuya, Y., Kusano, T., Endo, S., Takahashi, N., and Yamane, I. (1978). Reduced tumorigenicity by addition in vitro of Sendai virus. Eur. J. Cancer 14, 837-850. Mazelis, A. G., Albert, D., Crisa, C., Fiore, H., Parasaram, D., Franklin, B., Ginsberg-Fellner, E, and McEvoy, R. C. (1987). Relationship of stressful housing conditions to the onset of diabetes mellitus induced by multiple, sub-diabetogenic doses of streptozotocin in mice. Diabetes Res. 6, 195200. Mazze, R. I., Cousins, M. J., and Kosek, J. C. (1973). Strain differences in metabolism and susceptibility to the nephrotoxic effects of methoxyflurane in rats. J. Pharmacol. Exp. Ther. 184, 481-488. McClintock, M. K. (1998). On the nature of mammalian and human pheromones. Ann. N.Y. Acad. Sci. 855, 390-392. McEachron, D. L., Tumas, K. M., Blank, K. J., and Prystowsky, M. B. (1995). Environmental lighting alters the infection process in an animal model of AIDS. Pharmacol. Biochem. Behav. 51, 947-952. Mclntosh, J. C., Simecka, J. W., Ross, S. E., Davis, J. K., Miller, E. J., and Cassell, G. H. (1992). Infection-induced airway fibrosis in two rat strains with differential susceptibility. Infect. Immun. 60, 2936-2942. McKisic, M. D., Lancki, D. W., Otto, G., Padrid, P., Snook, S., Cronin, D. C., II, Lohmar, P. D., Wong, T., and Fitch, E W. (1993). Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells. J. Immunol. 150, 419-428. McKisic, M. D., Paturzo, E X., and Smith, A. L. (1996). Mouse parvovirus infection potentiates rejection of tumor allografts and modulates T cell effector functions. Transplantation 61, 292-299.

1176 McMaster, G. K., Beard, E, Engers, H. D., and Hirt, B. (1981). Characterization of an immunosuppressive parvovirus related to the minute virus of mice. J. Virol. 38, 317-326. McNair, D. M., and Timmons, E. H. (1977). Effects of Aspiculuris tetraptera and Syphacia obvelata on exploratory behavior of an inbred mouse strain. Lab. Anim. Sci. 27, 38-42. McSheehy, T. (1983). Overview of the state of the art in environmental monitoring. In "The Importance of Laboratory Animal Genetics, Health, and the Environment in Biomedical Research" (E. C. Melby, Jr. and M. W. Balk, eds.), p. 161. Academic Press, Orlando, Florida. Meisel, R. L., Hay, T. C., Del Paine, S. N., and Luttrell, V. R. (1990). Induction of obesity by group housing in female Syrian hamsters. Physiol. Behav. 47, 815-817. Meiser, J., Kinzel, V., and Jirovec, O. (1971). Nosematosis as an accompanying infection to plasmacytoma ascites in Syrian golden hamsters. Pathol. Microbiol. 37, 249-260. Menaker, M., and Eskin, A. (1966). Entrainment of circadian rhythms by sound in Passer domesticus. Science 154, 1579-1581. Mendoza, S. P., Lowe, E. L., Davidson, J. M., and Levine, S. (1978). Annual cyclicity in the squirrel monkey (Saimiri sciureus): The relationship between testosterone, fatting, and sexual behavior. Horm. Behav. 11, 295303. Mercier, F. J., and Denjean, A. (1996). Guinea-pig tracheal responsiveness in vitro following general anaesthesia with halothane. Eur. Respir. J. 9, 14511455. Mergenhagen, S. E., Notkins, A. L., and Dougherty, S. F. (1967). Adjuvanticity of lactic dehydrogenase virus: Influence of virus infection on the establishment of immunologic tolerance to a protein antigen in adult mice. J. Immunol. 99, 576-581. Metcalf, J. E, and Wilson, G. B. (1987). Use of mitogen-induced lymphocyte transformation to assess toxicity of aminoglycosides. J. Environ. Pathol. Toxicol. Oncol. 7, 27-37. Michaelides, M. C., and Schlesinger, S. (1974). Effect of acute or chronc infection with lactic dehydrogenase virus (LDV) on the susceptibility of mice to plasmacytoma MOPC-315. J. Immunol. 112, 1560-1564. Michaelides, M. C., and Simms, E. S. (1980). Immune responses in mice infected with lactic dehydrogenase virus. Antibody response to a T-dependent and a T-independent antigen during acute and chronic LDV infection. Cell, Immunol. 50, 253-260. Miller, G. E, Barnard, D. E., Woodward, R. A., Flynn, B. M., and Bulte, J. W. (1997). Hepatic hemosiderosis in common marmosets, Callithrix jacchus: Effect of diet on incidence and severity. Lab. Anim. Sci. 47, 138-142. Millican, R. C. (1963). Pseudomonas aeruginosa infection and its effects in nonradiation stress. Lab. Anim. Sci. 13, 11-19. Milligan, S. R., Sales, G. D., and Khirynkh, K. (1993). Sound levels in rooms housing laboratory animals: An uncontrolled daily variable. Physiol. Behav. 53, 1067-1076. Milone, M. C., and Fitzgerald-Bocarsly, P. (1998). The mannose receptor mediates induction of IFN-alpha in peripheral blood dendritic cells by enveloped RNA and DNA viruses. J. Immunol. 161, 2391-2399. Mims, C. A., and Wainwright, S. (1968). The immuno-depressive action of lymphocytic choriomeningitis virus in mice. J. Immunol. 1111, 717-724. Minneman, K. P., Lynch, H. J., and Wurtman, R. J. (1974). Relationship between environmental light intensity and retina-mediated suppression of rat pineal serotonin-N-acetyltransferase. Life Sci. 15, 1791-1796. Misslin, R., Herzog, E, Koch, B., and Ropartz, P. (1982). Effects of isolation, handling, and novelty on the pituitary-adrenal response in the mouse. Psychoneuroendcrinology 7, 217-221. Mitropoulos, K. A., Balasubcamaniam, S., Gibbons, G. E, and Reaves, B. E. A. (1972). Diurnal variation in the activity of cholesterol 7-hydroxylase in the livers of fed and fasted rats. FEBS Lett. 27, 203-206. Miyamoto, T., Taura, Y., Une, S., Yoshitake, M., Nakama, S., and Watanabe, S. (1992). Changes in blastogenic responses of lymphocytes and delayed type

NElL S. LIPMAN AND SCOTT E. PERKINS hypersensitivity responses after vaccination in dogs. J. Vet. Med. Sci. 54, 945-950. Mizuno, S., and Ishida, A. (1982). Selective enhancement of the cytotoxicity of the bleomycin derivative, peplomycin, by local anesthetics alone and combined with hyperthermia. Cancer Res. 42, 4726-4729. Mock, E. J., Norton, H. W., and Frankel, A. I. (1978). Daily rhythmicity of serum testosterone concentration in the male laboratory rat. Endocrinology 103, 1111-1121. Mohn, G., and Philipp, E. M. (1981). Effects of Syphacia muris and the anthelmintic fenbendazole on the microsomal monooxygenase system in mouse liver. Lab. Anim. 15, 89-95. Monahan, E. J., and Maxson, S. C. (1998). Y chromosomes, urinary chemosignals, and an agonistic behavior (offense) of mice. Physiol. Behav. 64, 123132. Morales, S. V. M., Llopis, G. A., Tejerizo, P. M. L., and Ferrandiz, E J. (1993). Concentration of nitrates in drinking water and its relationship with bladder cancer. J. Environ. Pathol. Toxicol. Oncol. 12, 229-236. Mordelet-Dambrine, M., Danel, C., Farinotti, R., Urzua, G., Barritault, L., and Huchon, G. J. (1992). Influence of Pneumocystis carinii pneumonia on serum and tissue concentrations of pentamidine administered to rats by tracheal injections. Am. Rev. Respir. Dis. 146, 735-739. Mori, I., Hayashi, T., Kitazima, S., and Yamamoto, H. (1992). Binding of asparaginase to mouse monocytes. Int. J. Exp. PathoL 73, 585-592. Morita, E., Kaneko, S., Hiragun, T., Shindo, H., Tanaka, T., Furukawa, T., Nobukiyo, A., and Yamamoto, S. (1999). Fur mites induce dermatitis associated with IgE hyperproduction in an inbred strain of mice, NC/Kuj. J. Dermatol. Sci. 19, 37-43. Morris, R. D., Audet, A. M., Angelillo, I. F., Chalmers, T. C., and Mosteller, F. (1992). Chlorination, chlorination by-products, and cancer: A metaanalysis. Am. J. Public Health 82, 955-964. Morseth, S. L., Dengerink, H. A., and Wright, J. W. (1985). Effect of impulse noise on water consumption and blood pressure in the female rat. Physiol. Behav. 34, 1013-1016. Moss, R. L., Flynn, R. E., Shi, J., Shen, X. M., Dudley, C., Zhou, A., and Novotny, M. (1998). Electrophysiological and biochemical responses of mouse vomeronasal receptor cells to urine-derived compounds: Possible mechanism of action. Chem. Senses 23, 483-489. Mougdil, G. C. (1986). Update on anaesthesia and the immune response. Can. Anaesth. Soc. J. 33, 554-560. Mrosovsky, N. (1988). Phase response curve for social entrainment. J. Comp. Physiol. 162, 35-46. Mucignat-Caretta, C., Caretta, A., and Baldini, E. (1998). Protein-bound male urinary pheromones: Differential responses according to age and gender. Chem. Senses 23, 67-70. Mulder, J. B. (1971). Animal behavior and electromagnetic energy waves. Lab. Anim. Sci. 21, 389-393. Murakami, H. (1971). Differences between internal and external environment of the mouse cage. Lab. Anim. Sci. 21, 680-684. Murthy, A. S. K., Brezak, M. A., Baez, A. G. (1970). Postcastrational adrenal tumors in two strains of mice: Morphologic, histochemical, and chromatographic studies. J. Natl. Cancer Inst. 45, 1211-1222. Musey, P. I., Adlercreutz, H., Gould K. G., Collins, D. C., Fotsis, T., Bannwart, C., Makela, T., Wahala, K., Brunow, G., and Hase, T. (1995). Effect of diet on lignans and isoflavonoid phytoestrogens in chimpanzees. Life Sci. 57, 655-664. Naiki, M., Takagaki, Y., and Fujiwara, K. (1965). Note on the change of transaminases in the liver and the significance of the transaminase ratio in experimental Tyzzer's disease of mice. Jpn. J. Exp. Med. 35, 305-309. Nair, V., and Casper, R. (1969). The influence of light on daily rhythm in hepatic drug metabolizing enzymes in rat. Life Sci. 8, 1291-1298. Namiki, M., Takayama, H., and Fujiwara, K. (1977). Viral growth in splenic megakaryocytes of mice experimentally infected with mouse hepatitis virus MHV-2. Jpn. J. Exp. Med. 47, 41-48.

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH Naot, Y., Merchav, S., Ben-David, E., and Ginsburg, H. (1979a). Mitogenic activity of Mycoplasma pulmonis. I. Stimulation of rat B and T lymphocytes. Immunology 36, 399-406. Naot, Y., Simon-Tou, R., and Ginburg, H. (1979b). Mitogenic activity of Mycoplasma pulmonis. II. Studies on the biochemical nature of the mitogenic factor. Eur. J. Immunol. 9, 149-151. Nash, R. E., and Llanos, J. M. E. (1971). Twenty-four hour variations in DNA synthesis of a fast-growing and a slow-growing hepatoma: DNA synthesis rhythm in hepatoma. J. Natl. Cancer Inst. 47, 1007-1012. National Research Council (1992). "Recognition and Alleviation of Pain and Distress in Laboratory Animals." National Academy of Sciences, Washington, D.C. National Research Council (1996). "Guide for the Care and Use of Laboratory Animals." National Academy of Sciences, Washington, D.C. Nelson, J. B. (1952a). Acute hepatitis associated with mouse leukemia. I. Pathological features and transmission of the disease. J. Exp. Med. 96, 293300. Nelson, J. B. (1952b). Acute hepatitis associated with mouse leukemia. II. Etiology and host range of the causal agent in mice. J. Exp. Med. 96, 301-305. Nelson, J. B. (1959). Emergence of hepatitis virus in mice infected with ascites tumor. Proc. Soc. Exp. Biol. Med. 102, 357-360. Nelson, J. B. (1960). The problems of disease and quality in laboratory animals. J. Med. Educ. 35, 34-43. Nelson, R. J., and Zucker, I. (1987). Spontaneous testicular recrudescence of Syrian hamsters: Role of stimulatory photoperiods. Physiol. Behav. 39, 616-617. Nerem, R. M., Levesque, M. J., and Cornhill, J. E (1980). Social environment as a factor in diet-induced atherosclerosis. Science 208, 1475-1476. Nettesheim, P., Schreiber, H., Cresia, D. A., and Richter, C. B. (1974). Respiratory infections in the pathogenesis of lung cancer. Recent Results Cancer Res. 44, 138-157. Nettesheim, P., Topping, D. C., and Jambasi, R. (1981). Host and environmental factors enhancing carcinogenesis in the respiratory tract. Ann. Rev. Pharmacol. Toxicol. 21, 133-163. Newberne, P. M., and McConnell, R. G. (1980). Dietary nutrients and contaminants in laboratory animal experimentation. J. Environ. Pathol. Toxicol. 4, 105-122. Newberne, P. M., Weigert, J., and Kula, N. (1979). Effects of dietary fat on hepatic mixed-function oxidases and hepatocellular carcinoma induced by aflatoxin B1 in rats. Cancer Res. 39, 3986-3991. Newton, W. M. (1978). Environmental impact on laboratory animals. Adv. Vet. Sci. Comp. Med. 22, 1-28. Nguyen, M. T., Camenisch, T., Snouwaert, J., Hicks, E., Coffman, T. M., Anderson, P. A. W., Malouf, N. N., and Koller, B. H. (1997). The prostaglandin receptor EP4 triggers remodelling of the cardiovascular system at birth. Nature 390, 78-81. Nicklas, W., Giese, M., Zawatzky, R., Kirchner, H., and Eaton, P. (1988). Contamination of a monoclonal antibody with LDH-virus causes interferon induction. Lab. Anim. Sci. 38, 152-154. Niederkorn, J. Y. (1985). Enhanced pulmonary natural killer cell activity during murine encephalitozoonosis. J. Parasitol. 71, 70-74. Niederkorn, J. Y., Shadduck, J. A., and Schmidt, E. C. (1981). Susceptibility of selected inbred strains of mice to Encephalitozoon cuniculi. J. Infect. Dis. 144, 249-253. Niederkorn, J. Y., Brieland, J. A., and Mayhew, E. (1983). Enhanced natural killer cell activity in experimental murine encephalitozoonosis. Infect. Immun. 41, 302-307. Niven, J. S. E, Gledhill, A. W., Dick, G. W. A., and An&ewes, C. H. (1952). Further light on mouse hepatitis. Lancet 2, 1061. Njaa, L. R., Utne, E, and Braeckkan, O. R. (1957). Effect of relative humidity on rat breeding and ringtail. Nature (Lond.) 180, 290-291. Noell, W. K., and Albrecht, R. (1971). Irreversible effects of visible light on the retina: Role of vitamin A. Science 172, 76-80.

1177 Nossaman, B. C., Amouzadeh, H. R., and Sangiah, S. (1990). Effects of chloramphenicol, cimetidine, and phenobarbital on and tolerance to xylazineketamine anesthesia in dogs. Vet. Hum. Toxicol. 32, 216-219. Notkins, A. L. (1971). Enzymatic and immunologic alterations in mice infected with lactic dehydrogenase virus. Am. J. Pathol. 64, 733-746. Notkins, A. L., and Scheele, C. (1964). Impaired clearance of plasma enzyme in normal and lactate dehydrogenase agent infected mice. Proc. Soc. Exp. Biol. Med. 117, 350-353. Notkins, A. L., Mergenhagen, S. E., Rizzo, A. A., Scheele, C., and Waldman, T. A. (1966). Elevated r-globulin and increased antibody production in mice infected with lactic dehydrogenase virus. J. Exp. Med. 123, 347-364. Nunoya, T., Itabashi, M., Kudow, S., Hayashi, M., and Tajima, M. (1977). An epizootic outbreak of sialodacryoadenitis in rats. Jpn. J. Vet. Sci. 39, 445450. Nyska, A., Maronpot, R. R., Eldridge, S. R., Haseman, J. K., and Hailey, J. R. (1997). Alteration in cell kinetics in control B6C3F~ mice infected with Helicobacter hepaticus. Toxicol. Pathol. 25, 591-596. Nyska, A., Leininger, J. R., Maronpot, R. R., Haseman, J. K., and Hailey, J. R. (1998). Effect of individual versus group caging on the incidence of pituitary and Leydig cell tumors in F344 rats: Proposed mechanism. Med. Hypotheses. 50, 525-529. Ohanian, E. V., and Iwai, A. J. (1980). Etiological role of cadmium in hypertension in an animal model. J. Environ. Pathol. Toxicol. 4, 229-241. Oldstone, M. B. A. (1990). Viruses as therapeutic agents. J. Exp. Med. 171, 2077-2089. Oldstone, M. B. A., and Dixon, E J. (1972). Inhibition of antibodies to nuclear antigen and to DNA in New Zealand mice infected with lactate dehydrogenase virus. Science 175, 784-786. Olovson, S. G. (1986). Diet and breeding performance in cats. Lab. Anim. 20, 221-230. O'Steen, W. K., and Anderson, K. V. (1972). Photoreceptor degeneration after exposure of rats to incandescent illumination. Z. Zellforsch. Mikrosk. Anat. 127, 306-313. O'Steen, W. K., Shear, C. R., and Anderson, K. V. (1973). Retinal damage after prolonged exposure to visible light. A light and electron microscopic study. Am. J. Anat. 134, 5-22. Oyama, T. (1973). Endocrine responses to anesthetic agents. Br. J. Anaesth. 45, 276-281. Oz, H. S., and Hughes, W. T. (1997). Pneumocystis carinii infection alters GTPbinding proteins in the lung. J. Parasitol. 83, 679-685. Padnos, M., and Molomut, N. (1973). Inhibition of mouse tumors and viruses by the M-P strain of LCM virus. In "Lymphocytic Choriomeningitis Virus and Other Arenaviruses" (E Lehmann-Grube, ed.), pp. 151-163. SpringerVerlag, New York. Pakes, S. P., Lu, Y. S., and Meunier, P. C. (1984). Factors that complicate animal research. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 649-665. Academic Press, Orlando, Florida. Papaioannou, V. E., and Fox, J. G. (1993). Efficacy of tribromoethanol anesthesia in mice. Lab. Anim. Sci. 43, 189-192. Paradisi, F., Graziano, L., and Maio, G. (1972). Histochemistry of glutamicoxaloacetic transaminase in mouse liver during MHV3 infection. Experimentia 28, 551-552. Parker, J. C. (1980). The possibilities and limitations of virus control in laboratory animals. In "Animal Quality and Models in Research" (A. Spiegel, S. Erichsen, and H. A. SoUeveld, eds.), pp. 161-172. Gustav Fischer Verlag, New York. Parker, L. E, and Radow, B. L. (1974). Isolation stress and volitional ethanol consumption in the rat. Physiol. Behav. 12, 1-3. Paylor, R., and Crawley, J. N. (1997). Inbred strain differences in prepulse inhibition of the mouse startle response. Psychopharmacology 132, 169180. Payvandi, E, Amrute, S., and Fitzgerald-Bocarsly, P. (1998). Exogenous and

1178 endogenous IL-10 regulate IFN-alpha production by peripheral blood mononuclear cells in response to viral stimulation. J. Immunol. 160, 58615868. Pearce, B. D., Hobbs, M. V., McGraw, T. S., and Buchmeier, M. J. (1994). Cytokine induction during T-cell-mediated clearance of mouse hepatitis virus from neurons in vivo. J. Virol. 68, 5483-5495. Pearson, D. J., and Taylor, G. (1975). The influence of the nematode Syphacia obvelata on adjuvant arthritis in rats. Immunology 29, 391-396. Peck, R. M., Eaton, G. J., Peck, E. B., and Litwin, S. (1983). Influence of Sendai virus on carcinogenesis in strain A mice. Lab. Anim. Sci. 33, 154156. Peng, X., Lang, C. M., Drozdowicz, C. K., and Ohlsson-Wilhelm, B. M. (1989). Effect of cage population density on plasma corticosterone and peripheral lymphocyte populations of laboratory mice. Lab. Anim. 23, 302-306. Penn, I. (1988). Tumors of the immunocompromised patient. Annu. Rev. Med. 39, 63-73. Percy, D. H., Hayes, M. A., Kocal, T. E., and Wojcinski, Z. W. (1988). Depletion of salivary gland epidermal growth factor by sialodacryoadenitis virus infection in the Wistar rat. Vet. Pathol. 25, 183-192. Perez, C., Canal, J. R., Dominguez, E., Campillo, J. E., Guillen, M., and Torres, M. D. (1997). Individual housing influences certain biochemical parameters in the rat, Lab. Anim. 31, 357-361. Perkins, S. E., and Lipman, N. S. (1995). Characterization and qualification of microenvironmental contaminants in isolator cages with a variety of contact beddings. Contemp. Top. Lab. Anim. Sci. 34, 93-98. Perkins, S. E., and Lipman, N. S. (1996). Evaluation of microenvironmental conditions and noise generation in three individually ventilated rodent caging systems and static isolator cages. Contemp. Top. Lab. Anim. Sci. 35, 61-65. Peters, L., and Kelly, H. (1977). Influence of stress and stress hormones on transplantability of a non-immunogenic syngeneic mouse tumor. Cancer 39, 1482-1488. Peterson, E. A. (1968). High frequency and ultrasonic over-stimulation of the guinea pig ear. J. Aud. Res. 8, 43-61. Peterson, E. A. (1980). Noise in the animal environment. Lab. Anim. Sci. 30, 422-439. Petrie, M. (1966). The occurrence of Nosema cuniculi (Encephalitozoon cuniculi) in the cells of transplantable, malignant ascites tumors and its effect upon tumor and host. Acta Pathol. Microbiol. Scand. 66, 13-30. Pettinger, W. A., Tanaka, K., Keeton, K., Campbell, W. B., and Brooks, S. N. (1975). Renin release, an artifact of anesthesia and its implications in rats (38597). Proc. Soc. Exp. Biol. Med. 148, 625-630. Pfau, C. J., Valenti, J. K., Jacobson, S., and Pevear, D. C. (1982). Cytotoxic Tcells are induced in mice infected with lymphocytic choriomeningitis virus strains of markedly different properties. Infect. Immun. 36, 598-602. Piazza, M., Piccianino, E, and Matano, F. (1965). Haematological changes in viral (MHV-3) murine hepatitis. Nature 205, 1034-1035. Pickering, R. G., and Pickering, C. E. (1984). The effect of diet on the incidence of pituitary tumours in female Wistar rats. Lab. Anim. 18, 298-314. Pierson, M., and Liebmann, S. L. (1992). Noise exposure-induced audiogenic seizure susceptibility in Sprague-Dawley rats. Epilepsy Res. 13, 35-42. Pierson, C. L., Johnson, A. G., and Feller, I. (1976). Effect of cyclophosphamide on the immune response to Pseudomonas aeruginosa in mice. Infect. Immun. 14, 168-177. Pilotto, L. S. (1995). Disinfection of drinking water, disinfection by-products, and cancer: What about Australia? Aust. J. Public Health 19, 89-93. Pincus, J. H. (1969). Carbon dioxide fixation in rat brain: Relationship to cerebral excitability. Exp. Neurol. 24, 339-347. Pittet, J. E, Hashimoto, S., Pian, M., McElroy, M. C., Nitenberg, G., and Wiener-Kronish, J. P. (1996). Exotoxin A stimulates fluid reabsorption from distal airspaces of lung in anesthesized rats. Am. J. Physiol. 270, 232241. Plaut, S. M., Alder, R., Friedman, S. B., and Ritterson, A. L. (1969). Social fac-

NEIL S. LIPMAN AND SCOTT E. PERKINS tors and resistance to malaria in the mouse: Effects of group vs. individual housing on resistance to Plasmodium berghei infection. Psychosom. Med. 31, 536-540. Plotnikoff, N. P., Murgo, A. J., Miller, G. C., Corder, C. N., and Faith, R. E. (1985). Enkephalins: Immuno-modulators. Fed. Proc. 44, 118-122. Poole, T. B., and Morgan, M. D. R. (1976). Social and territorial behavior of laboratory mice (Mus musculus L.) in small complex areas. Anim. Behav. 24, 476-480. Popper, C. W., Chuang C. C., and Kopin I. J. (1977). Plasma catecholamine concentrations in unanesthetized rats during sleep, wakefulness, immobilization, and after decapitation. J. Pharmacol Exp. Then. 202, 144-148. Port, C. D., Garvin, P. J., Ganote, C. E., and Sawyer, D. C. (1978). Pathologic changes induced by a euthanasia agent. Lab. Anim. Sci. 28, 4481. Porter, G., Lane-Petter, W., and Horne, M. (1963). Effects of strong light on breeding mice. J. Anim. Tech. Assoc. 14, 117-119. Pottratz, S. T., Reese, S., and Sheldon, J. L. (1998). Pneumocystis carinii induces interleukin 6 production by an alveolar epithelial cell line. Eur. J. Clin. Invest. 28, 424-429. Potts, R. C., Hassan, H. A., Brown, R. A., MacConnachie, A., Gibbs, J. H., Robertson, A. J., and Beck, J. S. (1983a). In vitro effects of doxycycline and tetracycline on mitogen-stimulated lymphocyte growth. Clin. Exp. Immunol. 53, 458-464. Potts, R. C., MacConnachie, A., Brown, R. A., Gibbs, J. H., Robertson, A. J., Hassan, H. A., and Beck, J. S. (1983b). Some tetracycline drugs suppress mitogen-stimulated lymphocyte growth but others do not. Br. J. Clin. Pharmacol. 16, 127-132. Prien, T., Traber, D. L., Linares, H. A., and Davenport. S. L. (1988). Haemolysis and artifactual lung damage induced by a euthanasia agent. Lab. Anim. 22, 170-172. Proust, J. J., Bucholz, M. A., and Nordin, A. A. (1985). A "lymphokine-like" soluble product that induces proliferation and maturation of B cells appears in the serum-free supernatant of a T cell hybridoma as a consequence of mycoplasmal contamination. J. Immunol. 134, 390-396. Pucak, G. J., Lee, C. S., and Zaino, A. S. (1977). Effects of prolonged high temperature on testicular development and fertility in the male rat. Lab. Anim. Sci. 27, 76-77. Pye, A. (1973). The destructive effect of intense pure tones on the cochleae of mammals. In "Disorders of Aural Function" (A. Taylor, ed.), pp. 89-96. Academic Press, London. Rabin, B. S., and Salvin, S. B. (1987). Effect of differential housing and time on immune reactivity to sheep erythrocytes and Candida. Brain Behav. Immun. 1, 267-275. Radzialowski, E M., and Bousquet, W. E (1968). Daily rhythmic variation in hepatic drug metabolism in the rat and mouse. J. Pharmacol. Exp. Then. 163, 229-238. Ramaley, J. A. (1972). Changes in daily serum corticosterone values in maturing male and female rats. Steroids 20, 185-197. Ramljak, D., Jones, A. B., Diwan, B. A., Perantoni, A. O., Hochadel, J. E, and Anderson, L. M. (1998). Epidermal growth factor and transforming growth factor-alpha-associated overexpression of cyclin D 1, Cdk4, and c-Myc during hepatocarcinogenesis in Helicobacter hepaticus-infected A/JCr mice. Cancer Res. 58, 3590-3597. Rao, G. N., and Lindsey, J. R. (1988). Ankylosis of hock joints in group caged male B6C3F1 mice. Lab. Anim. Sci. 38, 417-421. Rao, G. N., Ney, E., and Herbert, R. A. (1997). Influence of diet on mammary cancer in transgenic mice bearing an oncogene expressed in mammary tissue. Breast Cancer Res. Treat. 45, 149-158. Raszyk, J., Pokorna, Z., Strnadova, V., Gajd Askova, V., Ulrich, R., Jarosova, A., Salava, J., and Palac, J. (1995). Mutagenicity of stable dust and drinking water on swine and cattle farms. Vet. Med. (Praha) 40, 273-278. Reinhard, M. K., Hottendorf, G. H., and Powell, E. D. (1991). Differences in the sensitivity of Fischer and Sprague-Dawley rats to aminoglycoside nephrotoxicity. Toxicol. Pathol. 19, 66-71.

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH

Repacholi, M. H. (1997). Radiofrequency field exposure and cancer: What do the laboratory studies suggest ? Environ. Health Perspect. 1t)5, 1565 - 1568. Repacholi, M. H. (1998). Low-level exposure to radiofrequency electromagnetic fields: Health effects and research needs. Bioelectromagnetics 19, 1-19. Repacholi, M. H., and Greenebaum, B. (1999). Interaction of static and extremely low frequency electric and magnetic fields with living systems: Health effects and research needs. Bioelectromagnetics 20, 133-160. Repacholi, M. H., Basten, A., Gebski, V., Noonan, D., Finnie, J., and Harris, A. W. (1997). Lymphomas in E mu-Pim 1 transgenic mice exposed to pulsed 900 MHz electromagnetic fields. Radiat. Res. 147, 631-640. Report to the Congress by the Comptroller General of the United States (1981). Problems in assessing the cancer risks of low-level ionizing radiation exposure. US. General Accounting Office 2, 1-39. Rice, S. A., and Fish, K. J. (1987). Anesthetic considerations for drug metabolism studies in laboratory animals. Lab. Anim. Sci. 37, 520. Richardson, M., Fletch, A., Delaney, K., De Reske, M., Wilcox, L. H., and Kinlough-Rathbone, R. L. (1997a). Increased expression of vascular cell adhesion molecule-1 by the aortic endothelium of rabbits with Pasteurella multocida pneumonia. Lab. Anim. Sci. 47, 27-35. Richardson, M., De Reske, M., Delaney, K., Fletch, A., Wilcox, L. H., and Kinlough-Rathbone, R. L. (1997b). Respiratory infection in lipid-fed rabbits enhances sudanophilia and the expression of VCAM- 1. Am. J. Pathol. 151, 1009-1017. Richter, C. (1968). Inherent twenty-four hour and lunar clocks of a primate-the squirrel monkey. Commun. Behav. Biol. 1, 305-322. Riley, V. (1964). Synergism between a lactate dehydrogenase elevating virus and Eperythrozoon coccoides. Science 146, 921- 922. Riley, V. (1966). Spontaneous mammary tumors: Decrease of incidence in mice infected with enzyme-elevating virus. Science 153, 1657-1658. Riley, V. (1975). Mouse mammary tumors: Alterations of incidence as apparent function of stress. Science 189, 465-467. Riley, V., Campbell, H. A., and Stock, C. C. (1970). Asparaginase clearance: Influence of the LDH-elevating virus. Proc. Soc. Exp. Biol. Med. 133, 38-42. Riley, V., Braun, W., Ishizuka, M., and Spackman, D. (1975). Antibodyproducing cells: Virus-induced alteration of response to antigen. Proc. Natl. Acad. Sci. 73, 1707-1711. Riley, V., Spackman, D. H., Santisteban, G. A., Dalldorf, G., Hellstrom, I., Hellstrom, K. E., Lance, E. M., Rowson, K. E., Mahy, B. W., Alexander, P., Stock, C. C., Sjogres, H. O., Hollander, V. P., and Horzinek, M. C. (1978). The LDH virus: An interfering biological contaminant. Science 21)0, 124-126. Riviere, J. E., and Spoo, J. W. (1995). Aminoglycoside antibiotics. In "Veterinary Pharmacology and Therapeutics" (H. R. Adams, ed.), pp. 797-819. Iowa State Univ. Press, Ames. Robacker, K. M., Kulkarni, A. P., and Hodgson, E. (1981). Pesticide induced changes in the mouse hepatic microsomal cytochrome P-450-dependent monooxygenase system and other enzymes. J. Environ. Sci. Health 16, 529-545. Roberts, N. J. (1982). Different effects of influenza virus, respiratory syncytial virus, and Sendai virus on human lymphocytes and macrophages. Infect. Immun. 35, 1142-1146. Romeo, R. D., Parfitt, D. B., Richardson, H. N., and Sisk, C. L. (1998). Pheromones elicit equivalent levels of fos-immunoreactivity in prepubertal and adult male Syrian hamsters. Horm. Behav. 34, 48-55. Romero, J. A. (1976). Influence of diurnal cycles on biochemical parameters of drug sensitivity: The pineal gland as a model. Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 1157-1161. Ronco, P., Riviere, Y., Thouas, Y., Bandu, M. T., Guillon, J. C., Verroust, P., and Morel-Maroger, L. (1981). Lymphocytic choriomeningitis virus infection in the nude mouse. An immunopathological study. Immunology 43, 763770.

1179 Ronis, M. J., Badger, T. M., Shema, S. J., Roberson, E K., and Shaikh, E (1998a). Effects on pubertal growth and reproduction in rats exposed to lead perinatally or continuously throughout development. J. Toxicol. Environ. Health 53, 327-341. Ronis, M. J., Gandy, J., and Badger, T. (1998b). Endocrine mechanisms underlying reproductive toxicity in the developing rat chronically exposed to dietary lead. J. Toxicol. Environ. Health 54, 77-99. Rosen, D. D., and Berk, R. S. (1977). Pseudomonas eye infections in cyclophosphamide treated mice. Invest. Ophthalmol. Vis. Sci. 16, 649-652. Ross, M. H., and Bras, G. (1973). Influence of protein under- and over-nutrition on spontaneous tumor prevalence in the rat. J. Nutr. 103, 944-963. Ross, S. E., Simecka, J. W., Gambill, G. P., Davis, J. K., and Cassell, G. H. (1992). Mycoplasma pulmonis possesses a novel chemoattractant for B lymphocytes. Infect. Immun. 60, 669-674. Rought, S. E., Yau, P. M., Chuang, L. E, Doi, R. H., and Chuang, R. Y. (1999). Effect of the chlorinated hydrocarbons heptachlor, chlordane, and toxaphene on retinoblastoma tumor suppressor in human lymphocytes. Toxicol. Lett. 104, 127-135. Ruebner, B. H., and Hirano, T. (1965). Viral hepatitis in mice. Changes in oxidative enzymes and phosphatases after murine hepatitis virus (MHV-3) infection. Lab. Invest. 14, 157-168. Ruffolo, P. R., Margolis, G., and Kilham, L. (1966). The induction of hepatitis by prior partial hepatectomy in resistant adult rats infected with H-1 virus. Am. J. Pathol. 49, 795-824. Russell, R. J., Festing, M. E, Deeny, A. A., and Peters, A. G. (1993). DNA fingerprinting for genetic monitoring of inbred laboratory rats and mice. Lab. Anim. Sci. 43, 460-465. Rutten, A. A., and de Groot, A. P. (1992). Comparison of cereal-based diet with purified diet by short-term feeding studies in rats, mice, and hamsters, with emphasis on toxicity characteristics. Food Chem. Toxicol. 30, 601610. Saibaba, P., Sales, G. D., Stodulski, G., and Hau, J. (1996). Behaviour of rats in their home cages: Daytime variations and effects of routine husbandry procedures analysed by time sampling techniques. Lab. Anim. 30, 13-21. Sales, G. D. (1991). The effect of 22 kHz calls and artificial 38 kHz signals on activity in rats. Behav. Process. 24, 83-93. Sales, G., and Pye, J. D. (1974). "Ultrasonic Communications by Animals." Chapman and Hall, London. Sales, G. D., and Smith, J. D. (1980). Ultrasonic behavior and mother-infant interactions in rodents. In "Maternal Influences and Early Behaviour" (W. Smotherman and R. W. Bell, eds.), pp. 105-133. Spectrum Press, New York. Sales, G. D., Wilson, K. J., Spencer, K. E., and Milligan, S. R. (1988). Environmental ultrasound in laboratories and animal houses: A possible cause for concern in the welfare and use of laboratory animals. Lab. Anim. 22, 369-375. Saltarelli, C. G., and Coppola, C. P. (1979). Influence of visible light on organ weights of mice. Lab. Anim. Sci. 29, 319-322. Sanz, P., Rodriguez-Vicente, M. C., Villar, P., and Repetto, M. (1988). Uncontrollable atmospheric conditions which can affect animal experimentation. Vet. Hum. Toxicol. 30, 452-454. Sato, K., Ooi, H. K., Nonaka, N., Oku, Y., and Kamiya, M. (1995). Antibody production in Syphacia obvelata infected mice. J. Parasitol. 81, 559-562. Sato, W., Enzan, K., Mitsuhata, H., Mazaki, Y., Kayaba, M., and Suzuki, M. (1996). Effect of sevoflurane on release of TNF-alpha and IL-1 beta from human monocytes. Masui 45, 309-312. Schecter, A. J., Olson, J., and Papke, O. (1996). Exposure of laboratory animals to polychlorinated dibenzodioxins and polychlorinated dibenzofurans from commercial rodent chow. Chemosphere 32, 501-508. Scheid, M. P., Goldstein, G., and Boyse, E. A. (1975). Differentiation of T-cells in nude mice. Science 10, 1211-1213. Scheving, L. E., and Pauly, J. E. (1967). Daily rhythmic variations in blood coagulation times in rats. Anat. Rec. 157, 657-665.

1180 Scheving, L. E., Vedral, D. E, and Pauly, J. E. (1968). A circadian susceptibility rhythm in rats to pentobarbital sodium. Anat. Rec. 160, 741-749. Scheving, L. E., Burns, E. R., Pauly, J. E., and Tsai, T. H. (1978). Circadian variation in cell division of the mouse alimentary tract, bone marrow, and corneal epithelium. Anat. Rec. 191, 479-486. Scheving, L. E., Tsai, T. H., Powell, E. W., Pasley, J. N., Halberg, E, and Dunn, J. (1983). Bilateral lesions of superchiasmatic nuclei affect circadian rhythms in [3H]-thymidine incorporation into deoxyribonuleic acid in mouse intestinal tract, mitotic index of corneal epithelium, and serum corticosterone. Anat. Rec. 205, 239-249. Schindler, L., Engles, H., and Kirchner, H. (1982). Activation of natural killer cells and induction of interferon after injection of mouse hepatitis virus type 3 in mice. Infect. Immun. 35, 869-873. Schinkel, A. H., Smit, J. J., van Tellingen, O., Beijnen, J. H., Wagenaar, E., Van Deemter, L., Mol, C. A., Van Der Valk, M. A., Robanus-Maandag, E. C., te Riele, H. P. J., Berns, A. J. M., and Borst, P. (1994). Disruption of the mouse mdr 1 a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77, 491-502. Schoeb, T. R., Davidson, M. K., and Lindsey, J. R. (1982). Intracage ammonia promotes growth of Mycoplasma pulmonis in the respiratory tract of rats. Infect. Immun. 38, 212- 217. Schook, L. B., Carrick, L., Jr., and Berk, R. S. (1977). Experimental pulmonary infection in mice by tracheal intubation of Pseudomonas aeruginosa: The use of antineoplastic agents to overcome natural resistance. Can. J. Microbiol. 23, 823-826. Schreiber, H., Nettesheim, P., Lijinsky, W., Richter, C. B., and Walburg, H. E., Jr. (1972). Induction of lung cancer in germ free, specific pathogen free, and infected rats by N-nitroso-heptamethyleneimine: Enhancement of respiratory infection. J. Natl. Cancer Inst. 4, 1107-1114. Schulman, J. L., and Kilbourne, E. D. (1962). Airborne transmission of influenza virus infection in mice. Nature 195, 1129-1130. Schuster, G. S., Caughman, G. B., O'Dell, N. L. (1991). Altered lipid metabolism in parvovirus-infected cells. Microbios 66, 143 - 155. Schwimmbeck, P. L., Dryberg, T., and Oldstone, M. B. A. (1988). Abrogation of diabetes in BB rats by acute virus infection. Association of virallymphocyte interactions. J. Immunol. 140, 3394-3400. Seamer, J., Gledhill, A. W., Barlow, J. L., and Hotchin, J. (1961). Effect of Eperythrozoon coccoides upon lymphocytic choriomeningitis in mice. J. ImmunoL 86, 512-515. Sebesteny, A., and Hill, A. C. (1974). Hepatitis and brain lesions due to mouse hepatitis virus accompanied by wasting in nude mice. Lab. Anim. 8, 317326. Segovia, J. C., Bueren, J. A., and Almendral, J. M. (1995). Myeloid depression follows infection of susceptible newborn mice with the parvovirus minute virus of mice (strain i). J. Virol. 69, 3229-3232. Selgrade, M. K., Repacholi, M. H., and Koren, H. S. (1997). Ultraviolet radiation-induced immune modulation: Potential consequences for infectious, allergic, and autoimmune disease. Environ. Health Perspect. 105, 332334. Semple-Rowland, S. L., and Dawson, W. W. (1987a). Cyclic light intensity threshold for retinal damage in albino rats raised under 6 lux. Exp. Eye Res. 44, 643- 661. Semple-Rowland, S. L., and Dawson, W. W. (1987b). Retinal cyclic light damage threshold for albino rats. Lab. Anim. Sci. 37, 289-298. Serrano, L. J. (1971). Carbon dioxide and ammonia in mouse cages: Effect of cage covers, population, and activity. Lab. Anim. Sci. 21, 75-85. Shanks, N., Renton, C., Zalcman, S., and Anisman, H. (1994). Influence of change from grouped to individual housing on a T-cell-dependent immune response in mice: Antagonism by diazepam. PharmacoL Biochem. Behav. 47, 497-502. Shapiro, R. (1980). Chemical contamination of drinking water: What it is and where it comes from. Lab. Anim. 9, 45-51. Shapiro, J., Jersky, J., Katzav, S., Feldman, M., and Segal, S. (1981). Anesthetic

NEIL S. LIPMAN AND SCOTT E. PERKINS drugs accelerate the progression of postoperative metastases of mouse tumors. J. Clin. Invest. 68, 678-685. Sharon, I. M., Feller, R. P., and Burney, S. W. (1971). The effects of lights of different spectra on caries incidence in the golden hamster. Arch. Oral Biol. 16, 1427-1423. Shear, C. R., O'Steen, W. K., and Anderson, K. V. (1973). Effects of short-term low intensity light on the albino rat retina. An electron microscopic study. Am. J. Anat. 138, 127-132. Sheehan, P. M., Stokes, D. C., Yeh, Y. Y., and Hughes, W. T. (1986). Surfactant phospholipids and lavage phospholipase A2 in experimental Pneumocystis carinii pneumonia. Am. Rev. Respir. Dis. 134, 526-531. Shenaeva, T. A. (1990). Effects of vibration and noise on the reproductive function in experimental animals. Gig. Tr. Prof. Zabol. 9, 16-21. Sherrill, A., and Gorham, J. (1985). Bone marrow hypoplasia associated with estrus in ferrets. Lab. Anim. Sci. 35, 280-286. Shirakami, G., Magaribuchi, T., Shingu, K., Saito, Y., O'higashi, T., Nakao, K., and Mori, K. (1995). Effects of anesthesia and surgery on plasma endothelin levels. Anesth. Analg. 80, 449-453. Shoji, R., Murakami, U., and Shimizu, T. (1975). Influence of low intensity ultrasonic irradiation on prenatal development of two inbred mouse strains. Teratology 12, 227-232. Shomer, N. H., Dangler, C. A., Schrenzel, M. D., and Fox, J. G. (1997). Helicobacter bilis-induced inflammatory bowel disease in SCID mice with defined flora. Infect. Immun. 65, 4858-4864. Sibilia, M., and Wagner, E. F. (1995). Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 269, 234-238. Siegel, M. A., and Jensen, R. A. (1986). The effects of naloxone and cage size on social play and activity in isolated young rats. Behav. Neural. Biol. 45, 155-168. Siegfried, B., Frischknecht, H. R., and Waser, P. G. (1984). Defeat, learned submissiveness, and analgesia in mice: Effect of genotype. Behav. Neural. Biol. 42, 91-97. Silverman, J., and Adams, J. D. (1983). N-nitrosamines in laboratory animal feed and bedding. Lab. Anim. Sci. 33, 161-164. Simmons, M. I., Robie, D. M., and Jones, J. B. (1968). Effect of a filter cover on temperature and humidity in a mouse cage. Lab. Anim. 2, 113-120. Simpson, E. M., Linder, C. C., Sargent, E. E., Davisson, M. T., Mobraaten, L. E., and Sharp, J. J. (1997). Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16, 19-27. Simpson-Haidaris, P. J., Courtney, M. A., Wright, T. W., Goss, R., Harmsen, A., and Gigliotti, E (1998). Induction of fibrinogen expression in the lung epithelium during Pneumocystis carinii pneumonia. Infect. Immun. 66, 44314439. Sipes, I. G., and Gandolfi, A. J. (1991). Biotransformation of toxicants. In "Casarett and Doull's Toxicology, the Basic Science of Poisons" (M. O. Amdur, J. Doull, and C. D. Klaasen, eds.), pp. 88-126. Pergamon Press, New York. Sipowicz, M. A., Chomarat, P., Diwan, B. A., Anver, M. A., Awasti, Y. C., Ward, J. M., Rice, J. M., Kasprzak, K. S., Wild, C. P., and Anderson, L. M. (1997). Increased oxidative DNA damage and hepatocyte overexpression of specific cytochrome P450 isoforms in hepatitis of mice infected with Helicobacter hepaticus. Am. J. Pathol. 151, 933-941. Sklar, L. S., and Anisman, H. (1979). Stress and coping factors influence tumor growth. Science 205, 513-515. Skopets, B., Wilson, R. P., Griffith, J. W., and Lang, C. M. (1996). Ivermectin toxicity in young mice. Lab. Anim. Sci. 46, 111-112. Smiler, K. L., Stein, S., Hrapkiewicz, K. L., and Hiben, J. R. (1990). Tissue response to intramuscular and intraperitoneal injections of ketamine and xylazine in rats. Lab. Anim. Sci. 40, 60-64. Solomon, G. F. (1969). Stress and antibody response in rats. Int. Arch. Allergy Appl. Immunol. 35, 97-104. Spalding, J. F., Archuleta, R. F., and Holland, L. M. (1969a). Influence of the visible colour spectrum on activity in mice. Lab. Anim. Care 19, 50-54.

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH Spalding, J. E, Holland, L. M., and Tietjen, G. L. (1969b). Sex ratio of progeny from mice reared under various colour lighting conditions. Lab. Anim. Care 19, 602-604. Spearow, J. L., Doemeny, P., Sera, R., Leffler, R., and Berkley, M. (1999). Genetic variation in susceptibility to endocrine disruption by estrogen in mice. Science 285, 1259-1261. Stavinoha, W. B. (1993). Use of microwaves for rapid fixation of tissues in vivo. Scanning 15, 115-117. Steffan, A. M., Pereira, C. A., Bingen, A., Valle, M., Martin, J. P., Koehren, E, Royer, C., Gendrault, J. L., and Kirn, A. (1995). Mouse hepatitis virus type 3 infection provokes a decrease in the number of sunusoidal endothelial cell fenestrae both in vivo and in vitro. Hepatology 22, 395-401. Steplewski, Z., Goldman, P. R., and Vogel, W. H. (1987). Effect of housing stress on the formation and development of tumors in rats. Cancer Lett. 34, 257-261. St. Lezin, E., Simonet, L., Pravanec, M., Kurtz, T. W. (1992). Hypertensive strains and normotensive "control" strains. How closely are they related? Hypertension 19, 419-424. Streilein, J. W., Shadduck, J. A., and Pakes, S. P. (1981). Effects of splenectomy and Sendai virus infection on rejection of male skin isografts by pathogen free C57BL/6 female mice. Transplantation 32, 34-37. Stuhlman, R. A., and Wagner, J. E. (1971). Ringtail in Mystromys albicaudatus: A case report. Lab. Anim. Sci. 21, 585-587. Surbeck, H. (1995). Determination of natural radionuclides in drinking water: A tentative protocol. Sci. Total Environ. 173-174, 91-109. Taets, C., Aref, S., and Rayburn, A. L. (1998). The clastogenic potential of triazine herbicide combinations found in potable water supplies. Environ. Health Perspect. 106, 197-201. Taffs, L. E (1974). Some diseases in normal and immunosuppressed experimental animals. Lab. Anim. 8, 149-154. Takagaki, Y., Ito, M., Naiki, M., Fujiwara, K., Okugi, M., Maejima, K., and Tajima, Y. (1966). Experimental Tyzzer's disease in different species of laboratory animals. Jpn. J. Exp. Med. 36, 519-534. Takenaka, S., and Fujiwara, K. (1975). Effect of carbon tetrachloride on experimental Tyzzer's disease of mice. Jpn. J. Exp. Med. 45, 393-402. Takeyama, H., Kawashima, K., Yamada, K., and Ito, Y. (1979). Induction of tumor resistance in mice by L1210 leukemia cells persistently infected with HVJ (Sendai virus). Gann 70, 493-501. Tamura, T., and Fujiwara, K. (1979). IgM and IgG response to sheep red blood cells in mouse hepatitis virus infected nude mice. Microbiol. Immunol. 23, 177-183. Tamura, T., Machii, K., Ueda, K., and Fujiwara, K. (1978). Modification of immune response in nude mice infected with mouse hepatitis virus. Microbiol. Immunol. 22, 557-564. Tamura, T., Sakaguchi, A., Kai, C., and Fujiwara, K. (1980). Enhanced phagocytic activity of macrophages in mouse hepatitis virus infected nude mice. Microbiol. Immunol. 24, 243-247. Tamura, T., Sakaguchi, A., Ishida, T., and Fujiwara, K. (1981). Effect of mouse hepatitis virus infection on natural killer cell activity in nude mice. Microbiol. Immunol. 25, 1363-1368. Tardieu, M., Hery, C., and Dupuy, J. M. (1980). Neonatal susceptibility to MHV-3 infection in mice. II. Role of natural effector marrow cells in transfer to resistance. J. Immunol. 124, 418-423. Targowski, S. P., Klucinski, W., Babiker, S., and Nonnecke, B. J. (1984). Effect of ammonia on in vivo and in vitro immune responses. Infect. Immun. 43, 289-293. Tattersall, P., and Cotmore, S. E (1986). The rodent parvoviruses. In "Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research" (P. N. Bhatt, R. O. Jacoby, H. C. Morse III, and A. E. New, eds.), pp. 305-348. Academic Press, Orlando, Florida. Taura, Y., Ishi, K., Nagami, M., Mikasa, N., Nakaichi, M., and Nakama, S. (1995). Changes in lymphoproliferation and DTH responses after vaccination immediately before surgery in puppies. J. Vet. Med. Sci. 57, 899-904.

1181 Taurog, J. D., Leary, S. L., Cremer, M. A., Mahowald, M. L., Sandberg, G. E, and Manning, P. J. (1984). Infection with Mycoplasma pulmonis modulates adjuvant and collagen-induced arthritis in Lewis rats. Arthritis Rheum. 27, 943-946. Tavadia, H., Fleming, K., Hume, P., and Simpson, H. (1972). Circadian rhythmicity of plasma cortisol and PHA-induced lymphocyte transformation. Clin. Exp. Immunol. 22, 190-193. Taylor, N. S., Fox, J. G., and Yan, L. (1995). In vitro hepatotoxic factor in Helicobacter hepaticus, H. pylori, and other Helicobacter species. J. Med. Microbiol. 42, 48-52. Theiss, J. C., Shimkim, M. B., Stoner, G. D., Kniazeff, A. J., and Hoppenstand, R. D. (1980). Effect of lactate dehydrogenase virus on chemically induced mouse lung tumorigenesis. Cancer Res. 40, 64-66. Thigpen, J. E., Li, L. A., Richter, C. B., Lebetkin, E. H., and Jameson, C. W. (1987a). The mouse bioassay for the detection of estrogenic activity in rodent diets: I. A standardized method for conducting the mouse bioassay. Lab. Anim. Sci. 37, 596-601. Thigpen, J. E., Li, L. A., Richter, C. B., Lebetkin, E. H., and Jameson, C. W. (1987b). The mouse bioassay for the detection of estrogenic activity in rodent diets: II. Comparative estrogenic activity of purified, certified, and standard open closed formula rodent diets. Lab. Anim. Sci. 37, 602605. Thigpen, J. E., Lebetkin, E. H., Dawes, M. L., Richter, C. B., and Crawford, D. (1987c). The mouse bioassay for the detection of estrogenic activity in rodent diets. III. Stimulation of uterine weight by. dextrose, sucrose, and corn starch. Lab. Anim. Sci. 37, 606-609. Thomsen, A. R., Bro-Jorgensen, K., and Jensen, B. L. (1982). Lymphocytic choriomeningitis virus-induced immunosuppression: Evidence for viral interference with T-cell maturation. Infect. Immun. 37, 981-986. Thong, Y. H., and Ferrante, A. (1979). Inhibition of mitogen-induced human lymphocyte proliferative responses by tetracycline analogues. Clin. Exp. Immunol. 35, 443-446. Thong, Y. H., and Ferrante, A. (1980). Effect of tetracycline treatment on immunological responses in mice. Clin. Exp. Immunol. 39, 728-732. Threadgill, D. W., Dlugosz, A. A., Hansen, L. A., Tennenbaum, T., Lichti, U., Yee, D., La Mantia, C., Mourton, T., Herrup, K., Harris, R. C., Barnard, J. A., Yuspa, S. H., Coffey, R. J., and Magnuson, T. (1995). Targeted disruption of mouse EGF receptor: Effect of genetic background on mutant phenotype. Science 269, 230-234. Threadgill, D. W., Yee, D., Matin, A., Nadeau, J. H., and Magnuson, T. (1997). Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain. Mamm. Genome 8, 390-393. Tiensiwakul, P., and Husain, S. S. (1979). Effect of mouse hepatitis virus infection on iron retention in the mouse liver. Br. J. Exp. Pathol. 60, 161-166. Tisdale, W. A. (1963). Potentiating effect of K-virus on mouse hepatitis virus (MHV-S) in weanling mice. Proc. Soc. Exp. Biol. Med. 114, 774-777. Toolan, H. W., and Ledinko, N. (1968). Inhibition by H-1 virus of the incidence of tumors produced by adenovirus 12 in hamsters. Virology 35, 475478. Toolan, H. W., Rhode, S. L., III, and Gierthy, J. F. (1982). Inhibition of 7,12dimethylbenz(a)anthracene tumors in Syrian hamsters by prior infection with H-1 parvoviruses. Cancer Res. 42, 2552-2555. Torronen, R., Karenlampi, S., and Pelkonen, K. (1994). Hepa-1 enzyme induction assay as an in vitro indicator of the CYP1Al-inducing potencies of laboratory rodent diets in vivo. Life Sci. 55, 1945-1954. Toth, L. A., and January, B. (1990). Physiological stabilization of rabbits after shipping. Lab. Anim. Sci. 40, 384-387. Tsukamoto, K., Machida, K., Ina, Y., Kuriyama, T., Suzuki, K., Murayama, R., and Saiki, C. (1994). Effects of crowding on immune functions in mice. Nippon Eiseigaku Zasshi 49, 827-836. Tu, H., Diberadinis, L. J., and Lipman, N. S. (1997). Determination of air distribution, exchange, velocity, and leakage in three individually ventilated rodent caging systems. Contemp. Top. Lab. Anim. Sci. 36, 69-73.

1182

NEIL S. LIPMAN AND SCOTT E. PERKINS

factors affecting the response of laboratory animals to drugs. Fed. Proc., Tucker, H. A., Petitclerc, D., and Zinn, S. A. (1984). The influence of photopeFed. Am. Soc. Exp. Biol. 35, 1125-1132. riod on body weight gain, body composition, nutrient intake, and hormone Vessey, S. H. (1964). Effects of grouping on levels of circulating antibodies in secretion. J. Anim. Sci. 59, 1610-1620. mice. Proc. Soc. Exp. Biol. Med. 115, 225-255. Tudoric, N., Coon, R. L., Kampine, J. P., and Bosnjak, Z. J. (1995). Effects of Vincent, J. G., Veomett, R. C., and Riley, R. E (1955). Relation of the indigehalothane and isoflurane on antigen- and leukotriene D4-induced constricnous flora of the small intestine of the rat to post-irradiation bacteremia. tion of guinea pig trachea. Acta Anaesthesiol. Scand. 39, 1111-1116. J. BacterioL 69, 38-44. Tuli, J. S., Smith, J. A., and Morton, D. B. (1995a). Corticosterone, adrenal, and Virelizier, J. L., Virelizier, A. M., and Allison, A. C. (1976). The role of circuspleen weight in mice after tail bleeding, and its effect on nearby animals. lating interferon in the modifications of the immune responsiveness to Lab. Anim. 29, 90-95. mouse hepatitis virus (MHV-3). J. Immunol. 117, 748-753. Tuli, J. S., Smith, J. A., and Morton, D. B. (1995b). Stress measurements in mice Visintainer, M. A., Volpicelli, J. R., and Seligman, M. E. (1982). Tumor reafter transportation. Lab. Anim. 29, 132-138. jection in rats after inescapable or escapable shock. Science 216, 437Tzvetkov, D. (1993). Effect of vibrations on the organismBpossibilities for de439. velopment of non-specific diseases and their prognostication. Cent. Eur. J. Vitarella, D., James, R. A., Miller, K. L., Struve, M. E, Wong, B. A., and DorPublic Health 1, 1O- 15. man, D. C. (1998). Development of an inhalation system for the simultaneTzvetkov, D., Boev, M., and Baykoushev, B. (1992). Vibrations- discriminant ous exposure of rat dams and pups during developmental neurotoxicity models and possibilities for prognosticating specific and nonspecific effects studies. Inhal. Toxicol. 10, 1095-1117. on the organism. Ann. Occup. Hyg. 36, 253-264. Wada, J. A., and Asakura, T. (1970). Circadian alteration of audiogenic seizure Uhl, E. W., Moldawer, L. L., Busse, W. W., Jack, T. J., and Castleman, W. L. susceptibility in rats. Exp. NeuroL 29, 211-214. (1998). Increased tumor necrosis factor-alpha (TNF-alpha) gene expresWade, A. E., Holl, J. E., Hilliard, C. C., Molton, E., and Greene, E E. (1968). sion in parainfluenza type 1 (Sendai) virus-induced bronchiolar fibrosis. Alteration of drug metabolism in rats and mice by an environment of Am. J. Pathol. 152, 513-522. cedarwood. Pharmacology 1, 317-328. Urano, T., and Maejima, K. (1978). Provocation of pseudomoniasis with cyWagner, M. (1988). The effect of infection with the pinworm (Syphacia muris) clophosphamide in mice. Lab. Anim. 12, 159-161. on rat growth. Lab. Anim. Sci. 38, 476-478. U.S. Nuclear Regulatory Commission (USNRC) (1981). "Regulatory Guide: Wainberg, M. A., and Israel, E. (1980). Viral inhibition of lymphocyte mitoInstruction Concerning Risks from Occupational Radiation Exposure." genesis. I. Evidence for the nonspecificity of the effect. J. Immunol. 124, Regulatory Guide 8.29. U.S. Government Printing Office, Washington, 64 -70. D.C. Walker, E. A., Castegnaro, M., and Griciute, L. (1979). N-nitrosamines in the U.S. Nuclear Regulatory Commission (USNRC) (1987). "Regulatory Guide: diet of experimental animals. Cancer Lett. 6, 175-178. Instruction Concerning Prenatal Radiation Exposure." Regulatory Guide Wallace, M. E. (1976). Effects of stress due to deprivation and transport in dif8.13. U.S. Government Printing Office, Washington, D.C. ferent genotypes of house mouse. Lab. Anim. 10, 335-347. Utsumi, K., Maeda, T., Tarsumi, H., and Fujiwara, K. (1978). Some clinical and Waller, T., Morein, B., and Tabiansson, E. (1978). Humoral immune response epizootological observations of infectious sialoadenitis in rats. Exp. Anim. to infection with Encephalitozoon cuniculi in rabbits. Lab. Anim. 12, 14527, 283-287. 148. Utsumi, K., Ishikawa, T., Maeda, T., Shimizu, S., Tatsumi, H., and Fujiwara, K. Walzer, P. D., Kim, C. K., Linke, J., Pogue, C. L., Huerkamp, M. J., Chrisp, (1980). Infectious sialodacryoadenitis and rat breeding. Lab. Anim. 14, C. E., Lerro, A. V., Wixson, S. K., Hall, E., and Shultz, L. D. (1989). Out303-307. breaks of Pneumocystis carinii pneumonia in colonies of immunodeficient Utsumi, K., Maeda, K., Yokota, Y., Fukugawa, S., and Fujiwara, K. (1991). Remice. Infect. Immun. 57, 62-70. productive disorders in female rats infected with sialodacryoadenitis virus. " Wang, S. D., Huang, K. J., Lin, Y. S., and Lei, H. Y. (1994). Sepsis-induced Jikken Dobutsu 40, 361-365. apoptosis of the thymocytes in mice. J. Immunol. 152, 5014-5021. Vacha, J., Znojil, V., Pospisil, M., Hola, J., and Pipalova, I. (1994). Microcytic Ward, J. M., Collins, M. J., and Parker, J. C. (1977). Naturally occurring anemia and changes in ferrokinetics as late after-effects of glucan adminismouse hepatitis virus infection in the nude mouse. Lab. Anim. Sci. 27, 372tration in murine hepatitis virus-infected C57BL/10ScSnPh mice. Int. J. 376. Immunopharmacol. 16, 51- 60. Ward, J. M., Fox, J. G., Anver, M. R., Haines, D. C., George, C. V., Collins, Vadiei, K., Berens, K. L., and Luke, D. R. (1990). Isolation-induced renal funcM. J., Jr., Gorelick, P. L., Nagashima, K., Gonda, M. A., Gilden, R. V., tional changes in rats from four breeders. Lab. Anim. Sci. 40, 56-59. Tully, J. G., Russell, R. J., Benveniste, R. E., Paster, B. J., Dewhirst, E E., Vagell, M. E., and McGinnis, M. Y. (1998). The role of gonadal steroid recepDonovan, J. C., Anderson, L. M., and Rice, J. M. (1994a). Chronic active tor activation in the restoration of sociosexual behavior in adult male rats. hepatitis and associated liver tumors in mice caused by a persistent bacterHorm. Behav. 33, 163-179. ial infection with a novel Helicobacter species. J. Natl. Cancer Inst. 86, van der Veen, J., Poort, Y., and Birchfield, D. J. (1972). Effect of relative hu1222-1227. midity on experimental transmission of Sendai virus in mice. Proc. Soc. Ward, J. M., Anver, M. R., Haines, D. C., and Benveniste, R. E. (1994b). Exp. Biol. Med. 140, 1437-1440. Chronic active hepatitis in mice caused by Helicobacter hepaticus. Am. J. van Oorschot, R. A., Williams-Blangero, S., and Vande Berg, J. L. (1992). Pathol. 145, 959-968. Genetic diversity of laboratory gray short-tailed opossums (Monodelphis Warren, K. S., Rosenthal, M. S., and Domingo, E. O. (1969). Mouse hepatitis domestica): Effect of newly introduced wild-caught animals. Lab. Anim. virus (MHV3) infection in chronic murine schistosomiasis mansoni. Bull Sci. 42, 255-260. N.Y. Acad. Med. 45, 211-224. Van Winkle, T. J., and Balk, M. W. (1986). Spontaneous corneal opacities in labWarschkau, H., Yu, H., and Kiderlen, A. E (1998). Activation and suppression oratory mice. Lab. Anim. Sci. 36, 248-255. of natural cellular immune functions by Pneumocystis carinii. ImmunobiVargas-Rivera, J., Ortega-Corona, B. G., Garcia-Pineda, J., Carranza, J., Salazar, ology 198, 343-360. L. A., and Villareal, J. (1990). Influence of previous housing history on the Weihe, W. H. (1965). Temperature and humidity climatograms for rats and toxicity of amphetamine in aggregate mice. Arch. Invest. Med. (Mex). 21, mice. Lab. Anim. Care 15, 18-28. 65-69. Weihe, W. H. (1971). The significance of the physical environment for the Vessel, E. S., Lang, C. M., White, W. J., Passanati, G. T., Hill, R. N., Clemens, health and state of adaptation of laboratory animals. In "Defining the LabT. L., Lui, D. K., and Johnson, W. D. (1976). Environmental and genetic

29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH

oratory Animal," pp. 353-378. National Academy of Sciences, Washington, D.C. Weihe, W. H. (1973). The effect of temperature on the action of drugs. In "The Pharmacology of Thermoregulation" (P. Lomax and E. Schonbaum, eds.), pp. 409-425. Karger, Basel. Weihe, W. H. (1976). Influence of light on animals. In "Control of the Animal House Environment" (T. McSheehy, ed.), Laboratory Animal Handbook No. 7, pp. 63-76. Laboratory Animal Ltd., London. Weihe, W. H., Brezowsky, H., and Schwarzenbach, E R. (1961). Der Nachweiz einer Wirkung des Klimas und Wetters auf das Wachstum der Ratte. Pflugers Arch. Gesamte Physiol. Menschen Tiere. 273, 514-527. Weihe, W. H., Schidlow, J., and Strittmatter, J. (1969). The effect of light intensity on the breeding and development of rats and golden hamsters. Int. J. Biometeorol. 13, 69-79. Weinland, L. S. (1963). Production of abnormal hamster embryos with ultrasound. Proc. Penn. Acad. Sci. 37, 48-49. Weinreich, S., Capkova, J., Hoebe-Hewryk, B., Boog, C., and Ivanyi, P. (1996). Grouped caging predisposes male mice to ankylosing enthesopathy. Ann. Rheum. Dis. 55, 645-647. Weisbroth, S. H. (1982). Arthropods. In "The Mouse in Biomedical Research. Vol. II: Diseases" (H. L. Foster, J. D. Small, and J. G. Fox, eds.), pp. 385402. Academic Press, New York. Weiss, J., and Zimmermann, E (1999). Tribromoethanol (avertin) as an anaesthetic in mice [letter]. Lab. Anim. 33, 192-193. Welch, B. L., and Welch, A. S. (1969). Sustained effects of brief daily stress (fighting) upon brain and adrenal catecholamines and adrenal, spleen, and heart weights of mice. Proc. Natl. Acad. Sci. 64, 100-107. Welch, B. L., Brown, D. G., Welch, A. S., and Lin, D. C. (1974). Isolation, restrictive confinement, or crowding of rats for one year. I. Weight, nucleic acids, and protein of brain regions. Brain Res. 75, 71-84. Wells, A. B. (1970). The kinetics of cell proliferation in the tracheobronchial epithelia of rats with and without chronic respiratory disease. Cell Tissue Kinet. 3, 185-206. Welsh, R. M. (1978). Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice. II. Characterization of natural killer cell induction. J. Exp. Med. 148, 163-181. Welsh, R. M., Jr., and Doe, W. E (1980). Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice. Natural killer cell activity in cultured spleen leukocytes concomitant with T-cell dependent immune interferon production. Infect. Immun. 30, 473-483. Westerberg, S. C., Smith, C. B., Wiley, B. B., and Jensen, C. (1972). Mycoplasmavirus interrelationships in mouse tracheal organ cultures. Infect. Immun. 5, 840-846. Wheelock, E. E (1996). The effects of nontumor viruses on virus-induced leukemia in mice: Reciprocal interference between Sendai virus and Friend leukemia virus in DBA/2 mice. Proc. Natl. Acad. Sci. US.A. 55, 774-780. White, N. R., and Barfield, R. J. (1987). Role of the ultrasonic vocalization of the female rat (Rattus norvegicus) in sexual behavior. J. Comp. Psychol. 101, 73-81. Whitmore, S. P., Gilliam, A. E, Hendren, R. W., Lewis, S. E., Rao, G. N., and Whisnant, C. C. (1996). Genetic monitoring of inbred rodents from controlled production colonies through biochemical markers and skin grafting procedures. Lab. Anim. Sci. 46, 585-588. Wightman, S. R., Mann, P. C., and Wagner, J. E. (1980). Dihydrostreptomycin toxicity in the Mongolian gerbil, Meriones unguiculatus. Lab. Anim. Sci. 30, 71-75. Wilberz, S., Partke, H. J., Dagnaes-Hansen, E, and Herberg, L. (1991). Persistent MHV (mouse hepatitis virus) infection reduces the incidence of diabetes mellitus in non-obese diabetic mice. Diabetologia 34, 2-5. Wilkening, G. M., and Sutton, C. H. (1990). Health effects of nonionizing radiation. Med. Clin. North Am. 74, 489-507. Williams, D. I. (1971). Maze exploration in the rat under different levels of illumination. Anim. Behav. 19, 365-367.

1183 Williams, D. L., and Di Luzzio, N. R. (1980). Glucan-induced modification of murine viral hepatitis. Science 108, 67-69. Williams, G. M., and Weisburger, J. H. (1991). Chemical carcinogenesis. In "Casarett and Doull's Toxicology, the Basic Science of Poisons" (M. O. Amdur, J. Doull, and C. D. Klassen, eds.), pp. 127-200. Pergamon Press, New York. Williams-Blangero, S. (1993). Research-oriented genetic management of nonhuman primate colonies. Lab. Anim. Sci. 43, 535-540. Williams-Blangero, S., Blangero, J., Murthy, K. K., and Lanford, R. E. (1996). Genetic analysis of serum alanine transaminase activity in normal and hepatitis C virus-infected chimpanzees: An application of research-oriented genetic management. Lab. Anim. Sci. 46, 26-30. Wise, A. (1982). Interaction of diet and toxicity--the future role of purified diet in toxicological research. Arch. Toxicol. 50, 287-299. Wogan, G. N. (1968). Aflatoxin risks and control measures. Fed. Proc., Fed. Am. Soc. Exp. Biol. 27, 932-938. Wolstenholme, G. E. W., and O'Connor, M., eds. (1967). Effects of external stimuli on reproduction. Ciba Foundation Study No. 26. J. and A. Churchill Ltd., London. Wongwiwat, M., Sukapanit, S., Triyanod, C., and Sawyer, W. D. (1972). Circadian rhythm of the resistance of mice to acute pneumococcal infection. Infect. Immun. 5, 442-448. Wright, J. E, and Seibold, H. R. (1958). Estrogen contamination of pelleted feed for laboratory animals. Effects on guinea pig production. J. Am. Vet. Med. Assoc. 132, 258-261. Wu-Hsieh, B., Howard, D. H., and Ahmed, R. (1988). Virus-induced immunosuppression: A murine model of susceptibility to opportunistic infection. J. Infect. Dis. 158, 232-235. Wurtman, R. J. (1975). The effects of light on man and other mammals. Annu. Rev. Physiol. 37, 467-483. Wybran, J. (1985). Enkephalins and endorphins as modifiers of the immune system: Present and future. Fed. Proc. 44, 92-94. Yagil, R., Etzion, Z., and Berlyne, G. M. (1976). Changes in rat milk quantity and quality due to variations in litter size and high ambient temperature. Lab. Anim. Sci. 26, 33-37. Yamada, A., Osada, Y., Takayama, S., Akimoto, T., Ogawa, H., Oshima, Y., and Fujiwara, K. (1969). Tyzzer's disease syndrome in laboratory rats treated with adenocorticotropic hormone. Jpn. J. Exp. Med. 39, 505-518. Yang, C. S., Brady, J. E, and Hong, J. Y. (1992). Dietary effects on cytochromes P450, xenobiotic metabolism, and toxicity. FASEB J. 6, 737-744. Yoneda, K., and Waltzer, P. D. (1980). Interaction of Pneumocystis carinii with host lungs: An ultrastructural study. Infect. Immun. 29, 692-703. Yu, M. L., and Limper, A. H. (1997). Pneumocystis carinii induces ICAM-1 expression in lung epithelial cells through a TNF-alpha-mediated mechanism. Am. J. Physiol. 273, 1103-1111. Yuon, J. K., and Barski, G. (1966). Interference between lymphocytic choriomeningitis and Rauscher leukemia in mice. J. Natl. Cancer Inst. 37, 381388. Zbiesieni, B. M. (1980). Diurnal and Seasonal changes in ascorbic acid level in adrenal glands of laboratory mice. Bull. Acad. Pol. Sci. Ser. Sci. Biol. 27, 653-656. Zeller, W., Meier, G., Burki, K., and Panoussis, B. (1998). Adverse effects of tribromoethanol as used in the production of transgenic mice. Lab. Anim. 32, 407-413. Zhang, X. P., and List, W. E (1996). Effects of halothane, isoflurane, and sevoflurane on calcium-related contraction in porcine coronary arteries. Acta Anaesthesiol. Scand. 40, 815- 819. Zheng, W., Shen, H., Blaner, W. S., Zhao, Q., Ren, X., Graziano, J. H. (1996). Chronic lead exposure alters transthyretin concentration in rat cerebrospinal fluid: The role of the choroid plexus. Toxicol. Appl. Pharmacol. 139, 445-450. Zhou, A., and Moss, R. L. (1997). Effect of urine-derived compounds on cAMP accumulation in mouse vomeronasal cells. Neuroreport 8, 2173-2177.

1184 Zinkernagel, R. M., and Doherty, P. C. (1975). H-2 compatibility requirement for T-cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus. Different cytotoxic T-cell specificities are associated with structures coded for H-2K or H-2D. J. Exp. Med. 141, 14271436. Zinkernagel, R. M., and Doherty, P. C. (1979). MHC-restricted cytotoxic T cells: Studies on the biological role of polymorphic major transplantation

NElL S. LIPMAN AND SCOTT E. PERKINS antigens determining T-cell restriction-specificity function and responsiveness. Adv. Immunol. 27, 51-177. Zondek, B., and Tamari, I. (1967). Effects of auditory stimuli on reproduction. In "Effects of External Stimuli on Reproduction" (G. E. W. Wolstenholme and M. O'Connor, eds.), Ciba Foundation Study Group No. 26, pp. 4-19. J. and A. Churchill Ltd., London.

Chapter 30 Animal Models in Biomedical Research Fred W. Quimby

I.

II.

III.

an Animal Model? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transgenesis and Mutagenesis: Creating Models for the Future . . . . . C. Sources of Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Nature of Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hypothesis Testing and Serendipity . . . . . . . . . . . . . . . . . . . . . . . . . . B. Breakthroughs in Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. T a k i n g Advantage of Interspecies Similarities: Research on Obesity. History of Animal Use in Biomedical Research . . . . . . . . . . . . . . . . . . . . . What Is

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A.

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Early History

............................................

From

Animal models have been used to understand the laws of nature throughout the millennium; however, since the scientific method was conceived, their use has accelerated and our knowledge has greatly expanded. Animal models include prokaryotes, invertebrates, and vertebrates, and each has played a significant role in defining principles in the biological sciences. Due to the high conservation of DNA between homologous genes and similar physiologic characteristics, vertebrate animals have frequently been the choice of investigators developing new drugs and medical devices to improve human health. For exactly the same reasons, vertebrate animals themselves have often been beneficiaries of such research. This chapter explores facets of the history of biomedical science and hypothesizes a scientific revolution in biologyma revolution where the principles of physics, chemistry, mathematics, and computer science will merge with biology to create advances unthinkable before. In LABORATORY ANIMAL MEDICINE,

2nd edition

1197 1200 1200 1201 1204 1206 1206

Pasteur to Prusiner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Animals as Recipients of Animal Research . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.

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this revolution the lowly mouse will reign as the principal animal model for investigations.

I.

WHAT IS AN ANIMAL MODEL?

A.

Types of Models

1. Introduction There are many types of models used in biomedical research, e.g., in vitro assay, computer simulation, mathematical models,

and animal models, and although living vertebrate animals represent only a fraction of the models used, they have been responsible for many important advances in biology and medicine Copyright2002, ElsevierScience (USA). All rightsreserved. ISBN 0-12-263951-0

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(National Research Council [NRC], 1985). Invertebrate models have made a profound impact in the areas of neurobiology, genetics, and development and include the nematode Caenorhabditis elegans, protozoa, cockroaches, sea urchins, the fruit fly Drosophila melanogaster, Aplysia, and squid, among others. The sea urchin, for instance, contributed to the discovery of meiosis, events associated with fertilization, discovery of cell sorting by differential adhesion, basic control of cell cycling, and cytokinesis (NRC, 1985). Similar lists can be prepared for insects, squid, and other marine invertebrates. These nonvertebrate models have been previously reviewed and are not the major focus of this discussion (NRC, 1985; Jasny and Koshland, 1990; Huber et al., 1990; Schulhof and Miller, 1990; Woodhead, 1989). A model, whether animal or nonanimal, is meant to be a mimic or surrogate and not necessarily identical to the subject being modeled (NRC, 1998; Scarpelli, 1997). In this chapter it is assumed that the human biological system is the subject being modeled; however, it is noteworthy that many of the advances made through studies of animal models have been applicable to animals other than humans (see Section III,C). Conceptually, animals may model analogous processes (e.g., relating one structure or process to another) or homologous processes (e.g., reflecting counterpart genetic sequences). Homology-based modeling appears to be uniquely biological, whereas finding corresponding features between two animals (or physical processes) is commonplace in physics, mathematics, and engineering (NRC, 1985). Prior to the current interest in genomics, many animal models were selected as analogs of human processes or conditions (they appeared similar); today we recognize many to be true homologies. However, caution should be exercised in interpreting the benefit of an animal homolog of humans; the physiologic adaptations occurring during the evolution of certain species may make their homologs poor analogs. Similarly, convergent evolution may result in good analogs from genetically distant structures and processes. Another useful concept in modeling concerns one-to-one modeling versus many-to-many modeling. In one-to-one modeling of a normal (or abnormal) human process, a species is sought that demonstrates analogous behavior for several features of the process and no negative features. This species is then considered a good model for this process, and when used in investigation, fruitful information is expected (consider an infectious disease like salmonellosis where the signs and symptoms in a monkey may be identical to those in humans; or a genetic disease such as X-linked muscular dystrophy where the signs in dogs resemble those in humans). Many-to-many modeling results from analysis of a process in an organism where each component feature of that process is evaluated at several hierarchical levels, e.g., system, organ, tissue, cell, and subcellular levels. Then, at each level all the taxa (species) are noted in which analogous features appear. Using this approach, a new kind of epistemic structure emerges, and the matrix of biologi-

FRED W. QUIMBY

cal knowledge replaces the one-to-one model for reaching an understanding, particularly of complex processes (NRC, 1985; Office of Technology Assessment [OTA], 1986). An attempt has been made to illustrate the advantages of the many-to-many model in the discussion of models for aging (see Section I,B, 2,b). Studies comparing analogies (and homologies) between many taxa have also contributed greatly in the understanding of central nervous system function, embryogenesis, and endocrinology. The current interest in comparative genomics promises for the first time to bring new order to the study of biology, where comparing the structure of genes to the function of their products may give new insight into the evolutionary history of species and what it means to be human. Animal models are used to elucidate basic physiologic, biochemical, and genetic processes found in health and disease. Rarely will the model share all the clinical, pathologic, and functional characteristics of the disease with humans, and while desirable, this is not necessary for the model to make substantial contributions on disease pathogenesis, prevention, and treatment. The differences between animal models and humans afflicted with type 1 (juvenile) insulin-dependent diabetes mellitus, Duchenne muscular dystrophy, and pancreatic ductal adenocarcinoma have been recorded, yet these models have contributed greatly to the understanding of the molecular basis of, and treatments for, these disorders (NRC, 1998; Scarpelli, 1997). In this context it is important to note that despite the many different factors modifying the evolutionary history of humans and that of the mouse, resulting in numerous differences in morphology and metabolism, there remains an impressive degree of genetic conservation between these species, including high sequence homology for both genes and their translated proteins, which appear to have similar, if not identical, functions in the two species (Davisson et al., 1998). Syntenic maps that document the order of genes along chromosomes between these species also demonstrate impressive similarities. Such mapping has aided the identification of new genetic loci in both species and in part explains the organization of complex processes that are often shared by both species (Copeland et al., 1993; Nadeau et al., 1989; Davisson et al., 1998). The characterization of murine models of various dwarfing syndromes has lead to an understanding of dwarfing in humans and suggested potential therapies (NRC, 1998). 2.

Spontaneous and Induced Animal Models

Animal models can be classified as spontaneous or induced. Spontaneous models may be represented by normal animals with physiologic mechanisms similar to those of humans or by abnormal members of a species that arise naturally through mutation(s). In contrast, normal animals submitted to surgical or another manipulation resulting in an abnormal physiologic state are induced models. The single largest category of induced models is that arising through genetic manipulation. Currently,

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

one new such animal model is being described each week. Occasionally, investigators will refer to another category of animal model, the so-called negative model. This is an animal that fails to develop a particular disease, e.g., infectious disease, or fails to respond to specific physiologic stimuli. Often insights into the genetic constitution of these animals lead to the discovery of genes (and proteins) that impart resistance to disease or an induced disorder, e.g., autoimmunity. Some of the best-characterized models are those with naturally occurring mutations that lead to disorders similar to those in man. Among the best-known spontaneous models are the Gunn rat (hereditary hyperbilirubinemia), piebald lethal and lethal spotting strains of mice (aganglionic megacolon), nonobese diabetic mouse and BB Wistar rats (type 1 diabetes mellitus), New Zealand Black and New Zealand White mice and their hybrids (autoimmune disease), nude mice (DiGeorge syndrome), SCID mice (severe combined immunodeficiency), Watanabe rabbits (hypercholesterolemia), Brattleboro rats (neurogenic diabetes insipidus), obese chickens (autoimmune thyroiditis), spontaneously hypertensive rats (SHR-primary hypertension), dogs and mice with Duchenne X-linked muscular dystrophy, dogs with hemophilia A and B, swine with hyper-low-density lipoproteinemia and malignant hyperthermia, mink with Chediak-Higashi syndrome, cats with achalasia, gerbils with epilepsy, cattle with ichthyosis congenita and hyperkeratosis, and sheep with Dubin-Johnson syndrome (Andrews et al., 1979; also see Section I,C). It is clear even from this very brief list that the types of diseases and the breadth of species involved are great. Induced models have been used to unravel some of the most important concepts in physiology and medicine. Whereas surgical models in nonhuman primates have contributed greatly to the understanding of brain plasticity following sensory-input deprivation and the largely separate cognitive domain with its own perception, learning, and memory experiences found in each cerebral hemisphere (Merzenich, 1998; Florence et al., 1998; Jones and Pons, 1998), surgical models in dogs led to breakthroughs in organ transplantation; coronary bypass surgery; balloon angioplasty; replacement of heart valves; development of cardiac pacemakers; the discovery of insulin and the treatment of diabetes mellitus; fluid therapy and other treatments for shock, liver failure, and gallstones; and surgical resection of the intestines, including the technique of colostomy (Council on Scientific Affairs, 1989; Quimby, 1994a; Bay et al., 1995; Quimby et al., 1995). Additional useful models have been induced by diet or administration of drugs or chemicals. Combining the chemicals alloxan and streptozotocin was found to selectively destroy the beta cells of the islets of Langerhans, rendering rats, rabbits, and other animal models susceptible to insulin-dependent diabetes mellitus (Sisson and Plotz, 1967; Golob et al., 1970). This model in mice has recently demonstrated NAD depletion by poly(ADP-ribose) polymerase as a dominant metabolic event in islet cell destruction, which may

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be useful in developing therapeutic strategies for type 1 diabetes (Burkhart et al., 1999), However, for studies of disease pathogenesis, the spontaneous mutant NOD mouse and B B rat are more analogous (and perhaps homologs) to human type 1 diabetes (Atkinson and Leiter, 1999). In 1981, investigators discovered that the drug 1-methyl-4 phenyl-l,2,3,6-tetrahydropyridine (MPTP) produced clinical signs of Parkinson's disease when injected into nonhuman primates. Further studies confirmed that this chemical destroyed the dopamine-producing cells of the brain-re-creating the pathologic lesion in the human disease (Lewin, 1984). The model system has contributed greatly to our knowledge of the structure and function of the basal ganglia as well as the mechanism of neurodegeneration (Grtinewald and Beal, 1999) and to the evaluation of new therapies such as transplantation and pallidotomy (Tolwani et al., 1999). Diet-induced models have been responsible for discovery of most vitamins and the necessity for trace minerals as nutrients, as well as for exploration of the pathogenesis of many diseases, e.g, atheroslcerosis. Eijkman's observations of chickens with beriberi (thiamin deficiency) resulted in a cure for humans (and animals) and led to the discovery of vitamins (Eijkman, 1965; see Section II,A,2). In fact, dietary manipulations in the chicken alone have contributed to our knowledge of rickets (Kwan et al., 1989), vitamin A deficiency (Band et al., 1972), vitamin B 6 deficiency (Masse et al., 1989), zinc deficiency (O'Dell et al., 1990), Friedreich's ataxia (van Gelder and Belanger, 1988), fetal alcohol syndrome (Means et al., 1988), and atherosclerosis (Dauber, 1944; Pick et al., 1952). Often complex induced models are used by combining drugs, surgery, diet, and infectious agents. An excellent example is the hu-SCID mouse, where a natural mutation in the RAG1 gene prevents T- or B-cell antigen receptor rearrangements, resulting in a severe combined immunodeficiency. When this mouse is injected with human lymphocytes or stem cells, it adopts the immune system of humans (Carballido et al., 2000). Finally, injection of this reconstituted mouse with HIV- 1 virus leads to viral propagation and a small-animal model for the assessment of anti-HIV drugs (Mosier et al., 1991). a. From past advances in amphibian developmental biology to the current global decline in frogs. An example illustrating the importance of previous basic science discoveries using normal animals to the contemporary problems facing people today relates developmental biology in frogs to the current problem of associating frog deformities and population declines with environmental agents. The African clawed frog, Xenopus laevis, has been an important model for elucidating various aspects of embryogenesis by acting at the interface of molecular and developmental biology. The Xenopus system was responsible for the first isolation of a eukaryotic gene, the initial studies on gene amplification, the first demonstration of accurate transcription from a cloned gene, and the first isolation and characterization of a eukaryotic transcription factor (Dawid and Sargent, 1988).

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Together, these discoveries have led to a frog oocyte expression least one report demonstrated embryotoxicity and deformities system that allows for efficient translation of proteins from in- (skeletal) in larvae hatching from eggs exposed to unshielded jected mRNA, as well as a functional transcription factor assay. ambient UVB radiation (Blaustein et al., 1997), and synergism The original Spermann-Mangold experiment conducted in between UVB radiation and the fungal parasite Saprolegnia 1924 demonstrated that the earliest visible sign of gastrulation, ferax was shown to increase embryo mortality for several spedevelopment of the dorsal lip, served as a central organizer ca- cies of frogs (Kiesecker and B laustein, 1995). Large-scale frog pable of directing mesodermal and neural derivatives in the em- die-offs have been associated with infections, including by the bryo. Later it was discovered that members of the transforming fungus Batrachochytrium dendrobatidis (Berger et al., 1998; growth factor (TGF)-[~2 family, located in the egg and dispersed Morell, 1999) and iridoviruses (Daszak et al., 1999); however, by cytoplasmic flow before the first cleavage stage, bestow only infestation by the digenetic trematode Ribeiroia has been distinct properties on vegetal blastomeres that allow them to associated with frog deformities. The trematode infestations are induce directed differentiation, embryo polarity, and ordered associated with extra or missing hindlimbs, but they cannot exglobal structure during embryogenesis (Dawid and Sargent, plain the full range of deformities seen in Minnesota (Johnson 1988). This organized sequence of development was abrogated et al., 1999; Sessions et al., 1999). An ichthyophonus-like inby exposure of eggs to ultraviolet light. Amphibian, especially fection has been described in frogs from areas characterized frog, development has been widely studied because of the di- by high deformity rate (Mikaelian et al, 2000; Babbit, Bowser, versity of strategies used by anurans to accommodate larval and and Sower, personal communication, 2000). A causal relationadult forms in aquatic and terrestrial environments. In fact, ship between this disease and limb deformity remains to be changes in developmental patterns, especially those concerned demonstrated. One interesting study of frog deformities in Quebec linked with the timing of developmental events in larvae (heterochrony), have been responsible for numerous species, many hindlimb malformations to the use of agricultural pesticides captured from the wild, to be included in investigations (Han- (Ouellet et al., 1997). Since the 1920s, investigators have linked ken, 1999). The dramatic developmental changes associated various chemicals with deformities in adult or larval frogs; with metamorphosis, which include remodeling of virtually these include hyperactivity and hindlimb deformity in Rana every larval organ system in certain families, e.g., Rana, have temporaria caused by DDT (Cooke, 1973); curvature of the attracted enormous interest and resulted in excellent reviews digits and abnormal limb articulations in Rana sylvatica by (Rose, 1999; Duellman and Trueb, 1994; Fritzsch, 1990; Mc- thiosemicarbazide (Riley and Weil, 1987); spinal curvature, Diarmid and Altig, 1999). Even with this body of information, blister formation, and abnormal behavior in Microhyla ornata scientists and government agencies were unprepared for the by malathion (Pawar et al., 1983); stunted growth, microcephaly, and curved spine in Rana pipiens exposed to paraquat events observed in 1995. On August 8, 1995, a group of middle school students on a na- (Dial and Bauer, 1984); spinal deformities in Rana pipiens, ture trip in central Minnesota discovered a high percentage of Rana catesbeiana, and Xenopus laevis by dieldrin (Schuytema deformed northern leopard frogs, Rana pipiens, at a local farm et al., 1991); and spinal deformity accompanied by convulsive pond. The frogs, all recent metamorphs, had a variety of hind- and twitching behavior in Rana pipiens by the pyrethroid inseclimb deformities, from missing legs and legs of different sizes ticide esfenvalerate (Materna et al., 1995). Again, none of these to legs that failed to bend properly (Souder, 2000). Within days synthetic chemicals mimicked the deformities seen in frogs the Minnesota Pollution Control Agency began investigations, across North America. In the 1980s, developmental biologists and toxicologists toand within a month biologists from the University of Minnesota were involved. What began as a middle school outing evolved gether developed an assay using Xenopus laevis embryos and into one of the most puzzling episodes in the annals of field bi- larvae to document developmental abnormalities associated ology, scores of independent observations (ranging from Cali- with environmental chemicals (Fort et al., 1988; Bantle et al., fornia to Maine and southern Canada) involving multiple spe- 1991). The test is called frog embryo teratogenesis assay: Xenocies of frogs with deformities of forelimbs and hindlimbs, eyes, pus (FETAX). Preliminary studies of water and sediment from jaws, and skin coloration. Missing digits (ectrodactyly) and Minnesota ponds identified with frog deformities demonstrated whole limbs (ectromelia), as well as supernumerary hands and a component in both that was associated with embryo mortality entire limbs were observed. Over the following 5 years investi- and malformations in a dose-dependent fashion. This compogators documented decreased numbers of frogs, as well as local nent(s) was removed by activated carbon filters (but not by boilextinctions, and many believed the deformities were linked to the ing) and was responsible for a wide range of malformations generalized population decline of amphibians seen worldwide. involving the skeletal system, brain, eyes, tail, gut, jaw, and kidBy 1996, the Environmental Protection Agency (EPA) had ney. Malformations were not seen in water or sediments colalso become involved, and amphibian developmental biologists lected from Minnesota reference ponds lacking frog deformities began searching for a cause. Results of early investigations ex- (Burkhart et al., 1998). Two subsequent papers extend these posing frog eggs to ultraviolet light were mixed; however, at findings to include additional affected ponds from Minnesota

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and Vermont and pond sediment-specific developmental alterations, including hindlimb deformities in which Xenopus tadpoles mimicked the deformities seen in Rana sp. (Fort et al., 1999a). In addition, Chemicals causing differing abnormalities in developing frogs were isolated from affected pond sediments. Among these were substances shown to inhibit thyroxin activity, which delayed tail resorption (Fort et al., 1999b). Addition of thyroxin to FETAX assays employing these chemicals abolished the chemical-induced deformity in Xenopus larvae. It is becoming apparent that multiple environmental insults, acting singularly or together, are responsible for the numerous deformities seen in North American frogs. It is also obvious that while years of previous research in amphibian developmental biology greatly accelerated the search for environmental chemicals associated with deformities, very little is known about infectious agents and how they impact frog populations. Finally, only through a multidisciplinary approach to this problem, involving developmental biologists, infectious disease experts, field biologists, toxicologists, aquatic ecologists, geneticists, and population biologists, will the true significance of these myriad causative agents on local extinctions and global population decline be understood. b. From equine infectious anemia to AIDS. Knowledge gained from spontaneous and induced models of retroviral infection led to the early recognition of human T-cell leukemia virus (HTLV) and human immunodeficiency virus (HIV) in humans. At the time of this writing, HIV virus has infected 30 million people worldwide with over 250,000 new infections per month and more than 10 million deaths (Nathanson, 1998). While trivalent therapy (HAART) has greatly delayed the progression of disease in many individuals and dramatically reduced maternal transfer to the fetus and newborn, its widespread use has yet to be adopted. Many believe only vaccination will stop the current epidemic among humans. New hope for a safe and effective vaccine has arisen, due to the new understanding of the infection cycle of HIV, the protection of viral proteins that bind cell receptors by sugar residues, the role of chemokine receptors, and effectiveness of cytotoxic T lymphocytes (Robinson et al., 1999; Cafaro et al., 1999; Wyatt and Sodroski, 1998; Emerman and Malim, 1998). Nonhuman primate models were essential in these studies. Likewise, nonhuman primate models have documented the efficacy of monoclonal antibodies to prevent mucosal transmission to the newborn (Mascola et al., 2000; Baba et al., 2000). However, the current knowledge of HIV was constructed from a long history of animal research, much of it conducted by veterinarians. One of the earliest studies demonstrating that particles smaller than bacteria were responsible for disease was the discovery by Loftier and Frosch of "filterable agents" that caused foot-and-mouth disease in cattle. They, along with Sigurdsson, who was the first to demonstrate long latency between infection and disease in retroviruses, were veterinarians (Leader, 1975).

Retroviruses were initially grouped together because of their ability to reverse-transcribe their viral RNA into host DNA through the action of the enzyme reverse transcriptase, but they are now classified into three subfamilies: Oncornavirinae, Lentivirinae, and Spumavirinae. Oncornaviruses and lentiviruses cause both human and animal disease and have been the subject of many investigations using animal models. Oncornaviruses are known to infect and cause cancer in sheep, cattle, chickens, cats, monkeys, humans, and laboratory rodents (Salzman, 1986). Despite a long history of veterinary research leading to their isolation, composition, replication, and mechanisms of resistance, human oncornaviruses were only documented in 1979 with the discovery of human T-cell leukemia virus (HTLV) (Poiesz et al., 1980). Lentiviruses are known to infect sheep, goats, cattle, horses, cats, and monkeys, and many were well documented in the literature before HIV was discovered in 1984 (Barre-Sinoussi et al., 1983; Popovic et al., 1984; Gallo et al., 1984). However, the pattern of disease among animals infected with lentiviruses is quite different among species. While sheep develop pneumonia, goats develop arthritis and encephalitis, and horses develop anemia (Mosier, 1996; Tenorio et al., 1992; Gardner, 1997; Tashijan and Crusberg, 1989); cats and nonhuman primates share the immunodeficiency seen in humans (Gardner, 1989; Sparger et al., 1989; Theilen, 1988). The pioneering work by Robert Gallo (National Institutes of Health) and Luc Montagnier (Pasteur Institute) led to the isolation and description of the first human lentivirus (HIV). Since then several different strains and many variants of this virus have been described (Nielsen et al., 1993; Baiter, 1998). In his book "Virus Hunting: AIDS, Cancer, and the Human Retrovirus," Gallo states: "The work that influenced me came from William and Oswald Jarrett of Scotland and later Essex, Bill Hardy, Ed Hoover and others (each of whom did work on the cat leukemia virus); Peter Biggs in England and Creighton in the U.S. (chicken leukemia virus), Janice Miller, Carl Olson (U.S.) and M. van Der Marten (U.S.) and later Arsene Burney of Brussels (cow leukemia virus) and Tom Kawakami (Gibbon ape leukemia virus) as well as from some of the earlier writings of Ludwik Gross and others regarding 'mouse leukemia virus'" (1991). The work that so influenced Gallo was, in large part, the work of veterinarians using a host of animal models. This body of research has led to our present-day understanding of lentiviruses, the development of therapeutic drugs, and the evaluation of genetically engineered vaccines (McMichael and Hanke, 1999). 3.

Validation and the "Ideal" A n i m a l Model

Various authors have attempted to defne the "ideal" animal model. Features such as (a) similarity to the process being mimicked, (b) ease of handling, (c) ability to produce large litters, (d) economy of maintenance, (e) ability to sample blood and tissues sequentially in the same individual, (f) defined

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genetic composition, and (g) defined disease status are commonly mentioned (Migaki and Capen, 1984; Leader and Padgett, 1980; Dodds and Abelseth, 1980). Perhaps the most important single feature of the model is how closely it resembles the original human condition or process. As previously mentioned, a model is never identical to a human but should be analogous to the targeted condition, i.e., share similarities that are important in elucidating new information relevant to the human condition or process, with the exception of negative models (see Section I,A). Shapiro uses the term "validation" as a formal testing of the hypothesis that significant similarities exist between the model and the modeled (Shapiro, 1998). He argues that to be valid, the animal model should be productive of new insights into and effective treatments for the human condition being modeled. While this goal is ideal, it may be impossible to attain in the preliminary stages of model characterization. In addition, many models are used to investigate nonpathologic processes, and models that simulate the target process most closely or are most likely to yield useful information are preferred (see Section II). The National Research Council (NRC) has advised the National Center for Research Resources (NCRR) on some criteria to be used in establishing priorities for fields and models to support (NRC, 1998). Among the criteria listed are that the model (1) is appropriate for its intended use(s) (a specific disease model faithfully mimics the human disease and a model system is appropriate for the human system being modeled); (2) can be developed, maintained, and provided at reasonable cost in relation to the perceived or potential scientific values that will accrue from it; (3) is of value for more than one limited kind of research; (4) is reproducible and reliable, so results can be confirmed; and (5) is reasonably available and accessible. These seem to be prudent criteria to follow when a funding organization seeks the greatest benefit within the confines of a finite budget. These recommendations also fulfill most of the criteria of an "ideal" model. There are instances where the only model (or the best model) cannot be maintained and provided at a reasonable cost (e.g., chimpanzees, which are invaluable for several vaccine efficacy trials) or are directed at one limited kind of research (e.g., murine knockout model of cystic fibrosis or murine transgenic model of amyotrophic lateral sclerosis). These exceptions are recognized by the NRC committee.

B.

Transgenesis and Mutagenesis: Creating Models for the Future

1. Methods

a. Techniques affecting the function of genes. Prior to the 1980s, most animal models were discovered by screening large breeding colonies or depending on the chance observations of

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veterinary clinicians (see Section I,C). However, several techniques, generally applied to small rodents, led to a large increase in available animal models. When the drug N-nitroso-N-ethylurea (ENU) is injected into male mice, single base pair mutations are created in the germ cells. By breeding progeny and backcrossing mice, homozygotes for the mutated allele are obtained. Genes in mouse embryonic stem cells (ES) can be mutated by use of ENU. This process is random, and in many earlier investigations the mutated gene had not been cloned or identified; however, since the 1980s, many useful models of human disease have been so created in mice, including models for phenylketonuria (mutated phenylalanine hydroxylase gene), c~-thalassemia (a-globin), [3thalassemia (~-globin), osteopetrosis (carbonic anhydrase II), glucose-6-phosphate deficiency, tetrahydrobiopterin-deficient hyperphenylalaninemia (GTP-cyclohydrolase I), Duchenne muscular dystrophy (dystrophin), triose-phosphate isomerase deficiency, adenomatous intestinal polyposis coli, hypersarcosinemia (sarcosine dehydrogenase), erythropoietic protoporphyria (ferrochelatase), and glutathionuria (y-glutamyltranspeptidase) (Herweijer et al., 1997). Zebrafish, Danio rerio, have been used extensively for studies in development because their embryos are transparent, each clutch contains 50-100 embryos, and the fish are amenable to large-scale mutagenesis using compounds like ENU (Driever and Fishman, 1996). Distinct genes have different mutability rates; however, ENU is reported to induce genetic mutations at average induction rates of 1 in 1000. This estimate serves as the basis for large-scale genomic screens (Ntisslein-Volhard, 1994). Because earlier work provided much of the outline for early embryonic cell fate, lineage, and patterning and for nervous system development (Eisen, 1994); and methods for production of homozygous fish, using genetically impotent sperm to induce the maternal chromosomes of the egg to complete meiosis II and pressure to prevent the first cell division (Steisinger et al., 1981), mutant phenotypes can be rapidly screened by visual inspection under a dissecting microscope, with mutants bred to homozygosity in three generations. Irradiation was used as a germline mutagen dating back to the early 1920s. X-rays have been shown to cause small chromosomal deletions in mouse spermatogonia, postmeiotic germ cells, and oocytes (Takahashi et al., 1994). Examples of radiationinduced models in wide use include the beige mouse (bg), dominant cataract (Cat-2t), and cleidocranial dysplasia (Roths et al., 1999). Chlorambucil has also been used to produce deletions in postmeiotic germ cells; however, because chlorambucil and Xrays often produce large deletions, in practice it is difficult to recover large numbers of mutant mice. Since the 1980s, the most popular and productive method for altering gene function is via transgenesis. The first method, described by Gordon and Ruddle (1981), involved direct insertion of cloned genetic material into the pronucleus of a fertilized mouse egg. While this method is simple, can be performed in many species, and does not appear to have limits on the size of

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

DNA that can be injected, it is limited in that the site of integration is random. Pronucleus injection has been successfully used to create transgenic rabbits, sheep, and goats, in addition to mice and rats (Pinkert, 1997; Mullins and Mullins, 1996). Earlier it was discovered that infection of preimplantation mouse embryos with Moloney murine leukemia virus led to germline integration of the proviral DNA in offspring (Jaenisch, 1976), and by the early 1980s, construction of retrovirus packaging mutants allowed for the integration of foreign genes in the mouse germline (Mann et al., 1983). This method generally results in low gene expression of the inserted element, the insertion takes place with minimal disruption of host genetic sequences, and insertion is nonrandom (the sequences flanking retroviral insertion sites contain chromatin that is DNase I hypersensitive). Around the same time, mouse embryonic stem cell (ES) lines were first produced and maintained in culture (Martin, 1981). This discovery allowed investigators to insert genes by homologous recombination, using vectors that contain both positive and negative selectable markers. As a result, ES cells with a targeted mutation can be combined with a developing mouse embryo that is implanted into pseudopregnant surrogate mothers. Offspring are chimeras for the insertion and after selection and appropriate matings, result in founders that have germline expression of the mutation (see Chapter 28 for details). Mice submitted to this form of targeted mutagenesis may have a nonfunctional gene (knockout), have a gene with altered expression, or gain a new functional gene (knockin). Gene expression can also be altered in a tissue-specific fashion by modifying a coding sequence such that critical components of the targeted gene are flanked by loxP sequences, which are targets for bacteriophage Cre recombinase. Crossing mice with loxP-modified loci to transgenic-expressing Cre recombinase under control of a linease-specific promoter results in tissue-specific exon excision and ablation of gene function (Gordon, 1997; Nagy and Rossant, 1996). The list of mouse mutations generated by targeted mutagenesis is staggering (see ). When alterations to the germline occur in specific genes, the result may be a gain of function (as when an extra copy of a human gene is introduced into the mouse genome) or a loss of function (as when a point mutation is induced by homologous recombination, a deletion produced by irradiation or antisense/ ribozyme constructs targeted against specific genes introduced via transgenesis) (Cameron and Jennings, 1989; Katsuki et al., 1988). Because ENU induces intragenic point mutations, it is likely that both gain-of-function and loss-of-function mutations can be produced. b. Controlling gene expression in transgenic mice. A variety of clever techniques have been developed to control the expression of transferred genes in addition to lineage-specific expression described above. Transgenic animals made by pro-

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nuclear injection may have the DNA construct designed with tissue-specific regulatory sequences (i.e., promoters) or with ubiquitous promoters that lead to widespread gene expression. Mice and rats have been made using shuttle vectors in which a bacterial gene inserted into the mouse genome is susceptible to mutation. Once the mouse is exposed to a mutagen, the high molecular weight genomic DNA is isolated and a bacteriophage packaging extract is used to isolate the bacterial sequence. Analysis of bacterial sequences in phage particles can establish the mutation frequency of the original mutagen (Roths et al., 1999). Another modification of the standard microinjection method for producing transgenic mice is one in which large multilocus segments of human DNA were transferred into the mouse pronucleus in the form of yeast artificial chromosomes (YACs). The entire [3-globin multigene locus (248 kb) was cloned into yeast, and once integrated, this locus could be mutated at precise points by homologous recombination. After transferring YACs and mutated YACs into mice, the full developmental expression of epsilon, gamma, beta, and delta genes was observed since the YAC also contained the human locus control region that interacts with structural genes to ensure that the correct globin is produced at the proper time and place during development (Clegg et al., 1997). These YAC transgenic mice are free of the restrictions inherent in single-gene cloned DNA, e.g., the genomic organization is not disrupted around the structural gene; thus, higher levels of transcription and developmental regulation of gene expression can be studied. Another method, developed by Ktihn et al., (1995), allows for the controlled expression of a transferred gene by linking an interferon-responsive promoter to control the expression of Cre recombinase. Under these conditions, treating mice with interferon activated the Mx-cre transgene, allowing expression of Cre recombinase, which deleted the floxed target gene, DNA polymerase [3 (see discussion above for C r e - l o x P system). Here the target gene was eliminated in all interferon-responsive lineages. A modification of this system was employed by Utomo et al. (2000), which integrates the advantages of tetracyclinecontrolled gene expression and Cre r e c o m b i n a s e - l o x P sitemediated gene inactivation. In this system the reverse tetracycline responsive transactivator (rtTA), which is transcriptionally active only when bound by tetracycline (or doxycycline), is placed in cis configuration to the rtTA-inducible promotor, which directs the expression of Cre recombinase. Exposure of the mouse to doxycycline provides the temporal off-on switch for tissue-specific regulation of gene expression through the C r e - l o x P system. Another system that combines cell type-specific and temporal controls consists of a fusion between Cre and a mutated steroid ligand-binding domain of the receptor controlled by tissuespecific promoters, The chimeric protein becomes activated upon binding with synthetic ligands such as tamoxifen or RU486 (Schwenk et al., 1998).

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c. R e p r o d u c i n g identical individuals. For years scientists have created inbred strains of rodents by repeated brother X sister matings. After 20 such matings the offspring are considered an inbred strain. When a transgenic mouse is developed and the investigator wants the transgene expressed on another background, depending on whether the mutation is dominant, recessive, or recessive and lethal, the transgenic mouse can be crossed into a selected strain using one of the following techniques: backcross matings, cross-intercross matings using homozygotes, cross-intercross matings using heterozygotes, or cross-intercross matings using ovarian transplantation (NRC, 1989). Techniques for cloning individual mammals have been published in studies that followed the original work conducted by John Gurden in frogs nearly 30 years ago (Wilmut, 1998). Cells of the individual to be cloned are grown in culture such as fibroblasts, and their nuclei are removed and fused into enucleated eggs. The resulting embryos are transferred into surrogate mothers, and offspring are identical to the original animal used as a source of cultured cells. This technique has been successful for cloning sheep, goats, cattle, fish, and mice (Wilmut et al., 1997; Kato et al., 1998; Wakayama et al., 1998; Steisinger et al., 1981; Bagulsi et al., 1999) and offers a much more efficient way to propagate large numbers of identical individuals, including individuals expressing the product of a transgene in milk, which may be isolated and used as a therapeutic compound in humans, e.g., human Factor IX (Wilmut, 1998). In 1999, mice were cloned from ES cells rather than from the low-passage primary cell cultures used in the past. This technology offers researchers the opportunity to create new knockout mice in a single generation rather than the three generations currently required (Vogel, 1999). However, in each of these procedures, the resulting clones show mitochondrial heterogeneity; thus, they are genetic chimeras (Evans et al., 1999). Chan et al. (2000) reported on successful clonal propagation of rhesus monkeys by embryo splitting. An 8-cell embryo was split to produce a set of identical quadruplet embryos, each consisting of two blastomeres. Two blastomeres are then inserted into an empty zona pellucida, creating one set of quadruplets. A pair of quadruplet embryos was transferred into a surrogate female rhesus monkey that delivered a healthy cloned infant. d. Cryopreservation. With the rapid accumulation of mouse strains developed by transgenesis and chemical or radiation-induced mutagenesis, cryopreservation is an inexpensive and efficient alternative to maintaining living animals. Since the first successful embryo-freezing protocol was established in 1972 by Whittingham et al., cryopreservation programs for maintaining inbred strains by embryo freezing have become commonplace. In addition to 8-cell embryos, all stages of preimplantation embryos plus sperm and oocytes may be suc-

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cessfully frozen. While maintaining stocks with frozen sperm and oocytes is more economical, embryo freezing is warranted when the full inbred background containing the transgene is desired (Crister and Mobraaten, 2000; Agca, 2000; Rall et al., 2000). Details on cryopreservation protocols along with an estimate of costs have been recently reviewed (Mobraaten, 1999). 2.

Functional Genomics: Applications to Biomedical Research

The human genome, the full set of genes that defines humans, is composed of 80,000 or so genes and 3 billion base pairs. With support from the NIH, Department of Energy (DOE), the Wellcome Trust, and others, the project to sequence the entire human genome, known as the Human Genome Project (HGP), was launched on October 1, 1990, with an expected completion date of 2005. Advances in the technology available to this project have greatly accelerated the task, and the complete, first-draft copy was finished in 2000. The entire sequence is publicly available on GenBank (accessible at ), which in 1999 was already receiving over 200,000 queries a day. Over 39,000 species are represented in the database, and over 60,000 sequence-comparison searches are conducted each day (Collins, 1999). The purpose behind the endeavor was to have a new understanding of the genetic contributions to human disease and the development of new strategies for minimizing or preventing disease phenotypes. To accomplish these goals, the HGP focused on four major initiatives: (1) develop a genetic map that establishes 6000 markers spaced within 106 bp of each other to aid in family linkage studies, (2) construct a physical map with ordered sets of DNA, properly aligned to cover each chromosome, (3) position expressed sequence tags (ESTs) on the physical map to assist in locating unknown genes, and (4) and develop the dense map of DNA variants within the population, primarily identifying insertions and deletions of nucleotides, differences in the copy number of repeat sequences, and single nucleotide polymorphisms (SNPs). One SNP is thought to occur in every 300 bp. The SNPs falling within protein-coding regions of a locus are particularly informative because they are more likely to predict changes in gene function. By 1994, the first genetic map was completed. The physical map containing 41,000 aligned sequenced-tagged sites (STS) was completed in 1998. A map containing over 38,000 ESTs was available by 1999. In 2000, the first draft of the human genome was completed, and knowledge gained through the various maps listed above led to the rapid discovery of several new genes responsible for human disease, including those that regulate families of interleukins (Loots et al., 2000) and those responsible for hearing loss (Willems, 2000). Earlier, the sequencing of mutant genes in the mouse led to the elucidation of mutant human genes causing Prader-Willi syndrome and polycystic kidney disease (Gardner et al., 1992; Moyer et al., 1994).

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a. Comparative mapping. With the physical maps of the human genome now in hand, the time it takes to locate and identify a gene responsible for a disease inherited within a family has been reduced from years to weeks. In many instances, knowledge of these genetic variants has led to diagnostic tests that can be used to predict a person's increased risk of disease. Examples include the BRCA1 and BRCA2 genes and breast cancer, the hepatocyte nuclear factor 4c~ (HNF-~) in maturityonset diabetes of the young (MODY) type 1, the glucokinase (GCK) gene in MODY type 2, human Mut L homolog 1 (hMLH1) in hereditary nonpolyposis colon cancer, and ~-synclein in Parkinson's disease (Wooster et al., 1995; Yamagata et al., 1996; Froguel etal., 1993; Papadopoular et al., 1994). However, as Collins eloquently states: "Knowledge about the genetic control of cellular function will underpin future strategies to prevent or treat disease phenotypes" (1999). To better understand the function of expressed gene products (functional genomics), comparative gene mapping is critical. As the HGP began, several organisms with smaller genomes were identified for gene mapping. The entire map of Saccharomyces cerevisiae, a yeast with 6000 genes, was completed in 1996 (Goffeau et al., 1996) as was the map of Escherichia coli (Blattner et al., 1997). The map of the 19,000 genes of Caenorhabditis elegans was completed in 1998 (Chalfie, 1998; C. elegans Sequencing Consortium, 1998) and the entire map of Drosophila melanogaster was completed in 2000 (Adams et al., 2000). In fact, at the time of this writing the genomes of more than 28 microorganisms have been fully sequenced (Fraser et al, 2000). Adaptation of the dot-blot hybridization technique to DNA chips (stamp-sized silicon disks containing over 100,000 difference probes), so-called microarrays, has proven promising in approaching genome-scale studies of genetic variation. It is useful in the detection of gene expression (mRNA) in tissues and cells (e.g., the expression of cytochrome P450 genes), for the large-scale analysis of gene copy number and protein expression, and for making genomic comparisons across species (Hacia et al., 1998). Although the human genome map allows for positional candidate cloning to rapidly isolate altered genes associated with human disease (Collins, 1995), this technique may not elucidate the function of these genes. To understand the function of proteins encoded in the DNA sequence, a variety of other techniques can be useful, including (1) searching for homologs in other species where protein structure or function is known, (2) predicting protein function from structure, and (3) developing knockout animals to study function. In this context it is interesting to note that the fruit fly has orthologs to 177 of the 289 human disease genes (Rubin et al., 2000). b. Using multiple species comparisons: Research on aging. Although animal models have contributed greatly to our understanding of aging, we are still a long way from understanding the multitude of factors that contribute to long life. In 1989, Ma-

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soro pioneered studies that demonstrated increased longevity in rats on calorie-restricted diets. Investigators have also demonstrated a wide range of physiologic and endocrinologic changes apparent in rodents fed restricted diets compared to rodents fed ad libitum (Masoro, 2000). Many of these changes (e.g., lean body mass, reduced blood insulin levels, reduced blood glucose levels, reduced cholesterol and triglycerides) are themselves thought to be biological markers of fitness (Masoro, 1988, 1989). Why calorie restriction has such a dramatic effect on body function is unknown, and these observations have led others to question the relevance of calorie restriction in rodents to human longevity. However, three separate research groups have provided data on normal and calorie-restricted nonhuman primates, which had similar changes in biomarkers and reduced incidence of mortality associated with diseases such as diabetes, heart disease, and cancer (Couzin, 1998). While results on longevity in calorie-restricted nonhuman primates will require many more years of observation, these preliminary results suggest that in terms of the impact of calorie restriction on measures of fitness, primates reproduce the observations made in rodents. We now recognize that the life span of an individual is determined by the interactions between genes and the environment. We are just learning how those interactions lead to the cellular and molecular involution associated with aging. Although it is clear that individuals are subject to differences in genetic risks for age-related disorders such as Alzheimer's disease (AD), diabetes, heart disease, stroke, and cancer, it is much less clear what influence genes play on maximizing life span (Finch and Tanzi, 1997). Three categories have been proposed to classify candidate loci for human longevity: (1) genes with homologs that influence longevity in other species, (2) genes that mediate cellular maintenance and repair, and (3) genes that are associated with susceptibility to major age-related diseases (Sch~ichter et al., 1993). Using the nematode Caenorhabditis elegans, six induced mutations were found that extended life span by 40 to 100%. Each was found to increase resistance to stressors such as temperature, free radicals, and ultraviolet light (Friedman and Johnson, 1988; Larsen et al., 1995; Kenyon et al., 1993; Morris et al., 1996; Kimura et al., 1997). Age-1 appears to be associated with a phosphatidylinositol 3-kinase (Morris et al., 1996), whereas daf-2 is identified with an insulin receptor-like gene (Kimura et al., 1997). These two mutations are associated with the greatest increase in life span, both involve insulin-like signaling, and their effects are consistent with the extension of life span in food-restricted rodents (Sohal and Weindruch, 1996). The clock gene (clk) also plays a role in C. elegans, where mutations are associated with slower development, lengthened cell cycles, and modified behavior (Lakowski and Hekimi, 1996; Ewbank et al., 1997). Clk-1 encodes an 82-amino acid

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tandem repeat that is highly conserved in eukaryotes. The yeast homolog indirectly regulates the transcription of genes involved with energy metabolism. The wild-type clock gene in Drosophila and mice encodes transcription factors that are essential in the maintenance of circadian rhythms (Darlington et al., 1998; Gekakis et al., 1998). Double mutant C. elegans with daf-2/clk-1 have a fivefold increase in lifespan (Ewbank et al., 1997). The molecular pathways dependent on the expression of the transcriptional activator, clock, in mice and their relationship to longevity are now the focus of investigation (Okamura et al., 1999; Hardin and Glossop, 1999). In bakers' yeast (Saccharomyces cerevisiae) the SGS1 gene encodes a DNA helicase. Mutation of this gene causes premature aging in the yeast and redistribution of silent information regulator (sir)-3 protein from the telomers to the nucleolus (Kennedy et al., 1997; Sinclair et al., 1997). Loss-of-function mutations also occur in DNA (RecQ) helicase genes in humans, leading to impaired DNA replication and repair, decrease in telomere length, and the autosomal recessive adult-onset progeroid disorder known as Werner syndrome (Yu et al., 1997). Telomere length is inversely proportional to somatic cell proliferative capacity for all mammalian cells. Unlike in germline cells, where telomerase is constantly active, in somatic cells telomerase is not expressed (Kim et al., 1994). As a result, in somatic cells, repeats of the sequence TTAGGG/CCCTAA at the chromosome ends become shorter following each replication. One hypothesis holds that cell replicative senescence (the inability to further divide) occurs at some telomere length threshold. One in vitro experiment demonstrated that two different human cell types could be induced to greatly exceed their replicative life spans by stable transfection of the human telomerase gene (Bodnar et al., 1998). One in vivo experiment utilized telomerase deficient, m T R -/-, mice to evaluate the role of telomerase gene expression on hepatocyte proliferation and the hepatic response to chronic liver injury (cirrhosis). Following adenovirus-mediated transfer of the M T R gene (by tail vein inoculation) into m T R - / - mice, 85-100% of hepatocytes expressed M T R (but no expression was seen in spleen, consistent with hepatotropism of adenovirus). When transfected mice were compared to nontransfected controls n each undergoing partial hepatectomymthe transfected mice had increased numbers of regenerative hepatic nodules and greater numbers of mitotic figures compared to those in nontransfected M T R - / - mice. When both transfected and nontransfected mice were treated with carbon tetrachloride, the transfected mice had much less steatosis and fibrosis (cirrhosis) compared to nontransfected mice. In fact, transfected M T R - / - mice behaved like normal mice. These studies demonstrated that accelerated telomere loss was a factor leading to end-stage liver failure and cirrhosis and that telomerase gene therapy protects the liver against chronic liver disease induced surgically or by hepatotoxic drugs (Rudolph et al., 2000). Another silent information regulator, SIR2, encodes the S I R 2 P

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protein a NAD-dependent histone deacetylase in yeast. S I R 2 P is responsible for compressing chromatin and through this regulates access of many nuclear proteins to DNA and represses homologous recombination at the highly repetitive rDNA locus. Because accumulation of extrachromosomal rDNA circles is a major cause of aging in yeast, the function of S I R 2 P weighs heavily on yeast longevity. Lin et al. (2000) developed a method of imposing calorie restriction on yeast. They found the restriction was associated with a 2 0 - 4 0 % increase in life s p a n n s i m ilar to the effects of calorie restriction in mammals. Because S I R 2 P expression is dependent on NAD, its activity is linked to the energy status of the cell. The long-lived yeast had increased S I R 2 P activity and decreased rDNA circles, thus linking longevity to gene expression and energy status in the cell. These findings have placed new emphasis on the role of chromatin silencing on aging in model systems and humans (Campisi, 2000). Alzheimer's disease is a major age-related neurodegenerative disorder responsible for over 70% of all cases of late-onset dementia, afflicting over 4 million Americans and causing over 100,000 deaths per year in the United States (Martin, 1999; Shoulson, 1998). It is characterized by the deposition of amyloid [3-peptide (A[3) in plaques and in cerebral blood vessels and the development of neurofibrillary tangles (NFTs). However, both A[3 and NFT occur in the cerebral cortex of normal (nondementia) but aging individuals. Studies in human twins show a strong genetic influence in the development of AD, with concordance for AD in monozygous twins two-to threefold greater than that occuring among dizygous pairs (Gatz et al., 1997). A familial early-onset form of AD is caused by mutations in the [3-amyloid or presenilin genes. The [3-amyloid gene encodes a large protein, amyloid precursor protein (APP), a cellular transmembrane protein that gives rise to [3-amyloid, a fragment varying between 40 and 42 amino acids in length. Studies in rats have demonstrated rapid catabolism of infused A[342 to A[340 via neutral endopeptidase in normal rat brain (Iwata et al., 2000). Seven different mutations in the gene for APP have been found in human families with familial AD, and each led to increased production of the longer 1-42 [3-amyloid, which forms fibrillar aggregates that are neurotoxic. Far more common in early-onset AD are mutations in the gene for presenilin 1 and 2. The presenilins are found in the Golgi apparatus and endosomes and are postulated to interact with the y-secretase enzyme that cleaves APP to the longer 1-42 [3amyloid (Waggie et al., 1999; Martin, 1998). It has been demonstrated that the PS1 gene is identical to the tumor suppressor inhibited pathway clone 2 (TSIP2) gene and suggests that this protein is involved in the p53-induced apoptosis pathway, and possibly in the regulation of cancer-related pathways (Roperch et al., 1998). Mice transgenic for (and overexpressing) the 695-amino acid isoform of human Alzheimer [3-amyloid precursor protein had a 14-fold increase in A[31-42, numerous A[3 plaques in the brain, and impairment of memory in spatial reference and alternation

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

tasks by 9 months of age (Hsiao et al., 1996). In APP 23 transgenic mice there is also region-specific and age-dependent neuronal 10ss in the central nervous system (CNS) (Staufenbiel et al., 1998). Citron et al. (1997) transfected both cells and mice with several human presenilin genes associated with early-onset AD. All transfected cell lines produced greater amounts of 1-42 [3-amyloid as a result of altering the activity of 7- (but not c~- or [3-) secretase. Double transgenic mice (human PS 1 plus human APP695) expressing mutant presenilin genes deposited more A[31-42 in the brain than those expressing normal presenilin genes. Chui et al. (1999) examined the brains of aged transgenic and control mice differing in the expression of mutant presenilin 1 genes (PS 1). They discovered that despite the absence of plaques, transgenic mice had significantly more neuronal cell death, which was associated with increased intracellular A[3 in mutant transgenic mice compared to that in wild-type transgenic or age-matched control mice. They speculate that downstream events in the amyloid cascade, i.e., plaque formation, NFT, and inflammation, are not prerequisites for the induction of neuronal degeneration. It is also intriguing that apoptosis was induced in mutant presenilin 1 gene transfectants, an observation that links the previous association between PS 1 and TSIP 2 (and their role in the apoptosis cascade) and provides in vivo evidence for the previous observation that PS 1 mutations potentiate neuronal apoptosis induced by extracellular applied A[3-catenin (Zhang et al., 1998). The molecular events that regulate programmed cell death or apoptosis were first characterized in C. elegans with at least two different gene families, the caspase family of aspartate-specific proteases and the deathpromoting and death-suppressing proteins of the Bcl-2 protooncogene family. Homologous genes for these pathways occur in mammalian cells (Martin et al., 1998). Presenilins are substrates for a caspase-3 family protease during apoptosis, and the early-onset AD mutations in the PS2 gene increase susceptibility to apoptosis and further fragmentation of the PS2 protein. A homolog of the presenilin gene in C. elegans is SEL-12, the protein that facilitates the LIN-12 Notch receptor. In fruit flies, LIN-12 Notch receptor expression is modulated by catenins through the wingless (wg) signaling pathway (Axelrod et al., 1996). This observation regarding catenins in fruit flies may help explain how a nematode presenilin facilitates LIN-12 Notch receptor, and help shed light on the role of catenins in the endoproteolysis of PS 1 or PS2 in mammals and their contribution to neurodegeneration in AD (Finch and Tanzi, 1997). Other molecular studies have provided evidence for an alternative mechanism that links mutations in PS 1 and PS2 to initiation of apoptosis. Buxbaum et al. (1998) have isolated a new protein named calsenilin, a calcium-binding protein of the recovering family. Normally PS 1 and PS2 bind calsenilin, and it has been postulated that this interaction may protect against apoptosis. Overexpression of calsenilin in human neuroglioma cells, however, leads to alternative processing of PS2 in a man-

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ner similar to that seen during caspase-induced apoptosis. Further studies are necessary to determine whether the interaction between presenilins and calsenilin occurs physiologically and if so, determine the downstream consequences of this interaction. In all likelihood, transgenic mice expressing mutant PS 1 and PS2 will play an important role in these investigations. Should these exciting results linking mutant PS1/PS2 and A[31-42 to induction of neuronal apoptosis in animals be confirmed in AD patients, it will not only dispel a widely held theory on disease pathogenesis but open up opportunities to develop new therapies based on the interactions between presenilins, [3-catenin and other molecular mediators, and A[31-42 (Shoulson, 1998). c. Obstacles: Phenotyping and nanofabrication. Increasingly, discovery is being made of a considerable degree of conservatism in the activity of products expressed from homologous genes between species (see previous discussion on aging). This is particularly true when comparing mammalian species. However, it is also true that for many genetically engineered animals, particularly knockout mice, unexpected phenotypes are discovered. The mechanisms that result in these unexpected phenotypes are numerous and complex and serve to emphasize that in the postgenome era, work to understand the function of genes has only begun. In some instances knockout mice develop a phenotype quite different from that seen in humans with the same mutation. For instance, when mice are carriers of a deletion in the retinoblastoma (Rb) gene, they are prone to tumors; however, in contrast to humans where retinoblastoma is common, these mice develop tumors of the pituitary gland (Gordon, 1997). In the case of generating murine knockouts for transforming growth factor-J3 (TGF[3) genes, over 30 different murine phenotypes have been discovered by knocking out one of three structural genes (Pelton et al., 1991). This apparent nonredundancy in the TGF[3 family is due to a combination of mechanisms, including those involved with ligand processing, receptor interactions, and signaling pathways (Doetschman, 1999). Unlike the previous example, fibroblast growth factors (FGF) 1 and 2 have activities in numerous cell types and tissues; however, when FGF2 was knocked out, mice had no apparent abnormal phenotype (Zhou et al., 1998). This was unexpected since knockouts lacking Fgf 3, 4, 5, 7 or 8 have profound and unique phenotypic changes. Although living a normal, healthy life, Fgf2 -/- mice were later found to have increased platelet numbers and decreased cell density in the cerebral cortex, as well as changes in vascular tone and wound healing. Thus, unique phenotypes were eventually found for this knockout although the changes appear more important in adult life than in early development (Doetschman, 1999). In some knockout mice the lack of one gene product necessary for vertebral skeletal muscle development, MyoD or Myf5, results in rather normal muscle. While this appeared to be an example of gene redundancy, in fact it

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was an example of developmental compensation since the product of one expressed gene compensated for the lack of expression of the other (Ordahl and Williams, 1998). In other instances the mouse may not be the most appropriate species to exhibit phenotypic changes following transgenesis. Mice overexpressing the murine Ren-2 gene did not develop hypertension, while rats did (Mullins et al., 1990); likewise, mice harboring the human HLA-B27 gene did not develop inflammatory disease, whereas rats harboring the human HLA-B27 gene did (Hammer et al., 1990). Mice with a deleted hypoxanthine phosphoribosyltransferase (HPRT) gene never developed the Lesch-Nyhan syndrome seen in humans (Gordon, 1997). Strain background has also been found to profoundly change the phenotype of mice with a single gene knocked out. Although this has been demonstrated for many genes (and perhaps occurs with most), TGF[3 knockouts illustrate this point well, with modifier genes documented that affect the function of TGF[31 in preimplantation, yolk sac development, bowel and gastric inflammation, and colon tumor suppression (Doetschman, 1999). It has also been shown that knockouts on a mixed genetic background have a wider range of phenotypes. Other factors that play on the determination of phenotype in genetically engineered mice have been discussed by Roths et al. (1999). Pleiotropism is the production of multiple, often seemingly unrelated, phenotypic effects resulting from a single gene. The semidominant W locus encodes nonalbinotic white spotting, macrocytic anemia, and oocyte depletion. In addition, phenotypic similarities may be seen between mice bearing different knocked-out genes even when those genes are nonallelic. In this case, knocking out any critical gene in a pathway leading to a developmental defect may result in the same phenotype, e.g., mutations in either the Fas-encoding gene (lpr) or the Fas ligand (gld) results in lymphocyte hyperplasia (Murphy and Roths, 1977). Phenotypic divergence among mutant alleles at the same locus is also common, and among the mutant W alleles, changes in phenotype may be due to effects on transcription and RNA splicing, RNA stability and transport, translation, transport to the cell surface, or signal transduction (Roths et al., 1999). Many gene knockouts are manifested during gestation and may result in embryonic lethality or defective phenotypes at birth; however, a number of single mutations are expressed in mice only later in life. In some instances this is associated with reaching a critical threshold, such as the number of Purkinge cells in beige mice, which have progressive loss of these cells from birth to 12 months of age, when approximately 50% of Purkinge cell numbers are lost and signs of tremor, ataxia, and lethargy begin to occur. Taken together, these unexpected changes in phenotype place new emphasis on proper characterization of genetically engineered animals. Given the numerous mechanisms that can lead to different phenotypic expression of the same gene, it appears that the number of functional products will far exceed gene numbers and require the development of high throughput

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screening devices. In addition, the phenotype of the animal may well depend on a host of environmental factors that may influence the expression of genes and gene products. The laboratory animal specialist will be instrumental in the phenotyping process both in terms of controlling environmental variables and identifying those that may alter the phenotypic profile. In addition, meticulous attention must be given to development of a comprehensive screen that will help elucidate subtle abnormalities such as those originally missed in the evaluation of Fgf2 -/-. Wood (2000) has provided an overall process for assessment of phenotypes in transgenic animals based on a twotiered level of screening. The goal of a primary level assessment would be to find abnormalities, and the goal of the secondary level assessment would be to quantify and evaluate the abnormalities found in the primary assessment. The primary assessment would concentrate on clinical and pathologic (with clinical pathology) parameters and take into consideration the animal's activity, gross anatomy, reproduction, and life span, as well as a complete histopathologic and clinical pathologic examination designed to rule out the influence of pathogens. Environmental factors such as diet must also be taken into account. The secondary level assessment would involve evaluations of embryos, special pathology, biochemistry, physiology, and behavior. In all likelihood, dedicated centers will be established to conduct these specialized tests. The success of any secondary screen will depend on standardization, rapid screening, and automation. Sundberg and Boggess (1999) have also published a systematic approach to evaluation of mutant mice. Nanofabrication has led to the production of microchips used to establish microarrays in which tissue levels of specific mRNA can be detected (through binding of complementary DNA) to DNA probes in the microarray. This system provides high throughput for mRNA expression but depends on the selection of DNA probes utilized. Similarly, nanofabrication has led to microchips capable of cultivating and probing viable cells under in vitro conditions (Ghanem and Shuler, 2000). This same chip technology should be developed for measuring the expression of many proteins from tissues using the enzymelinked immunosorbent assay (ELISA) technology and adapting this tool for clinical chemistry and quantitating levels of peptides, hormones, and enzymes from blood. It is even possible to envision an automated microanalysis of hematologic parameters. These small-volume, high throughput screens, when coupled with microsurgical techniques for tissue harvesting, could satisfy the increasing demand for certain in vivo phenotypic analyses. Physiologic evaluations are also essential, and here nanofabrication of instruments especially for monitoring blood flow, pressure, tissue perfusion, and uterine contraction has made a major impact on cardiovascular monitoring (Doevendans et al., 1998). These technologies are augmented by a host of imaging techniques adapted to the mouse (Chien, 1996). However, work in this area must accelerate in order to more fully characterize the thousands of new mice engineered each year.

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30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

One of the greatest enigmas facing the biomedical research community is deciding what assays should be employed to phenotype the many divergent organ systems of mice. The National Institutes of Health (NIH) has convened expert panels to give direction in this endeavor, and publications are emerging from various research groups illustrating the techniques available for specific organ systems. A number of papers have addressed the issue of behavioral phenotyping transgenic mice, with special emphasis on several prominent areas of research (Crawley, 2000; McClearn and Vandenbergh, 2000; Nelson and Chiavegatto, 2000, Brown et al., 2000). Among the panels to be developed are those that deal with pain and distress, for these will guide all investigations by establishing humane end points (Carstens and Moberg, 2000; Dennis, 2000).

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S o u r c e s of A n i m a l M o d e l s

The number and types of resources available today to aid investigators in the selection of an animal model are vast, and any attempt to list them all would be foolhardy. However, there was a time when selecting the appropriate animal model relied solely on one's knowledge of its anatomy and physiology, and lists of anatomic, physiologic, and metabolic differences between species would guide in selection (OTA, 1986). Inbreeding mice to produce strains of genetic and physiologic consistency began with Ernest Castle, who first brought them into the laboratory in 1902 when he established the Bussey Institute. Clarence Cook Little developed the first inbred strain in 1909 (Davisson et al., 1998). The tradition of using (and developing) inbred strains of mice continued under George Snell (at the Jackson Laboratories), who discovered the major histocompatibility complex (MHC) in mice and was awarded the Nobel Prize in Medicine in 1980 (Quimby, 1994). As the laboratory produced more and more inbred strains and made them available to investigators (now over 259 strains being held), spontaneous mutants were quickly recognized and saved. To date, over 1000 spontaneous mutant mice have been recognized, and over 20 new mutations are documented each year in the Jackson Laboratory colonies (Davisson et al., 1998; Quimby, 1994b). Yet before 1960, it was difficult for investigators to find a reliable supply of laboratory animals. Analysis of abstracts published in the Federation Proceedings for the year 1960 show that cats and dogs were used in 37% of all physiologic studies, rats were used in 22%, and mice in less than 3% (Schmidt-Nielsen, 1961). Many of the dogs and cats used were likely to be pound animals. In fact, until the last decade most spontaneous models of inherited disease in animals were discovered by accident, e.g., veterinarians described new diseases in companion animals and livestock (Cornelius, 1969; Stormont, 1968; Lewis and Carraway, 1992), or spontaneous mutations in rodents and rabbits were identified by their attendants in large breeding colonies (Kondo and Watanabe, 1975; Sharp and Davisson, 1994). During the early 1960s, many of the infectious diseases of ro-

dents were identified; attempts to develop specific pathogenfree (SPF) animals by cesarean section and barrier isolation resuited in a reliable commercial supply of rats and mice by the 1970s (Quimby, 1994b). Similarly, research centers throughout the United States began reconstruction of animal facilities that employed the barrier concept, and standardization of caging and sanitation practices guaranteed the valuable models remained SPF, much of this due to documents written by the Institute for Animal Resources (ILAR) at the National Academy of Sciences (NAS) and funds made available from the National Center for Research Resources (NCRR) at the National Institutes of Health (NIH) (Quimby, 1994b). As research began to flourish in the 1970s, the need for animal models increased accordingly. Attempts to identify and preserve valuable models were undertaken by ILAR, but many important large-animal models disappeared as a result of the high cost of maintenance. In 1980, Gordon et al. published the first paper demonstrating the feasibility of pronuclear microinjection into a fertilized mouse egg; the term "transgenic" arose from these studies (Gordon and Ruddle, 1981). Brinster et al. (1981) showed that foreign genes so transferred could be efficiently expressed, although expression levels were difficult to control. Mouse embryonic stem cells were first produced in 1981 (Martin, 1981; Evans and Kaufman, 1981), and via a process called homologous recombination, foreign genes could be stably integrated into the murine genome or specific mouse genes could be rendered nonfunctional by mutation (knockouts) (Smithies et al., 1985). The long-awaited dream of medical scientists had arrived, a system to produce homologs of inherited diseases of humans in a small-animal model. The numbers of new animal models created this way is approximately 1 per day in early 2000. 1.

Organizations

Several organizations in the United States have taken the lead in providing information on (or access to) animal models. The ILAR (now renamed the Institute for Laboratory Animal Research) maintains two searchable databases: an Animal Models and Genetic Stocks (AMGS) Information Program and the International Laboratory Code Registryma listing of codes that identify an investigator, a laboratory, or an institution that needs rodents or rabbits. In addition, it publishes the quarterly I L A R Journal (devoted to research animal topics) and publishes a variety of books and other documents dealing with standards for animal care, education, and occupational health and conducts workshops on current animal-related topics. Noteworthy among these books is one dedicated to mammalian models for aging research (NRC, 1981) and another on models of thrombosis and hemorrhagic disease (NRC, 1976). Volume 39 (Nos. 2 and 3) of the I L A R Journal is dedicated to comparative gene mapping and contains the gene maps for over 16 species (phone: 202-3342590; ). The National Institutes of Health (NIH) has been the major

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funding agency for animal models of human diseases. It provides updated information on national and international efforts to produce animal models at <www.nih.gov/science/models>. The Office for Protection from Research Risks at the NIH also provides a comprehensive list of animal resource links, as well as special reports and regulations at . The National Agriculture Library at the U.S. Department of Agriculture operates the Animal Welfare Information Center (AWIC). This center manages the AGRICOLA database and provides literature searches on a wide variety of subjects related to the use of animals and alternatives in research. The quick bibliography series (qb90-09) lists 189 abstracts of books and articles between January 1979 and August 1989 on the subject of animal models of disease. Between January 1988 and January 1995 (qb95-14), 330 citations were found. Using the same retrieval system and database searching for animal model citations between January 1995 and January 2000, a total of 2486 citations were found. While this serves to illustrate the growth in use and publication of animal models, it represents only a fraction of the total publications describing animal models since both the terms "animal" and "model" had to be written in the title or abstract. Access to these quick bibliography series can be made through . Custom searches can also be arranged (phone: 301-504-6212; email: [email protected]). The American College of Laboratory Animal Medicine (ACLAM) was established in 1961 (previously known as the American Board of Laboratory Animal Medicine) with the expressed goal of encouraging education, training, and research in laboratory animal medicine and of certifying specialists in the field. An extensive series of texts has been published by ACLAM, covering the biology and use of many mammalian species, including rabbits (Manning et al., 1994), hamsters (VanHoosier and McPherson, 1987), guinea pigs (Wagner and Manning, 1976), rats (Baker et al., 1980), mice (Foster et al., 1982), and nonhuman primates (Bennett et al., 1995), as well as two volumes dedicated to spontaneous models of human disease (Andrews et al., 1979). The Armed Forces Institute of Pathology (AFIP) has contributed many articles on animal models in the journals Laboratory Animal Science and the American Journal of Pathology, and it publishes a handbook of models of human disease (Jones et al., 1972). Most companies providing laboratory rodent models for research also have websites containing detailed information on the models they provide; some are listed below under websites. 2.

Original Articles and Texts

Several journals contain major segments devoted to animal models, including ILAR Journal (email: [email protected]; see above); Contemporary Topics in Laboratory Science and Comparative Medicine (formerly Laboratory Animal Science), each

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published by the American Association for Laboratory Animal Science <www.aalas.org>; Lab Animal, published by Nature America, Inc. ; Experimental Animals, published by the Japanese Association of Laboratory Animal Science <www.jalas.or.jp/index>; and Laboratory Animals, published by Laboratory Animals, Ltd., the official publication of the Laboratory Animal Science Association of the United Kingdom <www.lal.org.uk>. Other European and U.S. journals that may deal with animal models have been identified (Quimby, 1994b; NRC, 1996). A wide variety of texts have been produced that cover the various animal models of human disease; space allows for only a few of them to be mentioned here. Readers should refer to other sections of this chapter for a more comprehensive listing. In addition to the bibliographies on animal models previously cited (see National Agriculture Library, AWIC, above), Hegreberg and Leathers (1981a,b) have cataloged both the naturally occurring and induced models available in the 1980s. Festing (1993a,b) has developed an index covering the sources of over 7000 stocks of laboratory animals worldwide and separately has recorded the origins, characteristics, and sources of inbred strains of mice. Earlier listings of inbred and genetically defined strains produced by the Federation of Societies for Experimental Biology cover rats and mice (Altman and Katz, 1979a) and hamsters, guinea pigs, rabbits, and chickens (Altman and Katz, 1979b), and they are still very useful. Lyon has published "Genetic Variants and Strains of the Laboratory Mouse" (Lyon, 1996). Gay (1965-1989) has edited nine volumes titled "Methods of Animal Experimentation" and Nathanielsz (1980-1987) has edited six volumes tided "Animal Models in Fetal Medicine"; both series elaborate on the proper use of induced models in research. The use of various animal species as both spontaneous and induced models appears in each species chapter in "The Experimental Animal in Biomedical Research" (Rollin and Kesel, 1995), and a separate text is devoted to induced models (Swindle and Adams, 1988). Individual species or groups of animals used as experimental models have been described for nonhuman primates (Dukelow, 1983; King et al., 1988; Joag, 2000), squirrel monkeys (Abee, 2000), swine (Stanton and Mersmann, 1986; Swindle et al., 1992; Mount and Ingram, 1971), domestic farm animals (Doyle et al., 1968; Lewis and Carraway, 1992); spontaneous and engineered mutant mice (Roths et al., 1999; Nomura, 1997; Gordon, 1997; Colbert and Klintworth, 1997), nude mice (Fogh and Giovanella, 1978, 1982), rats (Gill et al., 1989), fish (Powers, 1989; Ishikawa, 2000; Dooley and Zon, 2000; Paw and Zon, 2000; Zhu and Sun, 2000), birds (Konishi et al., 1989; Rose, 2000; Quimby et al., 1995; Medina and Reiner, 2000), and cetaceans (Wtirsig, 1989). The reader should also refer to each species chapter in this text for a general review of its uses in research. In addition, numerous reviews are available on the use of animal models for particular types of research, including fetal research (Hansen and Sladek, 1989), cytokine research (Durum

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and Muegge, 1998), autoimmune diseases (Cohen and Miller, 1994; Taylor, 1994; Antel and Owens, 1999; Kukreja and MacLaren, 1999), immunodeficiency diseases (NRC, 1989; Gershwin and Merchant, 1981; Sordat, 1984; Percy and Barta, 1993), gene therapy (Herweijer et al., 1997; Lee et al., 1999), hypertension (Bader et al., 1997), Lesch-Nyhan and other metabolic diseases (Jinnah and Breese, 1997; Haskins et al., 1992; Colbert and Klintworth, 1997; Ozaki et al., 1998), skin diseases (Norvell et al., 1997; Sundberg, 1994), amyotrophic lateral sclerosis (Dal Canto, 1997; Green and Tolwani, 1999; Sillevis and deJong, 1989), multiple sclerosis (Dal Canto, 1997; Gold et al., 2000; Wong et al., 1999a), peripheral neuropathies (Notterpek and Tolwani, 1999; Thomas, 1992), Parkinson's disease (Tolwani et al., 1999; Zigmond and Stricker, 1989), Alzheimer's disease (Price et al., 1998; van Leuven, 2000; Sommer et al., 2000; Guenette and Tanzi, 1999), Huntington's disease (Price et al., 1998; Brouillet et al., 1999), cerebral ischemia (Martin et al., 1998; Megyesi et al., 2000), corneal diseases (Smith et al., 1995; Cowell et al., 1999), retinal dystrophies (Flannery, 1999; Zack et al., 1999; Stone et al., 1999), myopia (Norton, 1999), myoclonus (Nguyen et al., 2000), cognition (Newsome and Stein-Aviles, 1999), behavior (Takahashi et al., 1994; Hunter et al., 2000), alcohol and drug abuse (Crabbe et al., 1994; Stewart, 2000), pain (Ren and Dubner, 1999; Ness, 1999; Brennan, 1999), nutrition (Beynen and West, 1988; Yamori, 1999; Montinaro et al., 1999; Wallace, 2000), atherosclerosis (Clarkson et al., 1988; Attie and Prescott, 1988; Clarkson and Klumpp, 1990; Narayanaswamy et al., 2000), hyperlipidemia (Sullivan et al., 1993; Breslow, 1993; Bauer, 1996), transplantation (Ladiges et al., 1990; Wenger et al., 2000; Locatelli et al., 2000), infectious diseases (Renegar, 1992; Gardner, 1997; Lee, 1999; Salzman, 1986; Wassom and Peper, 2000; Joag, 2000; Tsolis et al., 1999), neoplasia (Calnek, 1992; Kritchevsky, 1988; Kobaek-Larsen et al., 2000), pulmonary disease (Cantor, 1989; Chang, 2000; Forsythe and Ennis, 1999; Kon and Kay, 1999), hepatic disease (Newsome et al., 2000), ulcers (Lee, 2000), type 1 diabetes mellitus (Wong and Janeway, 1999; Wong et al., 1999a), type 2 diabetes mellitus (Ostenson et al., 1993; Kovacs et al., 1997; Bali et al., 1995; Shafrir et al., 1999; Reitman et al., 1999), and obesity (McCracken, 1988; Nicholls et al., 1996; Samad and Loskutoff, 1999). This is not meant to be an exhaustive list of either species used or the subject of their use, but rather a listing of some recent reviews that illustrate the diversity of subjects and printed material covering animal models. 3.

Websites

In addition to the websites maintained by ILAR, NIH, and AWIC (see Section I,C,1), numerous other websites have been established to disperse information on animal models available for research and genetic mapping resources, as well as more comprehensive publications• Vendors of laboratory animals maintain websites that de-

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scribe the characteristics of the animals they supply. The Jackson Laboratory <www/jax.org> database maintains a Lane List of Named Mutations and Polymorphic Loci, Neurology News, Ophthalmology News, and Dermatology News under the Animal Resources site. Within its Genetic Resources site is a searchable database on transgenic and targeted mutant mice, Mouse Mutant Resource, Neural Tube Defect Resource, Cytogenic Models Resource, Eye Models Resource, and Hearing Models Resource (Sharp and Davisson, 1994; Takahashi et al., 1994). Taconic Farms, Inc. <www.taconic.com> maintains a searchable database, Taconic Transgenic Models, providing information on resources available, as well as their R e s e a r c h Animal Review newsletter. Charles River Laboratories (email:[email protected]) also maintains a website <www.criver.com>. Internet resources for transgenic and targeted mutation research include TBASE, the transgenic/targeted mutation database maintained by Jackson Laboratories . TBASE also provides links to the following websites: Database of Gene Knockouts , and BioMedNet Mouse Knockout database . The Mouse Genome database integrates existing databases of mouse genetic information and includes mapping data, molecular probesm clones, strains, and allelic polymorphismsmas well as phenotypic data. The Encyclopedia of the Mouse Genome includes genetic linkage maps, cytogenic maps, and a text searchable tool to review information integrated from numerous centers. The Portable Dictionary of the Mouse Genome includes three separate estimates of gene position, accession numbers to GenBank sequences, data on homologs in human and other mammalian species, a complete data set on recombinant inbred strain-distribution patterns, and much more. Genetic and physical maps of the mouse genome are maintained by the Whitehead Institute at Massachusetts Institute of Technology . Other U.S. websites providing genetic information on rodents include the cybermouse project , the Gene Expression database , NCBI's Human/Mouse Homology relationship database , RATMAP , and the Mouse and Rat Research Home Page . European databases include the European Collaborative Interspecific Mouse Backcross , the Mouse Atlas Project and the Dysmorphic Human-Mouse homology database . The Japanese Animal Genome database can be accessed at .

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THE NATURE OF RESEARCH

Hypothesis Testing and Serendipity

1. The Progressive (and Sometimes Long) Route to Discovery The 1000-year period historically referred to as medieval or the Middle Ages was characterized by personal ambitions and bias and a preoccupation with religion and politics, which was rarely conducive to scientific discovery. Inquiry into the nature of things, fostered by Aristotle and Galen, ceased until the 1400s when once again interest in seeking facts to explain nature resumed. Realizing the situations that led to confound his predecessors, Francis Bacon (1620) proposed a process of scientific discovery based on a collection of facts and observations, followed by a systematic evaluation of these facts demonstrating them to be true. Distinction between the act and demonstration of discovery was precisely what was missing during the Middle Ages, when assertions were frequently made that the truth could be elicited from a small series of observations. Bacon's requirement for elimination of all those inessential conditions (which are not always associated with the phenomenon under study) was, in the end, unachievable, and the process of choosing facts was found to depend on individual judgment. However, Bacon did properly perceive the defects inherent in the scholastic method practiced before him, and he set the tenets for what would become the method of hypothesis testing. Arguably, the foundation for sorting fact from fiction in scientific investigations is based on hypothesis testing (a particularly weak aspect of Bacon's philosophy). Although it is never possible to directly prove a hypothesis by experimentation, but rather to disprove one (or more) alternative (null) hypotheses; history has documented the steady (although sometimes slow) progress toward understanding the world. That is not to say that certain observations made during the testing of one hypothesis have not led investigators in an altogether different direction. In fact, this happens with a certain consistency and at times results in knowledge as compelling as that originally sought by investigators. One may argue, and rightfully so, that hypothesis testing is an inefficient mechanism for discovery; however, this paradigm of generating a hypothesis based on known facts and designing experiments to disprove the hypothesis has been proven over time to generally produce meaningful and reproducible results.

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On the other hand, in looking at the outcome of one milestone in biology and medicine, such as coronary bypass surgery, and examining the history of the many facts necessary to allow experimenters to directly perform a bypass operation, it is astonishing how many individual feats and over how long a period of time it took to reach the final test. The earliest studies that contributed to the first successful bypass surgery in the 1970s go back to 1628 when Harvey described the circulation of frogs and reptiles; then in 1667, Hooke hypothesized (and later demonstrated) that pulmonary blood, flowing through lungs distended with air, could maintain the life of animals. These early observations had no impact on medicine until centuries later, primarily because other technologies necessary for successful application of extracorporeal oxygenation in humans, including antisepsis, anticoagulants, blood groups, anesthesia, etc., had not yet been discovered. Dogs played a critical role during this process of discovery and between 1700 and 1970 contributed knowledge on the differential pressures in the heart; measurements of cardiac output, cardiopulmonary function, and pulmonary capillary pressure; and development of heart chamber catheterization techniques, heart-lung pumps, angiography, indirect revascularization, direct autographs, saphenous vein grafts, balloon catheters, and floating catheters (Comroe and Dripps, 1974). While examining the history behind the 10 most important clinical advances in cardiopulmonary medicine and surgery, Comroe and Dripps (1976) selected 529 key articles (articles that had an important effect on the direction of research) in order to determine how these critical discoveries came about. They found that 41% of these articles reported work that had no relation to the disease it later helped to prevent, treat, or alleviate. This phenomenon probably contributes to the observation that few basic science discoveries, including those conducted using animals, are cited in seminal papers describing a clinical breakthrough. A recent series in the New England Journal of Medicine, called Clinical Implications of Basic Research, promises to correct this oversight (Wu, 2000). The idea that major clinical breakthroughs required a long history of basic science discoveries, often involving animals and often being conducted by individuals who were unaware of the ultimate application of this knowledge, continues to be true today. Rudolfo Llinas after reflecting on 47 years of research aimed at elucidating the nature of neurotransmission, much of it accomplished using the giant axons of squid, states: "In the end, our complete understanding of this process (synaptic transmission) will manifest itself not as a simple insight, but rather as an ungainly reconstruction of parallel events more numerous than elegant" (1999). Both Bacon and Mill, who followed him, believed it was the responsibility of scientists to find the "necessary and sufficient conditions" that describe phenomena. That exhaustive lists of circumstances had to be examined in the search of what was necessary and sufficient never concerned these philosophers

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(Bacon, 1620; Mill, 1843). Both saw virtue in experimentation, the collection of data under controlled circumstances. The scientific method practiced today evolved from the principles of Bacon and Mill and was refined by the middle of the nineteenth century. The method provides principles and procedures to be used in the pursuit of knowledge and incorporates the recognition of a problem with the accumulation of data through observation and experiment (empiricism) and the formulation and testing of hypotheses (Poincare, 1905). The method excludes the imposition of individual values, unsubstantiated generalizations, and deferments to higher authority as mechanisms for seeking the truth. It also subscribes to basing hypotheses only on the facts at hand and then rigorously testing hypotheses under various conditions. Hypotheses that appear to be true today may be disproved in the future as new conditions are imposed upon them and new technologies employed in the collection of data. Although great discoveries in biology and medicine have depended on the application of these principles, progress is still often slow. As hypotheses are proven incorrect, alternative hypotheses are sought and tested. Unexpected experimental results require careful consideration; and often the reasoned explanation of this data contributes information critical for the formulation of an alternative hypothesis. In the mid-1970s, a series of breeding experiments was conducted to test the hypothesis that systemic lupus erythematosus (SLE) resulted from a mutation passed between individuals through simple Mendelian inheritance. Dogs that spontaneously developed SLE were bred and their progeny tested (Lewis and Schwartz, 1971). Surprisingly, no offspring in three generations of inbreeding developed SLE, but over half the offspring developed other autoimmune diseases, including lymphocytic thyroiditis, Sj6grens syndrome, rheumatoid arthritis, and juvenile (type 1) diabetes (Quimby et al., 1979). After careful reexamination of the data, it was hypothesized that multiple, independently segregating genes were involved in the predisposition to autoimmunity and furthermore, that certain of these genes (class 1) would affect a key component in the immune system common to several autoimmune disorders, with other genes (class 2) acting to modify the expression of class 1 genes, producing a variety of different phenotypes (autoimmune disease syndromes) (Quimby and Schwartz, 1980). Data collected over the next 15 years, using techniques unavailable in the 1970s, have generally upheld this hypothesis and elucidated genetic mechanisms unimaginable at the time (Datta, 2000). 2.

Taking Advantage of Unexpected Findings

Serendipity also contributes to important discoveries. In 1889, a laboratory assistant noticed a large number of flies swarming about the urine of a depancreatized dog and brought it to the attention of VonMering and Minkowski. Minkowski discovered, on analysis, that the urine contained high concen-

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trations of sugar. This chance observation helped VonMering and Minkowski discover that the pancreas had multiple functions, one being to regulate blood glucose (Comroe, 1977). In the late 1800s, Christiaan Eijkman was sent to the Dutch Indies to study the cause of beriberi, a severe polyneuritis affecting residents of Java. While conducting studies, Eijkman noticed that chickens housed near the laboratory developed a similar disease. He tried and failed to transfer the illness from sick to healthy birds; however, shortly thereafter the disorder in chickens spontaneously cleared. Eijkman questioned a laboratory keeper about food provided the chickens and discovered that for economy, the attendant had previously switched from the regular chicken feed to boiled polished rice, which he obtained from the hospital kitchen. Several months later the practice of providing boiled rice to the chickens was discontinued, which correlated with disease recovery in the birds. This chance observation led Eijkman to conduct feed trials demonstrating that a factor missing in polished rice caused beriberi and that the disease could be cured by eating unpolished rice. These studies led to the discovery of the vitamin thiamin, and were the first to show that disease could be caused by the absence of something rather than the presence of something, e.g., bacteria or toxins (Eijkman, 1965). These examples reinforce the necessity for making careful observations, investigating unexpected findings, and designing careful follow-up experiments. Eijkman was awarded the Nobel Prize in Medicine in 1929, and Banting and Macleod received the Nobel Prize in 1923 for their discovery of insulin, made possible by the previous observations of VonMering and Minkowski (Leader and Stark, 1987).

B. 1.

Breakthroughs in Technology

Paradigm Shifts

In "The Structure of Scientific Revolutions," Kuhn (1970) makes a case for scientific communities sharing certain paradigms. Scientific communities consist of practitioners of a scientific specialty that share similar educations, literatures, communications, and techniques and as a result, frequently have similar viewpoints, goals, and a relative unanimity of judgment. Kuhn believes that science is not an objective progression toward the truth but rather a series of peaceful interludes, heavily influenced by the paradigms (call them theories) shared by the members of a scientific community and interrupted, on occasion, by intellectually violent revolutions that are associated with great gains in new knowledge. Revolutions are a change involving a certain sort of reconstruction of group (community) commitments. They usually are preceded by crisis (from within or outside the community) experienced by the community that undergoes revolution. Kuhn explains that scientific communities share a disciplinary

1202 matrix composed of symbolic generalizations (expressions, displayed without question or dissent by group members), beliefs in particular models, shared values, and exemplars (those concrete problem solutions that all students of community learn during their training). This disciplinary matrix is what provides the glue that keeps members of the community thinking (problem solving) alike. However, it is also what prevents members from taking high-stake chances and proposing new rules that counter prevailing opinion. Precisely when two members of a community disagree on a theory or principle because they realize that the paradigm no longer provides a sufficient basis for proof is the debate likely to continue in the form it inevitably takes during scientific revolutions. What happens during revolutions is that the similarity sets established by exemplars and other components of the disciplinary matrix can no longer neatly classify objects into similar groups. An example is the grouping of sun, moon, Mars, and Earth before and after Copernicus, where a convert to the new astronomy must now say (on seeing the moon), "I once took the moon to be a planet, but I was mistaken." As a result of the revolution, scientists with a new paradigm see differently than they did in the past and apply different rules, tools, and techniques to solve problems in the future (Kuhn, 1970). We are now at the threshold of such a revolution in biology. The crisis is beginning, and it takes the form of computerassisted biological modeling. Two diverse technologies are approaching maturity, computerized information processing and genetics (from recombinant DNA to functional genomics). They have converged as a result of the Human Genome Project where analysis of data provided by high throughput nucleotide sequencing (where the sequencing itself necessitated integrating the engineering fields of nanofabrication and computer sciences) requires computer-directed selection of intron-exon segments, protein modeling, associative screens matching models to existing protein families--so-called data mining (and thus linking structure to function)-- and rapid screens for nucleotide homology with other nucleotide (or amino acid) sequences in the same or another species. On the biological side of this research enterprise are techniques ranging from rapid throughput amino acid sequencing, X-ray crystallography (and other molecular imaging techniques contributing to structure), and genetic manipulation of biological models. On the computational side are analysis of gene expression using cDNA microassays, computer-assisted modeling (3-D imaging) from crystallographic images, and prediction of new molecular mimics (molecules that may simulate or inhibit the putative activities of another molecule). The end result is the new discipline of bioinformatics, which merges biology with mathematics, computer science, and engineering (Spengler, 2000). The merging of these technologies will be as profound in this century as were the technologies created by Galileo and van Leeuwenhoek in the seventeenth century. Clever combinations of technologies have led to novel methods for predicting protein function. One method called

FRED W. QUIMBY phylogenetic profiling (Pellegrini et al., 1999)" looks for the correlation of protein inheritance across species, while the second method, the Rosetta stone method, looks for correlation of protein domains across species. By use of these methods, proteins have been identified with particular complexes and pathways that suggest common functions for homologs in other species (see Section II,C). Likewise, comparing genomes of different species provides valuable information concerning noncoding regions in the DNA sequence. These correlations have revealed > 70% identity for noncoding regions in the mouse compared to human genomes (Spengler, 2000). The algorithm called Moving Average Point Analysis allows for global alignment of sequences, their comparison, and the display of their identity and has determined conserved sequences in mouse, dog, and human genomes. Likewise, programs have been written to evaluate complex physiologic phenomena, e.g., genes (and proteins) regulating DNA replication or simulation of chemical kinetic systems. But the revolution has just begun; the future shall fully integrate these various disciplines to achieve goals far more lofty than the association between DNA, protein structure, and function; bioinformatics will address complex mechanisms at the cellular, tissue, and population levels, as well as the interaction between genes, their products, and the environment. This level of investigation, unimaginable now, may lead to understanding the biological basis for learning and memory and a better understanding of how genes and the environment interact in processes such as obesity (see Section II,C). What is so revolutionary about integrating mathematics and computer science with biology? While not completely obvious now, the entire paradigm of hypothesis-driven research will change. Computers will make the comparisons, sort out the facts, and from them assist in generating hypotheses. For example, the new computer-assisted technology may input new nucleotide sequences from mouse chromosome 5 and after selecting for the coding region and Rosetta stone screening, demonstrate homology between the unknown sequence and a similar gene encoding the transmembrane receptor, Toll, in Drosophila. While Toll is known to affect dorsal-ventral polarity in Drosophila embryos, it also protects adults from attack by fungi. The cytoplasmic domain sequence of Toll has homology with the interleukin (IL)-1RI cytoplasmic domain in mammals. Human homologs are called Toll-like receptors (TLR), and mutations in TLR4, causing constitutive gene activation, led to expression of IL-1, IL-6, IL-8, and B7-1, proteins involved with inflammation and innate immunity. Both Toll (in Drosophila) and TLR4 share a signal transduction pathway involving activation of NF-×B (Wright, 1999; Medzhitov et al., 1997). One would thus postulate that disruption of the new mouse gene, through homologous recombination of ES cells, would lead to founders incapable of appropriate activation of NF-×B pathway and thus abrogation of the production of inflammatory cytokines essential in innate immunity. In fact, nature has already performed this experiment by creating a nonconservative point mutation in Tlr4 of C3H/HeJ

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30. ANIMAL MODELS IN BIOMEDICAL RESEARCH mice and a null mutation of T1P4 in C57BL10/ScCr mice (Poltorak et al., 1998; Qureshi et al., 1999). Both strains share an inability to properly respond to endotoxin, a lipopolysaccharide (LPS) in the cell walls of gram-negative bacteria, and in both strains genetic mapping has localized the defective gene (previously known as lps) to chromosome 5. Tlr4 is posulated to be a coreactor along with the LPS binding receptor, necessary for activation of signal transduction pathways leading to inflammatory cytokine production via NF-×B (Ulevitch, 1999). One may anticipate that much of the serendipity that characterized discovery in the past will be eliminated in this new age of bioinformatics, but that remains to be seen. The biologist ultimately must see if computer predictions are accurate and, rather than eliminate alternative hypotheses, test directly the consequences of knocking out or knocking in genes. For this work, the mouse will become the standard model of the future, having the advantage of similar physiology, similar genomic structure, high fecundity, and ease of maintenance. Before leaving the subject of scientific revolutions, it is prudent (and consistent with Kuhn's hypothesis) to ask if the members of the community of geneticists are witnessing a crisis. The answer is clearly yes. The crisis involves two different but related phenomena, information overload and speaking a com.mon language. The amount of information (call them facts) presented to geneticists today is overwhelming and is manifested by the proliferation of scientific journals, websites, and scientific meetings. In fact, the volume of data in various sequence data banks is so great that existing computers are often insufficient to analyze large data sets in reasonable time frames. There is also no easy way to transfer vast amounts of data between different institutions. As a result, individual investigators will have to become more focused and selective in the techniques they use and questions they ask and depend on collaboration with others to unravel complex or more generalized questions. Another generation of computers operating at higher speeds may make data analysis more efficient, and the Internet may ease the transfer of information between institutions. For the time being, anxiety is running high as scientists deal with the frustrations mentioned above (Malakoff, 1999). Equally critical is the interdependence between biologists, computer scientists, and engineers, each of whom uses a different language for expression. The full integration of several scientific communities into a single community has not been achieved, and frustrations are mounting as members of different communities attempt to work together. This problem was recognized by the Biomedical Information Science and Technology Initiative, which recommends a new paradigm in education where future biomedical scientists seek cross-disciplinary training (Malakoff, 1999). Several institutions in the United States have begun such programs where engineers, computer scientists, and biologists train together and gain the rudiments of each discipline.

2.

Making Use of Past Achievements

The time could not be more perfect for the next scientific revolution. If indeed the mouse (and possibly the rat) serves as the mammalian model that ultimately links gene and protein structure with function, it comes at a time when the discipline of laboratory animal science is reaching maturity. Since 1966, the year the Animal Welfare Act was passed into law, the world of biomedical research, in particular the use of rodent models, has dramatically changed. At that time spontaneous diseases of laboratory animals were the primary focus of articles published by Laboratory Animals and Laboratory Animal Science, and infectious diseases of laboratory mice and rats were prevalent, with 75% of institutions reporting respiratory disease in rats alone (Seamer and Chesterman, 1967). Several critical technologies, including the flexible-film isolator for cesariean rederivation of rodents (Trexler and Reynolds, 1957) and the defined bacterial "cocktail" (Schaedler et al., 1965), which was used to overcome intestinal complications frequent in germfree rodents and in which all eight strains have been defined by 165 rRNA sequence analysis (Dewhirst et al., 1999), were adopted early by a newly spawning industry to meet the demands of the scientific community (Foster, 1980). Professional societies were being formed in the United States and Europe to develop standards for laboratory animal care (Quimby, 1994). The Institute of Laboratory Animal Research (ILAR) of the National Academy of Sciences (NAS) held meetings devoted to laboratory animal housing and published a "Guide for Laboratory Animal Facilities and Care" (Hill, 1963), which served as the predecessor of the "Guide for the Care and Use of Laboratory Animals," a standard used by institutions throughout the United States (NRC, 1996). In the 1960s, the National Center for Research Resources (NCRR) of the National Institutes of Health (NIH) awarded grants to develop animal diagnostic/investigative laboratory programs to universities throughout the United States. These programs provided diagnostic support for the rapidly growing research animal enterprise and fostered research on disease pathogenesis and diagnostics. In the early 1970s, the NCRR developed a funding mechanism for development and improvement of institutional animal resources (Quimby, 1994b). Another program of the NCRR funded institutional training grants to educate and train competent laboratory animal veterinarians. The American College of Laboratory Animal Medicine (ACLAM) was established in 1961 to certify competency and encourage continued education (Quimby, 1995). During this same period, cage manufacturers met with scientists and laboratory animal specialists to design and construct optimum primary containment for laboratory animals, especially rodents. Although polycarbonate shoe-box cages became an early standard in the industry, new challenges to maintain rodents in germ-free environments (immunocompromised rodents) and contain agents used in infectious disease studies led to microisolator caging and the independently ventilated cage unit. The latter is becoming a

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standard in transgenic mouse facilities. Together with standardization of feeds and bedding, these changes have led to efficient, safe, and productive housing of the many SPF rodent models used today. Mouse genetics today is based on a long history of developing inbred strains, characterizing spontaneous mutations, characterizing phenotypic differences among strains, and mapping genetic loci. Equally important contributions have arisen as a result of recombinant DNA research and induced mutagenesis in the mouse, making it the ideal candidate for nonhuman mammalian genomics (see Section I,B for details). 3.

Looking at the Future

The impact of bioinformatics and its role in complementing functional genomics are likely to finally add to biology the same quantitative component that fostered 200 years of advances in theoretical physics and chemistry. As a better understanding is gained for each of the biochemical pathways controlling such fundamental processes as cell cycling, signal transduction, and transcription (including the proteins that modulate the activity of these biochemical pathways and the kinetics involved in these reactions), mathematical models will arise that may eventually predict certain outcomes. Eventually such models may anticipate host responses to such diverse entities as environmental chemicals, climatic change, and nutrition, but that is in the distant future. Certain fundamental changes are occurring already as new, more powerful tools are made available to scientists. The notion expounded by Robert Koch, which required the isolation and characterization of infectious agents from suspected cases and then re-creation of the illness by inoculation of the pure agent and its subsequent reisolation (the so-called Koch's postulates), is rapidly being replaced by the much simpler procedure of polymerase chain reaction (PCR) to identify microbes from tissues of infected individuals (Bradford, 1998). Genetic fishing using degenerate probes to identify putative agents of human (and animal) disease is more rapid and overcomes problems associated with low tissue concentration of the microbe and inability to culture certain organisms; however, occasionally mistakes are made. Although this technique will likely decrease the number of animals needed to validate a causative agent using Koch's postulates, recent studies have shown that in vitro model systems for investigating microbial evaluation (and in particular, mutations in bacteria conferring resistance and susceptibility to antibiotics) are not faithful replicas of the in vivo environment (Bull and Levin, 2000). As the number of infectious agents with complete genome sequences increase, new opportunities for investigation of microbial pathogenesis will arise (Strauss and Falkow, 1997). At this writing, complete genomes are available for Neisseria meningitidis (Tettelin et al., 2000), Escherichia coli (Collins et al., 1998), Helicobacter pylori (Alm et al., 2000), and C a m p y l o b a c t e r jejuni (Parkhill et al., 2000), and for 9 other pathogenic bacteria, with partial se-

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quences for another 28 bacterial pathogens (Weinstock, 2000). Technologies, including nucleic acid probe arrays and overlapping lambda clones of bacterial genomes, allow for rapid screening of thousands of bacterial genes temporally expressed during infection in in vivo models. Other rapid and specific technologies that can measure gene expression in the host under conditions of infections include in vivo expression technology (IVET), differential fluorescence induction (DFI), and the signature-tagged transposon method (STM), each designed to detect and follow specific virulence factors at discrete stages of interaction between mammalian host and the invading microorganism (Strauss and Falkow, 1997). Combining information on the genomes of the host and the pathogen will allow determination of genes for host resistance and susceptibility and provide the tools necessary to rapidly screen populations for individuals at risk. One example is the increased susceptibility to mycobacterial and salmonella infections in people who have IL-12R deficiency and reduced interferon (IFN)-y secretion but are otherwise healthy (deJong et al., 1998). Here the human findings were predicted based on published observations using IL-12R knockout mice (Magram et al., 1996; Wu et al., 1997; Flynn et al., 1993; Cooper et al., 1997). Information gathered through various genomics initiatives is already being used to improve gene therapy for inherited diseases and cancer (Geddes et al., 1997; Greenberg and Riddell, 1999; Ram et al., 1997; Roth et al., 1996), to modify organs and tissues for transplantation (Bracy et al., 1998), and to engineer vaccines against malaria and a host of other diseases (Hoffman et al., 1998; O'Donnell et al., 2000). In each case above, animal models were used to demonstrate efficacy. In addition, functional genomics is rapidly providing thousands of new molecular targets for future drug development, and animals remain as important models for efficacy and safety (Wetterau et al., 1998; Drews, 2000). After reflecting on future discoveries in science and the commotion that would follow the discovery of living things in other parts of our galaxy, Sir John Maddox continues: "But there will be more subtle surprises, which, of necessity, cannot be anticipated. They are the means by which the record of the past 500 years of science has been repeatedly enlivened. They are also the means by which the half-century ahead will enthrall the practitioners and change the lives of the rest of us" (1999).

CQ Taking Advantage of Interspecies Similarities: Research on Obesity

An example of a complex physiologic trait in which genetic, behavioral, and environmental components each play a role is that of obesity. Obesity, which is associated with decreased longevity in humans, is a complex disorder affecting 32% of adults and 40% of children in the United States (Campfield et al., 1998); it deals

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with the regulation of energy balance and fat mass, i.e., when intake exceeds expenditure, a state of positive energy balance is achieved, resulting in increased body weight. Some estimate that 40-70% of the variations in obesity-related phenotypes in humans are heritable (Allison et al. 1996; Comuzzie and Allison, 1998). Currently, three genome scanning efforts are underway that have obesity phenotypes as their central focus. Initial results suggest the existence of several genes that have an effect on obesity. Examples include a linkage region in human chromosome 2 that encompasses the gene for the prohormone proopiomelanocortin and a region on chromosome 8 that encompasses the gene for the [33-adrenergic receptor (Comuzzie and Allison, 1998). Efforts to map obesity genes in humans have been fueled by the discovery of genes affecting body weight and composition in animals, as well as by the large body of information available on feeding behavior and energy homeostasis collected from animal models (Woods et al., 1998). In the 1970s, it was discovered that cholecystokinin (CCK) regulated satiety in rats (Gibbs et al., 1973). However, while CCK had a potent effect on food intake, it had a limited effect on fat mass, implying that other factors affecting energy homeostasis were also essential for the long-term control of adiposity. Two central nervous system pathways control energy balance in response to body fat and include the anabolic neuropeptide Y axis (which stimulates food intake and promotes weight gain) and the catabolic hypothalamic melanocortin system (which reduces intake and promotes weight loss). Spontaneously mutant and knockout mice have contributed greatly to the current understanding of these two CNS pathways. Obese (ob/ob) and diabetic (db/db) mice were found to have single-gene defects affecting the expression of leptin (ob) and the function of the leptin receptor (db) (Zhang et al., 1994; Chua et al., 1996). Using these models, it was found that leptin is a hormone secreted by adipocytes that binds receptors in the hypothalamus (and cross the blood-brain barrier via receptors on brain capillary endothelial cells). Leptin was found to share many properties with insulin, the latter influencing secretion of the former. Both insulin and leptin reduce feed intake, body weight, and fat stores. Except in leptin-deficient obese mice, obese mammals have elevated plasma leptin levels and appear to be resistant to leptin-induced anorexia. Studies in the obese Zucker (fa/fa) rat, which has a mutation in the leptin receptor gene, suggest that the CNS effects of insulin require a functional leptin system (Woods et al., 1998). Human homologs for the defects seen in obese and diabetic mice have been characterized (Montague et al., 1997; Clement et al., 1998) and like the murine models, little effect is seen in obese humans following administration of exogenous leptin, except in those who are leptin-deficient (Himms-Hagan, 1999). Many other actions of leptin in mice are not seen in humans. Neuropeptide Y (NPY) is a neurotransmitter present throughout the brain; however, in the hypothalamus, NPY mediates effects on energy homeostasis. Central administration of NPY promotes positive energy balance and increased fat stores. Neu-

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ropeptide Y also reduces sympathetic nervous system outflow to brown adipose tissue and thus lowers energy expenditure while increasing enzymes necessary for lipogenesis of white fat; NPY is overexpressed in ob/ob and db/db mice, and its response is attenuated by leptin in ob/ob mice. Thus, leptin provides negative feedback to the NPY system (Woods et al., 1998). The NPY Y1 receptor knockouts have changes in fastinduced feeding behavior, cardiovascular responses to NPY, and energy expenditure, suggesting this receptor (as opposed to NPY Y5) is principally involved in the control of food intake (Pedrazzini et al., 1998). Melanocortins, peptides cleaved from proopiomelanocortin precursor, play a role in the central anabolic pathways where they participate in energy homeostasis and elicit anorexia in animal models (Sehioth, 1997). Genetic deficiency of the melanocortin 4 receptor in mice results in hyperphagia and obesity (Cone et al., 1996). However, studies in the ob/ob mouse have shown that melanocortin neurons are a target of leptin action. Corticotropin-releasing hormone (CRH) is a catabolic hypothalamic neuropeptide that also contributes to energy homeostasis. Although endogenous CRH is involved with stress and illness, central administration of CRH reduces food intake and body weight. Leptin administration increases and glucocorticoids inhibit CRH gene expression in animals. (Seeley et al., 1996). Furthermore, although overproduction of CRH has been implicated in anorexia, underproduction promotes the actions of glucocorticoids, which cause weight gain and obesity. As with other neuropeptides involved in central control of energy homeostasis, leptin and insulin are intimately involved in CRH metabolism and activity. Observations made in mice with defective expression of insulin receptors in the central nervous system show that these animals have increased body fat content and are predisposed to obesity (B~ning et al., 2000). Finally, administration of leptin or insulin to animals potentiates the reduction in meal size seen following CCK administration. Thus, the size of fat stores can influence feeding behavior, resulting in animals being more sensitive to meal-generated signals following bouts of excessive eating or after gaining excess weight. Although it is unclear how satiety signals integrate with central anabolic and catabolic hypothalamic pathways, it is clear that the influence of adipose tissue signals on food intake is not responsive to individual meals, but rather exerts its effects over several days. These findings from animal research have helped elucidate critical components in the control of energy homeostasis and feeding behavior. The complexity of this system, with its redundancies and numerous points of negative feedback, has contributed to a program that efficiently responds to weight loss and replenishes depleted fuel stores. While the mechanisms for defending feed stores have probably conferred a selected advantage to individuals (and populations of animals) possessing them, finding a simple antiobesity molecular target for drug interaction is not likely to be easy and will certainly involve

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changes in environmental components, e.g., modified diets and physical exercise. The goal of all antiobesity drugs is to induce and maintain a state of negative energy balance (Campfield et al., 1998). Generally they fall into one of four classes of compounds: inhibitors of energy intake (appetite suppressants), inhibitors of fat absorption, enhances of energy expenditure, and stimulators of fat mobilization. Currently, only a small group of compounds remains approved and available for treating obesity, and each acts centrally as an appetite suppressant to reduce food intake by modulating monoamine neurotransmitters (serotonin and norepinephrine, or norepinephrine alone) in the brain. These drugs work by inhibiting the reuptake of serotonin or norepinephrine. Reports of heart valve disease in obese patients taking inhibitors of serotonin reuptake have resulted in three (of five) of these drugs being withdrawn. As a result, there is keen interest in development of drugs targeting other central (or peripheral) pathways leading to alteration in energy balance. Based on animal experimentation, new molecular targets for antiobesity drugs include among others, ob receptor agonists, NPY receptor antagonists, melanocortin 4 receptor agonists, agouti-related peptide agonists, proopiomelanocortin antagonists, melanocyte-concentrating hormone receptor antagonists, CRH receptor antagonists, urocortin antagonists, and CCK-A receptor agonists - - all of which are designed to suppress appetite. Drugs designed to enhance energy expenditure include stimulators of uncoupling protein 2 (UCP2) and 3, proton transporters that are expressed in peripheral tissue and cause increased thermogenesis; stimulators of protein kinase A (PKA), and agonists of the [33-adrenergic receptor. Finally, drugs that stimulate fat mobilization include those that agonize the obreceptor, stimulate PKA, agonize the [~3-adrenergic receptor, or agonize the growth hormone receptor (Campfield et al., 1998). Further elucidation of the integrated network of neurotransmitters, hormones, and receptors controlling energy balance may help define which target above is most likely to produce negative energy balance without compromising other systems. Alternatively, future studies may obviate new molecular targets that can safely be modulated therapeutically to achieve weight loss in overweight individuals.

III.

HISTORY OF ANIMAL USE IN

BIOMEDICAL RESEARCH A.

Early History

Humans have a history of close interaction with animals that extends back over 20,000 years (with the domestication of poultry in China) and includes the domestication of buffalo, cattle, sheep, and dogs between 6000 and 10,000 years ago. The earli-

E

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est written records date to 2000 BC when Babylonians and Assyrians documented surgery and medications for humans and animals. True scientifc inquiry began in the intellectually liberal climate of ancient Greece where the teachings of Aristotle, Plato, and Hippocrates symbolized a move to understand natural phenomena without resorting to mysticism or demonology. In this environment, philosophy was conceived and wisdom was admired. Early animal experimentation was conducted in 304 BC by the anatomist Erasistratos, who demonstrated the relationship between food intake and weight gain in brids. In the second century AD, the physician Galen used a variety of animals to show that arteries contained blood and not air, as believed by his contemporaries. During this period, physicians carried out careful anatomic dissections, and on the basis on the comparative anatomy of animals and humans, accumulated a remarkable list of achievements, including a description of embryonic development; the establishment of the importance of the umbilical cord for fetal survival; and the recognition of the relationship between the optic nerves, which arise from the eyes, and the brain. The Greeks, and later the Romans, developed schools of higher learning (including medical schools), created museums, and documented their findings in libraries. Physicians from this period recognized that fever aided the healing process, recognized the inherited nature of certain disorders and classified them, and practiced intubation to prevent suffocation and ligation and excision for the treatment of hemorrhoids. This brief period of scientific inquiry in Europe gave way to the Middle Ages, a 1200-year period characterized by war, religious persecution, and unsavory politics. During the Middle Ages until the Rennaisance, the writings of ancient Greece and Rome remained the final word on science and medicine. Medical education was revived in tenth-century Salerno, Italy, but because of a prohibition on human dissection that lasted into the thirteenth century, animals were substituted for humans as models in the instruction of anatomy. Because no investigations took place, virtually no new discoveries in medicine were made. Imagine how handicapped these medieval physicians must have been. They still did not know that the filling of lungs with air was necessary for life, that the body was composed of many cells organized into tissues, that blood circulated and the heart served as its pump, and that blood traverses from arteries to veins in tissues via capillaries; these facts were revealed by Hook in 1667, Swammerdam in 1667, Van Leeuwenhoek in 1680, Harvey in 1628, Malpighi in 1687, and Pecquet in 1651, respectively, each using animals to demonstrate these basic principles. In part, this return to the process of scientific discovery was built on the foundations established by Francis Bacon, a foundation based on collecting facts, developing hypotheses, and attempting to disprove them via experimentation (see Section II,A,1). The pace of biomedical research increased during the 1700s as Priestley discovered that the life-promoting constituent of air

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was oxygen. Scientists such as Von Hailer, Spallanzani, Trembly, and Stevens, each using animals, discovered the relationship between nerve impulses and muscle contraction, recorded cell division, and associated the process of digestion with the secretions of the stomach. Hales made the first recording of blood pressure in a horse in 1733, Crawford measured the metabolic heat of an animal using water calorimetry in 1788, and Beddoes successfully performed pneumotherapy in animals in 1795 (although it was not until 1917 that Haldane would introduce modern oxygen therapy for humans). By 1815, Laennec had perfected the stethoscope, using animals. Despite these dramatic gains in medical knowledge, physicians were still not aware of the germ theory of disease (and of course could not avoid, prevent, or treat infections) (Quimby, 1994a).

B.

F r o m P a s t e u r to P r u s i n e r

In the 1860s, the French scientist Louis Pasteur discovered that microscopic particles, which he called vibrions (i.e., bacteria), were a cause of a fatal disease in silkworms. When he eliminated the vibrions, silkworms grew free of disease--the first demonstration of the germ theory of disease. In 1877, Pasteur turned his attention to two animal diseases, anthrax in sheep and cholera in chickens. In each disease he isolated the causative agent, reduced its virulence by exposure to high temperature, and showed that on injection the attenuated organism imparted protection against the disease. Pasteur referred to this process as vaccination (from Latin vacca, "cow") in homage to the English surgeon Edward Jenner, who discovered that injection of matter from cowpox lesions into humans protected them against smallpox. Pasteur went on to develop the first vaccine against rabies, in which the virus was attenuated by passage through rabbits. This vaccine was shown to impart protection in dogs and later in humans. Pasteur's work with microscopic organisms as agents of disease quickly led to two other important discoveries. John Lister, having read of Pasteur's discovery, hypothesized that these microorganisms were responsible for wound infections. He impregnated cloth with an antiseptic of carbolic acid and showed that when used as a wound dressing, the antiseptic prevented infection and gangrene. This led to the generalized use of antiseptics before surgery and sterilization of surgical instruments. In 1876, Robert Koch would demonstrate a technique for growing bacteria outside of an animal (in vitro) in pure culture. This would reduce the number of animals required to conduct research on infectious agents, and it allowed Koch to establish postulates for definitively associating a specific agent with a specific disease (see Section II,B,3). Using these postulates, Koch discovered the cause of tuberculosis, Mycobacterium tuberculosis, and he developed tuberculin used to identify infected animals and people. Between 1840 and 1850, Long and

Morton demonstrated the usefulness of ether as a general anesthetic first in animals and later in humans. 1.

Contributions to Inheritance

The second half of the nineteenth century began a new era in biology and medicine. In addition to such medical developments as vaccination, testing for tuberculosis, anesthesia, and blood transfusion, each of which depended on animal experimentation, two other events changed the direction of biological science forever. In 1859, the English naturalist Charles Darwin published "On the Origin of Species," in which he hypothesized that all life evolves by selection of traits that give one species an advantage over others. Around the same time, the Austrian monk Gregor Mendel used peas to demonstrate that specific traits are inherited in a predictable fashion. Nearly half a century later, the English biologist William Bateson reached the same conclusion by selectively breeding chickens and reported his result, as Mendel's work was being generally recognized. Mendel proposed two laws of heredity: first, that two different hereditary characters, after being combined in one generation, will again segregate in the next; and second, that hereditary characteristics assort in new daughter cells independently (Sourkes, 1966). Unfortunately, Bateson's investigations with chickens did not always give the numerical results of two independent pairs of characters. This led Sutton and Bovery, at the turn of the century, to conclude that the threadlike intracellular structures seen duplicating and separating into daughter cells carried the hereditary characters. Later Thomas Hunt Morgan, using cytogenetics and selected breeding in fruit flies, clearly demonstrated the phenomenon of genetic linkage (Morgan, 1928). Others went on the verify these observations in plants and animals. During the first half of the twentieth century, revelations concerning the discovery of nucleic acids by Kossel, using salmon sperm and human leukocytes (Sourkes, 1966); the structure of nucleotides by R A. Levine; and the structure of DNA by Watson, Crick, Wilkins, and Franklin depended on advances in chemistry and X-ray crystallography (Watson and Crick, 1953). In fact, it was the application of X-ray diffraction techniques that finally allowed scientists to deduce the double helical structure of DNA. When Watson and Crick saw Franklin's photographs, it galvanized them into action; by building models of the nucleotides and hypothesizing the points for hydrogen bonding between purines and pyrimidines, they quickly assembled the three-dimensional structure of DNA. Their insight into how the diffraction pattern correlated with helical symmetry allowed for a practical solution to a very complex and, until then, elusive problem. They reinforced the meaning of the term "great science," as expressed by Lisa Jardine, "Great science depends on remaining grounded in the real" (1999). There were 50 years between the isolation of "nuclein" in leukocytes by Kossel and the discovery of the double helical

1208 structure of DNA, which recognized that the pattern of purine and pyrimidine coupling contained the code for heritability. Likewise, there were 50 years between the hypothesis by Garrod in 1902 that family members with alkaptonuria had inherited a deficiency in a particular enzyme that metabolizes homogentisic acid and Beadle and Tatum's proof, using Neurospora, that indeed X-ray-induced genetic mutations affected the production of specific enzymes (Lederberg and Tatum, 1954). To a certain extent, these latter studies depended on the demonstration that bacteria (and other lower organisms) in fact contained genetic information that controlled protein synthesis in a manner similar to that in eukaryotes (Lwoff, 1953). This breakthrough provided the fuel for the revolution in molecular genetics, which included the biological synthesis of deoxyribonucleic acid (Kornberg, 1959) and the genetic regulation of protein synthesis (Jacob and Monod, 1961)meach depending on work in bacteria. Later, the details on the control of gene expression were heavily dependent on Escherichia coli and its well-studied lac operon, yeast, and the fruit fly, Drosophila melanogaster. No wonder these three organisms were among the first to have their entire genome sequenced. With the mechanisms in hand for synthesizing nucleotide sequences and creating constructs with precise regulatory sequences for insertion into the mammalian genome, another revolution is about to occur; however, this time the mouse will guide investigators interested in discovering the function of proteins encoded by new genes and unraveling the mechanisms behind inborn errors in humans (see Section II,B, 1). For their achievements in genetics and molecular biology, the following scientists have won the Nobel Prize: Thomas Morgan; Albrecht Kossel; George Beadle, Edward Tatum, and Joshua Lederberg; James Watson, Francis Crick, and Maurice Wilkins; Andre Lwoff, Francois Jacob, and Jacques Monod; and Severo Ochoa and Arthur Kornberg. After a century of research demonstrating that all "living" things replicate based on information encoded in DNA or RNA, imagine how heretical Stanley Prusiner's hypothesis appeared to the scientific community when he proposed an infectious disease agent composed entirely of protein (prions) that caused disease in humans and animals as a result of abnormal protein folding. Prusiner used many types of animals in pursuit of the prion, but laboratory hamsters and mice played a particularly important role, as did the disease-causing agent of scrapie in sheep (Liu et al., 1999; Supattapone et al., 1999). As in the scientific debate concerning the humoral versus cellular theory of immunity (see Section III,B,2,b), the scientific community continues to debate the prion hypothesis, and again as in the earlier debate, the Swedish Academy cast its vote by awarding Prusiner a Nobel Prize in 1997. 2.

Progress in the Field of Immunology

a. Origins. The concept of adaptive immunity, developing protection after exposure to an infectious agent or poison, dates back to at least 430 Bc when Thucydides writes of the plague of

FRED W. QUIMBY Athens, "Yet it was with those who had recovered from the disease that the sick and dying found most compassion. These knew what it was from experience and had now no fear themselves; for the same man was never attacked twice D never at least fatally" (1934). Despite this early recognition, the association of disease with infectious agents was missing. During the 1200s, the Black Death in Europe and the East was attributed to a conjunction of Mars, Saturn, and Jupiter; and later in the fifteenth century the appearance of syphilis in Europe was attributed to another conjunction of the same planets (Silverstein, 1989). Even during the Renaissance, when cities were adopting the principles of quarantine in the face of an epidemic, influenza was still ascribed to the influence of the stars, and mal-aria meant "bad air." It was not until the end of the nineteenth century that studies using animals allowed investigators such as Pasteur, Koch, Ehrlich, von Behring, and Metchnikoff to demonstrate the phenomenon of acquired immunity, the association between infectious agent and disease, the principles of vaccination, and the treatment of diphtheria (and tetanus) with antitoxins. It was not until the twentieth century that many other scientists unraveled the molecular basis for acquired immunity and extended its practical application to the fields of transplantation and vaccinology, and developed treatments for autoimmune, immunodeficiency, and hypersensitivity diseases, as well as treatments to prevent graft rejection and therapies for shock and cancer. b. Pioneers of humoral immunity. Emil von Behring (1854-1917) was a student of Robert Koch and went on to demonstrate that animals (guinea pigs, rabbits) vaccinated with diphtheria or tetanus organisms developed immunity to infection and to the detrimental effects of their toxins. In two manuscripts published in December 1890, he describes how he produced antitoxins to diphtheria and tetanus and how cell-free serum from immune animals protected nonimmune animals after passive transfer. Within 1 year the first human was successfully treated for diphtheria, and soon after, serum treatment came into general use. The death rate from diphtheria fell from 35 to 5%, and among those with laryngeal involvement, from 90 to 15%. Von Behring was awarded the Nobel Prize in 1901 (Sourkes, 1966). Another student of Koch was Paul Ehrlich, who by studying antisera made in animals (particularly guinea pigs) against plant and bacterial toxins developed a standardized test in 1897 to quantitate toxin and antitoxin. In doing so Ehrlich postulated the unique stereochemical relationship between active sites on antibody and antigen and introduced the concepts of antibody affinity and of functional domains on antibody molecules. Finally, he postulated that antibody formation was the cellular response to the binding of antigen to its surface receptors. He was awarded the Nobel Prize in 1908 along with Elie Metchnikoff (who discovered the antibacterial properties of phagocytes in a variety of animals) (Ehrlich, 1900). The mentor of both von Behring and Ehrlich, Robert Koch

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made many cohtributions, particularly in the new field of bacteriology; however, he also developed tuberculin and devised the standard tuberculin skin test, which was one of the first demonstrations of cellular immunity (Silverstein, 1989). He was awarded a Nobel Prize in 1905. In the mix with a group of scientists primarily devoted to the humoral theory of immunity, Elie Metchnikoff proposed a cellular theory of immunity. This was based on his observations of starfish phagocytic cells and their activity in the presence of bacteria. Furthermore, Metchnikoff theorized that immune activation produced a substance that heightened the activity of phagocytes. The work of Pasteur, Koch, Ehrlich, and von Behring all implicated humoral factors as protective in immunity, and the debate between humoral and cellular theories began. It did not help matters that during this debate, which lasted nearly 2 decades, the discoveries of bacterial agglutination (by Max von Gruber and Hurbert Durham), anaphylaxis (by Paul Portier and Charles Richet), the arthus phenomenon (by Maurice Arthus), and serum sickness (by Clemens von Pirquet and Bela Schick) all supported the humoral theory (and all involved animal research). Subsequently, the Nobel Prize would also be given to Charles Richet for his discovery of anaphylaxis, using dogs, and Jules Bordet for his discovery of complement, using guinea pigs (Bordet, 1909). Many years passed between Richet's discovery of experimental anaphylaxis and Bovet's discovery of histamine as a major mediator of that phenomenon. Bovet developed an ex v i v o assay based on exposing strips of sensitized animal uterine tissue to antigen. Later he developed antihistamines for the treatment of allergies such as asthma and atopy, based on the concept that synthetic molecules that resemble a metabolite of an active agent, e.g., histamine, may block or antagonize the effects of the active compound. Later, while studying the mode of action for curare in rabbits and dogs, he developed curare-like relaxants, tranquilizing drugs, and anesthetics. Daniel Bovet was awarded the Nobel Prize in 1957 (Sourkes, 1966). In retrospect, the arguments against a cellular theory seem antiquated, with the discovery of complement receptors on phagocytic cells, the role of complement as an opsonin for phagocytosis by Wright and Douglas, and even Koch's demonstration of the cellular infiltration at the site of a tuberculin reaction. Unfortunately, Metchnikoff was a lone voice in the cellularist doctrine, and in the end most early investigators of the twentieth century turned to investigations involving antibody. For nearly 50 years the important area of cellular immunity and the role of lymphocytes in immunity were placed on the back burner. This is an excellent example of Kuhn's contention that scientific communities tend to reinforce the familiar and reject hypotheses that are more controversial and require taking risks (see Section II,B,1; Kuhn, 1970). During the next 30 years investigators would study how immunologic (antibody) specificity was expressed and its biological implications (precipitation, agglutination, hemolysis, allergy), how immunologic specificity is structurally determined,

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and how the information for immunologic specificity is encoded. Major contributions were made by many, but perhaps none had a more profound effect on immunology (and especially medicine) than Karl Landsteiner. Born and educated in Vienna, he trained in the laboratories of Emil Fischer (in Wurzburg) and E. Bamberger (in Munich) before becoming an assistant to Max von Gruber (in Vienna). He traveled to Holland following World War I to be a pathologist at the Hague (19191922), and in 1922, he became a member of the Rockefeller Institute where he worked until his death in 1943. Landsteiner studied the phenomenon of red cell agglutination in humans and nonhuman primates. He discovered the ABO blood group and the isoagglutinins associated with them. He later discovered the MN and Rh factor blood groups. The consequences of this work allowed for proper typing of blood for transfusions (which has saved millions of lives from hemorrhage due to trauma, surgery, ulcer, ectopic pregnancy, bleeding after childbirth, and bleeding due to hemorrhagic disease, as well as by treatment for anemia and carbon monoxide poisoning). Although the MNP group is important primarily in forensic medicine, knowledge of the Rh factor and the antibody therapy developed from it has dramatically reduced the incompatability disease known as erythroblastosis fetalis (hemolytic disease of the newborn) (Landsteiner, 1945). Other major contributions made by Landsteiner include the demonstration of the first antitissue antibodies (antisperm), description of the first autoimmune disease--paroxysmal cold hemoglobinuria (with Donath in 1904)--the phenomenon of hapten inhibition, and the ability to passively transfer delayed hypersensitivity (with Merrill Chase in 1942). Landsteiner was one of the first to show that poliomyelitis and syphilis could be induced in nonhuman primates. Landsteiner was awarded the Nobel Prize in 1930. Other advances in immunology that centered primarily on antibodies and their function include the work of Tiselius and Kabat, classifying antibodies as high molecular weight gamma (y-) globulins; and the work of Porter and Edelman, using myeloma proteins of humans and mice, as well as guinea pig immunoglobulin, to demonstrate the basic structure of antibodies through selective chain cleavage with enzymes. This allowed for primary amino acid sequencing of antibodies, with the resulting acknowledgment of constant and variable regions, repeating domains, and sites for secondary biological activities, e.g., complement fixation (Silverstein, 1989). Once the structure of antibodies was known, many investigators began a search for the molecular (genetic) basis for proteins with constant and hypervariable regions. Calculations of the amount of DNA required to generate antibodies with all the diversity seen in animals and humans at times exceeded the size of the genome; therefore in 1965, Dreyer and Bennett proposed the existence of multiple variable region genes that could combine with the constant region gene to produce a unique isotype. Tonegawa and colleagues first discovered the presence of the constant region locus, plus multiple variable and joining region genes, which assemble such that a single variable region gene

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and a single joining region gene combine with the constant region gene to produce a unique light chain. With the assistance of Leroy Hood, Tonegawa later found a fourth gene cluster, called diversity, which contributed a single gene to form the longer heavy chain of immunoglobulin. This important work not only demonstrated how the enormous diversity of antibodies could be encoded in a compact segment of DNA, but also opened the doors to those searching for the elusive T-cell receptor. For their contributions, Porter and Edelman were awarded the Nobel Prize in 1972, and Tonegawa received the award in 1987. Two other groups made contributions involving antibodies that would revolutionize many areas of the biological sciences. For their contribution of the radioimmunoassay, a technique requiring specific antibodies made in animals (usually rabbits and goats), Rosalyn Yalow, Roger Guillemin, and Andrew Schally won the Nobel Prize in 1977. In 1984, the Nobel Prize was awarded to Cesar Milstein and Georges Kohler for their contribution of monoclonal antibodies and the hybridoma technique. These two assays, and the enzyme-linked immunosorbent assay (ELISA) that followed, revolutionized the detection of specific antigens in tissues and biological fluids and serve today as the basis for disease diagnosis. Monoclonal antibodies have a multitude of purposes, from disease diagnosis to the purification of proteins and therapy for cancer (Dickman, 1998). Of course, many other investigations concerning the role of antibodies in health and disease occurred during this period, including the discovery of immunoglobulin deficiency disease in 1952 by Ogden Bruton and the bursa of Fabricias as the site of antibody-producing "B" cells in the chick (the mammalian counterpart was fortunately found to be the bone marrow, thus preserving the B-cell designation). The discovery of mucosal immunoglobulin A and later discoveries that demonstrated the role of secretory piece and transepithelial transport of the molecule (much of this dependent on the use of various animals) were made, as well as the discovery of IgE by Ishizaka and Ishizaka in 1966; calculations on the size of the antibody combining site by Kabat; and the existence of idiotypes by Kunkel, Mannik, Williams, Oudin, Michel, Gell, and Kelus in 1964. However, 50 long years went by before another major investigation of the cellular theory of immunity was to occur, c. Pioneers o f cellular immunity. World War II stimulated research to improve the survivability of grafted tissues, particularly skin for wound victims. Peter Medawar was interested in tissue grafting and was the first to document that second grafts from the same donor to recipient were rejected more quickly than the first graft, whereas a third-party graft was not. This finding supported the view of immunologic specificity and the secondary response. He and colleagues Brent and Billingham established the field of transplantation biology. When Ray Owen documented that dizygotic cattle twins, which were red cell chimeras, were unable to reject each other's organs, the Australian physician MacFarlane Burnet proposed that im-

FRED W. QUIMBY

munologic responses arise late in animal development and that during early development, cells of the immune system would catalog available antigens as self (and thus not respond to them). Later in development, the introduction of new antigens would be considered nonself or foreign and elicit an immune response (Burnet, 1959). This hypothesis was studied by Medawar and colleagues subsequently by exposing neonates to antigens of another strain and then transplanting skin between these inbred strains as adults. He confirmed that early antigen exposure prevented the immune system from recognizing the antigen later as foreign; he called this acquired immunological tolerance (Billingham et al., 1953). These observations were among the first to be made in the new field of transplantation. They paved the way for characterizing the immune responses typical of rejection (which are primarily cellular in allograft rejection), the antigens that elicited these responses, and mechanisms for suppressing the response (such as acquired tolerance and immunosuppressive drugs). MacFarlane Burnet and Peter Medawar shared the Nobel Prize for Medicine in 1960. In the 1950s and 1960s, George Snell and Peter Gorer discovered the genetic locus in mice important in allograft (between individuals of the same species) rejection. The work of many who followed refined the information concerning this very complex locus, the major histocompatibility locus (MHC). However, this feat occurred because George Snell had the inventiveness to inbreed mice, thus generating strains (identical twins) and congenic lines that differed between one another by only a single gene. From this point onward, inbred strains and lines of mice would be the animal model of choice for those studying immunogenetics. They allowed for many of the studies in oncology, transplantation, and molecular biology. Among those who used inbred strains to study the MHC was Baruj Benacerraf of Harvard University. He studied the loci in mice that determined the immune response to synthetic polypeptides and named them immune response (IR) genes. It was later shown that the IR region fell into the MHC class 1 regionm a locus that controlled the intercommunication between various immunocytes. Jean Dausset, a French immunologist, discovered that the human leukocyte antigen (HLA) system in humans controlled graft rejection. These HLA antigens were later found to be encoded in the MHC. Benacerraf, Snell, and Dausset shared the Nobel Prize in 1980. Later, inbred strains of rats, rabbits, and guinea pigs would be available to aid future studies, and histocompatibility typing was extended to many species of animals. Also during the 1960s, Jacques Miller described the role of the thymus in immunity, the origin of the T cell, which would later be found to mediate graft rejection, modulate antibody synthesis, and directly participate in the eradication of infectious agents and cancer (Miller, 1961). By 1966, Claman, Chaperon, and Triplett, as well as Mitchell and Miller, had described T-cell subsets that discriminated between the types of T cells that assisted B cells in antibody production (helper cells) and those with direct cytotoxic activity. In 1970, Gershon and Kondo described suppressor T cells, which modified the activ-

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

ity of other lymphocytes. Now the availability of monoclonal antibodies was allowing scientists to detect surface markers on lymphocytes that could be used to distinguish functional subpopulations of lymphocytes, i.e., CD4 for helper cells and CD8 for cytotoxic lymphocytes. Availability of inbred strains also allowed investigators to evaluate immune mechanisms by passive transfer of subpopulations of cells between individuals. Early in these investigations, Peter Doherty and Rolf Zinkernagel discovered that certain immune responses were MHC-restricted, that is, the lymphocytes of one MHC type could not recognize foreign antigens presented by cells of another MHC type. This led to the discovery that antigen-presenting cells express foreign peptides together with MHC proteins on their surface. Cells presenting MHC II molecules stimulated CD4 + cells, and those presenting antigen together with MHC I molecules stimulated CD8 + cells. These discoveries explained how different cells of the immune system were selected to attack a particular foreign invader and would aid in the understanding of intrathymic education of newly developing T cells to become tolerant to self antigens (Zinkernagel and Doherty, 1997). For their achievements, Doherty and Zinkernagel were awarded the Nobel Prize in 1996. First described by Flanagen in 1966, the nude mouse occurred as a spontaneous mutation in which there is developmental failure of the thymic anlage, resulting in a mouse completely devoid of functional T cells. The scid mouse was described in 1983 by Bosma et al., and due to a mutation in r a g l gene encoding recombinase activity, this mouse failed to rearrange immunoglobulin or T-cell receptor genes and thus developed a combined (T- and B-cell) immunodeficiency. Use of these mice during the past 2 decades has greatly assisted in unraveling the cellular basis of immunity (Fogh and Giovanella, 1978, 1982; NRC, 1989; Carballido et al., 2000). As molecular techniques and the knockout technology became available, scientists probed deeply into the genetic mechanisms controlling immune function (Preckel et al., 1999; Pappu et al., 1999; Schaeffer et al., 1999; Philpott et al., 1992; DeTogni et al., 1994; Pages et al., 1999; Tamada et al., 2000). Once again, the mouse was the favored species for this work. The fine details on how animals (and humans) develop and maintain self-tolerance are unraveling and with them, new ideas for future therapy for autoimmune disease (see below). Likewise, the importance of a new class of immune modulators, the interleukins, has been recognized primarily through animal research. The ability to modulate levels of various interleukins has major implications for the treatment of inflammatory illnesses and shock; in the augmentation of immune responses against infectious agents, including parasites; and in the battle against cancer (Weinblatt et al., 1999; Hoffman et al., 1997; Tokman and Quimby, 1995). d. Transplantation biology. The reader must now be brought back to the 1960s and the pioneer work of George Snell, to pick up where Peter Medawar, of graft-rejection fame,

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left off. Several factors came together during this period to make the allografting of organs successful. First, the preservation and tissue typing of the donor organs were critical. Next, surgical manipulation of organs to allow prompt reestablishment of the blood supply was necessary. Finally, a mechanism of suppressing the cellular immune response was essential. Woodruff, a transplant surgeon in Edinburgh enthralled by the experiment of Medawar and colleagues, found a pair of dizygotic twins who shared each other's red cell types. He hypothesized that they had shared placental circulation and found, after cross-skin grafting them successfully, that humans, like cattle and mice, could develop acquired immunologic tolerance (Woodruff, 1959). During the first half of the twentieth century, Carres, Quinby, Dempster, and Simonsen each attempted renal autografts and allografts in nonimmunosuppressed dogs. Although autografts remained functional for longer periods than allografts, all transplants ceased to function due to lack of innervation, lymphatics, or both. It was the many investigations conducted by Joseph Murray during the early 1950s that resulted in a surgical technique that would leave autografted kidneys in dogs completely functional after 2 years (Murray et al., 1956). However, allografts were still quickly rejected. Lawrence (1959) was the first to liken the allograft rejection response to delayed hypersensitivity reactions. Attempts to suppress immune rejection with steroids, anticoagulants, or both, failed. Following trials in mice and rabbits (Main and Prehn, 1955), a protocol involving total body irradiation followed by bone marrow transplantation and kidney transplantation achieved variable success in human kidney graft recipients. In 1959, Schwartz and Dameshek reported on the ability of 6-mercaptopurine (6-MP) to prevent rabbits from producing an antihuman serum albumin antibody response. Calne (1960) in London used 6-MP to suppress the rejection of allografted canine kidneys with success, although the drug itself was toxic. Calne then urged G. H. Hitchings and G. B. Elion of Burroughs Wellcome Laboratories to become collaborators. New Wellcome drugs greatly improved allograft survival, with dogs surviving normally for years. Hitchings and Elion had developed the imidazole derivative of 6-MP, known as Imuran (azathioprine). This became the mainstay of transplant surgeons for the next 20 years (Murray, 1992). Following investigations in dogs, orthotopic liver allografts were performed in humans by Moore and Starzl. In 1960, Lower and Shumway developed a surgical technique for transplant of the heart in dogs. This was followed in 1970 by successful heart transplantation in man. Later achievements included transplantation of the bone marrow, pancreas, and portions of the intestinal tract. The advent of reagents to histocompatibility type donors and recipients reduced the immunologic barrier between them and led to more appropriate selection of grafts, with a resultant increase in functional longevity of the graft. Also, improved drugs, such as cyclosporin A, rapamycin, and FK506, targeted

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specific cellular events necessary for initiating the rejection process, causing fewer side effects and leaving the host must less immunologically compromised (Carpenter, 2000). Other drugs, such as mycophenolate mofetil and anti-IL-2R, are now in trials (Beniaminovitz et al., 2000). In addition, monoclonal antiCD3 antibodies have been used to prevent acute rejection since the early 1980s (Ortho Multicenter Transplant Study Group, 1985). However, the dream of transplant surgeons is to induce acquired immunologic tolerance in the recipient to the graft, what Medawar called "the Holy Grail." Starzl demonstrated early that kidney rejection in dogs treated with azathioprine could be reversed 88% of the time by injection with steroids (Marchioro et al., 1964). Similar rates were published for humans undergoing the same therapy. It was also shown that delayed hypersensitivity skin tests (against tuberculin, histoplasmin, coccidiodin) that were positive in the donor but not the recipient crossed over to the recipient about 77% of the time following a kidney transplant (Starzl, 1993). Microchimerism involving the survival of donor leukocytes in the body of the recipient following transplantation was proven using polymerase chain reaction (PCR) and persisted up to 29 years following transplantation (Starzl, 1993). Even more surprising was the observation that some liver graft recipients discontinued their immunosuppressive medication 1-6 years following transplantation and were normal 5-13 years later. These patients had achieved lasting immunologic tolerance, and all were shown to be lymphocyte chimeras. The movement of donor leukocytes out of the transplanted graft into immune compartments of the host had been demonstrated in animal models (Qian et al., 1994). It had also been shown that longstanding peripheral tolerance could be achieved in mice made chimeras by neonatal infusion of donor leukocytes (Silvers et al., 1975). These observations fostered the notion that for completely successful organ engraftment, four interrelated phenomena must occur in close temporal sequence: clonal deletion of the recipient immune (antigraft) response, clonal deletion of the donor's leukocyte response, maintenance of clonal exhaustion, and reduction in the immunogenicity of the transplanted organ over time (Starzl and Zinkernagel, 1998). However, it is not clear how these events are controlled. For some time it has been known that acute allograft rejection is mediated by CD4 + and CD8 + cells. The former are activated by binding foreign antigen in association with MHC class II molecules n i n this case, on the donated graft. For proper activation, costimulatory signals mediated via CD28 on T cells and B7 on antigen-presenting cells plus CD40 present on antigenpresenting cells binding CD40L on T cells must be engaged. Once activated, CD4 lymphocytes produce many cytokines that act in an autocrine fashion to stimulate more CD4 cells and a paracrine fashion to activate CD8 cells. Many of these cytokines, such as TNF, may be liberated by activated CD4 cells within the graft, causing rejection even without the cytotoxic effects of CD8 cells. These immune mechanisms were largely dis-

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covered through research on mice and rats (Sayegh and Turka, 1998). Investigators have demonstrated that blocking the costimulatory pathways for T cells in allografted animals (rodents and nonhuman primates) greatly prolongs graft survival and ablates the acute rejection phenomenon (Kirk et al., 1999). Although prevention of T-cell activation and cytokine release can explain acute graft survival, it does not really explain the long-term survival of grafts observed by Starzl in microchimeric animal and human transplant recipients. One explanation comes from the studies of Li and Wells, where peripheral allograft tolerance was established in mice by a combination of costimulatory blockade and the use of rapamycin. In this study, mice received an MHC-incompatible cardiac transplant plus monoclonal antibodies against CD40L (CD154) plus CTLAIg (which blocks CD28). In this case, heart grafts survived rejection, but skin grafts were rejected. When rapamycin is added to the regimen, permanent engraftment is seen for both heart and skin; however, if cyclosporin A (CsA) is substituted for rapamycin, neither graft survives rejection. The inclusion of CsA thus antagonized the tolerizing effects of both costimulatory blockade and rapamycin. Using cell labeling studies, these investigators found that costimulatory blockade alone inhibited proliferation of alloreactive T cells in vivo while allowing cell cycledependent T-cell apoptosis of proliferating T cells. Addition of rapamycin resulted in massive apoptosis of alloreactive T cells, but addition of CsA abolished T-cell proliferation and apoptosis. Subsequent studies demonstrated that the combination of blockade plus rapamycin did not induce tolerance in IL-2 or in bcl-XL transgenics. In both instances, these transgenic mice failed to be induced into T-cell apoptosis. Activation-induced cell death (by apoptosis) occurs when primed cells are repetitively activated by antigen and requires previous exposure to IL-2. Unlike CsA, rapamycin does not block IL-2 production, and it does not block antigen priming for apoptosis. The authors conclude that stable peripheral tolerance can be induced as long as alloreactive T cells are suppressed from initial cytokine production and eliminated by apoptosis (Li et al., 1999; Wells et al., 1999). How these observations fit into the microchimera model of Starzl is unknown, but one hypothesis is that the apoptotic alloreactive T cells trigger an immunoregulatory effect that serves to maintain the state of tolerance (Ferguson and Green, 1999). This is supported by the finding that apoptosis and immune tolerance are linked through the activation of immunoregulatory mechanisms mediated by inhibitory cytokines (ILl0, TGF-[3). In this model, tolerance is now stable because of a double hit--deletion and regulation. The initial blockade of acute rejection with immunosuppressive drugs coupled with the tremendous continuing antigenic stimulation caused by alloantigen-bearing donor cells in the lymphoid tissues of the host may lead to apoptosis of alloreactive cells and production of antigen-specific suppressor cells on a continuing basis. For their achievements in transplantation biology, Joseph

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

Murray and E. Donnall Thomas were awarded the Nobel Price in 1990. For their accomplishments in the field of pharmacology (including the development of drugs to treat high blood pressure and gastric ulcer, and immunosuppressant and antiviral drugs) Sir James Black, Gertrude Elion, and George Hitchings were awarded the Nobel Prize in 1988. e. Vaccinology. Intimately associated with advances in immunology were advances in vaccinology. Following in the footsteps of Jenner, Pasteur, and Bouquet, Calmette, and Guerin (BCG vaccine), Max Theiler was able to develop a mouse protection assay for yellow fever virus and attenuated viral strains in mice and chickens. Efficacy for these attenuated strains was demonstrated in monkeys and ultimately led to the first vaccine in the 1930s. Theiler was awarded a Nobel Prize in 1951 for his efforts (Strode, 1951). During the second half of the twentieth century, vaccines were developed and utilized to protect against diphtheria and pertussis (in the 1940s), poliomyelitis (in the 1950s), rubella or German measles (in the 1960s), pneumococcal, meningococcal diseases, measles, and mumps (in the 1970s), hepatitis A and B and Haemophilus influenzae (in the 1980s), and varicella or chicken pox and Lyme disease (in the 1990s). This history of vaccine development depended heavily on the use of animals, especially nonhuman primates (Hilleman, 1998). Despite this record and the hundreds of millions of human lives saved through vaccination, a safe, effective vaccine against HIV-1 and HIV-2 has been elusive. Vaccinologists are borrowing from all the concepts in modern immunology to devise recombinant vaccines that, via point mutation, immunize without leading to fully assembled virions, creating new vectors that will generate MHC class I-restricted cytotoxic T-lymphocyte responses, in addition to antibody responses and constructs that encode structural sequences for antibodyenhancing cytokines (Liu, 1998). Other approaches being used in vaccine development include polysaccharide protein conjugates for H. influenzae, Streptococcus, and Neisseria meningitidis; new adjuvants and delivery systems that would enhance mucosal immunity; edible-plant vaccines to inexpensively deliver vaccination without refrigeration to tropical countries; and therapeutic vaccines that make use of dendritic cells as vaccines for HIV (Liu, 1998). Once again, vertebrate animals are essential in evaluation of efficacy for each innovation (Seder and Gurunathan, 1999; Langermann, 1998; Guranathan et al., 1998; Hislop et al., 1998; Fehr et al., 1998; Marinaro et al., 1999). Two other experimental applications of vaccines have also arisen, based on new knowledge of the molecular basis of autoimmune disease and cancer. Based on prior work in mice, recombinant fusion DNA vaccines are now being evaluated in humans afflicted with lymphoma and myeloma (King et al., 1998; Falo and Storkus, 1998; Pardoll, 1998). In some instances, dendritic cells spiked with tumor-specific antigens are used as a vaccine against melanoma (Timmerman and Levy, 1998; Nestle et al., 1998). In other instances, monoclonal antibodies

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themselves are used to attack the tumor or enhance autologous T cells to attack the tumor (Houghton and Scheinberg, 2000; French et al., 1999; Dickman, 1998). New discoveries in the molecular pathogenesis of type 1 diabetes mellitus and multiple sclerosis, based on extensive analysis in animal models and humans, implicate clones of selfreactive T cells as mediators of these diseases (Bach et al., 1998; Sela, 1999; Wong et al., 1999b); as a result, the feasibility of eliciting T-cell responses against the self-reactive T-cell receptors is being evaluated (Weisman et al., 1996). Another approach to prevent autoimmune diseases in humans is based on two observations supported by animal studies; (1) genotyping family members at risk may identify candidates for disease before symptoms, and (2) certain autoantigens, when delivered by the oral route, induce tolerance (Lund et al., 1990; Miller et al., 1992; Bowman et al., 1994). Clinical trials using these approaches have people at risk of developing multiple sclerosis orally vaccinated with immunodominant peptides of myelin basic protein and those at risk of developing type 1 (juvenile) diabetes orally vaccinated against insulin or glutamic acid decarboxylase (Noseworthy, 1999). f Asthma. Much has been learned about allergic reactions since the discoveries by Richet and Bovet. The molecular pathogenesis of early and late responses, characterized by biphasic reactions mediated by IgE; release of various mediators, and the influx of different populations of cells, has been studied primarily in rats and mice (including knockouts) (Matsuoka et al., 2000). During the past 2 decades, new therapies, including deep bronchial inhalers to modulate the bronchoconstrictive response of inflammatory mediators, have aided in the reduction of signs and symptoms for those suffering from asthma. Clinical trials are underway in chronically asthmatic people, using a humanized mouse monoclonal anti-IgE antibody, and the first successes have been reported (Milgrom et al., 1999). Work that led to the development of this therapy involved studies in rats and cynomolgus monkeys (Fox et al., 1996). Of course, it would be impossible to recite, in any meaningful way, all the incredible accomplishments in biology and medicine that occurred during the twentieth century, using animal models. A former surgeon general of the United States has stated that every major achievement in medicine during this century has depended in some fashion on animal research. Over two thirds of all Nobel Prizes in Physiology and Medicine have been awarded to scientists who used vertebrate animals to accomplish their goals (Leader and Stark, 1987). In a survey of living Nobel laureates, 97% responded that animal experiments have been vital to the discovery and development of many advances in physiology and medicine, and 92% felt strongly that animal experiments are still crucial to the investigation and development of many medical treatments (Seriously Ill for Medical Research, 1998). The twentieth century saw an explosion of activity in every

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area of the biological sciences. Virtually all modern medical treatments and devices, including most that are now taken for granted, were developed through animal research. Advances in the science of cell biology, ecology, developmental biology, respiratory physiology, cardiovascular physiology, endocrinology, biochemistry, bacteriology, virology, parasitology, psychology, ethology, neurobiology, and nutrition and metabolism have enriched and supported the medical disciplines of cardiology, dermatology, surgery, orthopedics, pediatrics, anesthesiology, pharmacology, microbiology, psychiatry, neurology, dentistry, hematology, medical genetics, and women's health. Reviews documenting the progress in biomedical sciences are available (Singer and Underwood, 1962; Sourkes, 1966; Keen, 1914; Moore, 1995; Camac, 1959; Schmidt, 1959; Reiser, 1981; Bliss, 1982; McGehee, 1981; Comroe, 1983; Fox and Fox, 1988; Weisse, 1991; Robinson, 1976).

C.

Animals as Recipients of Animal Research

Because humans and nonhuman animals share common physiologic responses, many of the advances sought for humans through the use of animal models also benefit animals themselves. Humans and other vertebrates are susceptible to a large number of infectious diseases, and many disease agents infect both animals and humans. These are called zoonotic agents and are frequently passed between humans and other animals. Certain of these zoonotic diseases are prevented by vaccination in humans and animals, including rabies, anthrax, tetanus, and Lyme disease. Monkeys continue to be necessary in the evaluation of polio vaccine, although they may soon be replaced with transgenic mice bearing the human poliovirus receptor; in addition, human polio vaccine has been used to protect wild chimpanzees in East Africa (Quimby, 1994). Since the adoption of the Nuremberg Code in the 1950s, humans have been protected by first testing the safety of new drugs and medical devices in nonhuman animals (Spicker et al., 1988). As a result, an enormous amount of data is known on the safety and pharmacokinetics of these drugs in various animal species. It is therefore not surprising that drugs designed for human use, such as antibiotics, tranquilizers, steroids, sulfonamides, anesthetics, analgesics, chemotherapeutics, anticoagulants, antiparasitics, antiepileptics, and antihistamines, are all commonly used in veterinary practice (Quimby, 1998). Many surgical techniques intended for humans were first perfected in animals and subsequently used to treat animal disorders. Included among these techniques are repair of spinal cord, hip replacement, fracture repair, repair of congenital heart defects, treatment for burns, and organ transplantation. Nonhuman animals, particulary dogs, develop a variety of autoimmune and hematologic conditions that are identical to the human counterparts, and as a result, these animals benefit from blood transfusions, immunosuppressant therapy, purified blood

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components, and hormones, much like humans with the same disorders (NRC, 1994). These same animals benefit from both diagnostic procedures and, in some instances, surgical intervention originally designed for humans. In fact, dogs with disorders such as severe combined immunodeficiency, hemophilia A, and hemophilia B are currently the subjects of experimental gene therapy to evaluate its safety and efficacy before widespread deployment in humans. Pet animals are the beneficiaries of advances in biomedical imaging, and veterinary practitioners utilize X-ray machines, computed tomography, ultrasonography, and fiber-optic endoscopy for animal disease diagnosis. Knowledge of artificial insemination, semen evaluation and storage, egg incubation, and behavioral adaptation were employed in the successful captive breeding and reintroduction of the eastern peregrine falcon, which has now been removed from the endangered species list. Similarly, biomedical techniques such as in vitro fertilization are being used in zoological gardens to help restore populations of animals threatened with extinction. Occasionally, a compound designed for use in animals is discovered to aid in the treatment of human disease. Levamisole, developed as a cattle wormer, is now used together with the chemotherapeutic agent 5-fluorouracil for the treatment of colon cancer. Ivermectin, developed as a preventive for heartworm disease in dogs, is now being used to treat millions of people in the tropics infected with onchocerciasis (river blindness). This sharing of diagnostic methods, preventives, drugs, surgical techniques, and medical devices between humans and other animals adds credence to the philosophy of Sir William Osier, a physician and Dean of Johns Hopkins University School of Medicine and an outspoken advocate and practitioner of comparative medicine, that there is only "one medicine" (see Chapter 31 for further details) and calls into question the rationale of those who seek to eliminate the use of animals in biomedical research. This seems especially true if one considers that each year only half as many animals, of all species, undergo an experimental medical procedure as the number of pets in the United States that endure surgery for cosmetic reasons (Conn and Parker, 1998).

REFERENCES

Abee, C. R. (2000). Squirrel monkey (Saimiri spp.): Research and resources. ILAR J. 41, 2-9. Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., and the Drosophila Genome Sequence Team. (2000). The genome sequence of Drosophila melanogaster. Science 287, 2185-2195. Agca, Y. (2000). Cryopreservationof oocyte and ovarian tissue. ILAR J. 41, 207-220.

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

Allison, D. B., Kaprio, J., Korkeila, M., Koskenvuo, M., Neale, M. C., and Hayakawa, K. (1996). The heritability of body mass index among an international sample of monozygotic twins reared apart. Int. J. Obes. 20, 501506. Aim, R. A., Bina, J., Andrews, B. M., Doig, P., Hancock, R. E., and Trust, T. J. (2000). Comparative genomics of Helicobacter pylori: Analysis of the outer membrane protein families. Infect. Immun. 68, 4155-4168. Altman, P. L., and Katz, D. D., eds. (1979a). "Inbred and Genetically Defined Strains of Laboratory Animals. Part 1. Rats and Mice." Federation of American Societies for Experimental Biology, Bethesda, Maryland. Altman, P. L., and Katz, D. D., eds. (1979b). "Inbred and Genetically Defined Strains of Laboratory Animals. Part 2. Hamster, Guinea Pig, Rabbit, and Chicken." Federation of American Societies for Experimental Biology, Bethesda, Maryland. Andrews, E. J., Ward, B. C., and Altman, N. H., eds. (1979). "Spontaneous Animal Models of Human Disease," Vols. 1 and 2. Academic Press, New York. Antel, J. P., and Owens, T. (1999). Immune regulation and CNS autoimmune disease. J. Neuroimmunol. 100, 181-189. Atkinson, M. A., and Leiter, E. H. (1999). The NOD mouse model of type 1 diabetes: As good as it gets? Nat. Med. 5, 601-604. Attie, A. D., and Prescott, M. F. (1988). The spontaneous hypercholesteroemic pig as an animal model for human atherosclerosis. ILAR News 30, 5-12. Axelrod, J. D., Matsuno, K., Artavanis-Tsakonas, S., and Perrimon, N. (1996). Interaction between wingless and notch signalling pathways mediated by dishevelled. Science 271, 1826-1832. Baba, T. W., Liska, V., Hofmann-Lehmann, R., Vlasak, J., Xu, W., Aychunie, S., Cavacini, L. A., Posner, M. R., Katinger, H., Stiegler, G., Bernacky, B. J., Rizvi, T. A., Schmidt, R., Hill, L. R., Keeling, M. E., Lu, Y., Wright, J. E., Chou, T.-C. (2000). Human neutralizing monoclonal antibodies of the IqE1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nature Med. 6, 200-206. Bach, J. F., Koutouzov, S., and vanEndert, P. M. (1998). Are there unique autoantigens triggering autoimmune diseases? Immunol. Rev. 164, 139-155. Bacon, F. (1620). "Novum Organum: Aphorisms, concerning the Interpretation of Nature and the Kingdom of Man" (Vol. 28, pp. 105-198. Reprinted in Great Books, Encyclopaedia Britannica, Chicago, 1990). Bader, M., Paul, M., and Ganten, D. (1997). Transgenic animal models of hypertension. In "Biological Aspects of Disease: Contributions from Animal Models" (P. M. Innaccone and D. G. Scarpelli, eds.), pp. 165-200. Harwood Academic Publ., Amsterdam. Bagulsi, A., Behboodi, E., Melican, D. T., Pollock, J. S., Destrempes, M. M., Cammuso, C., Williams, J. L., Nims, S. D., Porter, C. A., Midura, P., Palacios, M. J., Ayres, S. L., Denniston, R. S., Hayes, M. L., Ziomek, C. A., Meade, H. M., Godke, R. A., Gavin, W. G., Overstrom, E. W., and Echelard, Y. (1999). Production of goats by somatic cell nuclear transfer. Nat. Biotechnol. 17, 456-461. Baker, H. J., Lindsey, J. R., and Weisbroth, S. H., eds. (1980). "The Laboratory Rat: Research Applications," Vol. 2. Academic Press, New York. Bali, D., Svetlanov, A., Lee, H. W., Fusco-Demane, D., Leiser, M., Li, B., Barzilai, N., Surana, M., Hou, H., and Fleischer, N. (1995). Animal model for maturity-onset diabetes of the young generated by disruption of the mouse glucokinase gene. J. Biol. Chem. 270, 21464-21467. Baiter, M. (1998). New HIV strain could pose health threat. Science 281, 14251426. Band, B. G., Bang, E B. and Foard, M. A. (1972). Lymphocyte depression induced in chickens on diet deficient in vitamin A and other components. Am. J. Pathol. 68, 147-154. Bantle, J., Dumont, N., Finch, R. A., and Linder, G. (1991). "Atlas of Abnormalities: A Guide for the Performance of FETAX." Oklahoma State Publ., Stillwater. Barre-Sinoussi, E, Chermann, J. C., Rey, E, Nugeyre, M. T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Brun-Vezinet, E, Rouzioux, C., Rozenbaum, W., and Montagnier, L. (1983). Isolation of a T-lymphotropic

1215 retrovirus from a patient at risk for acquired immunodeficiency syndrome (AIDS). Science 220, 868-871. Bauer, J. E. (1996). Comparative lipid and lipoprotein metabolism. Vet. Pathol. 25, 49-56. Bay, A., Carbone, L., Frank, W., and Quimby, F. (1995). Swine. In "The Experimental Animal in Biomedical Research" (B. E. Rollin, ed.), Vol. 2, pp. 167-193. CRC Press, Boca Raton, Florida. Beniaminovitz, A., Itescu, S., Leitz, K., Donovan, M., Burke, E. M., Groff, B. D., Edwards, N., and Mancini, D. M. (2000). Prevention of rejection in cardiac transplantation by blockade of the interleukin-2 receptor with monoclonal antibody. N. Engl. J. Med. 342, 613 - 619. Bennett, B. T., Abee, C. R., and Henrickson, R., eds. (1995). "Nonhuman Primates in Biomedical Research: Biology and Management." Academic Press, New York. Berger, L., Speare, R., Daszak, P., Green, D. F., Cunningham, A. A., and Goggin, C. L. (1998). Chytridiomycosis causes amphibian mortality associated with population declines in the rainforests of Australia and Central America. Proc. Natl. Acad. Sci. (USA) 95, 9031-9036. Beynen, A. C., and West, C. E., eds. (1988). "Use of Animal Models for Research in Human Nutrition." Karger, Basel. Billingham, R. E., Brent, L., and Medawar, P. B. (1953). Actively acquired tolerance of foreign cells. Nature 172, 603- 607. Blattner, F. R., Plunkett, G., Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., and Shao, Y. (1997). The complete genome sequence of Escherichia coli K-12. Science 277, 1453-1474. Blaustein, A. R., Kiesecker, J. M., Chivers, D. P., and Anthony, R. G. (1997). Ambient UV-B radiation causes deformities in amphibian embryos. Proc. Natl. Acad. Sci. US.A. 94, 13735-13737. Bliss, M. (1982). "The Discovery of Insulin." Univ. of Chicago Press, Chicago. Bloom, B. R. (1999). The future of public health. Nature 402, 63-64. Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C.-P., Morin, G. B., Harley, C. B., Shay, J. W., Liehtsteiner, S., and Wright, W. E. (1998). Extension of life-span by introduction of telomerase into normal cells. Science 279, 349-352. Bordet, J. (1909). "Studies In Immunity." Wiley, New York. Bosma, G. C., Custer, R. P., and Bosma, M. J. (1983). A severe combined immunodeficiency mutation in the mouse. Nature (Lond.) 301, 527-530. Bowman, D., Leiter, E., and Atkinson, W. (1994). Prevention of diabetes in the NOD mouse: Implications for therapeutic intervention in human disease. Immunol. Today 15, 115-120. Bracy, J. L., Sachs, D. H., and Iacomini, J. (1998). Inhibition of xenoreactive natural antibody production by retroviral gene therapy. Science 281, 18451847. Bradford, M. (1998). Nineteenth century rules of causation outdated? Science 282, 220. Brennan, T. J. (1999). Postoperative models of pain. ILAR J. 40, 129-136. Breslow, J. L. (1993). Transgenic mouse models of lipoprotein metabolism and atherosclerosis. Proc. Natl. Acad. Sci. US.A. 90, 8314- 8318. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Senear, A. W., Warren, R., and Palmiter, R. D. (1981). Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27, 223-231. Brouillet, E., Conde, F., Beal, M. F., and Hantraye, P. (1999). Replicating Huntington's disease phenotype in experimental animals. Prog. NeurobioI. 59, 427-468. Brown, R. E., Stanford, L., and Schellinck, H. M. (2000). Developing standardized behavioral tests for knockout and mutant mice. ILAR J. 41, 163-174. Briining, J. C., Gautam, D., Burks, D. J., Gillette, J., Schubert, M., Orban, P. C., Klein, R., Krone, W., Mtiller-Wieland, D., and Kahn, C. R. (2000). Role of brain insulin receptor in control of body weight and reproduction. Science 289, 2122-2125. Bull, J., and Levin, B. (2000). Mice are not furry petri dishes. Science 287, 1409.

1

2

1

6

F

R

Burkhart, J. G., Helgen, J. C., Fort, D. J., Gallagher, K., Bowers, D., Propst, T. L., Gernes, M., Magner, J., Shelby, M. D., and Lucier, G. (1998). Induction of mortality and malformation in Xenopus laevis embryos by water sources associated with field frog deformities. Environ. Health Perspect. 106, 841-848. Burkhart, V., Wang, Z.-Q., Radons, J., Heller, B., Herceg, Z., Stingl, L., Wagner, E. E, and Kolb, H. (1999). Mice lacking the poly(ADP-ribose) polymerase gene are resistant to pancreatic beta cell destruction and diabetes development induced by streptozocin. Nat. Med. 5, 314 -319. Burnet, F. M. (1959). "The Clonal Selection Theory of Acquired Immunity." Cambridge Univ. Press, London. Buxbaum, J., Choi, E. K., Luo, Y., Lilliehook, C., Crowley, A. C., Merriam, D. E., and Wasco, W. (1998). Calsenilin: A novel calcium-binding protein that interacts with the presenilins and regulates the level of a presenilin fragment. Nat. Med. 4, 1177-1181. Cafaro, A., Caputo, A., Fracasso, C., Maggiorella, M. T., Goletti, D., Baroncelli, S., Pace, M., Sernicola, L., Koanga-Mogtomo, M., Betti, M., Borsetti, A., Belli, R., Akerblom, L., Corrias, E, Butto, S., Heeney, J., Verani, P., Titti, E, and Ensoli, B. (1999). Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine. Nat. Med. 5, 643650. Calne, R. Y. (1960). The rejection of renal homografts: Inhibition in dogs by 6mercaptopurine. Lancet 1, 417-422. Calnek, B. W. (1992). Chicken neoplasia--a model for cancer research. Br. Poult. Sci. 33, 3-16. Camac, C. N. B. (1959). "Classics of Medicine and Surgery." Dover, New York. Cameron, E H., and Jennings, P. A. (1989). Specific gene suppression by engineered ribozymes in monkey cells. Proc. Natl. Acad. Sci. U.S.A. 86, 91399143. Campfield, L. A., Smith, E J., and Burn, P. (1998). Strategies and potential molecular targets for obesity treatment. Science 280, 1383 - 1387. Campisi, J. (2000). Aging, chromatin, and food restriction--connecting the dots. Science 289, 2062-2063. Cantor, J. O. (1989). "Handbook of Animal Models of Pulmonary Disease." CRC Press, Boca Raton, Florida. Carballido, J. M., Namikawa, R., Carballido-Perrig, N., Antonenko, S., Roncarolo, M.-G., and DeVries, J. E. (2000). Generation of primary antigenspecific human T- and B-cell responses in immunocompetent SCID-hu mice. Nat. Med. 6, 103-106. Carpenter, C. B. (2000). Improving the success of organ transplantation. N. Engl. J. Med. 342, 647-648. Carstens, E., and Moberg, G. P. (2000). Recognizing pain and distress in laboratory animals. ILAR J. 41, 62-71. C. elegans Sequencing Consortium (1998). Genome sequence of the nematode C. elegans: A platform for investigating biology. Science 282, 2012-2018. Chalfie, M. (1998). Genome sequencing. The worm revealed. Nature 396, 620621. Chan, A. W. S., Dominko, T., Luetjens, C. M., Neuber, E., Martinovich, C., Hewitson, L., Simerly, C. R., and Schatten, G. P. (2000). Clonal propagation of primate offspring by embryo splitting. Science 287, 317-319. Chang, T. W. (2000). The pharmacological basis of anti-IgE therapy. Nat. Biotechnol. 18, 157-162. Chien, K. R. (1996). Genes and physiology: Molecular physiology in genetically engineered animals. J. Clin. Invest. 98(11), 519-526. Chua, S. C. Jr., Chung, W. K., Wu-Peng, X. S., Zhang, Y., Liu, S. M., Tartaglia, L., and Leibel, R. L. (1996). Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science 271, 994-996. Chui, D., Tanahashi, H., Ozawa, K., Ikeda, S., Checler, E, Ueda, O., Suzuki, H., Araki, W., Inoue, H., Shirotani, K., Takahashi, K., Gallyas, E, and Tabira, T. (1999). Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation. Nature Med. 5, 560-564. Citron, M., Westaway, D., Xia, W., Carlson, G., Diehl, T., Levesque, G.,

E

D

W. QUIMBY

Johnson-Wood, K., Lee, M., Seubert, E, Davis, A., Kholudenko, D., Motter, R., Sherrington, R., Perry, B., Yao, H., Strome, R., Lieberberg, I., Rommens, J., Kim, S., Schenk, D., Fraser, P., Hyslop, P. S., and Selkoe, D. J. (1997). Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid [~-protein in both transfected cells and transgenic mice. Nat. Med. 3, 67-72. Clarkson, T. B., and Klumpp, S. A., (1990). The contribution of nonhuman primates to understanding coronary artery atherosclerosis in humans. ILAR News 32, 4 - 8 . Clarkson, T. B., Shively, C. A., and Weingand, K. W. (1988). Animal models of diet induced atherosclerosis. In "Use of Animal Models for Research in Human Nutrition" (A. C. Beynen and C. E. West, eds.), pp. 56-82. Karger, Basel. Clegg, C. H., Haugen, H. S., Stamatoyannopoulos, G., and Peterson, K. R. (1997). Studying the developmental regulation of the human [Lglobin locus in transgenic mice. Lab. Anim. 26, 43-46. Clement, K., Vaisse, C., Lahlou, N., Cabrol, S., Pelloux, V., Cassuto, D., Gourmelen, M., Dina, C., Chambaz, J., Lacorte, J. M., Basdevant, A., Bourneres, P., Lebouc, Y., Froguel, P., and Guy-Grand, B. (1998). A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 392, 398-401. Cohen, I. R., and Miller, A., eds. (1994). "Autoimmune Disease Models." Academic Press, San Diego. Colbert, M. C., and Klintworth, G. K. (1997). Murine models of genetic abnormalities: Of mice and man-made mice. In "Biological Aspects of Disease: Contributions from Animal Models" (P. M. Innaccone and D. G. Scarpelli, eds.), pp. 39-68. Harwood Academic Publ., Amsterdam. Collins, E (1999). Medical and societal consequences of the human genome project. N. Engl. J. Med. 341, 28-37. Collins, E S. (1995). Ahead of schedule and under budget: The Genome Project passes its fifth birthday. Proc. Natl. Acad. Sci. (USA) 92, 10821-10823. Collins, E S., Patrinos, A., Jordan, E., Chakravarti, A., Gesteland, R., Waiters, L., and members of the DOE and NIH planning groups (1998). New goals for the U.S. Human Genome Project: 1998-2003. Science 282, 682-689. Comroe, J. (1977). "Retrospectroscope: Insights into Medical Discovery." Von Gehr Press. Menlo Park, California. Comroe, J. H., (1983). "Exploring the Heart: Discoveries in Heart Disease and High Blood Pressure." Norton, New York. Comroe, J. H., Jr., and Dripps, R. D. (1974). Ben Franklin and open heart surgery. Circ. Res. 35, 661-669. Comroe, J. H., Jr., and Dripps, R. D. (1976). Scientific basis for the support of biomedical research. Science 192, 105-111. Comuzzie, A. G., and Allison, D. B. (1998). The search for human obesity genes. Science 280, 1374-1377. Cone, R. D., Lu, D., Koppula, S., Vage, D., Klungland, H., Boston, B., Chen, W., Orth, D. N., Pouton, C., and Kesterson, R. A. (1996). The melanocortin receptors: Agonists, antagonists, and the hormonal control of pigmentation. Recent Prog. Horm. Res. 51, 287. Conn, P. M., and Parker, J. (1998). Animal rights: Reaching the public. Science 282, 1417. Cooke, A. S. (1973). Response of Rana temporaria tadpoles to chronic doses of pp'-DDT. Copeia 1973, 647-652. Cooper, A. M., Magram, J., Ferrante, J., and Orme, I. A. (1997). Interleukin 12 is crucial to the development of protective immunity in mice intravenously infected with mycobacterium tuberculosis. J. Exp. Med. 186, 39-45. Copeland, N. G., Jenkins, N. A., Gilbert, D. J., Eppig, J. T., Maltais, L. J., Miller, J. C., Dietrich, W. E, Weaver, A., Lincoln, S. E., Steen, R. G., Stein, L. D., Nadeau, J. H., and Lander, E. S. (1993). A genetic linkage map of the mouse: Current applications and future prospects. Science 262, 57-66. Cornelius, C. E. (1969). Animal models--a neglected medical resource. N. Engl. J. Med. 281, 934-944. Council on Scientific Affairs (1989). Animals in research. J. Am. Med. Assoc. 261, 3602-3606.

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

Couzin, J. (1998). Low-calorie diets may slow monkey's aging. Science 282, 1018. Cowell, B. A., Wu, C., and Fleiszig, M. J. (1999). Use of an animal model in studies of bacterial corneal disease. ILAR J. 30, 43-50. Crabbe, J. S., Belknap, J. K., and Buck, K. J. (1994). Genetic animal models of alcohol and drug abuse. Science 264, 1715-1723. Crawley, J. N. (2000). Behavioral phenotyping of transgenic and knockout mice: Experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. ILAR J. 41, 136-143. Crister, J. K., and Mobraaten, L. E. (2000). Cryopreservation of mouse spermatozoa. ILAR J. 41, 197-206. Dal Canto, M. (1997). Animal models of human central nervous system diseases: Multiple sclerosis and amyotrophic lateral sclerosis. In "Biological Aspects of Disease: Contributions from Animal Models" (E M. Innaccone and D. G. Scarpelli, eds.), pp. 145-164. Harwood Academic Publ., Amsterdam. Darlington, T. K., Wager-Smith, K., Fernanda Ceriani, M., Staknis, D., Gekakis, N., Steeves, T. D. L., Weitz, C. J., Takahashi, J. S. and Kay, S. A. (1998). Closing the circadian loop: Clock-induced transcription of its own inhibitors per and tim. Science 280, 1599-1603. Daszak, E, Berger, L., Cunningham, A. A., Hyatt, A. D., Green, D. E., and Speare, R. (1999). Emerging infectious diseases and amphibian population declines. Emerg. Inf. Dis. 5, 735-748. Datta, S. K. (2000). Positive selection for autoimmunity. Nat. Med. 6, 259-261. Dauber, D. V. (1944). Spontaneous arteriosclerosis in chicken. Arch. Pathol. 88, 46-52. Davisson, M. T., Bradt, D. W., Merriam, J. J., Rockwood, S. E, and Eppig, J. T. (1998). The mouse gene map. ILAR J. 39, 96 - 131. Dawid, I. B., and Sargent, T. D. (1988). Xenopus laevis in developmental and molecular biology. Science 240, 1443-1448. deJong, R., Altare, E, Haagen, I.-A., Elferink, D. G., deBoer, T., Vriesman, E J. C. V. B., Kabel, E J., Draaisma, J. M. T., vanDissel, J. T., Kroon, E E, Casanova, J.-L., and Ottenhoff, T. H. M. (1998). Severe mycobacterial and salmonella infections in interleukin-12 receptor-deficient patients. Science 280, 1435-1438. Dennis, M. B., Jr. (2000). Humane endpoints for genetically engineered animal models. ILAR J. 41, 94-98. DeTogni, E, Goellner, J., Ruddle, N. H., Streeter, E R., Fick, A., Mariathasan, S., Smith, S. C., Carlson, R., Shornick, L. E, Strauss-Schoenberger, J., Russell, J. H., Karr, R., and Chaplin, D. D. (1994). Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science 264, 703 -707. Dewhirst, E E., Chien, C.-C., Paster, B. J., Ericson, R. L., Orcutt, R. E, Schauer, D. B., and Fox, J. G. (1999). Phylogeny of the defned murine microbiota: Altered Schaedler flora. Appl. Environ. Microbiol. 65, 3287-3292. Dial, N. A., and Bauer, C. A. (1984). Teratogenic and lethal effects of paraquat on developing frog embryos (Rana pipiens). Bull. Environ. Contain. Toxicol. 33, 592-597. Dickman, S. (1998). Antibodies stage a comeback in cancer treatment. Science 280, 1196 - 1197. Dodds, W. J., and Abelseth, M. K. (1980). Criteria for selecting the animal to meet the research need. Lab. Anim. Sci. 30, 460-463. Doetschman, T. (1999). Interpretation of phenotype in genetically engineered mice. Lab. Anim. Sci. 49, 137-148. Doevendans, E A., Daemen, M. J., deMuinck, E. D. and Smits, J. E (1998). Cardiovascular phenotyping in mice. Cardiovasc. Res. 39, 34-49. Dooley, K., and Zon, L. I. (2000). Zebrafish: A model system for the study of human disease. Curr. Opin. Genet. Dev. 10, 252-256. Doyle, R. E., Garb, S., Davis, L. E., Meyer, D. K., and Clayton, E W. (1968). Domestic farm animals in medical research. Ann. N. Y Acad. Sci. 14"7, 129204. Drews, J. (2000). Drug discovery: A historical perspective. Science 287, 19601964.

1217 Driever, W., and Fishman, M. C. (1996). The zebrafish: Heritable disorders in transparent embryos. J. Clin. Invest. 98, s41-s46. Duellman, W. E., and Trueb, L. (1994). Metamorphosis. In "Biology of Amphibians," Chap. 7, pp. 173-194. Johns Hopkins Univ. Press, Baltimore. Dukelow, W. R., ed. (1983). "Nonhuman Primate Models for Human Diseases." CRC Press, Boca Raton, Florida. Durum, S. K., and Muegge, K., eds. (1998). "Cytokine Knockouts." Humana Press, Totowa, New Jersey. Ehrlich, E (1900). On immunity with special reference to cell life. Proc. R. Soc. Lond. 66, 424-448. Eijkman, C. (1965). Antineuritic vitamin and beri-beri. In "Nobel Lectures in Physiology or Medicine, 1922-1941 ." pp. 199-207. Elsevier, Amsterdam. Eisen, J. S. (1994). Development of motoneuronal phenotype. Annu. Rev. Neurosci. 17, 1-30. Emerman, M., and Malim, M. H. (1998). HIV-1 regulatory/accessory genes: Keys to unraveling viral and host cell biology. Science 280, 18801883. Evans, M. J., and Kaufman, M. H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-156. Evans, M. J., Gurer, C., Loike, J. D., Wilmut, I., Schnieke, A. E., and Schon, E. A. (1999). Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep. Nat. Genet. 23, 90-93. Ewbank, J. J., Barnes, T. M., Lakowski, B., Lussier, M., Bussey, H., and Hekimi, S. (1997). Structural and functional conservation of the Caenorhabditis elegans timing gene, Clk-1. Science 275, 980-983. Falo, L. D., and Storkus, W. J. (1998). Giving DNA vaccines a helping hand. Nat. Med. 4, 1239-1240. Fehr, T., Naim, H. Y., Bachmann, M. E, Ochsenbein, A. E, Spielhofer, E, Bucher, E., Hengartner, H., Billeter, M. A., and Zinkernagel, R. M. (1998). T-cell independent IgM and enduring protective IgG antibodies induced by chimeric measles viruses. Nat. Med. 4, 945-956. Ferguson, T. A., and Green, D. R. (1999). T cells are just dying to accept grafts. Nat. Med. 5, 1231-1235. Festing, M. E W. (1993a). "International Index of Laboratory Animals," 6th ed. Author, Leicester, United Kingdom. Festing, M. E W. (1993b). Origins and characteristics of inbred strains of mice. Mouse Genome 91, 393-542. Finch, C. E., and Tanzi, R. E. (1997). Genetics of aging. Science 228, 402-411. Flanagan, S. E (1966). "Nude" a new hairless gene with pleiotropic effects in the mouse. Genet. Res. 8, 295-309. Flannery, J. G. (1999). Transgenic animal models for the study of inherited retinal dystrophies. ILAR J. 40, 51-58. Florence, S. L., Taub, H. B., and Kaas, J. H. (1998). Large-scale sprouting of corticol connections after peripheral injury in adult macaque monkeys. Science 282, 1117-1121. Flynn, J. L., Chan, J., Triebold, K. J., Dalton, D. K., Stewart, T. A. and Bloom, B. R. (1993). An essential role for interferon y in resistance to mycobacterium tuberculosis infection. J. Exp. Med. 178, 2249-2254. Fogh, J., and Giovanella, B. C., eds. (1978). "The Nude Mouse in Experimental and Clinical Research," Vol. 1. Academic Press, New York. Fogh, J., and Giovanella, B. C., eds. (1982). "The Nude Mouse in Experimental and Clinical Research," Vol. 2. Academic Press, New York. Forsythe, E, and Ennis, M. (1999). Adenosine, mast cells, and asthma. Inflamm. Res. 48, 301-307. Fort, E J., Delphon, C. R., Powers, R., Helems, R., Gonzapez, R., and Stover, E. L. (1988). Development of automated methods of identifying toxicants in the environment. Bull. Environ. Contam. Toxicol. 54, 104-111. Fort, D. J., Propst, T. L., Stover, E. L., Helgen, J. C., Levey, R. B., Gallagher, K., and Burkhart, J. G. (1999a). Effects of pond water, sediment, and sediment extracts from Minnesota and Vermont, USA, on early development and metamorphosis of Xenopus. Environ. Toxicol. Chem. 18, 2305-2315. Fort, D. J., Rogers, R. L., Copley, H. E, Bruning, L. A., Stover, E. L., Helgen, J. C., and Burkhart, J. G. (1999b). Progress toward identifying causes of

1218 maldevelopment induced in Xenopus by pond water and sediment extracts from Minnesota, USA. Environ. Toxicol. Chem. 18, 2316-2324. Foster, H. L. (1980). The history of commercial production of laboratory rodents. Lab. Anim. Sci. 30, 793-798. Foster, H. L., Small, J. D., and Fox, J. G., eds. (1982). "The Mouse in Biomedical Research: Experimental Biology and Oncology," Vol. 4. Academic Press, New York. Fox, E., and Fox, D. M. (1988). "AIDS: The Burdens of History." Univ. of California Press, Berkeley and Los Angeles. Fox, J. A., Hotaling, T. E., Struble, C., Ruppel, J., Bates, D. J., and Schoenhoff, M. B. (1996). Tissue distribution and complex formation with IgE of an anti-IgE antibody after intravenous administration in cynomolgus monkeys. J. Pharmacol. Exp. Ther. 279, 1000-1008. Fraser, C. M., Eisen, J. A., and Salzberg, S. L. (2000). Microbial genome sequencing. Nature 406, 799-803. French, R. R., Chan, H. T. C., Tutt, A. L., and Glennie, M. J. (1999). CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat. Med. 5, 548-552. Friedman, D. B., and Johnson, T. E. (1988). A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility. Genetics 118, 75-86. Fritzsch, B. (1990). The evolution of metamorphosis in amphibians. J. Neurobiol. 21, 1011-1021. Froguel, E, Zouali, H., Vionnet, N., Velho, G., Vaxillaire, M., Sun, E, Lesage, S., Stoffel, M., Takeda, J., and Passa, E (1993). Familial hypoglycemia due to mutations in glucokinase: Definition of a subtype of diabetes mellitus. N. Engl. J. Med. 328, 697-702. Gallo, R. (1991). "Virus Hunting: AIDS, Cancer, and the Human Retrovirus." HarperCollins Publ., New York. Gallo, R. C., Salahuddin, S. Z., Popovic, M., Sheaver, G. M., Kaplan, M., Haynes, B. E, Palker, T. J., Redfield, R., Oleske, J., Safai, B., White, G., Foster, E, and Markham, E D. (1984). Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224, 500-503. Gardner, M. B. (1989). SIV infected rhesus macaques: An AIDS model for immunoprevention and immunotherapy. Adv. Exp. Med. Biol. 251, 279-293. Gardner, M. B. (1997). AIDS models. In "Biology Aspects of Disease: Contributions from Animal Models" (E M. Innaccone and D. G. Scarpelli, eds.), pp. 247-303. Harwood Academic Publ. Amsterdam. Gardner, J. M., Nakatsu, Y., Gondo, Y., Lee, S., Lyon, M. E, King, R. A., and Brilliant, M. H. (1992). The mouse pink-eyed dilution gene: Association with human Prader-Willi and Angelman syndromes. Science 257, 11211125. Gatz, M., Pedersen, N. L., Berg, S., Johansson, B., Johansson, K., Motimer, J. A., Posner, S. E, Viitanen, M., Winblad, B., and Ahlbom, A. (1997). Heritability for Alzheimer's disease: The study of dementia in Swedish twins. J. Gerontol. A Biol. Sci. Med. Sci. 52, Ml17-M125. Gay, W. I., ed. (1965-1989). "Methods of Animal Experimentation," Vol. 1, 1965; Vol. 2, 1965; Vol. 3, 1968; Vol. 4, 1973; Vol. 5, 1974; Vol. 6, 1981; Vol. 7, Part A, 1986; Vol. 7, Part B, 1986; Vol. 7, Part C, 1989. Academic Press, New York. Geddes, B. J., Harding, T. C., Lightman, S. L. and Uney, J. B. (1997). Long-term gene therapy in the CNS: Reversal of hypothalamic diabetes insipidus in the Brattleboro rat by using an adenovirus expressing arginine vasopressin. Nat. Med. 12, 1402-1404. Gekakis, N., Staknis, D., Nguyen, H. B., Davis, E C., Wilsbacher, L. D., King, D. E, Takahashi, J. S., and Weitz, C. J. (1998). Role of the clock protein in mammalian circadian mechanism. Science 280, 1564-1569. Gershwin, M. E., and Merchant, B., eds. (1981). "Immunologic Defects in Laboratory Animals," Vols. 1 and 2. Plenum Press, New York. Ghanem, A., and Shuler, M. L. (2000). Combining cell culture analogue reactor designs and PBPK models to probe mechanisms of naphthalene toxicity. Biotechnol. Prog. 16, 334-345.

FRED W. QUIMBY Gibbs, J., Young, R. C., and Smith, G. E (1973). Cholecystokinin decreases food intake in rats. J. Comp. Physiol. Psychol. 84, 488-495. Gill, T. J., III, Smith, G. J., Wissler, R. W., and Kunz, H. W. (1989). The rat as an experimental animal. Science 254, 269-276. Gold, R., Hartung, H. E, and Toyka, K. V. (2000). Animal models for autoimmune demyelinating disorders of the nervous system. Mol. Med. Today 6, 88-91. Golob, E. K., Rishi, S., Becker, K. L., and Moore, C. (1970). Streptozoticin diabetes in pregnant and nonpregnant rats. Metabol. 19, 1014-1021. Gordon, J. W. (1997). Transgenic animal models of disease. In "Biological Aspects of Disease: Contributions of Animal Models" (E M. Iannaccone and D. G. Scarpelli, eds.), pp. 15-37. Harwood Academic Publishers, Amsterdam. Gordon, J. W., and Ruddle, E H. (1981). Integration and stable germline transmission of genes injected into mouse pronuclei. Science 214, 1244-1246. Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A., and Ruddle, E H. (1980). Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. US.A. 77, 7380-7384. Green, S. L., and Tolwani, R. J. (1999). Animal models for motor neuron disease. Lab. Anim. Sci. 49, 480-495. Greenberg, E D., and Riddell, S. R. (1999). Deficient cellular immunity--finding and fixing the defects. Science 285, 546-551. Grfinewald, T., and Beal, M. E (1999). NOS knockouts and neuroprotection. Nat. Med. 5, 1354-1355. Guenette, S. Y., and Tanzi, R. E. (1999). Progress toward valid transgenic mouse models for Alzheimer's disease. Neurobiol. Aging 20, 201-211. Gurunathan, S., Prussin, C., Sacks, D. L., and Seder, R. A. (1998). Vaccine requirements for sustained cellular immunity to an intracellular parasitic infection. Nat. Med. 4, 1409-1414. Hacia, J. G., Brody, L. C., and Collins, E S. (1998). Applications of DNA chips for genomic analysis. Mol. Psychiatry 3, 483-492. Hammer, R. E., Maika, S. D., Richardson, J. A., Tang, J. E, and Taurog, J. D. (1990). Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human B2-m: An animal model of HLA-B27 associated human disorders. Cell 63, 1099-1112. Hanken, J. (1999). Larvae in amphibian development and evolution. In "The Origin and Evolution of Larval Forms" (B. K. Hall and M. H. Wake, eds.), Chap. 3., pp. 61-96. Academic Press, San Diego. Hansen, J. T., and Sladek, J. R., Jr. (1989). Fetal research. Science 246, 775782. Hardin, E E., and Glossop, N. R. J. (1999). The CRYs of flies and mice. Science 286, 2460-2461. Haskins, M. E., Otis, E. J., Hayden, J. E., Jezyk, E E, and Stramm, L. L. (1992). Hepatic storage of glycosaminoglycans in feline and canine models of mucopolysaccharidoses I, VI, VII. Vet. Pathol. 29, 112-119. Hegreberg, G., and Leathers, C., eds. (1981a). "Bibliography of Induced Animal Models of Human Disease." Washington State Univ. Press, Pullman. Hegreberg, G., and Leathers, C., eds. (198 lb). "Bibliography of Naturally Occurring Animal Models of Human Disease." Washington State Univ. Press, Pullman. Herweijer, H., Harding, C. O., Hagstrom, J. E., and Wolff, J. (1997). Animal models in gene therapy. In "Biological Aspects of Disease: Contributions from Animal Models" (E M. Innaccone and D. G. Scarpelli, eds.), pp. 6 9 92. Harwood Academic Publ., Amsterdam. Hill, B. E (1963). Proceedings of the symposium on research animal housing. Lab. Anim. Care 13, 221-442. Hilleman, M. R. (1998). Six decades of vaccine developmentma personal history. Nat. Med. 4, 507-514. Himms-Hagen, J. (1999). Physiological roles of the leptin endocrine system: Differences between mice and humans. Crit. Rev. Clin. Lab. Sci. 36, 575655. Hislop, A. D., Good, M. E, Mateo, L., Gardner, J., Gatei, M. H., Daniel, R. C. W., Meyers, B. V., Lavin, M. E, and Suhrbier, A. (1998). Vaccine-induced

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

cytotoxic T lymphocytes protect against retroviral challenge. Nat. Med. 4, 1193-1196. Hoffman, S. L., Crutcher, J. M., Puri, S. K., Ansari, A. A., Villinger, E, Franke, E. D., Singh, P. P., Finkelman, E, Gately, M. K., Dutta, G. P., and Sedeyah, M. (1997). Sterile protection of monkeys against malaria after administration of interleukin- 12. Nat. Med. 3, 80- 83. Hoffman, S. L., Rogers, W. O., Carucci, D. J., and Venter, J. C. (1998). From genomics to vaccines: Malaria as a model system. Nat. Med. 4, 1351-1353. Hooke, R. (1667). An account of an experiment made by M. Hooke, of preserving animals alive by blowing through their wings with bellows. Philos. Trans. R. Soc. Lond. 2, 539-540. Houghton, A. N., and Scheinberg, D. A. (2000). Monoclonal antibody therapies--a "constant" threat to cancer. Nat. Med. 4, 373-374. Hsiao, K., Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y., Younkin, S., Yang, E, and Cole, G. (1996). Correlative memory deficits, A[3 elevation, and amyloid plaques in transgenic mice. Science 274, 99-102. Huber, I., Masler, E. P., and Rao, B. R., eds. (1990). "Cockroaches as Models for Neurobiology: Applications in Biomedical Research," Vol. 1, 2, and 3. CRC Press, Boca Raton, Florida. Hunter, A. J., Nolan, P. M., and Brown, S. D. (2000). Towards new models of disease and physiology in the neurosciences: The role of induced and naturally occurring mutations. Hum. Mol. Genet. 9, 893-900. Ishikawa, Y. (2000). Medakafish as a model system for vertebrate developmental genetics. Bioessays 22, 487-495. Iwata, N., Tsubuki, S., Takaki, Y., Watanabe, K., Sekrguchi, M., Hosoki, E., Kawashima-Morishima, M., Lee, H.-J., Hama, E., Sekine-Aizawa, Y., and Saido, T. C. (2000). Identification of the major A[31-42-degrading catabolic pathway in brain parenchyma: Suppression leads to biochemical and pathological deposition. Nat. Med. 6, 143-150. Jacob, E, and Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318-356. Jaenisch, R. (1976). Germline integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. U.S.A. 73, 1260-1264. Jardine, L. (1999). "Ingenious Pursuits: Building the Scientific Revolution." Nan A. Talese. Doubleday, New York. Jasny, B., and Koshland, D. E., Jr., eds. (1990). "Biological Systems: Papers from Science, 1988-1989." American Association for the Advancement of Science, Washington, D.C. Jinnah, H., and Breese, G. R. (1997). Animal models for Lesch-Nyhan Disease. In "Biological Aspects of Disease: Contributions from Animal Models" (P. M. Innaccone and D. G. Scarpelli, eds.), pp. 93-144. Harwood Academic Publ. Amsterdam. Joag, S. V. (2000). Primate models of AIDS. Microbes Infect. 2, 223-229. Johnson, P. T. J., Lunde, K. B., Ritchie, E. G., and Launer, A. E. (1999). The effect of trematode infection on amphibian limb development and survivorship. Science 284, 802-804. Jones, E. G., and Pons, T. P. (1998). Thalamic and brainstem contributions to large-scale plasticity of primate somatosensory cortex. Science 282, 11211126. Jones, T. C., Hackel, D. B. and Migaki, G. (1972). "Handbook: Animal Models of Human Disease." Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Kato, Y., Tani, T., Sotomaru, Y., Kurokawa, K., Kato, J. Y., Doguchi, H., Yasue, H., and Tsunoda, Y. (1998). Eight calves cloned from somatic cells of a single adult. Science 282, 2095-2098. Katsuki, M., S ato, M., Kimura, M., Yokoyama, M., Kobayashi, K., and Nomura, T. (1988). Science 241, 593-595. Keen, W. W. (1914). "Animal Experimentation and Medical Progress." Houghton Mifflin, Boston. Kennedy, B. K., Gotta, M., Sinclair, D. A., Mills, K., McNabb, D. S., Murthy, M., Pak, S. M., Laroche, T., Gasser, S. M., and Guarente, L. (1997). Redistribution of silencing proteins from telomeres to the nucleo-

1219 lus is associated with extension of life span in S. cerevisiae. Cell 89, 381-391. Kenyon, C., Chang, J., Gensch, E., Rudner, A., and Tabtiang, R. (1993). A C. elegans mutant that lives twice as long as wild type. Nature 366, 461464. Kiesecker, J. M., and Blaustein, A. R. (1995). Synergism between UV-B radiation and a pathogen magnified amphibian embryo mortality in nature. Proc. Natl. Acad. Sci. U.S.A. 92, 11049-11052. Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B. West, M. D., Ho, P. L. C., Coviello, G. M., Wright, W. E., Weinrich, S. L., and Shay, J. W. (1994). Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011-2015. Kimura, K. D., Tissenbaum, H. A., Liu, Y., and Ruvkun, G. (1997). Daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277, 942-946. King, C. A., Spellerberg, M. B., Zhu, D., Rice, J., Sahota, S. S., Thompsett, A. R., Hamblin, T. J., Radl, J., and Stevenson, E K. (1998). DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma. Nat. Med. 4, 1281-1286. King, E A., Yarbrough, C. I., Anderson, D. C., Gordon, T. R, and Gould, K. G. (1988). Primates. Science 240, 1475-1483. Kirk, A. D., Burkly, L. C., Batty, D. S., Baumgartner, E., Berning, J. D., Buchanan, K., Fechner, J. H., Germond, R. L., Kampen, R. L., Patterson, N. B., Swanson, S. J., Tadaki, D. K., TenHoor, C. N., White, L., Knochtle, S. J., and Harlan, D. M. (1999). Treatment with humanized monoclonal antibody against CD154 prevents acute allograft rejection in nonhuman primates. Nature Med. 6, 686-693. Kobaek-Larsen, M., Thorup, I., Diederichsen, A., Fenger, C., and Hoitinga, M. R. (2000). Review of colorectal cancer and its metastasis in rodent models: Comparative aspects with those of humans. Comp. Med. 50, 16-26. Kon, O. M., and Kay, A. B. (1999). Anti-T cell strategies in asthma. Inflamm. Res. 48, 516-523. Kondo, J., and Watanabe, Y. (1975). A heritable hyperlipemic rabbit. Exp. Anim. (Tokyo) 24, 89-94. Konishi, M., Emlen, S. T., Ricklefs, R. E., and Wingfield, J. C. (1989). Contributions of bird studies to biology. Science 246, 465-469. Kornberg, A. (1959). The biological synthesis of deoxyribonucleic acid. Les Prix Nobel en 1959. Kovacs, P., Voigt, B., Berg, S., Vogt, L., and Kloting, I. (1997). WOK.1W rats. A potential animal model of the insulin resistance syndrome. In "Lipids and Syndromes of Insulin Resistance: From Molecular Biology to Clinical Medicine" (pp. 94-99). New York Academy of Sciences, New York. Kritchevsky, D. (1988). Dietary effects in experimental carcinogenesis: Animal models. In "The Use of Animal Models in Human Nutrition" (A. C. Beynen and C. E. West, eds.), pp. 174-185. Karger, Basel. Ktihn, R., Schwenk, E, Aguet, M., and Rajewsky, K. (1995). Inducible gene targeting in mice. Science 269, 1427-1429. Kuhn, T. S. (1970) "The Structure of Scientific Revolutions." 2nd ed. Univ. of Chicago Press, Chicago. Kukreja, A., and MacLaren, N. K. (1999). Autoimmunity and diabetes. J. Clin. Endocrinol. Metab. 84, 4371-4378. Kwan, A. P. L., Dickson, I. R., Freemont, A. J., and Grant, M. E. (1989). Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage. J. Ceil Biol. 109, 1849-1855. Ladiges, W. C., Storb, R., and Thomas, E. D. (1990). Canine models of bone marrow transplantation. Lab. Anim. Sci. 40, 11-15. Lakowski, B., and Hekimi, S. (1996). Determination of life-span in Caenorhabditis elegans by four clock genes. Science 272, 1010-1013. Landsteiner, K. (1945). "Specificity of Serological Reactions." Dover, New York. Langermann, S. (1998). Site-directed immunogenesis. Nat. Med. 4, 547-548. Larsen, P. L., Albert, P. S., and Riddle, D. L. (1995). Genes that regulate both

1220

development and longevity in Caenorhabditis elegans. Genetics 139, 1567-1583. Lawrence, H. S. (1959). Homograft sensitivity: An expression of the immunologic origins and consequences of individuality. Physiol. Rev. 39, 811859. Leader, R. (1975). Veterinary medicine. MD 19, 77-88. Leader, R. W., and Padgett, G. A. (1980). The genesis and validation of animal models. Am. J. Pathol. 101, 511-516. Leader, R. W., and Stark, D. (1987). The importance of animals in biomedical research. Perspect. Biol. Med. 30, 470-485. Lederberg, J., and Tatum, E. L. (1954). Sex in bacteria: Genetic studies 19451952. Science 118, 169-175. Lee, A. (1999). Animal models of Helicobacter infection. Mol. Med. Today 5, 500-511. Lee, A. (2000). Animal models of gastroduodenal ulcer disease. Baillieres Best Pract. Res. Clin. Gastroenterol. 14, 75-96. Lee, B., Dennis, J. A., Healy, E J., Mull, B., Pastore, L., Yu, H., AguilarCordova, E., O'Brien, W., Reeds, E, and Beaudet, A. L. (1999). Hepatocyte gene therapy in a large animal: A neonatal bovine model of citrullinemia. Proc. Natl. Acad. Sci. US.A. 96, 3981-3986. Lewin, R. (1984). Brain enzyme is the target of drug toxin. Science 225, 14601462. Lewis, R. M., and Schwartz, R. S. (1971). Canine systemic lupus erythematosus. Genetic analysis of an established breeding colony. J. Exp. Med. 134, 417-421. Lewis, S. M., and Carraway, J. H. (1992). Large animal models of human disease. Lab. Anim. 21, 22-31. Li, Y., Li, X. C., Zheng, X. X, Wells, A. D., Turka, L. A., and Strom, T. B. (1999). Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T-cells and induction of peripheral allograft tolerance. Nature Med. 5, 1298-1302. Lin, S.-J., Defossez, E-A., and Guarente, L. (2000). Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 2126-2128. Liu, M. A. (1998). Vaccine developments. Nat. Med. 4, 515-519. Liu, H., Farr-Jones, S., Ulyanov, N. B., Llinas, M., Marqusee, S., Groth, D., Cohen, E E., Prusiner, S. B., and James, T. L. (1999). Solution structure of Syrian hamster prion protein rPrP (90-231). Biochemistry 38, 53625377. Llinas, R. R. (1999). "The Squid Giant Synapse: A Model for Chemical Transmission." Oxford Univ. Press, New York. Locatelli, E, Rondelli, D., and Burgio, G. R. (2000). Tolerance and hematopoietic stem cell transplantation 50 years after Burnet's theory. Exp. Hematol. 28, 479-489. Loots, G. G., Locksley, R. M., Blankespoor, C. M., Wang, Z. E., Miller, W., Rubin, E. M., and Frazer, K. A. (2000). Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons. Science 288, 136-148. Lund, T., O'Reilly, L., Hutchings, E, Kanagawa, O., Simpson, E., Gravely, R., Chandler, E, Dyson, J., Picard, J. K., Edwards, A., Kioussis, D., and Cooke, A. (1990). Prevention of insulin-dependent diabetes mellitus in non-obese diabetic mice by transgenes encoding modified I-A [3-chain or normal I-E a-chain. Nature 345, 727-729. Lwoff, A. (1953). Lysogeny. Bacteriol. Rev. 17, 269-337. Lyon, M. E, ed. (1996). "Genetic Variants and Strains of the Laboratory Mouse," 3rd ed. Oxford Univ. Press, New York. Maddox, J. (1999). The unexpected science to come. Sci. Am. 280, 62-67. Magram, J., Connaughton, S. E., Warrier, R. R., Carvajal, D. M., Wu, C.-Y., Ferrante, J., Stewart, C., Sarmiento, U., Faherty, D. A., and Gately, M. K. (1996). IL-R-deficient mice are defective in IFN y production and type 1 cytokine responses. Immunity 4, 471- 481. Main, J. M., and Prehn, R. T. (1955). Successful skin homografts after administration of high dose x radiation and homologous bone marrow. J. Natl. Cancer Inst. 15, 10235-10239.

FRED W. QUIMBY Malakoff, D. (1999). NIH urged to fund centers to merge computing and biology. Science 284, 1742. Mann, J. R., Mulligan, R. C., and Baltimore, D. (1983). Construction of retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33, 153-159. Manning, E J., Ringler, D. H., and Newcomer, C. E., eds. (1994). "The Biology of the Laboratory Rabbit," 2nd ed. Academic Press, San Diego. Marchioro, T. L., Axtell, H. K., LaVia, M. F., Waddell, W. R., and Starzl, T. E. (1964). The role of adrenocortical steroids in reversing established homograft rejection. Surgery 55, 412-417. Marinaro, M., Boyaka, E N., Jackson, R. J., Finkelman, F. D., Kiyono, H., Jirillo, E., and McGhee, J. R. (1999). Use of intranasal IL-12 to target predominantly Thl responses to nasal and Th2 responses to oral vaccines given with cholera toxin. J. Immunol. 162, 114-121. Martin, G. R. (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. US.A. 78, 7634-7638. Martin, J. B. (1999). Molecular basis of the neurodegenerative disorders. N. Engl. J. Med. 340, 1970-1980. Martin, L. J., Portera-Cailliau, C., Ginsberg, S. D., and A1-Abdulla, N. A. (1998). Animal models and degenerative disorders of the human brain. Lab. Anim. 27, 18-25. Mascola, J. R., Stiegler, G., VanCott, T. C., Katinger, H., Carpenter, C. B., Hanson, C. E., Beary, H., Hayes, D., Frankel, S. H., Birx, D. L., and Lewis, M. G. (2000). Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nature Med. 6, 207-210. Masoro, E. (1988). Physiological system markers of aging. Exp. Gerontol. 23, 391-397. Masoro, E. (1989). Overview of the effects of food restriction. Prog. Clin. Biol. Res. 287, 27-35. Masoro, E. (2000). Caloric restriction and aging: An update. Exp. Gerontol. 35, 299-305. Masse, E, Vuilleumier, J. E, and Weiser, H. (1989). Pyridoxine status as assessed by the concentration of B6-aldehyde vitamers. Int. J. Vitam. Nutr. Res. 59, 344-351. Materna, E. J., Rabeni, C. E, and LaPoint, T. W. (1995). Effects of the synthetic pyrethroid insecticide, esfenvalerate, on larval leopard frogs (Rana spp.). Environ. Toxicol. Chem. 14, 613- 622. Matsuoka, T., Hirata, M., Tanaka, H., Takahashi, Y., Murata, T., Kabashima, K., Sugimoto, Y., Kobayashi, T., Ushikubi, F., Aze, Y., Eguchi, N., Urade, Y., Yoshida, N., Kimura, K., Mizoguchi, A., Honda, Y., Nagai, H., and Narumiya, S. (2000). Prostaglandin D2 as a mediator of allergic asthma. Science 287, 2013-2017. McClearn, G. E., and Vandenbergh, D. J. (2000). Structure and limits of animal models: Examples from alcohol research. ILAR J. 41, 144-152. McCracken, K. J. (1988). Obesity--role of animal models. In "Comparative Nutrition" (K. Blaxter and I. MacDonald, eds.), pp. 163-184. J. Libby, London. McDiarmid, R. W., and Altig, R., eds. (1999). "Tadpoles: The Biology of Anuran Larvae." Univ. of Chicago Press, Chicago. McGehee, H. (1981). "Research and Discovery in Medicine: Contributions from Johns Hopkins." Johns Hopkins Univ. Press, Baltimore. McMichael, A. J., and Hanke, T. (1999). Is an HIV vaccine possible? Nat. Med. 5, 612-614. Means, L. W., Burnett, M. A., and Pennington, S. N. (1988). The effect of embryonic ethanol exposure on detour learning in the chick. Alcohol 5, 305-311. Medina, L., and Reiner, A. (2000). Do birds possess homologues of mammalian primary visual, somatosensory, and motor cortices? Trends Neurosci. 23, 1-12. Medzhitov, R., Preston-Hurlburt, E, and Janeway, C. A., Jr. (1997). A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394-397.

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

Megyesi, J. E, Vollrath, B., Cook, D. A., and Findlay, J. M. (2000). In vivo animal models of cerebral vasospasm: A review. Neurosurgery 46, 448460. Merzenich, M. (1998). Long-term change of mind. Science 282, 1062-1063. Migaki, G., and Capen, C. C. (1984). Animal models in biomedical research. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), 1st ed., Chap. 24, pp. 667-695. Academic Press, New York. Mikaelian, I., Ouellet, M., Pauli, B., Rodrigue, J., Harshbarger, J. C., and Green, D. M. (2000). Ichthyophonus-like infection in wild amphibians from Quebec, Canada. Dis. Aquat. Org., in press. Milgrom, H., Fick, R. B., Su, J. Q., Reimann, J. D., Bush, R. K., Watrous, M. L., and Metzger, W. J. (1999). Treatment of allergic asthma with monoclonal anti-IgE antibody. N. Engl. J. Med. 341, 1966-1973. Mill, J. S. (1843). "A System of Logic, Ratiocinative and Inductive, Being a Connected View of the Principles of Evidence and the Methods of Scientific Investigation" (Reprinted by Univ. of Toronto Press, Toronto, 1974). Miller, A., Lider, O., Roberts, A. B., Sporn, M. B., and Weiner, H. L. (1992). Suppressor T cells generated by oral tolerization to myelin basic protein suppresses both in vitro and in vivo immune responses by the release of transforming growth factor [3 after antigen-specific triggering. Proc. Natl. Acad. Sci. U.S.A. 89, 421- 425. Miller, J. E A. P. (1961). Immunological function of the thymus. Lancet 2, 748-749. Mobraaten, L. E. (1999). Cryopreservation in a transgenic program. Lab. Anim. 28, 15-20. Montague, C. T., Farooqi, I. S., Whitehead, J. P., Soos, M. A., Rau, H., Wareham, N. J., Digby, J. E., Mohammed, S. N., Hurst, J., Cheetham, C. H., Earley, A. R., Barnett, A. H., Prins, J. B., and O'Rahilly, S. (1997). Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 387, 903-908. Montinaro, V., Gesualdo, L., and Schena, E P. (1999). The relevance of experimental models in the pathogenetic investigation of primary IgA nephropathy. Ann. Med. Interne (Paris) 150, 99-107. Moore, E D. (1995). "A Miracle and a Privilege." Joseph Henry Press, Washington, D.C. Morell, V. (1999). Are pathogens felling frogs? Science 284, 728-734. Morgan, T. H. (1928). "The Theory of the Gene." Yale Univ. Press, New Haven, Connecticut. Morris, J. Z., Tissenbaum, H. A., and Ruvkun, G. A. (1996). A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans. Nature 382, 536-539. Mosier, D. E. (1996). Small animal models for acquired immune deficiency syndrome (AIDS) research. Lab. Anim. Sci. 46, 257-265. Mosier, D. E., Gulizia, R. J., Baird, S. M., Wilson, D. B., Spector, D. H., and Spector, S. A. (1991). Human immunodeficiency virus infection of humanPBL-SCID mice. Science 251, 791-794. Mount, L. E., and Ingram, D. L. (1971). "The Pig as a Laboratory Animal." Academic Press, New York. Moyer, J. H., Lee-Tischler, M. J., Kwon, H.-Y., Schrick, J. J., Avner, E. D., Sweeney, W. E., Godfrey, V. L., Cacheiro, N. L. A., Wilkinson, J. E., and Woychik, R. P. (1994). Candidate gene associated with a mutation causing recessive polycystic kidney disease in mice. Science 264, 1329-1333. Mullins, L. J., and Mullins, J. J. (1996). Transgenesis in the rat and larger mammals. J. Clin. Invest. 98(11), 537-540. Mullins, J. J., Peters, J., and Ganten, D. (1990). Fulminant hypertension in transgenic rats harboring the mouse Ren-2 gene. Nature 344, 541-544. Murphy, E. D., and Roths, J. B. (1977). A single gene model for massive lymphoproliferation with autoimmunity in new mouse strain MRL. Fed. Proc. 36, 1246-1250. Murray, J. E. (1992). Biological spinoffs of organ transplantation. Science 256, 1411-1416. Murray, J. E. Merrill, J. P., Harrison, J. H., Wilson, R. E., and Dammin, G. J. (1963). Prolonged survival of human-kidney homografts by immunosuppressive drug therapy. N. Engl. J. Med. 268, 1315-1323.

1221 Nadeau, J. H., Eppig, J. T., and Reiner, A. H. (1989). "Linkage and Synteny Homologies between Mouse and Man." Jackson Laboratory, Bar Harbor, Maine. Nagy, A., and Rossant, J. (1996). Targeted mutagenesis: Analysis of phenotype without germline transmission. J. Clin. Invest. 98, s31-s35. Narayanaswamy, M., Wright, K. C., and Kanderpa, K. (2000). Animal models for atherosclerosis, restenosis, and endovascular graft research. J. Vasc. Interv. Radiol. 11, 5-17. Nathanielsz, E W., ed. (1980-1987). "Animal Models in Fetal Medicine," Vol. 1, 1980; Vol. 2, 1982; Vol. 3, 1982; Vol. 4, 1984; Vol. 5, 1986; Vol. 6, 1987; Elsevier, Amsterdam. Nathanson, N. (1998). Harnessing research to control AIDS. Nat. Med. 4, 879881. National Research Council (NRC) (1976). "Animal Models of Thrombosis and Hemorrhagic Diseases." National Academy Press, Washington, D.C. National Research Council (NRC) (1981). "Mammalian Models for Research on Aging." National Academy Press, Washington, D.C. National Research Council (NRC) (1985). "Models for Biomedical Research." National Academy Press, Washington, D.C. National Research Council (NRC) (1989). "Immunodeficient Rodents. A Guide to Their Immunobiology, Husbandry, and Use." National Academy Press, Washington, D.C. National Research Council (NRC) (1994). "Laboratory Animal Management: Dogs." National Academy Press, Washington, D.C. National Research Council (NRC) (1996). "Guide for the Care and Use of Laboratory Animals." National Academy Press, Washington, D.C. National Research Council (NRC) (1998). "Biomedical Models and Resources: Current Needs and Future Opportunities." National Academy Press, Washington, D.C. Nelson, R. J., and Chiavegatto, S. (2000). Aggression in knockout mice. ILAR J. 41, 153-162. Ness, T. J. (1999). Models of visceral nociception. ILAR J. 40, 119-128. Nestle, E O., Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer, R., Burg, G., and Schadendorf, D. (1998). Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat. Med. 3, 328-332. Newsome, E N., Plevris, J. N., Nelson, L. J., and Hayes, E C. (2000). Animal models of fulminant hepatic failure: A critical evaluation. Liver Transpl. 6, 21-31. Newsome, W. T., and Stein-Aviles, J. A. (1999). Nonhuman primate models of visually based cognition. ILAR J. 40, 78-91. Nguyen, B. Q., Kanthasamy, A. G., and Truong, D. D. (2000). Animal models of myoclonus: An overview. Mov. Disord. lg(Suppl. 1), 22-25. Nicholls, R. D., Glenn, C. C., Jong, M. T. C., Saitoh, S,, Mascari, M. J., and Driscoll, D. J. (1996). "Molecular Pathogenesis of Prader-Willi Syndrome." Louisiana State Univ. Press, Baton Rouge. Nielsen, C., Pedersen, C., Lundgren, J. D., and Gerstoft, J. (1993). Biological properties of HIV isolates in primary HIV infection: Consequences for the subsequent course of infection. AIDS 7, 1035-1040. Nomura, T. (1997). Practical development of genetically engineered animals as human disease models. Lab. Anim. Sci. 47, 113-117. Norton, T. T. (1999). Animal models of myopia: Learning how vision controls the size of the eye. ILAR J. 40, 59-77. Norvell, S., Bornslaeger, E., and Green, M. (1997). Transgenic mouse models of human epidermal diseases. In "Biological Aspects of Disease: Contributions from Animal Models" (E M. Innaccone and D. G. Scarpelli, eds.), pp. 201-246. Harwood Academic Publ., Amsterdam. Noseworthy, J. H. (1999). Progress in determining the causes and treatments of multiple sclerosis. Nature 399, A40-A47. Notterpek, L., and Tolwani, R. J. (1999). Experimental models of peripheral neuropathies. Lab. Anim. Sci. 49, 588-599. Nusslein-Volhard, C. (1994). Of flies and fishes. Science 266, 572-574. O'Dell, B. L., Conley-Harrison, J., Browning, J. D., Besch-Williford, C., Hempe, J. M., and Savage, J. E. (1990). Zinc deficiency and peripheral neuropathy in chicks. Proc. Soc. Exp. Biol. Med. 94, 1-11.

1

2

2

2

F

R

O'Donnell, R. A., Saul, A., Cowman, A. E, and Crabb, B. S. (2000). Functional conservation of the malaria vaccine antigen MSP-119 across distantly related Plasmodium species. Nat. Med. 6, 91-95. Office of Technology Assessment (OTA) (1986). "Alternatives to Animal Use in Research, Testing, and Education." U.S. Government Printing Office (OTA-BA-273), Washington, D.C. Okamura, H., Miyake, S., Sumi, Y., Yamaguchi, S., Yasui, A., Muijtjens, M., Hoeijmakers, J. H. J., and van der Horst, G. T. J. (1999). Photic induction of mPerl and mPer2 in Cry-deficient mice lacking a biological clock. Science 286, 2531-2534. Ordahl, C. E, and Williams, B. A. (1998). Knowing chops from chuck: Roasting myoD redundancy. Bioessays 20, 357-362. Ortho Multicenter Transplant Study Group (OMTSG) (1985). A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cardaveric renal transplants. N. Engl. J. Med. 313, 337-342. Ostenson, C. G., Khan, A., and Efendic, S. (1993). Impaired glucose-induced insulin secretion: Studies in animal models of spontaneous NIDDM. Adv. Exp. Med. Biol. 334, 1-11. Ouellet, M., Bonin, J., Rodrigue, J., DesGranges, J.-L., and Lair, S. (1997). Hindlimb deformities (ectromelia, ectrodactyly) in free-living anurans from agricultural habitats. J. Wild. Dis. 33, 95-104. Ozaki, K., Fujimori, H., Nomura, S., Nishikawa, T., Nishimura, M., Pan-Hou, A. H., and Narama, I. (1998). Morphologic and hematologic characteristics of storage pool deficiency in beige rats (Chediak-Higashi syndrome). Lab Anim. Sci. 48, 502-506. Pages, G., Guerin, S., Grall, D., Bonino, E, Smith, A., Anjuere, E, Auberger, E, and Pouyssegur, J. (1999). Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Science 286, 1374-1377. Pappu, R., Cheng, A. M., Li, B., Gong, Q., Chiu, C., Griffin, M., White, M., Sleckman, B. E, and Chan, A. C. (1999). Requirement for B cell linker protein (BLNK) in B cell development. Science 286, 1949-1954. Pardoll, D. M. (1998). Cancer vaccines. Nat. Med. 4, 525-531. Parkhill, J., Wren, B. W., Mungall, K., Ketley, J. M., Churcher, C., Basham, D., Chillingworth, T., Davies, R. M., Feltwell, T., Holroyd, S., Jagels, K., Karlyshev, A. V., Moule, S., Pallen, M. J., Penn, C. W., Quail, M. A., Rajandream, M. A., Rutherford, K. M., van Vliet, A. H., Whithead, S., and Barrell, B. G. (2000). The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403, 665668. Paw, B. H., and Zon, L. I. (2000). Zebrafish: A genetic approach in studying hematopoiesis. Curr. Opin. Hematol. 7, 79-84. Pawar, K. R., Ghate, H. V., and Katdare, M. (1983). Effect of malathion on embryonic development of the frog Microhyla ornata (Dumeril and Bibron). Bull. Environ. Contam. Toxicol. 31, 170-176. Pedrazzini, T., Seydoux, J., Kunstner, E, Aubert, J.-E, Grouzman, E., Beermann, E, and Brunner, H.-R. (1998). Cardiovascular response, feeding behavior, and locomotor activity in mice lacking the NPY Y1 receptor. Nat. Med. 4, 722-726. Pellegrini, M., Marcotte, E. M., Thompson, M. J., Eisenberg, D., and Yeates, T. O. (1999). Assigning protein functions by comparative genome analysis: Protein phylogenetic profiles. Proc. Natl. Acad. Sci. U.S.A. 96, 4285- 4288. Pelton, R. W., Saxena, B., Jones, M., Moses, H. L., and Gold, L. I. (1991). Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: Expression patterns suggest multiple roles during embryonic development. J. Cell Biol. 115, 1091-1105. Percy, D. H., and Barta, J. R. (1993). Spontaneous and experimental infections in scid and scid/beige mice. Lab. Anim. Sci. 43, 127-132. Philpott, K. L., Viney, J. L., Kay, G., Rastan, S., Gardiner, E. M., Chae, S., Hayday, A. C., and Owen, M. J. (1992). Lymphoid development in mice congenitally lacking T cell receptor ~[3-expressing cells. Science 256, 14481452. Pick, R., Stamler, J., and Katz, L. (1952). The inhibition of coronary atherosclerosis by estrogen in cholesterol-fed chicks. Circulation 6, 276-282.

E

D

W. QUIMBY

Pinkert, C. A. (1997). The history and theory of transgenic animals. Lab. Anim. 26, 29-34. Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A., Minna, J. E., and Gallo, R. C. (1980). Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. U.S.A. 77, 7415-7419. Poincare, H. (1905). "Science and Hypothesis." Walter Scott Publ. Co., London. Poltorak, A., He, X., Smirnova, I., Liu, M.-Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., RicciardiCastagnoli, P., Layton, B., and Beutler, B. (1998). Defective LPS signaling in C3H/HeJ and C57BL/10 ScCr mice: Mutations in Tlr4 gene. Science 282, 2085-2088. Popovic, M., Sarngadharan, M. G., Reed, E., and Gallo, R. C. (1984). Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 497-500. Powers, D. A. (1989). Fish as model systems. Science 246, 352-361. Preckel, T., Fung-Leung, W. P., Cai, Z., Vitiello, A., Salter-Cid, L., Wingvist, O., Wolfe, T. G., Von Horrath, M., Angulo, A., Ghazal, P., Lee, J. D., Fourie, A. M., Wu, Y., Pang, J., Ngo, K., Peterson, P. A., Fruh, K., and Yang, Y. (1999). Impaired immunoproteasome assembly and immune responses in PA 28 -/- mice. Science 286, 2162-2165. Price, D. L., Sisodia, S. S., and Borchett, D. R. (1998). Genetic neurodegenerative diseases: The human illness and transgenic models. Science 282, 1079-1083. Qian, S., Demetris, A. J., Murase, N., Rao, A. S., Fung, J. J., and Starzl, T. E. (1994). Murine liver allograft transplantation: Tolerance and donor cell chimerism. Hepatology 19, 916- 924. Quimby, F. W. (1994a). Armadillos to zebra fish: Animals in the service of medicine. In "Medical and Health Annual, 1994," pp. 100-123. Encyclopaedia Britannica, Chicago. Quimby, F. W. (1994b). Twenty-five years of progress in laboratory animal science. Lab. Anim. 28, 158-171. Quimby, F. W. (1995). The role of attending veterinarians in laboratory animal welfare. J. Am. Vet. Med. Assoc. 206, 461-465. Quimby, F. W. (1998). Benefits to veterinary medicine from animal research. Appl. Anim. Behav. Sci. 59, 183-192. Quimby, F. W., and Schwartz, R. S. (1980). Etiopathogenesis of systemic lupus erythematostls. In "Pathobiology Annual 1978" (H. L. Ioachin, ed.), Vol. 8, pp. 35-59. Raven Press, New York. Quimby, F. W., Schwartz, R. S., Poskitt, T., and Lewis, R. M. (1979). A disorder in dogs resembling Sj6grens syndrome. Clin. Immunol. Immunopathol. 12, 471-476. Quimby, F. W., Schat, K., Lucio-Martinez, B., Ludders, J., and Van Tienhoven, A. (1995). Poultry. In "The Experimental Animal in Biomedical Research" (B. E. Rollin, ed.), Vol. 2, pp. 195-249. CRC Press, Boca Raton, Florida. Qureshi, S. T., Lariviere, L., Leveque, G., Clemont, S., Moore, K. J., Gros, P., and Malo, D. (1999). Endotoxin-tolerant mice have mutations in Toll-like receptor-4 (Tlr-4). J. Exp. Med. 189, 615-625. Rail, W. F., Schmidt, P. M., Lin, X., Brown, S. S., Ward, A. C., and Hansen, C. T. (2000). Factors affecting the efficiency of embryo cryopreservation and rederivation of rat and mouse models. ILAR J. 41, 221-227. Ram, Z., Culver, K. W., Oshiro, E. M., Viola, J. J., DeVroom, H. L., Otto, E., Long, Z., Chiang, Y., McGarrity, G. J., Muul, L. M., Katz, D., Blaese, R. M., and Oldfield, E. H. (1997). Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat. Med. 3, 1354-1361. Reiser, S. J. (1981). "Medicine and the Reign of Technology." Cambridge Univ. Press, Cambridge. Reitman, M. L., Mason, M. M., Moitra, J., Gavrilova, O., Marcus-Samuels, B., Eckhaus, M., and Vinson, C. (1999). Transgenic mice lacking white fat: Models for understanding human lipoatrophic diabetes. Ann. N.Y. Acad. Sci. 892, 289-296.

30. ANIMAL M O D E L S IN BIOMEDICAL R E S E A R C H

Ren, K., and Dubner, R. (1999). Inflammatory models of pain and hyperalgesia. ILAR J. 40, 111-118. Renegar, K. B. (1992). Influenza virus infections and immunity: A review of human and animal models. Lab. Anim. Sci. 42, 222-232. Riley, E. E., and Weil, M. R. (1987). The effects of thiosemicarbazide on development in the wood frog, Rana sylvatica. II. Critical exposure length and age sensitivity. Ecotoxicol. Environ. Saf 13, 202-207. Robinson, D. (1976). "The Miracle Finders: The Stories behind the Most Important Breakthroughs of Modern Medicine." McKay, New York. Robinson, H. L., Montefiori, D. C., Johnson, R. E, Manson, K. H., Kalish, M. L., Lifton, J. D., Rizvi, T. A., Lu, S., Hu, S. L., Mazzara, G. E, Panicali, D., Herndon, J., Glickman, R., Candido, M. A., Lydy, S. L., Wyand, M. S., and McClure, H. M. (1999). Neutralizing antibody independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations. Nat. Med. 5, 526-534. Rollin, B. E., and Kesel, M. L., eds. (1995). "The Experimental Animal in Biomedical Research," Vol. 2. CRC Press, Boca Raton, Florida. Roperch, J.-E, Alvaro, V., Prieur, S., Tuynder, M., Nemani, M., Lethrosne, E, Piouffre, L., Gendron, M.-C., Israeli, D., Dausset, J., Oren, M., Amson, R., and Telerman, A. (1998). Inhibition of presenilin 1 expression is promoted by p53 and p21 wAF-aand results in apoptosis and tumor suppression. Nat. Med. 4, 835-838. Rose, C. S. (1999). Hormonal control in larval development and evolution-amphibians. In "The Origin and Evolution of Larval Forms" (B. K. Hall, and M. H. Wake, eds.), Chap. 6, pp. 167-216. Academic Press, San Diego. Rose, S. E (2000). God's organism? The chick as a model system for memory studies. Learn. Memory 7, 1-17. Roth, J. A., Nguyen, D., Lawrence, D. D., Kemp, B. L., Carrasco, C. H., Ferson, D. Z., Hong, W. K., Komaki, R., Lee, J. J., Nesbitt, J. C., Pisters, K. M. W., Putnam, J. B., Schea, R., Shin, D. M., Walsh, G. L., Dolormente, M. M., Han, C.-I., Martin, F. D., Yen, N., Xu, K., Stephens, L. C., McDonnell, T. J., Mukhopadhyay, T., and Cai, D. (1996). Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat. Med. 2, 985-990. Roths, J. B., Foxworth, W. B., McArthur, M. J., Montgomery, C. A., and Kier, A. B. (1999). Spontaneous and engineered mutant mice as models for experimental and comparative pathology: History, comparison, and developmental technology. Lab. Anim. Sci. 49, 12-34. Rubin, G. M., and Lewis, E. B. (2000). A brief history of Drosophila's contributions to genome research. Science 287, 2216-2218. Rudolph, K. L., Chang, S., Millard, M., Schreiber-Agus, N., and DePinho, R. A. (2000). Inhibition of experimental liver cirrhosis in mice by telomerase gene delivery. Science 287, 1253-1258. Salzman, L. A., ed. (1986). "Animal Models of Retrovirus Infection and Their Relationship to AIDS." Academic Press, Orlando, Florida. Samad, E, and Loskutoff, D. J. (1999). Hemostatic gene expression and vascular disease in obesity: Insights from studies of genetically obese mice. Thromb. Haemost. 82, 742-747. Sayegh, M. H., and Turka, L. A. (1998). The role of T-cell costimulatory activation pathways in transplant rejection. N. Engl. J. Med. 338, 1813-1820. Scarpelli, D. G. (1997). Animal models of disease: Utility and limitations. In "Biological Aspects of Disease: Contributions from Animal Models" (E M. Iannaccone and D. G. Scarpelli, eds.), pp. 1-13. Harwood Academic Publ., Amsterdam. Sch~ichter, E, Cohen, D., and Kirkwood, T. B. (1993). Prospects for the genetics of human longevity. Hum. Genet. 91, 519-526. Schaedler, R. W., Dubos, R., and Costello, R. (1965). Association of germfree mice with bacteria isolated from normal mice. J. Exp. Med. 122, 77-82. Schaeffer, E. M., Debnath, J., Yap, G., McVicar, D., Liao, X. C., Littman, D. R., Sher, A., Varmus, H. E., Lenardo, M. J., and Schwartzberg, E L. (1999). Requirement for Tec kinases RIK and Itk in T cell receptor signalling and immunity. Science 284, 638-641.

1223 Schmidt, J. E. (1959). "Medical Discoveries--Who and When." Thomas, Springfield, Illinois. Schmidt-Nielsen, B. (1961). Choice of experimental animals for research. Fed. Proc. 20, 902-906. Schulhof, J., and Miller, L. (1990). Insect models: Clarity through simplicity. Lab. Anim. 19, 54-59. Schuytema, G. S., Nebeker, A. V., Griffis, W. L., and Wilson, K. N. (1991). Teratogenesis, toxicity, and bioconcentration in frogs exposed to dieldrin. Arch. Environ. Contam. Toxicol. 21, 332-350. Schwartz, R. S., and Dameshek, W. (1959). Drug-induced immunologic tolerance. Nature 183, 1682-1683. Schwenk, E, Kuhn, H., Angrand, E O., Rajewsky, K., and Stewart, A. E (1998). Temporally and spatially regulated somatic mutagenesis in mice. Nucleic Acids Res. 26, 1427-1433. Seamer, J., and Chesterman, E C. (1967). A survey of disease in laboratory animals. Lab. Anim. 1, 117-139. Seder, R. A., and Gurunathan, S. (1999). DNA vaccines--designer vaccines for the 21st century. N. EngL J. Med. 341, 277-278. Seeley, R. J., van Dijk, G., Campfield, L. A., Smith, E J., Burn, E, Nelligan, J. A., Bell, S. M., Baskin, D. G., Woods, S. C., and Schwartz, M. W. (1996). Intraventricular leptin reduces food intake and body weight of lean rats but not obese Zucker rats. Horm. Metab. Res. 28, 664-668. Sehioth, H. B., Muceniece, R., Larsson, M., and Wikberg, J. E. S. (1997). The melanocortin 1, 3, 4, or 5 receptors do not have a binding epitope for ACTH beyond the sequence of alpha-MSH. J. EndocrinoL 155, 73-78. Sela, M. (1999). Specific vaccines against autoimmune diseases. C.R. Acad. Sci. IlL 322, 933-938. Seriously Ill for Medical Research (SIMR) (1998). "Centenary Survey of Nobel Laureates in Physiology or Medicine." SIMR, Dunstable, United Kingdom. Sessions, S. K., Franssen, R. A., and Horner, V. L. (1999). Morphological clues from multilegged frogs: Are retinoids to blame? Science 284, 800-802. Shafrir, E., Ziv, E., and Mosthaf, L. (1999). Nutritionally induced insulin resistance and receptor defect leading to beta-cell failure in animal models. Ann. N. Y Acad. Sci. 892, 223-246. Shapiro, K. J. (1998). Looking at animal models: Both sides of the debate. Lab. Anim. 27, 26-29. Sharp, J. J., and Davisson, M. T. (1994). The Jackson Laboratory induced mutant resource. Lab. Anim. 23, 33-40. Shoulson, I. (1998). Experimental therapeutics of neurodegenerative disorders: Unmet needs. Science 282, 1072-1074. Sillevis, S. E, and deJong, J. M. (1989). Animal models of amyotrophic lateral sclerosis and the spinal muscular atrophies. J. Neurol. Sci. 91, 231-258. Silvers, W., Elkins, W., and Quimby, E W. (1975). Cellular basis of tolerance in neonatally induced mouse chimeras. J. Exp. Med. 142, 1312-1315. Silverstein, A. M. (1989). "A History of Immunology." Academic Press, San Diego. Sinclair, D. A., Mills, K., and Guarente, L. (1997). Accelerated aging and nucleolar fragmentation in yeast sgsl mutants. Science 277, 1313-1316. Singer, C., and Underwood, E. A. (1962). "A Short History of Medicine," 2nd ed. Oxford Univ. Press, Oxford. Sisson, J. A., and Plotz, E. (1967). Effect of alloxan diabetes on maternal and fetal serum lipids in the rabbit. Exp. Mol. Pathol. 6, 274-282. Smith, R. S., Linder, C. C., and Sundberg, J. E (1995). Mouse models for studying cataracts. Ophthal. News, July 1995, 1-11. Smithies, O., Grett, R. G., Boggs, S. S., Koralewski, M. A., and Kucherlapati, R. S. (1985). Insertion of DNA sequences into the human chromosome [3-globin locus by homologous recombination. Nature 317, 230-234. Sohal, R. S., and Weindruch, R. (1996). Oxidative stress, calorie restriction, and aging. Science 273, 59-63. Sommer, B., Sturchler-Pierrat, C., Abramowski, D., Wiederhold, K. H., Calhoun, M., Jucker, M., Kelly, E, and Staufenbiel, M. (2000). Transgenic approaches to model Alzheimer's disease. Rev. Neurosci. 11, 47-51.

1224

Sordat, B., ed. (1984). "Immune-Deficient Animals." Karger, Basel. Sounder, W. (2000). "A Plague of Frogs." Hyperion, New York. Sourkes, T. L. (1966). "Nobel Prize Winners in Medicine and Physiology 1901-1965." Abelard-Schuman, London. Sparger, E. E., Luciw, P. A., Elder, J. H., Yamamoto, J. K., Lowenstine, L. J., and Pedersen, N. C. (1989). Feline immunodeficiency virus is a lentivirus associated with AIDS-like disease in cats. AIDS 3, $43-$49. Spengler, S. J. (2000). Bioinformatics in the informational age. Science 287, 1223 -1227. Spicker, S. E, Alon, I., deVries, A., and Engelhardt, H. T. (1988). "The Use of Human Beings in Research." Kluwer Academic Publ., Dordrecht, The Netherlands. Stanton, H. C., and Mersmann, H. J. (1986). "Swine in Cardiovascular Research," Vols. 1 and 2. CRC Press, Boca Raton, Florida. Starzl, T. E. (1993). Chimerism after whole organ transplant. Guthrie J. 62, 49-53. Starzl, T. E., and Zinkernagel, R. M. (1998). Antigen localization and migration in immunity and tolerance. N. Engl. J. Med. 339, 1905-1912. Staufenbiel, M., Calhoun, M. E., Wiederhold, K. H., Abramowski, D., Phinney, A. L., Probst, A., Sturchler-Pierrat, C., Sommer, B., and Tucker, M. (1998). Neuron loss in APP transgenic mice. Nature 395, 755-756. Steisinger, G., Walker, C., Dower, N., Knauber, D., and Singer, E (1981). Production of clones of homozygous diploid zebrafish. Nature 291, 293-296. Stewart, J. (2000). Pathways to relapse: The neurobiology of drug- and stressinduced relapse to drug-taking. J. Psychiatry Neurosci. 25, 125-136. Stone, J., Maslim, J., Valter-Kocsi, K., Mervin, K., Bowers, E, Chu, Y., Barnett, N., Provis, J., Lewis, G., Fisher, S. K., Bisti, S., Gargini, C., Cervetto, L., Merin, S., and Peer, J. (1999). Mechanisms of photoreceptor death and survival in mammalian retina. Prog. Retin. Eye Res. 18, 689735. Stormont, C. (1968). Perspectives in the development of new animals for research. Lab. Anim. Care 18, 298-304. Strauss, E. J., and Falkow, S. (1997). Microbial pathogenesis: Genomics and beyond. Science 276, 707-712. Strode, G. K., ed. (1951). "Yellow Fever." McGraw-Hill, New York. Sullivan, M. P., Cerda, J. J., Robbins, E L., Burgin, C. W., and Beatty, A. J. (1993). The gerbil, hamster, and guinea pig as rodent models for hypeflipidemia. Lab. Anim. Sci. 43, 575-578. Sundberg, J. P. (1994). "Handbook of Mouse Mutations with Skin and Hair Abnormalities, Animal Models, and Biomedical Tools." CRC Press, Boca Raton, Florida. Sundberg, J. P., and Boggess, D., eds. (1999). "Systematic Approach to Evaluation of Mouse Mutations." CRC Press, Boca Raton, Florida. Supattapone, S., Bosque, P., Muramoto, T., Wille, H., Aagaard, C., Peretz, D., Nguyen, H. O., Heinrich, C., Torchia, M., Safar, J., Cohen, E E., De Armond, S. J., Prusiner, S. B., and Scott, M. (1999). Prion protein of 106 residues creates an artificial transmission barrier for prion replication in transgenic mice. Cell 96, 869-878. Swindle, M. M., and Adams, R. J. (1988). "Experimental Surgery and Physiology Induced Animal Models of Human Disease." Williams and Wilkins, Baltimore. Swindle, M. M., Moody, D. C., and Phillips, L. D. (1992). "Swine as Models in Biomedical Research." Iowa State Univ. Press, Ames. Takahashi, J. S., Pinto, L. H., and Vitaterna, M. H. (1994). Forward and reverse genetic approaches to behavior in the mouse. Science 264, 1724-1733. Tamada, K., Shimozaki, K., Chapoval, A. I., Zhu, G., Sica, G., Flies, D., Boone, T., Hsu, H., Fu, Y.-X., Nagata, S., Ni, J., and Chen, L. (2000). Modulation of T-cell-mediated immunity in tumor and graft-versus-host disease models through the LIGHT co-stimulatory pathway. Nat. Med. 6, 283 -289. Tashijan, R. J., and Crusberg, T. C. (1989). Equine infectious anemia as an AIDs animal model. J. Equine Vet. Sci. 9, 105-110. Taylor, P. C. (1994). "The Use of SCID Mice in the Investigation of Human Autoimmune Disease." CRC Press, Boca Raton, Florida.

FRED W. QUIMBY Tenorio, E., Ermal, R., Giri, S., and Brooks, D. (1992). Caprine arthritis encephalitis virus (CAEV) infection in goats as a model for rheumatoid arthritis. Lab. Anim. 21, 33-37. Tettelin, H., Saunders, N. J., Heidelberg, J., Jeffries, A. C., Nelson, K. E., Eisen, J. A., Ketchum, K. A., Hood, D. W., Peden, J. F., Dodson, R. J., Nelson, W. C., Gwinn, M. L., DeBoy, R., Peterson, J. D., Hickey, E. K., Haft, D. H., Salzberg, S. L., White, O., Fleischmann, R. O., Dougherty, B. A., Mason, T., Ciecko, A., Parksey, D. S., Blair, E., Cittone, H., Clark, E. B., Cotton, M. D., Utterback, T. R., Khouri, H., Qin, H., Vamathevan, J., Gill, J., Scarlato, V., Masignani, V., Pizza, M., Grandi, G., Sun, L., Smith, H. O., Frazer, C. M., Moxon, E. R., Rappuoli, R., and Venter, J. C. (2000). Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287, 1809-1815. Theilen, G. (1988). Studies of animal oncology with relevance to biomedical advances. J. Am. Vet. Med. Assoc. 193, 1124-1130. Thomas, P. K. (1992). Diabetic neuropathy: Models, mechanisms, and mayhem. Can. J. Neurol. Sci. 19, 1-7. Thucydides (1934). "The Peloponnesian War" (trans. R. Crawley), p. 112. Modern Library, New York. Timmerman, J. M., and Levy, R. (1998). Melanoma vaccines: Prim and proper presentation. Nat. Med. 4, 269-270. Tokman, M. G., and Quimby, F. W. (1995). Pathogenesis of experimental toxic shock syndrome: Effect of IL-2 on the induction of hypotension and cytokine synthesis. Shock 3, 145-151. Tolwani, R. J., Jakowec, M. W., Petzinger, G. M., Green, S., and Waggie, K. (1999). Experimental models of Parkinson's disease: Insights from many models. Lab. Anim. Sci. 49, 363-371. Trexler, P. C., and Reynolds, L. I. (1957). Flexible film apparatus for rearing and use of germ-free animals. Appl. Microbiol. 5, 6-11. Tsolis, R. M., Kingsley, R. A., Townsend, S. M., Ficht, T. A., Adams, L. G., and Baumler, A. J. (1999). Of mice, calves, and men. Comparison of the mouse typhoid model with other samonella infections. Adv. Exp. Med. Biol. 473, 261-274. Ulevitch, R. J. (1999). Endotoxin opens the Toll gates to innate immunity. Nat. Med. 5, 144 - 145. Utomo, A. R. H., Nikitin, A. Y., and Lee, W.-H. (2000). Temporal, spatial, and cell type specific control of Cre-mediated DNA recombination in transgenic mice. Nat. Biotechnol. 17, 1091-1096. van Gelder, N. M., and Belanger, F. (1988). Embryonic exposure to high taurine: A possible nutritional contribution to Friedreich's ataxia. J. Neurosci. Res. 20, 383-388. Van Hoosier, G. L., and McPherson, C. W., eds. (1987). "Laboratory Hamsters." Academic Press, New York. van Leuven, F. (2000). Single and multiple transgenic mice as models for Alzheimer's disease. Prog. Neurobiol. 61, 305-312. Vogel, G. (1999). Mice cloned from cultured stem cells. Science 286, 2437. Waggie, K. S., Kahle, P. J., and Tolwani, R. J. (1999). Neurons and mechanisms of neuronal death in neurodegenerative diseases: A brief review. Lab. Anim. Sci. 49, 358-362. Wagner, J. E., and Manning, P. J., eds. (1976). "The Biology of the Guinea Pig." Academic Press, New York. Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R., and Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369-374. Wallace, J. M. (2000). Nutrient partitioning during pregnancy: Adverse gestational outcome in overnourished adolescent dams. Proc. Nutr. Soc. 59, 107-117. Wassom, K., and Peper, R. L. (2000). Mammalian microsporidiosis. Vet. Pathol. 37, 113-128. Watson, J. D., and Crick, F. H. C. (1953). A structure for deoxyribose nucleic acid. Nature 171, 737-743. Weinblatt, M. E., Kremer, J. M., Bankhurst, A. D., Bulpitt, K. J., Fleischmann, R. M., Fox, R. I., Jackson, C. G., Lange, M., and Burge, D. J. (1999). A trial of etanercept, a recombinant tumor necrosis factor receptor: Fc fusion pro-

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH tein, in patients with rheumatoid arthritis receiving methotrexate. N. Engl. J. Med. 340, 253-259.

Weinstock, G. M. (2000). Genomics and bacterial pathogenesis. Emerg. Infect. Dis. 6, 496-504. Weisman, A., Ruiz, P. J., Hirschberg, D. L., Gelman, A., Oksenberg, J. R., Brocke, S., Mor, E, Cohen, I. R., and Steinman, L. (1996). Suppressive vaccination with DNA encoding a variable region gene of the T cell receptor prevents autoimmune encephalitis and activates Th2 immunity. Nat. Med. 2, 899-905. Weisse, A. (1991). "Medical Odysseys: The Different and Sometimes Unexpected Pathways to Twentieth-Century Medical Discoveries." Rutgers Univ. Press, New Brunswick, New Jersey. Wells, A. D., Li, X. C., Li, Y., Walsh, M. C., Zheng, X. X., Wu, Z., Nunez, G., Tang, A., Sayegh, M., Hancock, W. W., Strom, T. B., and Turka, L. A. (1999). Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nature Med. 5, 1303 - 1307. Wenger, D. A., Raft, M. A., Luzi, P., Datto, J., and Costantina-Ceccarini, E. (2000). Krabbe disease: Genetic aspects and progress toward therapy. Mol. Genet. Metab. 70, 1-9. Wetterau, J. R., Gregg, R. E., Harrity, T. W., Arbeeny, C., Cap, M., Connolly, E, Chu, C.-H., George, R. J., Gordon, D. A., Jamil, H., Jolibois, K. G., Kunselman, L. K., Lan, S.-J., Maccagnan, T. J., Ricci, B., Yan, M., Young, D., Chen, Y., Fryszman, O. M., Logan, J. V. H., Musial, C. L., Poss, M. A., Robl, J. A., Simpkins, L. M., Slusarchyk, W. A., Sulsky, R., Taunk, P., Magnin, D. R., Tino, J. A., Lawrence, R. M., Dickson, J. K., and Billar, S. A. (1998). An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits. Science 282, 751-754. Whittingham, D., Leibo, S., and Mazure, P. (1972). Survival of mouse embryos frozen to -192°C and -269°C. Science 178, 411-414. Willems, P. J. (2000). Genetic causes of hearing loss. N. Engl. J. Med. 342, 1101-1108. Wilmut, I. (1998). Cloning for medicine. Sci. Am. 279, 58-63. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., and Campbell, K. H. S. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810- 813. Wong, E S., Dittel, B. N., and Janeway, C. A. (1999a). Transgenes and knockout mutations in animal models of type 1 diabetes and multiple sclerosis. Immunol. Rev. 169, 93-104. Wong, E S., and Janeway, C. A. (1999). Insulin-dependent diabetes mellitus and its animal models. Curr. Opin. Immunol. 11, 643-647. Wong, E S., Karttunen, J., Dumont, C., Wen, L., Visintin, I., Rlip, I. M., Shastri, N., Pamer, E. G., and Janeway, C. A. (1999b). Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organspecific cDNA library. Nat. Med. 5, 1026-1031. Wood, P. A. (2000). Phenotype assessment: Are you missing something? Comp. Med. 50, 12-15. Woodhead, A. D., ed. (1989). "Nonmammalian Animal Models for Biomedical Research." CRC Press, Boca Raton, Florida. Woodruff, M. F. A., and Lennox, B. (1959). Lancet 2, 476. Woods, S. C., Seeley, R. J., Porte, D., Jr., and Schwartz, M. W. (1998). Signals that regulate food intake and energy homeostasis. Science 280, 13781382. Wooster, R., Bignell, G., Lancaster, J., Swift, S., Seal, S., Mangion, J., Collins, N.,

1225 Gregory, S., Gumbs, C., Micklem, G., Barfoot, R., Hamoudi, R., Patel, S., Rice, C., Biggs, P., Hashim, Y., Smith, A., Connov, E, Arason, A., Gudmundsson, J., Ficenec, D., Kefsell, D., Ford, D., Tonin, P., Bishop, D. T., Spurr, N. K., Ponder, B. A. J., Eeles, R., Peto, J., Devilee, P., Cornelisse, C., Lynch, H., Narod, S., Lenoir, G., Egilsson, V., Barkadottir, R. B., Easton, D. F., Bentley, D. R., Futreal, P. A., Ashworth, A., and Stratton, M. R. (1995). Identification of the breast cancer susceptibility gene BRCA2. Nature 378, 789-792. Wright, S. D. (1999). Toll, a new piece in the puzzle of innate immunity. J. Exp. Med. 189, 605-609. Wu, G. D. (2000). A nuclear receptor to prevent colon cancer. N. Engl. J. Med. 342, 651-653. Wu, C.-Y., Ferrante, J., Gately, M., and Magram, J. (1997). Characterization of IL- 12 receptor [31 chain (IL- 12R [31)-deficient mice. IL- 12R[31 is an essential component of the functional mouse IL-12 receptor. J. Immunol. 159, 1658-1665. Wtirsig, B. (1989). Cetaceans. Science 244, 1550-1559. Wyatt, R., and Sodroski, J. (1998). The HIV-1 envelope, glycoproteins: Fusogens, antigens, and immunogens. Science 280, 1884-1888. Yamagata, K., Furuta, H., Oda, O., Kaisaki, P. J., Menzel, S., Cox, N. J., Fajans, S. S., Signorini, S., Stoffel, M., and Bell., G. J. (1996). Mutations in the hepatocyte nuclear factor-4a gene in maturity onset diabetes of the young (MODY 1). Nature 384, 458-460. Yamori, Y. (1999). Implication of hypertensive rat models for primordial nutritional prevention of cardiovascular diseases. Clin. Exp. PharmacoL Physiol. 26, 568-572. Yu, L., Miki, T., Nakura, J., Oshima, J., Kamino, K., Rakugi, H., Ikegami, H., Higaki, J., Edland, S. D., Martin, G. M., and Ogihara, T. (1997). Association of a polymorphic variant of the Werner helicase gene with myocardial infarction in a Japanese population. Am. J. Med. Genet. 68, 494-498. Zack, D. J., Dean, M., Molday, R. S., Redmond, T. M., Nathans, J., Stone, E. M., Swaroop, A., Valle, D., and Weber, B. H. (1999). What can we learn about age-related macular degeneration from other retinal diseases? Mol. Vis. 3, 30-37. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J. M. (1994). Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425-432 [Erratum, Nature 374, (1995) 479]. Zhang, Z., Hartmann, H., Do, V. M., Abramowski, D., Sturchler-Pierrat, C., Staufenbiel, M., Sommer, B., van de Wetering, M., Clevers, H., Saftig, P., Strooper, B. D., He, X., and Yankner, B. A. (1998). Destabilization of [3catenin by mutations in presenilin-1 potentiates neuronal apoptosis. Nature 395, 698-702. Zhou, M., Sutliff, R. L., Paul, R. J., Lorenz, J. N., Hoying, J. B., Haudenschild, C. C., Ying, M., Coffin, J. D. Kong, L., Kramas, E. G., Luo, W., Boivin, G. P., Duffy, J. J., Pawlowski, S. A., and Doetschman, T. (1998). Fibroblast growth factor 2 control of vascular tone. Nat. Med. 4, 201-207. Zhu, Z. Y., and Sun, Y. H. (2000). Embryonic and genetic manipulation in fish. Cell Res. 10, 17-27. Zigmond, M. J., and Stricker, E. M. (1989). Animal models of parkinsonism using selective neurotoxins: Clinical and basic implications. Int. Rev. Neurobiol. 31, 1-79. Zinkernagel, R. M., and Doherty, P. C. (1997). The discovery of MHC restriction. Immunol. Today 18, 14-17.

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Chapter 31 Research in Laboratory Animal and Comparative Medicine Henry J. Baker and J. Russell Lindsey

I.

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VI.

LABORATORY ANIMAL MEDICINE, 2nd edition

Introduction .................................................

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Historical Perspective

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Comparative Medicine .....................................

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R e s e a r c h as a F o u n d a t i o n of the Specialty

The Research Process

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The Research Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Research Design

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A n i m a l Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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R e p o r t i n g R e s e a r c h Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Research Training ............................................

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F r o m the D.V.M. to L a b o r a t o r y A n i m a l Veterinarian

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C h a r a c t e r i s t i c s of L a b o r a t o r y A n i m a l M e d i c i n e T r a i n i n g

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G r a d u a t e E d u c a t i o n and L a b o r a t o r y A n i m a l M e d i c i n e . . . . . . . . . . . .

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S u p p o r t for T r a i n i n g

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Research Resources ...........................................

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Animal Resources

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Research Laboratories ..................................... Diagnostic Laboratories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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L i b r a r y and Electronic Literature S e a r c h i n g . . . . . . . . . . . . . . . . . . . .

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S u p p o r t for R e s e a r c h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Grantsmanship ...........................................

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Public Sources of S u p p o r t for R e s e a r c h

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Private Sources of S u p p o r t for R e s e a r c h . . . . . . . . . . . . . . . . . . . . . . . Intellectual P r o p e r t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Summary ...................................................

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References ..................................................

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Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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HENRY J. BAKER AND J. RUSSELL LINDSEY

I.

A.

INTRODUCTION

Historical Perspective

Laboratory animal medicine is the only specialty of the American Veterinary Medical Association in which diplomates are expected to practice their specialty almost entirely in biomedical research institutions and who are constantly involved in the research process. Consequently, it is reasonable to expect that laboratory animal specialists, particularly diplomates of the American College of Laboratory Animal Medicine (ACLAM), should be trained thoroughly in research and capable of functioning as research scientists. The founders of ACLAM recognized the importance of research skills for laboratory animal specialists and incorporated this key element into all aspects of standards that define training and experience necessary for certification in the specialty. Today, ACLAM's requirements for training, application for membership, certification examination, educational programs, and recertification emphasize scholarship and achievement in research relevant to the specialty (see ACLAM website ). In addition to an appreciation for research, which is needed to function successfully in an intensive research environment, the very foundation of the specialty and its future viability depends on the acquisition of new knowledge that will improve the use of animals in science. For example, 30 years ago it was common for suppliers of laboratory mice to be unable to fill orders during the summer because rotavirus infection, known as epizootic diarrhea of infant mice (EDIM), killed entire populations of neonatal laboratory mice. Solution of this vexing problem required the pioneering work of laboratory animal scientists, who discovered elegantly simple methods that prevented these zoonotics that were so disruptive to biomedical research (Kraft et al., 1964). Today, the exclusion of such pathogens is expected by laboratory animal specialists and investigators alike, and zoonotics of this type would not be tolerated by the scientific community. Abundant examples can be cited of research and development contributions of a highly productive cadre of research scientists within and outside of ACLAM that have been responsible for the high level of sophistication in animal experimentation enjoyed today. Although laboratory animal scientists can take much of the credit for research and development responsible for the current state of high-quality laboratory animal resources, an equally sustained effort will be required in the future if progress is to be made in meeting the laboratory animal challenges that accompany each advance in biomedical research. It is the responsibility of laboratory animal specialists to assure that the commitment, resources, and talent are available and applied to this important mission of the specialty, if laboratory animal medicine and science are to endure and flourish.

B.

Comparative Medicine

The conceptual basis for comparative medicine can be found in the visionary statements of some of the greatest innovators in biology and medicine at the turn of the twentieth century. Rudolf Virchow (1821-1902) captured the essence of comparative medicine in the much-quoted statement: "Between animal and human medicine there is no dividing line, nor should there be. The objective is different, but the experience obtained constitutes the basis of all medicine." Sir William Osier (18491919), Chairman of Medicine and Dean of the Johns Hopkins School of Medicine, was not only a notable proponent, but also a practitioner of the comparative approach, which he called "one medicine." In the 1870s and 1880s, he published numerous articles on comparative pathology of nematodes in dogs, hog cholera, echinococcosis, bovine tuberculosis, and parasites of pork (Harvey et al., 1989). Even though Claude Bernard (1813-1878) is reviled by modern opponents of animal research, he is considered to be the founder of experimental physiology and expressed his strong conviction about comparative medicine in 1865: "I not only conclude that experiments made on animals from the physiological, pathological, and therapeutic points of view have results that are applicable to theoretical medicine, but I think that without such comparative study of animals, practical medicine can never acquire scientific character." The concept of comparative anatomy is easily understood as the study of similar and dissimilar morphology of two or more species, but the dominion of comparative medicine may not be as clear or intuitive. In essence, comparative medicine includes characterization of similarities and differences in disease processes affecting humans and other animals. In the same way that the scope of comparative medicine is not generally understood, practitioners of the specialty are also not well defined. Obviously, successful application of the comparative approach to science requires knowledge of disease processes in both animals and humans. For veterinarians, this means at least an introduction to human diseases and in-depth study of the literature pertaining to specific diseases. Conversely, those trained in medicine or dentistry must gain a fundamental understanding of animal diseases and animal modeling. To some, comparative medicine represents the academic and research specialty companion to laboratory animal medicine. Although a division of emphasis can be made in this way, it serves no useful purpose and tends to segregate rather than integrate these companion, mutually complementary activities. The need for research is as critical for improving the suitability and health of animals used in research as it is for discovering new animal models of human diseases. Therefore, in this chapter, use of these terms will be overlapping and not meant to distinguish between research or training activities in animal care or modeling. Although laboratory animal scientists have embraced the concept of comparative medicine and advanced it more than most other medical

31. RESEARCH IN LABORATORYANIMALAND COMPARATIVEMEDICINE specialties, it is certainly not an exclusive domain, and continued recognition and acceptance by the biomedical community for its place as a veterinary specialty will demand sustained, noteworthy contributions to the field. Unfortunately, the recent increased emphasis on regulation of animal research has so seriously distracted laboratory animal specialists from participation in productive research that a dangerous trend is in progress, which, if allowed to progress unchecked, could disenfranchise laboratory animal veterinarians from leadership in comparative medicine. In spite of the lack of clarity about the boundaries and participants of comparative medicine, the value of this approach to medical science cannot be disputed. Clarence Cook Little's effort early in the twentieth century to genetically "purify" the mouse as a model for cancer research exemplifies the power of the comparative approach (Little, 1913; also see Mobraaten and Sharp, 1999). The eventual success in achieving genetic homozygosity in laboratory mice and the largely unanticipated occurrence of a multitude of spontaneous diseases, including cancer, in these strains may have contributed more to advancing the health of humans and animals than any other achievement in comparative medicine in the twentieth century. Inbred mice have been key to understanding cancer, susceptibility to infections, inherited diseases of all types, and the basic nature of the genome in health and disease. There are numerous examples of contributions made by laboratory animal scientists that have significantly altered biomedical research using animals (see Chapter 30 and McPherson and Mattingly, 1999). With the advent of powerful research technologies, particularly those of molecular biology, opportunities abound to continue contributions to comparative medicine. Laboratory animal specialists have unlimited opportunities, and in fact an obligation, to contribute to this incredibly exciting and important part of the specialty.

C.

Research as a Foundation of the Specialty

Subspecialization is not recognized or endorsed by ACLAM. However, there appears to be an informal trend toward division of laboratory animal scientists who emphasize clinical and service activities and those who concentrate their effort on research. This division of interests and activities has been accentuated by an increasing emphasis on regulatory and compliance issues raised by the U.S. Department of Agriculture's enforcement of the Animal Welfare Act, the National Institutes of Health (NIH) requirements to comply with the U.S. Public Health Service policies on animal welfare, accreditation of animal care programs by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), and institutional policies and procedures such as

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the Institutional Animal Care and Use Committee review and approval of all research projects using animals. One justification that has been advanced to support this inclination toward division of effort is that success in today's highly competitive research arena requires more than part-time participation, which is usually required by specialists who have a substantial commitment to clinical and service activities. The hazard of this arrangement is development of a two-tiered system that encourages separation of interests, discourages interaction, and can be a basis for adversarial attitudes. In the long run, the specialty and its members would suffer from such divisions for a variety of reasons. First, the further the distance between colony management and disease problems, which clinicians know best, and research to solve those problems, the greater the likelihood that research will become less relevant to advancing laboratory animal science. Second, the proportion of specialists who participate in research is directly related to the emphasis placed on scientific advancement of the specialty. If a high proportion of specialists isolate themselves from research, the specialty is in danger of losing a pillar of its founding precepts. Eventually, complete loss of research productivity by any specialty presages conversion of a profession to a trade. When the profession is practiced in academic and research institutions, this contrast is accentuated, and the loss of acceptance as a peer group could be rapid. Regrettably, movement in this direction is currently in progress, and there does not appear to be a concerted effort to slow or reverse the trend. Although the ACLAM and other organizations representing laboratory animal medicine recognize the importance of clinical medicine, the specialty must aggressively preserve, encourage, and enhance participation of specialists in productive research, if the scientific character of the specialty is to be maintained. Institutions that are unable to employ more than one specialist severely restrict opportunities for participation in research. In contrast, at institutions where several specialists share responsibilities, opportunities for research are much improved. Therefore, larger institutions must share greater responsibility for assuring research productivity of the specialty.

II.

THE RESEARCH PROCESS

A.

The Research Cycle

The research process can be considered to be a cycle of activities starting with a statement of a problem or question and ending with an answer to that question. The steps intervening between the beginning and ending activities include (1) searching the scientific literature for existing information that addresses

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HENRY J. BAKER AND J. RUSSELL LINDSEY

B. R e s e a r c h Design the problem, (2) critically assessing relevant literature to distinguish between valid conclusions based on sound data and errors Skillful research design is fundamental to all successful exin experimental execution or interpretation, (3) formulating a hypothesis or statement that addresses the problem and that will periments. A comprehensive discussion of the elements of good be tested, (4) designing one or more experiments to test the hy- experimental design is beyond the scope of this chapter, and the pothesis, (5) conducting the experiments according to a well- reader is referred to excellent publications on this subject defined experimental protocol, (6) evaluating results, (7) reach- (Navia, 1977). Good design starts with a clearly stated research ing conclusions by interpretation of the results, and (8) sharing problem amenable to testing by a series of manipulations that results and conclusions through presentations and publications. will result in clear differences between experimental and conNote that of these eight major steps, only one (step 5) involves trol groups. From the outset, methods to control confounding physically working in the laboratory. This disproportionate ra- variables independent of the subject parameter must be emtio of "thinking" and "doing" is contrary to the common per- ployed if small differences between experimental and control ception of the research process, but the investment in thinking groups are to be distinguished. Of all the subjects in experiand planning cannot be overestimated in importance (Navia, mental design of research using animal subjects, control of biological variables should be the dominion and expertise of the 1977). Discovery and characterization of naturally occurring animal laboratory animal specialist. This topic is worthy of extensive models of human diseases and other types of clinical investiga- discussion, and the reader is referred to Chapter 29 of this voltion may follow a different course of research activities than ume, "Factors That May Influence Animal Research," for com"hypothesis-driven" research, but although the emphasis may be prehensive descriptions of environmental factors that can different, the basic precepts remain the same. The fundamental influence research results and can be controlled by skillful pardifference is that testing a hypothesis by experimental manipu- ticipation by laboratory animal veterinarians. Selection of the lation of subjects allows for greater control of all variables other most appropriate animal subject or model is also a key element than the single parameter being studied. Clinical investigations in research design and the natural domain of the laboratory anmay not permit such elegant control of confounding variables. imal specialist. To a large extent, this requires an encyclopedic Nevertheless, clinical research can yield results as informative knowledge of the biology of several species, genetics of experand valuable as experimental designs. In the past, discovery of imental animals, detection of occult infections, and naturally disease models happened by serendipitous opportunities pre- occurring or induced animal models of human diseases. Chapsented to individuals with a mind-set prepared to recognize and ter 30 of this volume addresses some of the issues of animal pursue the opportunity. In contrast, genetic engineering using modeling. Other publications include "Spontaneous Animal transgenic technology has made it possible to selectively insert Models of Human Disease" in the ACLAM/Academic Press seforeign genes or disable endogenous genes ("knockout") in ries (Andrews et al., 1979; Desnick et al., 1982) and the Armed mice. In fact, this powerful new technology makes it possible to Forces Institute Registry of Comparative Pathology "Animal design and test hypotheses using cleverly modified transgenic Models of Human Disease" (Capen et al., 1985). These refermice. In addition to new professional opportunities created by ences provide a starting point that should be supplemented by transgenic technology, there is an increasing need to character- exhaustive search of the literature for details pertaining to a ize transgenic mice using histopathology, biochemistry, and specific research objective. molecular biology. These professional opportunities should attract specialists in comparative medicine and pathology. AlC. A n i m a l Subjects though transgenic animal models provide enormously important research opportunities, it is important to recognize that Unless the research topic involves the development of in vitro there are many examples of mice modified in this way that remethods to replace animal use in research, then animal subjects spond very differently than human and animal patients with corbecome a central theme for research pursued by laboratory aniresponding inherited diseases. Transgenic mice may show no clinical signs of disease or have lethal consequence of the gene mal medicine practitioners and trainees. The process of condeletion (Phaneuf et al., 1996). To be certain, these differences ducting animal research involves basic skills and issues that all in the response of mice can be instructive by identifying alter- research scientists deal with continuously but that are critically nate metabolic pathways that enable mice to circumvent patho- important for laboratory animal veterinarians to master. The logical consequence of a mutation. However, the experiments of special skills and insights required for successful research innature (naturally occurring mutations and other diseases in do- volving animals include (1) understanding the biology of spemestic animals) remain a valuable source for modeling the hu- cies in the context of their use in specific types of research, man condition and remain worthy of pursuit when they are en- (2) selecting appropriate experimental animal subjects, (3) developing skills required for manipulating animals in expericountered (see Chapters 28 and 30).

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31. RESEARCH IN LABORATORY ANIMAL AND COMPARATIVE MEDICINE

mental situations, (4) assuring that the animals to be used are healthy and not subjected to conditions that might confound the research objectives, (5) practicing humane treatment of animals, (6) and satisfying regulatory requirements. The literature that addresses many of these issues is widely dispersed but must be searched for specifics about selecting and using animals in biomedical research. The ACLAM text series published by Academic Press is an excellent starting point to find such information (see ACLAM website for a listing of titles). In addition to locating and understanding pertinent literature, firsthand experience gained through conducting research is an essential element of a well-rounded training program. Laboratory animal scientists are on the front lines of institutional commitment to compassionate and humane use of animals in research. Good science and good ethics demand that investigators select the least-invasive and most humane methods of animal research. Eaboratory animal veterinarians must be leaders in this vital research issue through collaboration or consultation. This can be accomplished only by careful consideration of research objectives and innovation to achieve these objectives with the least risk of pain or discomfort to animal subjects. Proceedings of an ACLAM symposium on the topic of "Advancing Science and Animal Welfare in the 21st Century" addresses advances made in the development of more humane methods of animal experimentation (Kraus, 2001).

D.

Reporting Research Results

Arguably, sharing research results through presentations and publications is the most important step in the research process. If results are not shared with others in the field, then the effort expended is completely wasted, other than personal gratification gained by the investigator performing the research! Value of the reporting process includes (1) formally organizing data and formulating conclusions; (2) providing an opportunity for critical review of results by others, such as meeting attendees, manuscript reviewers, editors, and readers; (3) assisting other investigators in developing better research designs or preventing unnecessary duplication of research; and (4) enabling granting agencies to recognize competence in a field for which research support may be sought. Careful consideration should be given when selecting meetings where results are reported and journals where manuscripts may be published to maximize the distribution of information to the segment of the scientific community who are most likely to be interested in and influenced by a study. In the laboratory animal and comparative medicine community, scientific meetings sponsored by ACLAM, American Association for Laboratory Animal Science (AALAS), and American Veterinary Medical Association (AVMA) are particularly relevant. Journals serving the specialty include Comparative Medicine (formerly Laboratory Animal Science), the

American Journal of Veterinary Research, the American Journal of Pathology, the American Journal of Medical Genetics (Animal Models series), Comparative Immunology, Microbiology and Infectious Diseases, Veterinary Immunology and Immunopathology, Journal of Comparative Pathology, Research in Veterinary Science, etc. Information that may have high impact on specific disease specialities should be submitted for presentation at meetings and publication in journals serving that particular discipline.

III.

A.

RESEARCH TRAINING

From the D.V.M. to Laboratory Animal Veterinarian

The traditional veterinary professional curriculum provides an outstanding foundation for development of laboratory animal specialists, but does not include the specialized knowledge and skills required for successful participation in clinical laboratory animal medicine or research. Veterinary students can augment their introductory course in laboratory animal medicine by serving externships, preceptorships, and summer fellowships at institutions that have departments of comparative medicine or laboratory animal medicine. Experience gained from these short-term training opportunities helps to solidify emerging interests in the specialty and provide insights into the culture of the specialty that cannot be gained from the lecture room. Such short-term experiences also provide faculty of laboratory animal medicine training institutions with the opportunity to gain firsthand knowledge about students who may proceed to seek extensive postdoctoral training in the specialty. Other types of research opportunities can be found at academic departments of medical and veterinary schools, research centers, national primate centers, and industrial settings. Detailed information about institutions offering short-term training experiences that are supported by NIH can be found at under listings for "ShortTerm Training Awards for Students of Health Professional Schools," and under "Institutional Training Awards," "Regional Primate Research Centers," and "Laboratory Animal Science Centers." Individual colleges of veterinary medicine and major pharmaceutical firms should be contacted for information about summer research fellowships, preceptorships, and similar preD.V.M. training opportunities that they may offer.

BO Characteristics of Laboratory

Animal Medicine Training Graduate veterinarians aspiring to become laboratory animal medicine specialists should seek a well-designed postdoctoral

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training program in an institution that provides a curriculum that meets the ACLAM standards. Even though on-job experience is an acceptable alternative to formal training to qualify for taking the ACLAM specialty board examination, it is less efficient and is usually not as comprehensive as formal training programs. A list of institutions offering formal training in laboratory animal medicine that are able to meet standards recognized by ACLAM can be found in the ACLAM website address cited. These should be viewed as minimal standards, since most training programs offer opportunities in addition to criteria prescribed for preparation to be certified by ACLAM. Potential applicants for training should inquire about the characteristics of each program being considered. Points of interest should include (1) experience of the institution and faculty in training laboratory animal specialists; (2) emphasis of the program on research, clinical, and administrative skills; (3) clinical expertise of the faculty; (4) opportunity to earn a graduate degree; (5) number of trainees currently in the program; (6) names and current positions of former trainees; (7) availability and amount of training stipends; (8) division of effort between clinical, service, research, and didactic activities; (9) institutional resources, such as diagnostic laboratories, as well as numbers and types of species being used in research; and (10) research strengths of training faculty, as Well as those of the entire institution. Didactic courses offered by a candidate-training institution should be compared with the core of knowledge identified by ACLAM as required for certification. Resources at a training institution that are critical for a good training experience include laboratories equipped with modern research instrumentation, diverse species used as experimental subjects, diagnostic laboratories staffed by qualified experts in laboratory animal clinical and anatomical pathology, a working library with substantial holdings pertaining to animal research, experimental surgery support laboratories, and adequate veterinary-assistant technical support. After receiving formal information about training programs of interest, applicants should schedule a telephone conversation with the appropriate contact person listed on the ACLAM website and discuss items of special interest or those needing clarification. A personal visit to the department or division offering training, which includes discussions with faculty and trainees, is highly advisable. A formal application process and interview are normally required by most training programs. Recommendations from individuals who know an applicant well and who have experience in the specialty can be very influential in the competition for training positions. Because of the unique role that laboratory animal specialists play in biomedical research, all training programs must include instruction and experience in both clinical and research topics, regardless of the intention of the trainee to emphasize one activity or the other after training. In fact, the credentials requirements of ACLAM demand evidence of a broad exposure to both activities. It is not necessary that both training activities occur

HENRY J. BAKERAND J. RUSSELL LINDSEY concurrently. Actually, some programs prefer to start postdoctoral training by emphasizing clinical experience for the first year. Since the trainee contributes substantially to the service activities of the institutional animal resources program, institutional support for the trainee's stipend during this time is well justified. All postdoctoral fellows supported by the U.S. Public Health Service (PHS) are required to devote at least 75-80% of effort to research training for the term of this award. Therefore, clinical training must be emphasized before or after, but not during PHS-sponsored training. The number and diversity of animals used in research can be key elements in assuring a broad training experience. In most institutions, rodents constitute the largest populations of research animals, but exposure to other species such as primates, lagomorphs, birds, amphibia, etc., is equally important. Even though graduate veterinarians are well versed in the health care of dogs and cats, the special considerations in housing and care of these species can be unique and unfamiliar. For example, the chapter by Griffin and Baker on use of cats as experimental animals (Chapter 12) emphasizes the unique aspects of research and breeding-colony management, many of which are not taught in the veterinary curriculum. The importance of interacting with other trainees with common aspirations, difficulties, and solutions to everyday problems cannot be overemphasized. Therefore, programs that have a cadre of veterinarians in training will provide a rich training environment. During training, the more senior trainees become increasingly influential and helpful to those trainees with less experience. Informal and formal meetings of trainees from a single institution or neighboring institutions can be a key characteristic of an excellent environment for training in laboratory animal medicine. Group interaction becomes especially important in preparation for specialty board examinations. Mentoring by an experienced laboratory animal veterinarian during the training period is an exceedingly important part of the training process. The mentor is responsible for providing a broad overview of the specialty, giving personal insights about current events and issues, protecting the trainee from political or intrapersonal incidents, assuring that there is balance in time commitments between varying duties competing for attention, monitoring the trainee's progression in acquiring skills and knowledge, and being an advocate for the trainee in the transition from training to first full-time professional appointment. The laboratory animal specialist may or may not be the trainee's research supervisor, but if possible, the research activity should be integrated closely with the other activities of the training program. If the research experience occurs in a laboratory distant from the laboratory animal medicine faculty, a clearly defined schedule and plan must be in place to assure that the requisite integration is achieved. It is not satisfactory for the research experience to be far afield of laboratory animal or comparative medicine in terms of goals pursued, insights gained, and skills acquired.

31. RESEARCH IN LABORATORYANIMALAND COMPARATIVEMEDICINE C.

Graduate Education and Laboratory Animal Medicine

Earning a graduate degree should be an option, but not necessarily a requirement of specialty training. Graduate education adds many didactic course requirements, examinations for admission to candidacy, and preparation of a dissertation, which are very time-consuming, rigorous, and not necessarily relevant to the goal of becoming a laboratory animal specialist. If an institution offers a graduate degree, and if the trainee is eager to pursue graduate education, then the additional demands of the graduate program must be discussed with the training faculty and mentor to be certain that all of the varied and sometime conflicting demands of the total training program are accommodated and resolved from the outset. The trainee must understand that an additional 3 to 5 years of time commitment are usually required to achieve the combination of specialty and Ph.D.-level graduate training, and 1 to 3 additional years for M.S.-level training. Benefits of a graduate degree include (1) organized, compulsory, in-depth training in research; (2) recognition for having accomplished a rigorous research training experience; and (3) an earned academic degree that is universally accepted as evidence for expertise in research and that some academic institutions regard as necessary for advancement in rank. D. I.

Support for Training

Pre- and Postdoctoral Training

As discussed earlier, salary or stipend support for trainees must comply with specific guidelines concerning the objectives of particular training grants or awards. For example, National Research Service Awards (NRSAs) funded by the National Institutes of Health (NIH) are intended to support only research training, but allow up to 20-25% effort in other academic activities. Therefore, if a substantial portion of effort is directed to development of clinical skills or performance of clinical service, then salary support must not be derived from NIH training grants (institutional or individual training grants). Because institutions benefit directly from trainee participation in clinical care of research animals, it is appropriate for salary support to come from institutional sources. In fact, a common practice is to appoint trainees to full-time clinical duties for the first year of experience and to convert after that time to a predominantly research-based training program. This allows the trainee to gain important skills and experience in clinical care, become familiar with the training department and institution as a whole, and learn about research laboratories that could be considered for research experience. A system that allows trainees to focus their attention and effort on either clinical or research activities can be helpful in this early stage of professional development. The

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NIH offers programs of support for research training, which encompass all stages of research career development. A complete listing of these programs and details, such as training program goals, eligibility, application procedures, etc., can be obtained from the website . The Comparative Medicine Division of the National Center for Research Resources (NCRR) of the NIH provides support for training veterinarians who wish to pursue post-D.V.M, training in research pertaining to laboratory animal science and comparative medicine. Specific programs that are commonly used for training in this specialty are National Research Service Award Short-Term Institutional Research Training Grants (T35), National Research Service Award to Individual Postdoctoral Fellowships (F32), National Research Service Award Institutional Research Training Grants (T32), Mentored Clinical Scientist Development Award (K08), and the Mentored Research Scientist Development Awards (K01). As described in the introductory paragraph of this section, a brief (1-3 months) exposure to the specialty can be extremely useful for students who are interested in gaining firsthand experience and insights. These awards are made to the training institution, and students interested in participating in this type of experience should inquire directly to institutions offering this training (see listing at ). Postdoctoral (postD.V.M.) training can be supported through institutional training grants, or individuals may apply directly for support. The primary distinction between these awards is that appointment can be made by institutions immediately, while the individual awards require at least a few years of research experience and development of a research training plan by the applicant and mentor prior to application. A listing of the institutions that are currently funded to provide this training, instructions for preparation of an application for individual training grants, and eligibility requirements for these awards can be found online at the NIH Comparative Medicine website . 2.

Advanced Research Career Development Awards

In the context of scientists who earn the Ph.D., a "postdoctoral" experience is intended to add to their research training, which was the primary goal of their graduate education. This postdoctoral experience is designed to allow progression from a mentored student to an independent scientist. The D.V.M. who enters a postdoctoral training program usually has had little or no research experience on which to build. Consequently, research training starts at the postdoctoral stage rather than refining research skills previously acquired. In addition, the postdoctoral experience for laboratory animal specialists divides attention to skill building between both clinical and research areas, further diluting time and attention available to become proficient in research. Nonetheless, the D.V.M. trainees

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bring to the postdoctoral stage of their career extensive education and experience in medical sciences. Therefore, the acquisition of strength in research is an achievable goal but may require more time than the Ph.D. "postdoc." The extensive protected time necessary for laboratory animal specialists to gain this critically important in-depth research experience requires longterm (3-5 years)salary and research support. Fortunately, such funding mechanisms exist to assist in research training beyond that supported by T32 or F32 awards. The Comparative Medicine Division of the NCRR, NIH offers Special Emphasis Research Career Awards (SERCA) in pathology and comparative medicine, also known as the Mentored Research Scientist Development Award (K01) and Mentored Clinical Scientist Development Award (K08) (see and ). Candidates for these awards must be graduate veterinarians who have completed fundamental training in the specialty, particularly clinical training. Institutions nominate candidates who demonstrate exceptional potential for achievement in research and provide the required rich training environment that will assure the success of the research trainee. These awards are given with the expectation that the first 3 years will be focused on mentored research training and the final 2 years will be devoted increasingly to independent research projects. In addition to salary support, these awards provide research funds. Competition for these awards is keen and requires a candidate with exceptional potential in research, an institutional environment that can support and facilitate this type of training, and a project proposal that is meritorious. Although these awards are relatively recent and few in number, their impact on increasing the numbers and qualifications of research scientists in laboratory animal science is already apparent. More extensive use of this excellent training mechanism should be a high priority for the specialty.

IV.

RESEARCH RESOURCES

A.

Animal Resources

Meritorious research cannot be performed without support from a variety of resources. Of these, animal resources are central to productive research in laboratory animal and comparative medicine. Characteristics of desirable animal resources include (1) a sound animal resource-management program that addresses the health and welfare of animals and the needs of investigators; (2) facilities that are adequate in size, design, and maintenance to accommodate the required species and number of animals needed by the institution's research faculty; (3) an animal science program that provides high-quality domiciliary

care of animals; (4) ability to accommodate special needs, such as barrier exclusion of pathogens and isolation of animals used in hazardous experiments at biosafety level 2 and higher; (5) surgery and support laboratories; (6) gnotobiotic laboratory; (7) transgenic laboratory; and (8) breeding colonies for production of special strains or stocks, including perpetuation of inherited diseases. One measure of the overall quality of animal resources is accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). This does not necessarily mean that resources that have not sought or achieved accreditation are deficient, and accreditation may not address the specific needs of investigators. Therefore, judgment must be used in assessing the adequacy of animal resources for support of research. Since laboratory animal specialists can wear two hats, as investigator and animal resource manager, the animal resources should be especially attuned and responsive to the needs of laboratory animal scientists as well as investigators in other disciplines.

B.

Research Laboratories

Research laboratories are second only to animal resources in importance and must be available and equipped to serve the needs of laboratory animal scientists. These laboratories may overlap in function and capability with the diagnostic resource discussed below. Basic laboratory functions and equipment should include (1) analytical biochemistry, (2) molecular biology, (3) mammalian cell culture, (4) microbial culture, (5) physiological function tests, (6) radioisotope handling and detection, (7) computer resources with software for date acquisition and statistical analysis, (8) gross and microscopic pathology, and (9) photographic capability. Special procedures that should be available through institutional shared resources include (1) flow cytometry; (2) electron microscopy; (3) quantitative densitometry; (4) DNA synthesis, sequencing, and detection; (5) gross and microscopic digital imaging and quantitation; (6) protein synthesis and sequencing; (7) body-imaging modalities, such as X-ray, bone densitometry, and ultrasonography; (8) statistical support; (9) confocal or other types of quantitative microscopy; (10) biohazard isolation laboratory; and (11) computer graphics and digital projection. In some cases, specialized research resources may be accessed through interinstitutional agreements. Examples of such specialized resources include regional primate research centers, specialized rodent resources, invertebrate resources, and special animal colonies. The scientific literature provides guidance to investigators who specialize in maintaining and using unique colonies of animals or who perform procedures such as cryopreservation of embryos or sperm (also see the Institute for Laboratory Animal Research, Animal Models and Genetic Stocks Information Program ).

31. RESEARCHIN LABORATORYANIMALAND COMPARATIVEMEDICINE

C.

Diagnostic Laboratories

Laboratories that provide clinical and pathological support necessary to maintain healthy laboratory animal colonies are a fundamental resource found in most large-animal care and use programs. Such laboratories also provide valuable research support because in the process of evaluating health problems, excellent research opportunities are likely to be revealed. In addition, these laboratories provide many of the analytical procedures that are commonly used in laboratory animal and comparative research projects. Professionals who operate these laboratories are often laboratory animal and/or comparative pathology specialists who are highly qualified to collaborate in research with others and serve as principal investigators of independent projects. In fact, in most institutions with these diagnostic resources, there is close integration with academic departments of laboratory animal medicine or comparative medicine, as well as with the animal services programs. These laboratories can also play a pivotal role in training veterinary students and postdoctoral students in laboratory animal diseases.

D.

Library and Electronic Literature Searching

Recent advances in computer-assisted searching of the scientific literature have virtually solved the difficult problem of finding and obtaining publications pertaining to highly circumscribed or specialized research topics. This is especially important for the laboratory animal specialist because topics of interest frequently are published in a variety of biomedical science journals and monographs that do not focus on laboratory animal science or comparative medicine. As discussed earlier, understanding work reported by others in the scientific literature is a critically important prerequisite to initiating a new investigation. Therefore, electronic computer-searching capability is an absolutely essential resource for research. Bibliographicsearching software is designed to allow investigators to perform successful searches themselves; however, assistance of a skilled librarian can be extremely helpful in both searching for citations and acquiring the corresponding articles. Vendors such as Ovid, EBSCO, and Cambridge Scientific Abstracts provide libraries as well as individuals with powerful online literature search programs or platforms. Each vendor includes various subject databases accessible through its platforms. For example, some of the databases pertinent to biomedical and veterinary sciences provided by Ovid are Journals@Ovid Full Text, Medline, AGRICOLA, Biological Abstracts, International Pharmaceutical Abstracts, and CAB. Vendors also provide access to full-text versions of articles of a limited number of journals. CARL is a vendor providing literature search capabilities using one database that crosses all subject areas. In addition, it provides a rapid and economical

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document delivery service. Literature search limited to specific journals or publishers can be accessed through online services such as Wiley Interscience Journal Finder , Springer Link Information Services , IDEAL (International Digital Electronic Library) , and Science Direct . These services usually allow free searching of their journals for citations and their corresponding abstracts. To obtain a full version of the article, users must subscribe to the service or pay a fee per article. Institutional libraries usually subscribe to one or more of these services and provide free access to their pa-. trons. Some services are now beginning to allow free access to full-text articles of older issues of their journal collection. Other sources of free full-text articles are Highwire Press and BioMedNet , although this list undoubtedly will increase in time. Citations accessed electronically can be saved and stored electronically in personal bibliographic databases, such as Reference Manager, Endnote, and Procite. Details about these products, marketed by the Institute for Scientific Information (ISI), can be found at . These personal bibliographic databases allow for organizing citations electronically to a journal's specifications when preparing manuscripts for publication. Enormous time and effort are saved by this advancement because it eliminates the laborious reorganization of references during manuscript revisions and avoids opportunities for typographical errors. Research scientists must make full use of these new library resources to be competitive in research today.

V.

SUPPORT FOR RESEARCH

A.

Grantsmanship

The term "grantsmanship" refers to the sum total of all strategies and procedures used to successfully compete for funding to support research and training. Success or failure in securing funding depends largely on (1) knowing where to apply, so as to focus an application toward an agency interested in the specific objectives of the application; and (2) writing a persuasive description of the research problem, a logical approach to its solution, and a high probability that the outcome of the proposed project will materially advance understanding of the topic's field. The skills required for writing a successful application have some similarities to those for writing a scientific paper, but there are distinct differences. First, the scientific paper describes work completed and includes supporting data. To be competitive, the research grant application must have preliminary data to demonstrate feasibility, but the reviewers are

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required to make judgments about the applicant's ability to perform the proposed experiments and evaluate the results. The second major difference is that the grant application includes a request for financial support that must be justified for specific expenditures. Grantsmanship must be learned by the budding laboratory animal scientist since future research pursuits depend on this skill as much as on the research skills practiced in the laboratory. The process of learning skills of grantmanship is much like that of other aspects of research, that is, through association with a mentor who has mastered the skills and demonstrated this mastery by successful competition for grant support. Initially, a basic understanding of the process can be found in introductory guides to grant writing. Guides of this type can be found in "Proposal Writer's Guide" by Don Thackery , "Guide for Writing a Funding Proposal" by S. Joseph Levine <wysiwyg://15/http:// www.canr.msu/edu/ace/dissthes/proposal.htm>, and "Guide to Grant and Proposal Writing" by Ann Martinez . In addition to understanding the objectives and requirements of granting agencies, it is also essential to understand the policies and procedures of the institution that will host the grant and provide administrative and business support. These details can usually be found through an institution's Office of Sponsored Programs. Two particularly complete and informative websites of such programs can be found at Pennsylvania State University and the University of Michigan . Although the information on these sites pertains to details for those particular institutions, much of the information is of general interest and very informative to grant applicants from other institutions.

B.

Public Sources of Support for Research

As detailed elsewhere in this chapter, the NIH, particularly the Comparative Medicine Division of the NCRR, is a major source of public support for research in laboratory animal medicine, comparative medicine, and comparative pathology. Investigators who study animal models of human diseases, or who wish to develop specialized resources for animal research or train students in the specialty should understand all of the various granting mechanisms available through this program. The online descriptions are a good starting point for current information that can be supplemented with direct consultation with staff, who are identified on the website . Comparative Medicine staff members are eager to assist potential applicants interested in one of their granting mechanisms, and they provide important advice about the intent of grant solicitations, format of the application process, and other admin-

istrative details, but they are not able to provide opinions about the merits of a proposal. The large variety and number of granting mechanisms provided by the various institutes of the NIH can be daunting to the new investigator. Nevertheless, familiarity with these mechanisms is essential for successful preparation and direction of applications to the appropriate agency within the NIH. Details about each of these mechanisms are available directly from the NIH through its website or from the NIH Office of Extramural Grants , which provides the latest information on funding opportunities, grant policies, receipt deadlines, etc. The NIH also provides information about past and current grants awarded on various topics (CRISP: Computer Retrieval of Information on Scientific Projects), which can be helpful in locating other investigators working on research of mutual interest.

C.

Private Sources of Support for Research

Research funding can be obtained from foundations and societies that focus on specific research goals. Excellent sources of information about funding from foundations can be found at websites for the Foundation Center and the Grant Advisor . Personal contact with an individual knowledgeable about the goals, policies, and procedures of a granting agency is always an important strategy but is absolutely mandatory for applications to private foundations. For laboratory animal medicine, grants are awarded by the American College of Laboratory Animal Medicine. Other organizations include the American Cancer Association, the Muscular Dystrophy Association, the American Heart Association, etc. Organizations that are less focused on a single disease or organ system include the Geraldine Dodge Foundation, the American Kennel Club, and the Mark Morris Foundation. Commercial firms that depend on research conducted under contract with academic institutions will search for academic-commercial partnerships, which are usually designed to accomplish a specific goal. Commercial contracts frequently are highly focused on product development, but some of the larger firms have foundations that fund projects that may not be product-oriented. Institutional grant-information offices and administration offices should be asked to provide information and periodic announcements of granting opportunities on topics of specific interest. Some of these offices subscribe to grant-alert services, such as Community of Science and Science Wise Alert . Each user provides the service with a profile of research interests, and the service automatically filters out the specific grant opportunities that pertain to those interests and emails the investigator directly with details of selected grant solicitations.

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31. RESEARCH IN LABORATORY ANIMAL AND COMPARATIVE MEDICINE

D.

Intellectual Property

During the past decade, academic institutions have become acutely aware of the enormous value of research findings produced by their faculty that have potential for commercialization. The current estimate is that the top 160 research universities in the United States spend more that $24 billion on research annually, and this research leads to 3500 patents per year. On the other hand, commercial firms are constantly searching for new findings that can be exploited commercially. Unfortunately, academic scientists have been slow to understand the need for protection of intellectual property, which they and their university own. Human and animal health care are prime topics for discoveries with high potential for valuable intellectual property. Most institutions have an office of industrial relations that serves the faculty by soliciting information about discoveries of potential value, assisting in evaluating the commercial value of research findings, gaining patent protection, and finding commercial firms who are interested in funding research that may be needed to bring an idea to market or who may be interested in licensing more advanced discoveries for commercialization. Laboratory animal research scientists are no less likely to make discoveries of commercial value and must become aware of all issues related to protecting intellectual property.

VI.

SUMMARY

Research is a foundation of laboratory animal and comparative medicine that has contributed enormously to the state of sophistication in animal experimentation currently enjoyed by biomedical scientists. Progress in biomedical science continues to advance at an ever increasing rate, making it imperative that laboratory animal and comparative medical specialists continue to contribute new knowledge required to solve complex problems introduced by new research concepts and technology. Although these major advances in biomedical science create problems, they also provide exciting opportunities for laboratory animal medicine specialists to demonstrate their expertise in animal experimentation and animal modeling. Regulatory and administrative distractions must not allow laboratory animal medicine to lose its momentum and reputation as a major contributor to the science of animal research. The next generation of specialists must accept the responsibility to become pre-

pared intellectually and gain the research skills needed to lead the specialty successfully into the twenty-first century. Fortunately, there are sufficient specialists who are successful research scientists and are willing to mentor trainees. Institutions with organized training programs offer excellent opportunities for those entering the field to become skilled in the art of research. Financial support during training is available to competitive candidates. Therefore, all of the essential ingredients required to sustain progress in developing research talent are available. All that remains is a commitment by those established in the specialty and those entering the specialty to maintain the scientific character of laboratory animal medicine.

REFERENCES

Andrews, E. J., Ward B. C., and Altman, N. H., eds. (1979). "Spontaneous Animal Models of Human Disease." Academic Press, New York. Bernard, C. (1865). "An Introduction to the Study of Experimental Medicine" (trans. H. C. Green. Reprinted by Dover, New York, 1957). Capen, C. C., Jones, T. C., and Migaki, G., eds. (1985). "Animal Models of Human Disease." Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Desnick, R. J., Patterson, D. E, and Scarpelli, D. G. (1982). "Animal Models of Inherited Metabolic Diseases." Alan R. Liss, New York. Harvey, A. M., Brieger, G. H., Abrams, S. A., and McKusick, V. A. (1989). "A Model of Its Kind: A Centennial History of Medicine at Johns Hopkins." Johns Hopkins Univ. Press, Baltimore. Kraft, L. M., Pardy, R. R, Pardy, D. A., and Zwickel, H. (1964). Practical control of diarrheal disease in a commercial mouse colony. Lab. Anim. Care 14, 16. Kraus, L. (2001). "Laboratory Animal Medicine: Advancing Science and Animal Welfare in the 21 st Century." American College of Laboratory Animal Medicine, Chester, New Hampshire. Little, C. C. (1913). Experimental studies of the inheritance of color in mice. Carnegie Institute Publ. 179, 17-102. McPherson, C. W., and Mattingly, S. E, eds. (1999). "50 Years of Laboratory Animal Science." American Association for Laboratory Animal Science, Memphis. Mobraaten, L. E., and Sharp, J. J. (1999). Evolution of genetic manipulation of laboratory animals. In "50 Years of Laboratory Animal Science" (C. W. McPherson and S. E Mattingly, eds.), pp. 129-135. American Association for Laboratory Animal Science, Memphis. Navia, J. M. (1977). The design of experiments. In "Animal Models in Dental Research," pp. 13-27. Univ. of Alabama Press, Tuscaloosa. Phaneuf, D., Wakamatsu, N., Huang, J. Q., Borowski, A., Peterson, A. C., Fortunato, S. R., et al. (1996). Dramatically different phenotypes in mouse models of human Tay-Sachs and Sandhoff diseases. Hum. Mol. Genet. 5, 1-14.

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Chapter 32 Laboratory Animal Behavior Kathryn A. L. Bayne, Bonnie V. Beaver, Joy A. Mench, and David B. Morton

I. II.

III. IV. V. VI.

Introduction .................................................

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Rodents

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A.

Mice ...................................................

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B.

Rats

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C.

Guinea Pigs

D.

Hamsters

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Rabbits .....................................................

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Laboratory Dogs and Cats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Nonhuman Primates

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Farm Animals

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References ..................................................

Increased awareness among the community using laboratory animals of the need for objective information regarding animal behavior resulted from federal regulations calling for promoting the "psychological well-being" of nonhuman primates. Although the literature was rich with scientific studies documenting the behavior of many species of primates in free-ranging conditions and the relatively extensive housing conditions in zoos, little was known about their behavioral profiles in the laboratory. In consequence, attention was focused on captive primate behavior and ways to enrich their environment to increase the breadth of normal behaviors exhibited and reduce or eliminate atypical behaviors. Federal regulations also called for laboratories to provide the opportunity for exercise for dogs. Again, a great deal of information was known about abnormal canine behavior, essentially due to the occurrence of undesirable/abnormal behaviors in companion dogs. Thus, new work LABORATORY ANIMAL MEDICINE, 2nd edition

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was done to evaluate the behavioral effects of providing canines the opportunity for exercise. The publication of the seventh edition of the National Research Council's (NRC) "Guide for the Care and Use of Laboratory Animals" (NRC, 1996) expanded the concern for laboratory animal behavior and environmental enrichment to all species used in research, testing, and education. This chapter presents a comprehensive overview of the species-typical and atypical behaviors in common laboratory animals. Where evidence has been shown in the literature, topics covered for each species include types of behavior problems, the effects of these problems on research, and environmental enrichment techniques. "It is difficult to determine to what extent behavior altered by captivity is simply another adaptive change to a different environment rather than an indication of adversity" (Shepherdson, 1998). Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

I.

INTRODUCTION

The study of laboratory animal behavior has increased steadil~r over the last decade. In the United States, this trend was initially focused on species for which there was a regulatory requirement to consider normalizing behavior, e.g., the U.S. Department of Agriculture requirement to promote the psychological well-being of nonhuman primates--the 1991 Animal Welfare Regulations (AWRs). With the advent of the seventh edition of the "Guide" (NRC, 1996), more emphasis has been placed on addressing the structural, social, and activity elements in all laboratory animals' environments. The European counterpart of the "Guide," Council Directive 86/609/EEC (European Directive, 1986), states that "any restriction on the extent to which an experimental animal can satisfy its physiological and ethological needs shall be limited to the absolute minimum." Indeed, the Europeans have made significant progress in addressing the behavioral needs of numerous laboratory animal species vis-a-vis environmental enrichment techniques. The material presented in this chapter summarizes the state of knowledge of animal behavior for a number of commonly used laboratory animals and provides suggestions for promoting normal behavior. Scientists should be concerned about the behavioral state of animals kept in laboratories, not only for ethical reasons but for reasons of science, as behavioral abnormalities may be accompanied by physiological or immunological variations from the norm, thereby potentially confounding research data. For example, it is now clear that the central nervous system has a significant direct effect on the immune system independent of corticosteroids (Dantzer and Kelley, 1989; Kingston and Hoffman-Goetz, 1996). Moreover, there is a wealth of literature on the effects on behavior of enriched and impoverished environments showing that they affect brain development, memory, learning ability, problem solving, and social interactions with humans and other animals. Enriched environments can also mitigate the effects of undernutrition and old age; promote recovery from brain trauma; and alter drug responses, tumor latency, LD 50%, and the development of athersclerosis (see, e.g., Chance, 1957; DePass et al., 1986; Renner and Rosenzweig, 1987; Claassen, 1994; Kempermann et al., 1997). Three important methodologies have been used to help determine what animals require, and each has its own advantages and limitations (Mason et al., 1998; Dawkins, 1990). The first is simply to observe what animals do and to prepare an ethogram of those behaviors based on the time they spend carrying them out and the time at which they perform those behaviors. One can then compare time budgets in different environments, with perhaps the natural species ethogram being the "gold standard" (see, e.g., Lawlor, 1984; Stauffacher, 1997b; Poole, 1992, 1998). This approach can also be used to determine whether and how animals interact with an environment and objects in that environment, such as furniture and toys (Mench, 1994). Also

with this type of approach, one can measure physiological variables or use psychometric tests (such as the open field test for anxiety, attack latency) and then see how the results deviate from those obtained in other environments (see Broom and Johnson, 1993; Cooper and Hendrie, 1994). The disadvantage of this approach is that the standard for what is "normal" may be subjectively based on the observer's knowledge and experience. The second approach is to offer animals a choice of environments or aspects of an environment to see which they prefer to spend their time in--so-called preference testing. This has an honorable tradition going back to the work of Craig (1918). In preference tests, animals have free access to different choices, and the amount of time spent with each choice is measured. However, the results should be treated cautiously (Duncan, 1978; Dawkins, 1990; Fraser, 1996) as animals may not indicate what is in their long-term interests and can choose only from the environments offered. Also, an animal's choice may vary with experience. The third method to assess animal needs builds on preference testing by determining how hard the animal will work to reach a certain environment and comparing this effort with other behaviors, i.e., testing the strength of their preferences. Animals will continue to work hard for essentials like food and water but make less effort for different substrates, environments, or social interactions. In this manner it may be possible to rank the relative importance of various activities in the behavioral repertoire to separate needs from wants from luxuries (see Dawkins, 1990, 1992). Limitations to this test are based on the inherent variability in the stimuli used to test the animals and in making equivalent comparisons between them. Behavior problems encountered in captive animals can be classified broadly and simply as "qualitative" or "quantitative" aberrant expressions of behavior (Erwin and Deni, 1979). This classification scheme implies either that species-typical behavior is modified in the amount expressed, such as an excessive amount of time spent in the behavior or the absence of the behavior, or that the behavior itself has been modified such that it is expressed in response to an atypical stimulus or is directed to an inappropriate target. In either case, a mismatch between the response and the stimulus is evident (Bayne, 1996). Fox (1968) has stated that a behavior that initially began as a means for the animal to adapt to the conditions that elicited the behavioral change frequently becomes maladaptive and can "become emancipated or released independent of the o r i g i n a l . . , stimuli." Crockett (1998) includes altered activity cycles in the list of behavioral measures that may reflect inadequate well-being. The effects of stereotypies on health and well-being are less clear-cut than for many other kinds of abnormal behaviors, such as self-biting (Mason, 1991; Lawrence and Rushen, 1993; Mench and Mason, 1997). For example, there is conflicting evidence as to whether the performance of stereotypic behavior results from stress or actually reduces stress levels (Ladewig

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32. LABORATORY ANIMAL BEHAVIOR

et al., 1993). Stereotypies are a heterogenous category of behaviors that can include a variety of locomotor, postural, or gestural patterns. Their development can be influenced by a number of factors, such as neurological predispositions, exposure to stressors, impoverished environmental conditions, and frustration of the motivation to perform particular behaviors. Once they are established, stereotypies can be difficult to stop. Since there is general agreement that stereotypies generally indicate that the animal's environment is or has been inadequate in some way, a better approach is to minimize or prevent the development of stereotypies by providing opportunities for the expression of species-typical behaviors (Duncan et al., 1993). For a broader perspective on environmental factors likely to impinge on animal welfare and science, see Clough (1982) and Rose (1994). The goals of enrichment are to decrease the incidence of abnormal behaviors and to increase the diversity of normal behaviors, and the evaluation of any enrichment can be carried out in any of the three ways described above (Bayne et al., 1992c; Beaver, 1989; Benn, 1995; Hart, 1994; Markowitz and Gavazzi, 1995; Newberry, 1995; Poole, 1998; Scharmann, 1991; Stauffacher, 1997a). However, it has to be remembered that simply changing an animal's behavior pattern does not necessarily mean the change is for the better. It may simply result in one stereotypic behavior being substituted for another. Therefore, a basic understanding of the species-typical behavior of the animal and an ongoing program to evaluate the effects of an enrichment program are key to improving the behavioral wellbeing of laboratory animals. This chapter will review some fundamental behaviors of the more common laboratory animals and link these to environmental enrichments that can improve animal well-being and provide a more refined animal model for research.

II.

RODENTS

Rats and mice comprise the most common mammals used in research in most countries (see Her Majesty's Stationery Office, 1997). Other rodents used in research include gerbils, guinea pigs, hamsters, voles, and various wild species. The systems of husbandry used for these animals have changed little since they were first kept in laboratories, but in the last 10 years or so, efforts have been made to make their immediate caging environment more in keeping with their behavioral needs. Rodents have been kept in captivity for at least a century and some species far longer. Despite this, they retain many of their natural characteristics and instincts that evolved in their wild ancestors, as evidenced from studies of their behaviors when they are placed in more "natural" habitats (e.g., Boice, 1977). This may generate ideas about how to modify their environ-

ments to suit them better and so reduce any adverse effects they may feel (Brain, 1992). However, the fact that there are several hundred strains of rodents (specifically, rats and mice, both inbred and outbred) raises the question of whether the environmental requirements are the same for all strains. Some general principles and guidelines for some of the commonly used species of rodents are described below.

A.

Mice

Mice show a range of behaviors (Brain et al., 1989; Jennings et al., 1998), but those that cause most concern involve stereotypies such as wire gnawing and jumping and aggression (particularly among male mice of certain strains). Expression of aggression, stereotypies, and natural behaviors may be influenced by cage design, cage furniture, and substrate. It may also be desirable to satisfy some select natural behaviors, such as nest building. 1.

Influences on Behavior

Mice kept in traditional cages may gnaw on the cage wire, jump and circle around the tops of cages, chew, and dig (e.g., Tuli, 1993; Wiirbel et al., 1996; Hobbs et al., 1997). Significant differences between strains in the kinds of behaviors expressed have been observed. For example, nude mutants jump more than the parent wild-type strain (Zur:ICR), which in turn, gnaw more; and DBA/2 mice exhibit more eating, grooming, and exploring than do CD-1 or B6CBAF1/J strains (Hobbs et al., 1997). Some behaviors may be modified through the provision of nesting materials, and empty water bottles and tunnels can be used as resting areas (Hobbs et al., 1997; Ward and DeMille, 1991). Wiirbel and colleagues (1998) also found that providing a cardboard tube significantly reduced wire gnawing in ICR mice. The tube provided shelter, resulting in increased resting periods, which probably indicated a feeling of security. In another study, mice (BALB/c and Crl:NMRI strains) kept in an enriched environment (polyvinyl chloride [PVC] tube, nesting material, and a metal grid) showed more climbing and eating than the controls kept in standard caging; but there were strain differences in that B ALB/c mice spent more time than NMRI climbing and moving about, but NMRI spent more time resting and grooming (Van de Weerd, 1996). Barbering (hair nibbling and whisker chewing) in mice is not uncommon and usually manifests as an area of hair loss over the upper back (shoulder and neck); the whisker bed is not as commonly affected (Hauschka, 1952; Long, 1972; Thornberg et al., 1973; Litterst, 1974). Barbering is considered to be a social behavioral effect with the dominant mouse being the perpetrator, and several common strains of mice are affected (e.g., SwissWebster, C57BL, C3H, CDF). Sarna et al. (2000) studied this effect in c57BL/6 mice and found that separating the mice

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

reversed the condition, and that when mice were rehoused together (in pairs), the barbering recurred as a result of mutual grooming. The recipients were passive in accepting barbering, and even pursued conspecifics for more grooming. Brain analysis showed differences between the barbers and the barbered animals, and the results were discussed in relation to social dominance and the consequences of barbering on brain function. Another form of noninfective alopecic (little erythema or swelling) hair loss occurs when animals push their noses through the hopper bars to access food. In these cases, the alopecia is around the muzzle. Aggression in male mice can hardly be called an aberrant behavior, but it is certainly a welfare problem for the animals lower in the hierarchy, resulting in bites to the tail, rump, ears, and shoulders. It can also occur between females and between sexes (e.g., when defending a litter). There are wellcharacterized strain differences, with inbred C57BL/10 and DBA/2, outbred Swiss, and TO mice considered more agonistic, whereas CBA/Ca, C3H/He mice are typically more docile (Jones and Brain, 1987). Aggressiveness has been shown to be affected by intrauterine position of the pups and is thought to be related to local placental transfer of hormones between the sexes (Vom Saal, 1991). Mice use olfactory clues, rather than sight or sound, to establish a pecking order. Disturbing the cage environment, by cleaning for example, can therefore precipitate a bout of fighting while scent marking is carried out and the order reestablished (Hurst et al., 1993). Blom (1993) found that while NMRI mice preferred clean to soiled cages that had not been cleaned for 4 to 6 days, 2-day soiled cages were preferred to clean ones. This may have implications for routine husbandry of animals and would be interesting to reevaluate in ventilated cages. Aggression has been shown to be influenced by strain, age, and prior encounters. Participation in aggressive encounters can affect levels of pituitary hormones, including the reproductive gonadotropins (animals may become infertile), adrenocorticotropic hormones (ACTH), as well as catecholamine levels in the adrenal medulla (Brain, 1990). Corticosterone levels can be higher in defeated subordinate mice, especially when they are kept in noncomplex conditions, such as bedded polycarbonate cages (Durschlag and Stauffacher, 1996), but can also be higher in dominant animals with a large territory to defend (Bishop and Chevins, 1989). Any enrichment to reduce aggression has to be carefully designed, as simply providing objects that mice can occupy may increase the amount of territory to defend and so promote aggression (Haemisch et al., 1994). However, if adequate space is provided in addition to shelters, the degree of injury may be reduced by allowing the opportunity for submissive posturing rather than overt fighting (Durschlag and Stauffacher, 1996). Other ways shown to successfully reduce aggression include housing mice together before puberty is reached, using more docile strains, and replacing conspecifics as soon as possible if they have to be separated, since even the removal of an

animal for 24 hr can increase the level of aggression on its return (Brain, 1997). 2.

Environmental Enrichment

Enriched environments do not appear to have any long-term effects on the performance of mice in open field tests, their docility, corticosterone levels, or adrenal gland weights. However, there is evidence that mice kept singly have compromised immune systems (Schwartz et al., 1974) and develop tumors faster than when kept in groups (Riley, 1981). Considerable work has recently been undertaken to look at what sorts of environments and substrates mice choose and how hard they will work to access them. The traditional housing system for rodents has been wire-bottom cages, but these are increasingly being replaced, when appropriate, by solid-bottom cages. This raises the question of what sort of bedding material is appropriate and liked by the animals. Given a choice, mice avoid wire-bottom cages, preferring solid-bottom cages with shredded filter paper (Blom, 1993, B lom et al., 1996), which they will work hard to obtain (Roper, 1973). When a cage was constructed as half mesh and half solid, the mice used the mesh half more as a latrine and rested in the solid part, a tendency confirmed in later studies where mice in enriched cages urinated in certain locations, unlike animals kept in standard conditions (Van de Weerd, 1996). Furthermore, in rank order, mice preferred shredded filter paper to wood chips to sawdust, and there were no strain differences in these preferences between BALB/c and C57BL/6 mice. B lom et al. (1996) attributed the preference for the paper to the higher irritancy of fine sawdust particles, but mice were also able to manipulate the paper and build nests, which might have contributed to their preference. When nesting materials were specifically tested, it was found that paper was preferred to wood products (i.e., paper tissues, towels, and strips to wood wool or shavings), and combinations of nesting materials were preferred. Certainly, pregnant and lactating females should be given nesting materials that they can manipulate, but it appears that all mice like to build nests (Van de Weerd et al., 1997). Sherwin (1996a) further emphasizes the potential importance to the animal of being able to build its own nest out of appropriate materials rather than being given one ready-made, and that this may be a behavioral need. It is important to note that some bedding materials may cause problems for neonatal mice because the materials may be very absorbent, thereby potentially dehydrating the pups (Hessler and Moreland, 1984). Van de Weerd (1996) evaluated the use of nest boxes and found that mice preferred a cage with a box to one without, but still preferred a nesting material on a grid floor to a grid floor with a nest box. However, findings in relation to whether mice choose dark or light nest boxes; open, closed, or meshed ones; or metal rather than plastic are at variance and may be straindependent, as the choice may reflect the balance of olfactory

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32. LABORATORY ANIMAL BEHAVIOR

to visual or tactile cues (see Buhot, 1989). Strain differences appear to exist in the design of the nest built. For example, in BALB/c and C57BL strains, the shape of the nest differed--the BALB/c's nest was domed and the C57BL's nest was bowlshaped (Van de Weerd, 1996). Sherwin (1996a) found that mice were prepared to work as hard, within the imposed cost of having to traverse 30 cm of shallow water, to reach a running wheel, deep sawdust (6 to 7 cm), a conspecific (unfamiliar male of the same strain), or increased space or shelter (plastic cup), as for food. The author interpreted these findings to indicate that the animals place a value on all of these items. Standard environments for group-housed mice have also been supplemented with cans (although rusting and physical hazards are concerns), glass bottles, plastic pipes, soft wood pieces, and cardboard tubes. Such environments, when compared with standard caging, have been shown to reduce aggression (providing that objects are renewed to remove olfactory clues; see Ambrose and Morton, 1997), emotionality, anxiety (open field test, freezing), and adrenal gland weight, and to promote exploratory activity (hole board tests, cage emergence time) and alertness (rearing) (Chamove, 1989; Ward and DeMille, 1991; Van de Weerd et al., 1994; Van de Weerd, 1996). However, a strain difference is apparent, with BALB/c mice appearing to be more anxious than C57BL mice. Another interesting finding is that standard-caged animals sleep huddled together, whereas enriched animals sleep in groups of two or three (Van de Weerd, 1996). Preferences for type of shelter have been studied in some detail, and it was found that individually housed mice prefer to sleep in loose sawdust rather than in tubes, and that there is no overall preference for shape, opacity, or openness of tube (Sherwin and Nicol, 1995; Sherwin, 1996b).

B. 1.

Rats

Influences on Behavior

Rats are the second most common rodent used in research laboratories, yet few studies on their behavioral needs and enrichment have been done, although many caretakers are intuitively looking at ways in which the animals' environments can be made more stimulating. Rats are nocturnal animals that show a range of natural active behaviors, such as exploration, inquisitiveness, digging, aggression, rearing, climbing, and jump/ pounce/roll/wrestle/hold behaviors, etc., in play, with a welldefined circadian rhythm (Blanchard et al., 1975, 1988; Flannelly and Lore, 1977; Silverman, 1978; Cowan, 1983; Weihe, 1987; Lore and Schultz, 1989; Batchelor, 1994). Choice tests have confirmed that they are inquisitive about novel environments (e.g., Hughes, 1968) and that they are gregarious animals (Latane, 1969; Latane et al., 1972). They are social animals that can live in well-defined, compatible groups (Barnett, 1967).

It is well known among caretakers and pet owners that when rats are kept in isolation, they tend to become more aggressive and may exhibit increased susceptibility to disease (Hatch et al., 1963; Baer, 1991). Hatch and colleagues (1963) observed that not only did the rats become more intractable, but their adrenal and thyroid glands increased in weight, their spleen and thymus decreased in weight, and their tolerance to chemical toxicity decreased, as shown by a reduced LDs0. Damon et al. (1986) showed that the LDs0 was also reduced if animals were not acclimated to a new environment, such as a metabolic cage, and acclimation has been shown to stabilize urinary excretion of steroids (Gomez-Sanchez and Gomez-Sanchez, 1991). Such noticeable effects are evident even after a few days and can be affected by overcrowding as well as isolation, resulting in increased corticosterone levels (Capel et al., 1980a,b; Holson et al., 1991). Brain and Benton (1979) have argued that it is not easy to determine whether these effects are truly due to isolation, as the variance could also be due to factors such as individual responses, strain, sex, and previous housing conditions. Work by Hurst and colleagues (1998) examining social sexual strategies suggests that the effects of social isolation may be different for males and females. Single housing of females had much less effect on time budgeting and corticosteroid levels than for males, e.g., tail chasing was less for females than males, but escape behaviors such as bar chewing were higher. Rose (1993) concludes that there is abundant evidence that individual housing of rats produces significant behavioral changes that impair physical and psychological fitness for research. The wealth of literature on the effects on behavior of enriched and impoverished environments has also shown substantial effects on brain development, including morphological and physiological aspects, synapse density, memory, learning ability, problem solving, brain responses to undernutrition and old age, recovery from brain trauma, and altered corticosterone levels (e.g., see Renner and Rosenzweig, 1987; Claassen, 1994; Kempermann et al., 1997; Hurst et al., 1998), and interestingly, increased benzodiazepine receptor binding (Wadham and Mortin, in press). Restraint is also an adverse condition for rats and has been shown not only to increase corticosteroids but to predispose animals to developing gastric ulcers (Gamello et al., 1986). The phenomenon is restrainer-dependent, with different types of restraint altering heart rate, blood pressure, and body temperature to varying degrees (Gardiner and Bennett, 1977; Wadham, 1996). 2.

Preferred Environments

Blom and colleagues (1995) determined on the basis of preference tests that females preferred cages with low heights (80 mm as opposed to heights of 320 mm; time spent in each was 29.9% compared to 19.2%), and males preferred the lowest height (38.6% in 80 mm) but also spent considerable time in higher cages (25.3% in 320 mm). Low light intensities were

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

chosen by all rats regardless of whether they were albino or pigmented strains (Wistar Cpb:WU and a pigmented Wistar hybrid), although the albino animals spent more time in the reduced light. When grid floor mesh size was examined, female Wistar rats preferred smaller rather than larger mesh (i.e., 10 mm x 10 mm, compared to 10 mm x 30, 50, or 80 mm; see Blom, 1993). When kept on grid floors, rats also appear to like cage furniture such as plastic tubes and raised wooden platforms, and they tend to use any form of shelter to take cover in and to climb on (Bradshaw and Poling, 1991; Scharmann, 1991; Batchelor, 1993, 1994; Manser et al., 1998a). Old mouse boxes placed in rat cages are used as hides and climbing objects. The rats gnaw holes at the corners, which provide at least two escape routes, and will turn the box on its edge or upside down (D. B. Morton, personal observation). Rats like to gnaw on wooden sticks (Orok-Edem and Key, 1994; Chmiel and Noonan, 1996) as well as furniture, which may pose a theoretical confounding variable for toxicology studies. Rats choose to spend more time in more complex environments (Denny, 1975). Rats prefer to work for their food, and thus will press a lever rather than eat food placed in front of them (Carder and Berkowitz, 1970; Singh, 1970) and will also remove the husk from a sunflower seed rather than eat one that is already prepared (Shettleworth and Jordan, 1986). The type of flooring preferred by rats is coincident with their long-term health, as solid floors are preferred to grid floors (Van de Weerd et al., 1996), and it is generally observed that grid floors are associated with superficial foot lesions leading to ulceration, inflammation, pain, and swelling (Kohn and Barthold, 1984). Regardless of previous experience of flooring type, rats spend more time resting on solid floors compared with the wire floors (88% :12%), but wire floors are nearly equally used during the rats' active period (55%: 45%) and for defecation and urination. Furthermore, a comparison of rats kept on the two types of flooring revealed no differences in weight gain, food or water consumption, ease of handling, and many physiological parameters, including immune function and catecholamine, testosterone, and corticosteroid levels (Manser, 1992; Nagel and Stauffacher, 1994; Manser et al., 1995; Stauffacher, 1997a; Van de Weerd et al., 1996). Therefore, there would seem to be no reason not to use solid-bottom cages given the increased potential for animal well-being. Moreover, rats will work (by lifting a weighted barrier) as hard to reach solid-bottom floors as they will to reach a novel environment (Manser et al., 1996). Solid-bottom cages require bedding, and choice tests have shown that rats prefer cages with wood shavings and paper bedding to those with sawdust (Blom et al., 1996; Van de Weerd et al., 1996). Paper bedding may also be more acceptable in toxicological studies. B lom et al. (1993, 1996) showed that rats preferred materials with large fibrous particles, that were manipulable, and that may produce ultrasound by particles rubbing against each other (see also Manser et al., 1998a). The type of nest box preferred by rats has also been investi-

gated. Manser et al. (1998a,b) found that rats spend considerable time in the nest (60 to 80% of the time) and that they will work harder to reach a cage with a nest box, whether or not it contains nesting material, than they will to reach an empty cage. Manser and colleagues went on to develop an opaque plastic nesting box with a roof and three sides as a practical piece of furniture, incorporating the preferred features. This box was not chewed and was easy to clean.

C. 1.

G u i n e a Pigs

Influences on Behavior

Compared with work on rats and mice, there has been little experimental work on guinea pigs in terms of their preferences and abnormal behavior patterns. Guinea pigs are social and crepuscular animals (active at dawn and dusk) but appear not to burrow. Manning and colleagues (1984) found that guinea pigs like to lie beside each other. Although not normally aggressive in the laboratory, they can be under certain conditions, e.g., when males are in the presence of females (Sachser and Lick, 1991). When disturbed, guinea pigs may squeak and rush for cover, but they also show freezing behavior in response to a stressor (Fara and Catlett, 1971; Suthedand and Festing, 1987), which may mistakenly be taken to indicate that they do not find the situation particularly aversive. Fears may be communicated through ultrasound vocalization. Guinea pigs do not appear to sleep for long periods of time but rather take short rests (White et al., 1989). Sachser (1986) looked at certain anatomical and physiological parameters when guinea pigs either were kept in colonies or were singly caged. He found that singly caged animals showed evidence of atrophy of the reproductive organs, with males having lower testosterone levels, which would lead to lower accessory gland weight. The singly caged guinea pigs also had lower corticosteroid levels and adrenal gland weights. 2.

Preferred Environments

Since they are social animals that like to be in contact with conspecifics, it is not unreasonable to think that guinea pigs would prefer to be kept in groups, but as stated previously, little experimental work has been carried out. White et al. (1989; White, 1990) suggested that guinea pigs did not utilize the whole of the cage area; however, Scharmann (1991) found that when the cage was enriched through the addition of hay and straw, then they burrowed and hid in it. Furthermore, Scharmann observed that guinea pigs spent some time chewing on the bars and hoppers of the cage. This behavior could be stopped by adding pieces of wood, which gave the guinea pigs something else to gnaw on. Whether this simply displaces one type of oral stereotypy with another is not clear. As guinea pigs are

32. LABORATORYANIMAL BEHAVIOR rather timid animals, they like cage or pen furniture that enables them to hide, such as boxes or plastic pipes (Noonan, 1994; Meyer, 1995).

D.

Hamsters

Hamsters have a reputation for fighting when housed in groups, but stable groups can be established if animals are grouped early in their lives. Little work has been done on what these animals prefer, but one study has shown, based on occupation times, that they prefer solid-bottom cages with bedding, regardless of their age or sex (Arnold and Estep, 1994). In the solid-bottom cage environment, hamsters spent more time engaged in behaviors such as sleeping, grooming, gnawing, eating, hoarding, and exploring. However, that preference could be influenced by experience. Forty percent of those reared on wire spent more of their time on wire mesh (see Arnold and Estep, 1990), although most chose the solid-bottom cages. Hamsters spend much of their time asleep (41%), older animals sleeping more than younger ones (44% compared to 37%). Hamsters reared on bedding rarely chose wire on which to sleep (<0.008%). Hoarding and gnawing may be stereotypic, and these workers found that those hamsters reared on wire-bottom cages hoarded more, and those reared on bedded, solid-bottom cages gnawed more, primarily when in the wire cages.

llI.

1.

RABBITS

Influences on Behavior

Because of cage size constraints in the laboratory environment, only a limited range of the numerous behaviors of rabbits are generally observed (Batchelor, 1991; Gunn and Morton, 1995; Love, 1994). Concomitant with this reduced variety of behavioral expression is the potential for undesirable or abnormal behaviors to develop. Among those observed in laboratory conditions are bar chewing (Raje and Stewart, 1997; Morton et al., 1993), which can result in broken teeth; excessive grooming leading to denuded areas and possible gastrointestinal problems; and psychogenic polydipsia with secondary polyuria (Potter and Borkowski, 1998). Several other stereotypies have been described, such as head swaying or weaving, vertical sliding of the nose between cage bars, pushing on cage parts with the head, pawing or digging at the cage floor or food hopper, and rapid circling (Morton et al., 1993). Excessive passive behavior, manifested as sitting in a hunched posture for a prolonged period of time or sitting with the head lowered in the cage corner, is also described as undesirable stereotyped behavior (Morton et al., 1993). These authors further describe individually housed

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animals as exhibiting increased inactivity, increased lying down, and incomplete behavior patterns or movements, as compared to group-housed rabbits. This observation has also been noted by Podberscek et al. (1991), who identified more "maintenance" behaviors in caged rabbits and more comfort behaviors (e.g., grooming) in penned rabbits. Although the incidence of abnormal behaviors is reduced in group-housed rabbits (Morton et al., 1993), aggression between animals is of sufficient concern (especially between bucks) to warrant careful consideration of the appropriate method of housing selected. Not unexpectedly, many abnormal behaviors expressed by rabbits are derivative of naturally occurring behaviors. For example, repetitive digging at the cage may be related to the activity of rabbits excavating a burrow. Circling behavior can be expressed in two normal contexts: (1) an aggressive encounter, or (2) courtship (Morton et al., 1993). The behavior shown by rabbits that put their head down in a cage corner is not unlike rabbits that lower their head toward an approaching conspecific. 2.

Environmental Enrichment

Similarly, environmental enrichment techniques that appear to be the most successful for rabbits are those that provide the animals with the opportunity to express a greater range of species-typical behaviors. For example, objects on which rabbits can chew are very desirable. A variety of objects are available, including plastic toys, Nylabones, Gumaknots, Booda Yapples, empty cardboard boxes, and food items to gnaw on (e.g., Bunny Blocks). Soda cans, Wiffle balls, sections of PVC pipe, balls, and suspended metallic items (e.g., washers) have been used to encourage nudging, playing, and investigation behaviors. Objects are frequently suspended in the cage to induce postures akin to the natural rearing position of rabbits. Rabbits are, however, capable of destroying cage objects made of plastic or thin metal and ingesting the material. Therefore, for reasons of health of the animal and scientific objectives, sturdy metal chew objects may be preferred. 3.

Social Housing

In general, social housing is more successful if immature animals are used. Groups of female rabbits can be formed, and the hierarchy established remains relatively stable. Groups of male rabbits may also be established, although the bucks are typically castrated (Raje and Stewart, 1997; Podberscek et al., 1991). It should also be nol~ed that there are reports of strain differences in aggressive behavior, which may influence the success of group housing rabbits (e.g., Dutch rabbits are more aggressive than New Zealand Whites) (Morton et al., 1993). Group-housed rabbits may be provided climbing structures; wood shavings or other appropriate floor covering to encourage foraging; and tunnels, buckets, barrels, or boxes for escape and hiding behaviors. The expression of chasing, jumping, gamboling, rearing, and

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

other behaviors requiring space for their performance is facilitated in group-housing conditions that are adequately enriched and in which the animals have established compatible relationships. It may not always be possible to determine the rank relationship between dyads of rabbits (Turner et al., 1997). However, social hierarchies for groups of mature does may be stable for long periods of time (e.g., 30 months as reported by Turner et al., 1997) with no differences detected between high- and low-ranking animals in several immunocompetence measures. Hence, the authors proposed that group housing may be an appropriate means of maintaining antibody-producing animals. Similarly, Whary et al. (1993) determined no significant difference in growth rate, humoral immunity, delayed-type hypersensitivity response, adrenal gland weight, or circulating corticosterone level between group-housed and single-caged does. The authors did observe a higher feed intake and lower lymphocyte count in group-housed rabbits. As there was no significant difference in growth rate despite the higher food consumption level, the authors postulated a better feed-conversion to bodyweight rate. Also, since the significantly lower white blood cell count occurred during only 1 week of observation and did not reflect a typical stress profile (leukocytosis and increased corticosterone level), it was not considered of biological relevance.

IV.

LABORATORY DOGS A N D C A T S

Dogs have been associated with humans for about 12,000 years and cats for approximately one-fourth that time (Beaver, 1992, 1999). In both cases, these species have filled many roles for humans, from companion to hunter to laboratory animal. For each role the animal fills, there are unique sets of behavioral criteria that need to be identified. In the case of laboratory animals, including dogs and cats, validity of data gathered may be called into question because of behavioral factors, including caretaker interactions (Beaver, 1989; Benn, 1995). The Animal Welfare Regulations (AWRs) mandate that laboratory animal facilities address certain criteria for cage size for dogs and cats and exercise opportunities for dogs (Benn, 1995; Hetts, 1991); but standards were based on "feel good, best guess" criteria rather than scientifically based behavioral studies that specifically evaluated various housing environments. Only after the AWRs took effect were controlled studies attempted. 1.

Cage Size and Activity

Activity levels of dogs housed in cages or runs have been reported, but studies are somewhat difficult to compare because authors were evaluating different parameters. For example, in cages of approximately 1 m 2, purpose-bred beagle dogs were reported to travel 55 m in 1 hr (Hughes et al., 1989a). These dogs

spent only 8% of a 12 hr photoperiod in motion (Hughes et al., (1989a). Hite et al. (1977) reported dogs living in approximately the same size cage spending 12.7% of the observation time sitting, and 6.6% of the time lying. Beagles housed in runs of approximately 1.25 m x 2.5 m spend 69% of their time lying or sleeping, compared to 74% of the time when the same dogs are caged instead (Neamand et al., 1975). The actual 24 hr time distribution of activities of beagles housed individually in runs has been described (Beaver, 1999). Passive behaviors such as sitting and lying down occupied 73.6% of the 24 hr period, with other activities (e.g., grooming, standing, walking) occupying the balance of the time. Most studies of dog activity were actually designed to compare the behaviors of beagles kept in different-sized cages or runs (Bebak and Beck, 1993; Hite et al., 1977; Hughes et al., 1989a; Neamand et al., 1975). Overall, there is no difference in the amount of aggression, play, or activity levels in differentsized cages (Bebak and Beck, 1993; Hite et al., 1977; Hughes et al., 1989a; Neamand et al., 1975). Dogs show little interest in exercise when allowed free time in runs or an exercise area (Campbell et al., 1988). Activity levels peak when people are present, even without significant direct interaction (Campbell et al., 1988; Hughes et al., 1989a). Physiological parameters do not differ among dogs under long-term confinement, dogs that have access to daily exercise periods, and dogs given forced exercise on treadmills (Clark et al., 1991; Tipton et al., 1974; Newton, 1972). Activity levels during cage confinement have not been reported for cats. Observations suggest that they rest more than dogs when confined to cages (Stermann et al., 1965). When kept as a group in a cat room, they still have personal space requirements and will spend time exploring, although this latter behavior is somewhat age-dependent. For cats, activity levels usually peak in the morning when caretakers arrive and are also high for approximately 30 min before and after. 2.

Social Interaction

Dogs are social animals and would normally spend approximately three-fourths of their time close to other pack members (Beaver, 1981; Fox et al., 1975). This strong tendericy for social interaction means that individually housed dogs will spend as much time as possible interacting with other dogs when released into an exercise area together (Campbell et al., 1988). Dogs penned in chain-link runs will lie next to one another, touching through the fence. When resting areas are specifically provided in runs, it is best to determine where the dog wants to rest so that the bench or mat can be appropriately placed. Dogs that are group-housed interact with each other and investigate the pen floor more often, so activity levels for these dogs are usually higher (Hubrecht et al., 1992; Hubrecht, 1993; Hughes et al., 1989a). For group-housed dogs, it is desirable to maintain stable

32. LABORATORYANIMAL BEHAVIOR groups. The introduction of new members or removal of one from an established pack means that social orders must be readjusted (NRC, 1994). The most significant switch would be combining dogs for the creation of a completely new group. Dominance mounting, ear sniffing, and communicative posturing are evident for several days while individuals determine what their social position is in the new pack. This can be stressful to some individuals and may affect data. Although unusual, overt fighting between animals can occur, particularly if the animals are not well socialized. Removal of the problem animal from the group tends to restore balance to the dominance rankings. Cats are housed in group environments even though feline social behavior can be more strained by forced interactions (Kessler and Turner, 1997). With abundant food and resting spots, cats can seek their own comfort level with conspecifics. Some will interact regularly with one or two close associates; others will remain primarily to themselves (Beaver, 1992). The introduction of new cats into a group can be very disruptive, so a long period should be allowed for adjustments. It is also important to keep in mind that when a cat has been removed from a group, even for only a few hours, if it brings back a strange odor, severe aggression toward it may occur. This can be significant and last long enough to prevent the cat from ever rejoining the group. 3.

Types of Behavior Problems

a.

Stereotypies

Although activity levels do not vary greatly between individually housed and group-housed dogs, the type of activity might (Bebak and Beck, 1993). Solitary dogs show more repetitive locomotor behaviors (Hubrecht et al., 1992). The development of stereotypic activity patterns is dependent on a number of factors besides how the animal is kept. Genetics, social factors, dietary energy, individual stress level, and environmental conditions may contribute to the development of these atypical activities (Fox, 1965). Stereotypic pacing, manifested by the dog walking back and forth along the front of a cage or run, is one of the more common problems. Usually the animal will throw its head up as it comes to the end of its left-to-right or right-to-left motion. Continuous circling is another stereotypy dogs can show. Prevention of a problem behavior is usually easier than dealing with it after it is well developed. Appropriate enrichment can increase the complexity of behavior shown and help prevent undesirable behaviors from beginning (Hubrecht, 1993). b.

Psychogenic Stress Responses

Several behaviors of dogs and cats are associated with events that are apparently stressful to the animal. In clinical behavioral medicine, they are so pathognomonic that stress intervention is initiated even if a specific stressor cannot be identified. Two major behavior problems in dogs are aggression and destruc-

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tiveness. Destructive behavior has been associated with both a lack of environmental stimulation and a lack of tolerance to being enclosed (referred to as "barrier frustration") (Houpt, 1998). Acral lick dermatitis (lick granulomas) in dogs and psychogenic alopecia in cats are the result of a normal behavior shown in excess. The condition in cats is more common in individually housed cats and will often disappear if that cat is placed with at least one other compatible cagemate (DeLuca and Kranda, 1992). Psychogenic polydipsia may first be noticed as polyuria. Urine spraying of vertical surfaces is a feline marking behavior associated with the invasion of territory or a perceived stress within that territory. Abrupt changes in cat litter type can result in 50% of the cats refusing to use the litter box and an increased incidence of spraying. Half of the cats may begin to use the new litter within a week, but that still leaves as many as 25% that eliminate outside the box. Environmental enrichment has been successfully used to reduce the incidence of psychogenic stress responses (Beaver, 1981, 1989; Benn, 1995; Markowitz and Gavazzi, 1995). Group housing for dogs, elimination of the addition of new members to group-housed cats, increased human contacts, play toys, social-interaction time for individually housed dogs, obedience lessons for dogs, trick learning, cage puzzles to earn treats or food, visually interesting activities in the room of cages, individual hiding areas like boxes or resting shelves for cats, food choices, and prey-chasing simulation for cats are but a few ideas. c.

Excessive Vocalization

Laboratory dogs and cats may vocalize excessively. Some research facilities use debarking surgery. This may control the noise, but it does not address the behavioral aspects of the problem. The vocalizations and simultaneous increases in activity are usually associated with the sounds of humans entering the kennel/cage area. Eventually the behaviors are reinforced by the human feeding or interacting with the cat or dog (Askew, 1996; Juarbe-Dfaz, 1997). The undesired behavior has just been rewarded. Strategies that do not reinforce the undesired behavior include social housing the animals, increasing the presence of personnel in the kennel/cage area at times other than feeding, desensitizing the animals by making the noises that trigger the animals' excessive vocalizations without the resuiting presentation of food, and rewarding quiet behaviors instead of noisy ones. 4.

Other Behaviors of Laboratory Dogs and Cats

There are many other behaviors commonly shown by laboratory dogs and cats that are uncommon in their pet counterparts. These behaviors are not considered abnormal, as their expression is often influenced by the structural features of the animals'

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

environment. Cats often lie in the litter box, usually after it has been cleaned. Both dogs and cats may play in or tip water bowls or food bowls. They apparently create their own games with things that move. Scratching posts for cats allow them to express a normal behavior and may reduce their need to perform other foot-oriented behaviors.

V.

NONHUMAN PRIMATES

Nonhuman primate behavior has been the subject of increased interest and attention due to federal regulations passed in 1985. Regulatory language published by the U.S. Department of Agriculture calling for an "environment to promote the psychological well-being of nonhuman primates" (AWRs) sparked several years of intensive research effort focused on identifying environmental factors that would improve the mental/behavioral health of captive primates. Extensive bibliographies have been published subsequently (Reinhardt and Reinhardt, 1998; Animal Welfare Information Center, 1992), which deal exclusively with this topic. 1.

Types of Behavior Problems

A number of theories have been advanced to explain the spontaneous occurrence of abnormal behavior in nonhuman primates, including genetics, boredom, frustration, redirected aggression, disease, and neurological developmental abnormality. Although the occurrence of developmentally induced abnormal behavior is well documented in nonhuman primates (Harlow and Harlow, 1962; Mitchell, 1968; Sackett, 1965), many of the other possible etiologies of aberrant behavior tend to be supported mostly by anecdotal evidence. In general, however, the consensus appears to be that if the abnormal behavior is not due to a genetic tendency and is not developmentally induced (e.g., isolation rearing), then a lack of the appropriate level of stimulation (cognitive, social, etc.) is considered key to the manifestation of aberrant behavior. While it could be argued that inappropriate environmental stimulation is a causative factor, it may simply be correlative. For example, it is unclear if a reduction in the amount of time spent in food processing (compared to the free-ranging state) may cause increased oral behavior in a primate, e.g., oral exploration of the environment, such as cage licking, polydipsia, uriposia (urine drinking), masturbation, or self-biting, due to an appetitive drive that is not satisfied; or if the occurrence of these behaviors simply may be correlated with a reduction in feeding behaviors because of the increased time available to the animal to engage in these activities. The expression of abnormal behavior displayed by the nonhuman primate may also depend in part on the housing of the animal.

For example, macaques may pace around the perimeter of a cage or large enclosure, but a combination of stereotypic pacing and somersaulting may be more likely to occur in a smallersized cage. The literature on captive primate behavior is replete with examples of manifestations of atypical captive animal behavior. Although the significance of the expression of atypical behaviors is highly variable, a rating scale has been developed for rhesus monkeys, which ranks behaviors according to how detrimental they are to the animal (Bayne and Novak, 1998). Table I describes some of the basic abnormal behaviors observed in captive primates. 2.

Effects of Behavior Problems on Research

Nonhuman primates are used in a variety of research endeavors, including cancer, heart disease, neurological disorders, AIDS research, vision, nutrition, reproduction, and dental research. A disturbance in the normal physiology or behavior of these animal models can have far-reaching effects on the advancement of science in these and other areas of study. Primates living in captive conditions can manifest abnormal immunological, hormonal, cardiovascular, and behavioral parameters. 3.

Environmental Enrichment

Many abnormal behavior patterns can become highly entrenched, with their elimination described in only limited instances of intense remediation programs (O'Neill, 1989). Reduction (but not elimination) of some aberrant behaviors has been achieved by employing specific environmental enrichment

Table I

Examples of Abnormal Behaviors Reported in Nonhuman Primates Self-directed Grooming/plucking hair Self-sucking (digits, hair, nipple, tongue/cheek) Self-clasping Eye poking ("saluting") Self-manipulation (e.g., masturbation) Uriposia Auto-aggression (self-biting, slapping, head banging) Regurgitation Other-directed Excessive aggression Repetitive cage biting/licking/manipulation Stereotypic use of cage manipulanda/cage furniture Polydipsia Coprophagy Nondirected Stereotypic locomotion Floating limb

32. LABORATORY ANIMAL BEHAVIOR

techniques targeted to particular behaviors (Bayne et al., 1991). For example, primates that engage in excessive grooming may benefit from a grooming board constructed of artificial shearling to deflect some of the grooming activity to another object (Bayne et al., 1991). A primate that engages in self-biting may redirect this behavior to a Kong toy. Animals that exhibit excessive repetitive locomotion may be distracted from this activity by offering a suitably appealing food item in a foraging device (Bayne, 1992c). Although some consideration should be given to implementing enrichment techniques that can help resolve specific abnormalities detected in the animal, the optimum enrichment program should, in general, encompass a diversity of approaches to increasing the complexity of the animal's home environment. a.

Enclosure Size, Design, and Furnishings

The size, design, and complexity of the primary enclosure can profoundly impact the well-being of the primate occupant. Although current regulations and guidelines vary slightly in the precise floor space and vertical height recommended (AWRs, NRC, Council Directive 86/609/EEC), the goal of providing the animal with sufficient space to engage in some degree of species-typical locomotion and postures is common between them. To that end, primate cages should accommodate the increased cage height necessary for arboreal species. Indeed, the adequacy of caging that does not accommodate tail length of perched animals has been questioned (Poole, 1991). Similarly, the amount of floor space provided to the caged primate has received considerable attention. Initially, the sentiment was propounded that more floor space was automatically "better" for the animal. Subsequent research, however, did not consistently substantiate that claim (e.g., Bayne and McCully, 1989; Line et al., 1991a). Although in some species more floor space reduced the occurrence of stereotypic behavior (Paulk et al., 1977; Boot et al., 1985), in other cases the provision of more vertical height had the same result (Watson and Shively, 1996). Sometimes, simply moving the animal from the lower-tiered cage to the upper tier can influence the behavior of the animal (Scott, 1991). Unfortunately, some reports of improved wellbeing ostensibly resulting from increased cage space are confounded by the concurrent provision of cage complexities (e.g., Kitchen and Martin, 1996). Cage design has evolved dramatically from the "turkey cage" style common in the 1960s and 1970s to modular units with removable walls and floors, and walls with colored pictures (e.g., Erwin and Landon, 1992). Cage additions, such as tunnels, have been suggested as a way to modify the cage environment to provide the animals with additional cage space, access to different views in the holding room, and closer proximity to other animals (Rumbaugh et al., 1991). Access to large exercise cages has also been proposed as another means of offering variety

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from the primary enclosure, an opportunity to engage in social interactions and for the expression of greater locomotion concomitant with a reduction in stereotypic behavior (Bryant et al., 1988; Kessel and Brent, 1995; Wolff and Ruppert, 1991; Kessel-Davenport and Brent, 1994). Cages have been modified to include visual barriers (Reinhardt et al., 1991a; Reinhardt and Reinhardt, 1991), windows, and "grooming contact bars" (Crockett et al., 1997) to give primates more control over their social environment. A variety of cage furnishings have also been described. These typically provide the animal with greater opportunity to express a range of postural adjustments. Many cage furnishings are more easily incorporated into the group-housing enclosure simply due to the size constraints of the single-cage environment. However, additions to cage enclosures that have been reported include swings (Bayne et al., 1989), ladders, perches (Watson, 1991; Williams et al., 1988), shelves, tunnels, Primahedrons, and nest boxes (Scott, 1991). Both synthetic and natural materials have been used successfully (e.g., branches, rope, and PVC). The size and complexity of the cage furnishings will depend on the size of the enclosure. Frequently, a simple metal bar is incorporated into the squeeze-back apparatus of the single cage as a perch. However, telephone poles and entire trees have also been provided as perches and climbing structures to captive primates in large outdoor enclosures (Maki and Bloomsmith, 1989). In group-housing enclosures, shelves are typically mounted at staggered heights to minimize hierarchically based aggression between animals. Complex pathways have been created for group-housed callitrichids, using rope as artificial vines (Snowdon and Savage, 1991). The importance of perching to captive primates is underscored by the observation that it is frequently the most-used enrichment device in a variety of "nonnutritive'7nonsocial enrichments (Bayne et al., 1994; Reinhardt, 1994a). Perches allow primates to choose between different elevations in the cage and may provide a sense of increased security as a consequence of the animal being off the cage floor. Cage furnishings such as tunnels the animals can run through, sit in, or sit on have additional value, as they provide a "visual break" that can arrest an aggressive chase sequence. They also can provide shy animals with a place that is out of sight to other animals and personnel. Some primates (e.g., marmosets) use elbow-shaped PVC pipes for denning purposes. In large enclosures, concrete culverts have been used as tunnels. Pools of water have been provided to primates housed in larger enclosures. Although sanitation and animal safety concerns must be addressed, such pools have been implemented successfully with juvenile macaques (e.g., National Institutes of Health), providing the animals with the opportunity to swim, splash, and "fish" for objects in the pool. Toddler play pools and horse troughs are among the containers that have been adapted for use in primate housing.

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

F o o d Strategies

In free-ranging conditions, foraging behaviors of nonhuman primates can occupy from 7 to 65% of the diurnal activity budget (Milton, 1980; Herbers, 1981; Strier, 1987; Malik and Southwick, 1988; Marriott, 1988). Indeed, it has been described as the "single most time-consuming behavior" during some seasons of the year (Malik and Southwick, 1988). Increasing the amount of time primates spend engaged in foraging-like activities (searching patterns, food processing, and consumption) can be accomplished by (1) hiding the food and requiring the animal to search for it (Anderson and Chamove, 1984; Boccia, 1989; Scott, 1991); (2) requiring the animal to solve a puzzle or task to access the food (Rosenblum and Smiley, 1984; Line and Houghton, 1987; Gust et al., 1988; Bloom and Cook, 1989; Scott, 1991); (3) providing food that requires processing time (Bloomsmith, 1992; Smith et al., 1989); or (4) reducing the size of the food item so that the time spent in obtaining an appreciable quantity of food is extended (Bayne et al., 1991, 1992c). Many of these approaches also promote the animal's expression of cognitive skills (such as problem solving) and fine-tuned motor skills. The diet of the animal can be broadened by the inclusion of food treats. The density, flavor, shape, and color of the treat are dimensions that can introduce variety into an otherwise monotonous diet. However, as recommended in the "Guide for the Care and Use of Laboratory Animals" (NRC, 1996), the diet should be nutritionally balanced. Any reduction in the wellbeing of the animal by engendering obesity through excessive food-treat provisioning is strongly discouraged. As with any other enrichment technique, the health and well-being of the animal and common sense should prevail.

c.

Manipulanda

Increasing the complexity of the cage environment by providing "toys" has been widely implemented. Although the inclusion of objects for primates to manipulate is a provision of the AWRs, the actual benefit to the animal of including manipulanda in the cage may be inconsistent. The species, sex, and age of the animal and type of toy can influence the animal's response to the item (Crockett et al., 1989; O'Neill, 1988; Line et al., 1991b; Weld et al., 1991). A criticism of the use of simple toys as enrichment devices is based on the finding that the animals' interest in them wanes quickly, although "use" of the toys tends to stabilize at a low level (e.g., B loomsmith et al., 1990; Bayne, 1989; Line and Morgan, 1991). It has been suggested that perhaps a schedule of toy removal and reintroduction would prolong the animals' interest and use of the objects (Crockett et al., 1989; Paquette and Prescott, 1988). Manipulanda both commercially manufactured and fabricated in-house are used. There is some preliminary evidence to suggest that among a va-

riety of rubber and plastic objects presented, balls are manipulated the least by some monkeys, while a wishbone-shaped toy was most favored (Weld et al., 1991). Thus, early judgments on the success of a toy as an enrichment should be avoided until a variety of objects have been evaluated. Other enrichment techniques that generally come under the category of manipulanda are mirrors and grooming boards. Although studies evaluating mirror viewing have tended to be rather short-term, the pattern of initial high use progressing to a low rate of use seems to be consistent (Collinge, 1989; O'NeillWagner et al., 1997). In chimpanzees, mirror use actually resuited in an increase in agonistic behavior and a decrease in affiliative behavior (Lambeth and Bloomsmith, 1992). Grooming boards made of artificial shearling have been shown to reduce stereotypical behavior in macaques (rhesus and cynomolgus) and baboons (Bayne et al., 1991; Lam et al., 1991; Pyle et al., 1996). The devices encouraged both grooming and foraging behavior in the primates. 4.

Social Behavior

Despite the inherent diversity of members of the order Primate, most species used in the research environment live in social units. In free-ranging conditions, these social units may be composed of monogamous pairs (e.g., owl monkeys), groups that are highly structured (e.g., baboons), groups that are less cohesive in their membership (e.g., squirrel monkey troops have been described as an "aggregate," or readily "subdivided" into smaller groups; see Rosenblum and Coe, 1977; Thorington, 1968), or groups that are organized along matrilines (e.g., rhesus monkeys), etc. It is this basic social nature of nonhuman primates that has engendered the approach that the "best" method of providing environmental stimulation is another animal (e.g., Love, 1995). Although concerns about animal health and safety, personnel safety, accessibility to specific animals, variability in research data, and cost have been factors that have discouraged widespread implementation of social housing, regulatory requirements and federal policy essentially describe the default means of housing as a social environment, with any deviation from this necessitating a justification based on the health and well-being of the animal or a research requirement (AWRs). More important, however, studies have shown that singly housed primates manifest greater abnormal behavior than do socially housed animals (e.g., Bayne et al., 1992b); higher levels of self-grooming and lower levels of activity (Eaton et al., 1994); and higher "tension-related" behaviors (Baker, 1996). Fundamental to successful social housing is a knowledge of the species' natural behavior, inclusive of its social organization. Reports on the success of social housing of laboratory primates vary. Line et al. (1990b) reported the death of 1 female, hospitalization of 1 male, and injuries to 8 other animals out of

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32. LABORATORY ANIMAL BEHAVIOR

13 aged rhesus monkeys in the period after grouping. Jensen et al. (1980) reported a mortality rate of 11% in bonnet

macaques due to injury over a 3-month period following group formation and 9% mortality in rhesus monkeys over the first 6 months following group formation. Conversely, Kaplan et al. (1980) were able to reduce mortality resulting from fighting by identifying ongoing social disruption (removal of some animals from the group with the introduction of new animals) and then modifying management practices. Bayne et al. (1995) reviewed the injury records for three research facilities housing rhesus macaques and cynomolgus monkeys. They determined that 12% of the records evaluated for group-housed monkeys involved a wounding incident. The authors concluded that grouphoused females acquired wounds and required wound management more frequently than group-housed males, and that group-housed animals that were wounded were likely to be injured again, although most wounds required only minor treatment. McCully et al. (1992) similarly reported only minor wounding among 30 adult rhesus monkeys housed in groups of 3 to 8 animals (5 groups of adult males and 1 group of adult and subadult females with 1 adult male). A number of factors can influence the success of group formation, such as species, rearing experience, group size (e.g., Erwin, 1979; Mclntyre and Petto, 1993), and method of group formation (Bernstein and Mason, 1962). The formation of pairs of primates, while also having some risk associated with it, appears to be more practical in the laboratory environment and more readily achieved than group housing. For cynomolgus monkeys, compatible female-female pairs have been described by Line et al. (1990a) and Crockett et al. (1994). However, formation of compatible male-female and familiar male-male pairs using cages equipped with grooming-contact bars has also been described (Crockett et al., 1997). Other successful pairs described in the literature include female baboons (Jerome and Szostak, 1987), male bonnet macaques (Coe and Rosenblum, 1984), adult male and adult female rhesus monkeys (e.g., Reinhardt, 1988; Reinhardt, 1994b), and male and female stumptail macaques (Reinhardt, 1994c). The extent to which social housing influences the physiology of the research primate has not been fully evaluated; however, there is evidence that some parameters are modified. Coe (1991) determined that natural killer cell activity decreased following the introduction of females into the housing area in which both dominant and subordinate male rhesus monkeys were pairhoused. He further demonstrated that the testosterone levels in the dominant partner of pair-housed male squirrel monkeys were consistently higher than those of the subordinate partner (Coe, 1991). Gonzalez et al. (1982) reported that plasma cortisol levels were lower in pair-housed female squirrel monkeys than in individually or group-housed females. They further reported that increases in cortisol levels following a stressor (handling and anesthesia) were smaller in pair-housed females.

Capitanio (1998) has described a higher mortality in simian immunodeficiency virus (SIV)-infected macaques that were exposed to unstable social affiliations shortly after inoculation of the virus. He suggests that both the hypothalamic-pituitaryadrenal and the sympathetic-adrenal-medullary systems are influenced by social instability-induced stress. Coelho et al. (1991) found that baboons given visual, tactile, and auditory contact between wire-mesh walls with a familiar companion had lower blood pressure than when they were housed alone or with unfamiliar animals. Reinhardt et al. (1988) found that the body weights of female rhesus monkeys that were pair-housed did not vary significantly in the first month following pair housing as compared to baseline weights, although subordinate animals did exhibit a significant weight gain in the second month following pair housing. Reinhardt et al. (199 lb) further demonstrated that there was no significant difference in the mean serum cortisol level between the dominant and subordinate partners of rhesus monkey pairs. Stanton et al. (1985) similarly identified a "social buffering" in squirrel monkeys, wherein cortisol secretion in response to a fear-inducing stimulus was highest in individually housed animals, was attenuated somewhat by the presence of a single cagemate, and was at baseline level when the monkeys were housed with several other squirrel monkeys. 5.

Novelty and Predictability

Many environmental enrichment programs depend to a large degree on the provision of manipulanda (toys) to the primates. More than one study has shown, however, that the animals' interest in the toy, while initially high, declines rapidly (Millar et al., 1988). Some programs have attempted to circumvent this response by rotating the types of objects provided to the animals with the goal of sustaining a higher interest in them (e.g., National Institutes of Health, 1991). However, species differences (Fragaszy and Mason, 1978) and age differences (Millar et al., 1988) in responses of primates to novel objects have been described. For example, titi monkeys responded to a novel object with distress and arousal, while squirrel monkeys did not; and older callitrichid offspring generally contacted novel objects first and for longer periods than either parents or younger offspring. The objects to be used in an enrichment program should be chosen based on not only their sanitizability and their safety, but also on their relative efficacy. Sambrook and Buchanan-Smith (1996) determined that guenons who showed an interest in novel objects preferred those that were responsive (made a noise when manipulated) and showed no preference for visually complex objects over visually simplistic objects. Predictability in an animal's environment is often linked to the animal having some control over certain environmental elements. Indeed, Fragaszy and Adams-Curtis (1991) propose that

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

having control over the environment is more important to the animal than novelty. In some species, animals are less stressed if they can predict when an unpleasant stimulus is going to occur or when they can control its occurrence with their behavior (Shepherdson, 1998). Yet, Novak and Drewson (1989) note that too much predictability in the environment may be "boring" for animals. Chamove and Moodie (1990) have demonstrated that novelty can stimulate exploratory behavior and elicit arousal behavior that has beneficial effects. However, novelty can also be a stressor; therefore, an enrichment plan that incorporates novelty should be evaluated carefully.

6.

Personnel Interactions

The potential impact of personnel on research animal wellbeing is quite significant. Animal care staff should be trained in the general behavior of the species for whom they provide care. In particular, they should be cognizant of simple facial and postural signals that communicate messages to the primates (e.g., a direct stare is interpreted as an aggressive behavior by macaques, and a yawn may be interpreted as an open-mouth threat). Although few objective studies have been conducted on the effect of positive personnel interaction with laboratory primates, Bayne et al. (1993) observed that increasing personnel time with primates in bouts of 2 min, 3 times a week (while distributing food treats), resulted in a significant reduction of certain undesirable behaviors (e.g., repetitive locomotion and other stereotypic behaviors) and increase in some positive behaviors (e.g., grooming). O'Neill (1989) suggests that stability in personnel is very important, and that certain characteristics, such as patience and kindness, are essential, while Wolfle (1996) states that staff should be carefully selected and should be "caring and compassionate." Choosing personnel with suitable characteristics for work with research animals can start as early as the initial interview (Mandrell, 1996) by assessing the individual's attitude toward animals in general and animal research in particular. Mandrell (1996) suggests that personnel in leadership roles should set the example for animal treatment by demonstrating a "genuine caring attitude." The interactions are two-way, of course. The primates are dependent on personnel for food, water, and a healthy environment. However, staff can also become very attached to the primates in their care, and work assignments (e.g., euthanasia being the most extreme example) may need to be accommodated if a relationship of attachment has developed between an employee and a particular animal. Nevertheless, the program of nonhuman primate environmental enrichment can also be enriching for the animal care staff as they become more engaged in designing enrichment methodologies and learning more about the animals in their care (Roberts 1989).

VI.

FARM A N I M A L S

Farm animal behavior has been studied quite extensively in the last few decades, both because behavior influences production traits and because of the increasing concern in developed countries about the welfare of intensively farmed animals. These studies have spawned several recent books on farm animal behavior that, although primarily written for application to the commercial production situation, also provide information useful for structuring behavioral environments and dealing with behavioral problems in the laboratory setting (e.g. Price, 1987; Appleby et al., 1992a; Lynch et al., 1992; Phillips, 1993; A1bright and Arave, 1997; Fraser and Broom, 1997; Houpt, 1998; Gonyou and Keeling, in press). Farm animals have been domesticated and selected for desirable production traits for thousands of years. Indirectly, they have also been selected for their behavioral adaptability to a range of social and physical environments. Nevertheless, farm animals can and do display behavioral problems in agricultural and laboratory settings. Domestication and selection have resulted largely in quantitative rather than qualitative changes in behavior (Price, 1998). Although the frequency and intensity of expression of particular behaviors have been changed, the repertoire of behavior is generally strikingly similar to that characteristic of ancestral species. More "naturalistic" environments can therefore be helpful in minimizing some behavioral problems. However, genetic factors should not be overlooked, since some behavioral problems arise primarily as a consequence of selection for production traits while others are exacerbated by such selection (Grandin, 1998). For example, turkeys can no longer mate, not because mating behavior has been changed by selection but because the size and breast conformation of the male makes mating physically impossible. Some genetic stocks of animals may show exaggerated fear responses or be particularly difficult to handle, both of which can lead to problems during interactions between animals and personnel in the laboratory. Transgenic farm animals may also have special behavioral problems and needs (Mench, 1999).

I.

Types of Behavior Problems

A variety of behavior problems can be observed in farm animals (Fraser and Broom, 1997). These include stereotypic behaviors, so-called vices (defined as problems that cause economic losses to producers), other aberrant or problematical social behaviors, apathy or lethargy, and fearfulness. Most of the stereotypic behaviors seen in farm animals are oral in nature. Examples are bar biting or licking in livestock; rock or tether chewing and sham chewing by sows; spot picking by poultry; excessive manipulation of waterers by both livestock and poul-

32. LABORATORYANIMAL BEHAVIOR try, which sometimes results in polydipsia; and tongue rolling by cattle. Locomotor-type stereotypies are less common but can sometimes be observed. For example, hens will pace, often for several hours, in the period before egg laying if no nesting material is available (Appleby et al., 1992a). This behavior appears to arise due to frustration of nest-building behavior (Duncan and Wood-Gush, 1972; Wood-Gush, 1972). A number of abnormal behaviors can result in injury or death to the animal or to the animal's conspecifics. For example, sows can injure themselves by repeated head rubbing or banging, a behavior that is considered to be a stereotypy (Fraser and Broom, 1997), while calves may groom themselves excessively and ingest large quantities of hair, which accumulates in the rumen and clogs the rumen openings. Excessive ingestion of wood or litter by farm animals can also lead to impaction in the digestive tract. Behaviors that cause injury to other animals include cannibalism of her young by a parturient sow (sow savaging); feather pecking (allopecking) and cannibalism by poultry; anal massage, belly nosing, and tail biting by pigs; sucking of another animal's ears, navel, scrotum, prepuce, or udder by cattle; and wool eating by confined sheep. Other abnormal or problematical social behaviors include excessively frequent or severe aggression (directed either toward other animals or humans), sexual dysfunction, and poor maternal behavior. Behavior problems that cause injury are typically dealt with in agricultural production settings by permanently altering the animals, for example, by beak-trimming poultry, tail-docking pigs, and dehorning cattle. Another behavioral problem in farm animals is the suppression of activity and alertness. Farm animals kept in barren surroundings may spend lengthy periods of time motionless (Wiepkema et al., 1983), and sows assume an apathetic-appearing posture called dog sitting (Fraser, 1975). Furthermore, Broom (1986) found that closely confined, individually housed sows were extremely unresponsive to stimuli other than those associated with feeding, compared with group-housed sows. Although there has been a great deal of research on many of these problems, in many cases the etiology, and hence the remedy, is still unclear. Feather pecking and cannibalism in poultry and other captive birds are examples of common and persistent behavioral abnormalities that seem to have multifactorial causes (Mench and Keeling, 2001). Feather pecking is the pecking and pulling at the feathers of another bird; cannibalism is the pecking and tearing of the skin and underlying tissues of another bird (Keeling, 1994). Feather pecking ranges from allopreening and gentle feather pecking, apparently normal behaviors that cause little or no feather damage, to severe feather pecking and pulling that are thought to be abnormal (Blokhuis, 1989; Keeling, 1994; Savory, 1995). In addition, there are two types of cannibalism, the first involving skin damage that occurs as a result of feather pecking and the other involving pecks directed to the cloaca (vent pecking). Cloacal cannibalism often results in the

1253

death of the cannibalized bird and is probably unrelated to feather pecking (Allen and Perry, 1975). Many factors influence the incidence of feather pecking and cannibalism, including light intensity, position of the enclosure in the building, housing system, strain, group size, type and availability of food, and housing density (Hughes and Duncan, 1972; Fraser and Broom, 1997). Of particular importance is the absence of litter material that can be used for foraging, exploration, and dustbathing (Blokhuis, 1986; Vestergaard and Lisborg, 1993). Food deficiencies can also be a trigger (Hughes and Duncan, 1972). In other cases the etiology of a particular problem is more straightforw.ard. Young piglets may manipulate the belly of other piglets, using their snout in the same way that they would use their snout to massage the udder of the sow (van Putten and Dammers, 1976). This behavior can cause inflammation on the ventral surface of the piglet and also affect weight gain (Fraser, 1978). Belly nosing occurs when piglets are weaned much earlier than the normal weaning age of 8-12 weeks, a common practice (Fraser, 1978).

2.

Effect of Behavior Problems on Research

As alluded to above, many behavior problems have demonstrated consequences for the animal's health and well-being, and thus can negatively affect research. For example, even in the absence of cannibalism, having feathers pulled out by another bird is painful for chickens (Gentle and Hunter, 1990), and the resulting poorer plumage condition impairs thermoregulation (Tauson and Svensson, 1980). Wool-bitten sheep may develop skin lesions and are also more susceptible to helminth parasites, suggesting that they are immunocompromised (Lynch et al., 1992). Even seemingly benign behavioral problems can have health consequences. Dog sitting by sows kept on solid floors contributes to the development of urinary tract infections, and it has been estimated that dog sitting is associated with serious clinical sequelae in about 2% of cases (Fraser and Broom, 1997). Even when animals are not displaying behavioral problems, providing opportunities for the performance of natural behaviors can promote health and physiological normality (Mench, 1988a). For example, hens provided with perches have increased strength in leg and wing bones, which is important because they can develop osteoporosis due to the heavy calcium demands associated with egg laying (Hughes and Appleby, 1989; Knowles and Broom, 1990). Providing perching opportunities may also lead to improved foot health, decreased fearfulness, and a reduction in pacing (Brastaad, 1990; Appleby et al., 1992b; Duncan et al., 1992; Abrahamsson and Tauson, 1993). Providing perches may even help control feather pecking and improve feather cover (Brastaad, 1990), since hens are less

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

likely to be pecked while perching than when standing on the floor (Wechsler and Huber-Eicher, 1988). Another example is the provision of either nutritive or nonnutritive artificial teats to weaned calves. Not only does this decrease cross-sucking, but the calves show increased secretion of insulin and digestive enzymes when they are allowed to suckle shortly after receiving a milk meal (de Passill6 et al., 1993; Morrow-Tesch, 1997). Other effects of increasing behavioral opportunities for farm animals are discussed in the sections that follow.

3.

Environmental Enrichment

a.

Enclosure Size and Furnishings

Farm animals can be kept in a variety of different types of enclosures in the laboratory setting, ranging in size from small cages or pens to extensive outdoor paddocks. The appropriate enclosure type and size will depend on a variety of factors, including the age, sex, and reproductive status of the animal; the type of research; the social environment; and in more extensive enclosures, the terrain and hence availability and distribution of resources such as food, water, cover, and shade (Curtis, 1983; NRC, 1996; FASS, 1999). Some types of enclosure furnishings discussed in this section may be practical only in certain types of enclosures. Appropriate enclosure furnishings for chickens include perches, nest boxes, and some type of cover. The advantages of perches for chickens have already been described. Laying-type chickens housed in both cages and floor pens use perches extensively, particularly at night (Brastaad, 1990; Appleby, 1995; Tauson, 1984). Heavier meat-type (broiler) chickens may use low perches, but to a much lesser extent (Hughes and Elson, 1977). Perching experience when chicks are young is important for the proper development of perching behavior (Faure and Jones, 1982a,b; Appleby et al., 1992a). Disadvantages of perches are that they are associated with keel-bone deformities, more cracked eggs, and long or twisted claws (Appleby et al., 1992b, 1993). In caged hens, claw problems can be greatly reduced by placing an abrasive strip on the feeder, which reduces claw length when hens scratch during feeding (Tauson, 1986). Ideally, enough perch space should be provided for all birds to perch simultaneously. Chickens will perch on a variety of surfaces, including wire mesh, metal, wood, padded vinyl, or plastic (Muiruri et al., 1990; Appleby et al., 1992b; Faure and Jones, 1982a,b). However, plastic is the least-preferred surface. Hens prefer larger-diameter, rectangular perches that are slightly rough-surfaced for gripping, although perch preferences are influenced by previous experience. In cages, perches should be installed at the rear of the cage and far enough above the floor to prevent birds from becoming trapped and to allow manure to be trampled through the floor efficiently. Hens will use many different types of nest boxes, but their preferences depend on a number of factors, including previous

experience (Appleby et al., 1992a). One of the most important factors appears to be the type of nesting material used inside the box. Hens prefer loose, moldable nesting material and prefer to build a nest rather than use a preshaped nest (Hughes et al., 1989b; Duncan and Kite, 1989). Because poultry are fearful of humans (Duncan, 1992), it is helpful to provide them with some form of cover in addition to the cover afforded by nest boxes. Chickens housed in pens use areas with cover more often than areas without cover, and show more relaxed behaviors (resting and preening) in the covered areas. A striped panel providing 67% cover is effective, and is preferred to solid, transparent, or less fully striped panels (Newberry and Shackelton, 1997). Enclosure furnishings per se are less critical for livestock, although goats will use climbing surfaces. However, it is important that feeders, waterers, and flooring surfaces or walking areas for farm animals be designed to accommodate natural feeding, drinking, and movement patterns (Baxter et al., 1983; NRAES-84, 1995). Poor flooring surfaces are major contributors to injury and lameness (Webb and Nilsson, 1983), and badly designed feeders can also cause injury (Taylor, 1995). For caged or crated animals, enclosure design can also be critical. Tauson (1985) found that laying-hen cages with certain floor slopes, bar orientations, cage-locking devices, and bar spacing caused birds to become trapped and injured during feeding and other behaviors. b.

Food Strategies

Except when kept on pasture, farm animals are typically fed formulated, concentrated feeds. Farm animals can certainly self-select nutritious diets, but providing food variety is not commonly used as a means of behavioral enrichment for farm animals, except that poultry may be given supplemental oyster shell and grit and livestock may be given salt blocks and hay. Chickens and pigs are omnivores and would normally spend a large part of their day foraging for diverse types of food (Stolba and Wood-Gush, 1989; Dawkins, 1989). Ruminants similarly spend a large part of their day grazing and selectively ingest particular types of plants. Many oral and locomotor-type behavior problems in farm animals seem to be associated, at least in part, with the lack of foraging opportunities associated with the feeding of concentrated feeds or with the use of feed restriction to control body weight (Kyriazakis and Savory, 1997). Oral behaviors by sows directed toward the pen or pen features (Terlouw et al., 1991), spot pecking and excessive aggression by broiler-breeder chickens (Mench, 1988; Kostal et al., 1992), wool chewing and slat biting by sheep (Cooper et al., 1994), and tail biting by pigs (Fraser, 1987) are examples. Increasing foraging opportunities by providing a material like straw, or by diluting the diet, can sometimes decrease these undesirable behaviors (NCrgaard-Nielsen et al., 1993; van Putten and van de Burgwal, 1990). As described above, giving young, and espe-

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32. LABORATORY ANIMAL BEHAVIOR

cially recently weaned, calves suckling stimuli can decrease cross-sucking. Feeders that require animals to work for their food are sometimes successful in reducing these problems (Young et al., 1994), but these devices can also cause frustration and result in poor condition (Lindberg and Nicol, 1994). Undesirable oral behaviors that are stimulated by foraging can be further exacerbated if there are nutritional deficiencies. For example, birds may ingest feathers that they pull, and nutritional causes have been suggested as a reason for the persistence of this behavior (Blokhuis, 1989). c.

Manipulanda

Farm animals manipulate objects primarily with their mouths, beaks, muzzles, or snouts; so behaviors directed toward manipulanda may be closely related to foraging and feeding behaviors, as well as to some social behaviors, like biting. This does not always result in improvements in welfare. For example, sows may simply redirect oral stereotypies like bar biting, which seem to be caused by feed restriction rather than confinement, to hanging manipulanda (Terlouw et al., 1991) and even to rocks if the sows are housed outdoors (Dailey and McGlone, 1997). In general, manipulanda are only effective as long-term enrichment stimuli if they have biological relevance to the animal (Newberry, 1995). Pigs will root in an earth-filled trough initially, but this behavior diminishes with time (Appleby and Wood-Gush, 1988). Straw that is renewed daily elicits sustained interest, however, probably because of the combination of novelty and palatability (Fraser et al., 1991). The effects of manipulanda on behavior and performance traits have been evaluated primarily for pigs and chickens. Pigs readily interact with hanging manipulanda, like movable bars, tires, chains, rubber hoses, and rope, although they seem to prefer soft, pliable manipulanda (Pearce et al., 1989; Apple and Craig, 1992; Pearce and Paterson, 1993). They will also nose a ball placed in their enclosure, but eventually lose interest unless food is dispensed when the ball is rolled (Young et al., 1994), indicating that the ball elicits foraging behavior. Providing manipulanda has resulted in improvements in growth in certain genotypes of pigs but not in others (Schaefer et al., 1990; Hill et al., 1998). The presence of manipulanda can decrease aggression and fearfulness in growing pigs (Pearce et al., 1989; Schaefer et al., 1990). The value of providing poultry with manipulanda is less clear. Some research has shown that providing chickens with objects to manipulate or interact with decreases aggression, mortality, and fearfulness and increases growth rate (Jones et al., 1980; Reed et al., 1993; Gvaryahu et al., 1994; Bubier, 1996; Jones, 1996). However, in other studies such objects have been found to increase fearfulness or aggression (Nicol, 1992; Lindberg and Nicol, 1994). In addition, chickens seem to habituate quickly to some manipulanda and stop interacting with them (Gao et al., 1994; Sherwin, 1993).

One type of manipulable object, bedding, can stimulate many behaviors. Pigs will use straw for nest building and foraging (Arey et al., 1991; Arey, 1993). The provision of straw bedding has been reported to increase udder comfort and reduce stereotypies and sedentary behaviors in sows, and increase play behavior in young pigs (Fraser, 1975; Petersen et al., 1995; Spoolder, 1995). Bedding materials are particularly important for nest building, although sows will instead direct nest building behaviors toward objects like hanging tassels (Widowski and Curtis, 1990), and this does seem to satisfy at least some of the motivational components of nesting behavior. Being able to engage in nest-building behavior probably reduces stress on the sow, and this in turn leads to reduced farrowing times and improved piglet viability (Cronin et al., 1996). However, if nesting material is still available after farrowing, sows appear to develop stronger bonds with their piglets, and providing bedding may reduce piglet crushing in some farrowing systems (Herskin et al., 1998). Bedding is also used by chickens for nest building and dustbathing. Dustbathing regulates the amount of lipids on the feathers and reduces feather damage (van Liere and Bokma, 1987; NCrgaard-Nielsen et al., 1993). Chickens prefer fine materials like peat or sand for dustbathing, but will also use wood shavings (Petherick and Duncan, 1989; van Liere et al., 1990; Sanotra et al., 1995). 4.

Social Behavior

In natural environments, feral farm animals (and their nondomestic ancestors) live in small or moderate-sized groups. The basic social unit in these groups comprises adult females and their offspring. Males associate with the female group during the breeding season, but during the nonbreeding season either are usually relatively solitary or form small bachelor groups. However, animals farmed commercially are now kept in groups that may differ significantly from this pattern, for example, single-sex or single-age groups that may be considerably larger or smaller than the feral norm. Farm animals are generally tolerant of a range of social conditions, but behavioral problems may arise under certain circumstances. These include excessive aggression or dominance-related problems, poor parenting, sexual dysfunction, and abnormal behaviors that lead to injury of other animals or that result, at least in part, from being housed in social isolation or deprived of maternal stimulation. These problems are more common in confined farm animals and can be exacerbated by many nonsocial factors, such as floor, feeder, and waterer space; enclosure configuration, including lack of "escape" areas for subordinate animals; and lack of general environmental stimulation. Social behavior in farm animals has been studied extensively, and because of the many different types of social environments in which farm animals may be housed in the laboratory, it is beyond the scope of this chapter to discuss social housing at length. Detailed information about the social behavior and

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

behavioral problems of each species of farm animal, as well as possible remedies for problems, can be found in a recent book edited by Gonyou and Keeling (2001). However, it is the general opinion that social stimulation is important for farm animals, and housing methods should address this when possible (Federation of Animal Science Societies, 1999). 5.

Novelty, Predictability, and Exploration

Having some degree of predictability is important in farm animals. For example, Carlstead (1986) found that pigs that were fed at unpredictable intervals were more aggressive than pigs that were given a reliable signal (e.g., a bell) when food was about to be delivered. However, lack of environmental challenge associated with highly predictable environments that lack novel stimuli has been implicated as a major cause of apathy and boredom in confined animals (Wemelsfelder and Birke, 1997). Novelty may be attractive to farm animals because it enables them to gain information about their environment, that is, it serves anexploratory function (Mench, 1998b). Hens placed in an enclosure that contains abundant resources still spend a proportion of their time exploring an empty tunnel attached to their enclosure (Nicol and Guilford, 1991). Pigs will similarly explore a pen adjacent to their home pen (Wood-Gush e t al., 1990), particularly if it contains a novel object (Wood-Gush and Vestergaard, 1991). However, as Wemelsfelder (1993) points out, it is not enough for animals simply to be exposed to n o v e l t y - - t h e y must also be allowed to interact with it in some biologically meaningful way. Newberry (1999) found that chickens would readily and repeatedly enter pens adjacent to their home pen. They strongly preferred an adjacent pen containing the same resources as those in the home pen (food, water, heat, and wood shavings), but adjacent pens that contained either frequently changed novel objects or supplemental resources (peat moss, a bale of straw, and an elevated platform) were also used. Empty adjacent pens were least preferred. In addition to interacting with novel stimuli, animals should also be able to avoid them if they so choose. If animals cannot control their exposure to a novel stimulus, it may cause fear instead of eliciting exploration (e.g., Murphy, 1977). Rearing conditions are important with respect to an animal's response to novelty and predictability. Chicks raised in visually complex environments, for example, show less fear of novelty than chicks raised in visually plain environments (Broom, 1969). 6.

Personnel Interactions

Large farm animals can be extremely dangerous to humans. An understanding of farm animal behavior is critical to appropriate handling. Principles of livestock handling to minimize injury and stress to both animals and humans are discussed in Grandin (1993). Poultry can also cause injuries by pecking or

scratching, and handling seems to be particularly stressful for them because of their general fear of humans (Duncan, 1992). Poultry should be handled gently in an upright position to minimize distress and struggling (Kannan and Mench, 1996), and laying hens should be removed from their enclosures particularly carefully because of their bone fragility (Knowles and Wilkins, 1998). Farm animal-personnel interactions are discussed in detail in a recent book by Hemsworth and Coleman (1998). Farm animals that are socialized by regular and gentle handling not only are less fearful of humans but may grow more quickly and/or have higher rates of milk production and improved immune competence. Even fairly brief visual contact with humans without handling can decrease fear responses, at least in chickens (Jones, 1996).

REFERENCES

Abrahamsson, E, and Tauson, R. (1993). Effect of perches at different positions in conventional cages for laying hens of two different strains. Acta Agric. Scand. 43, 228-235. Albright, J. L., and Arave, C. W. (1997). "The Behaviour of Cattle." CAB International, Wallingford, Oxon, United Kingdom. Allen, J. and Perry, G. C. (1975). Feather pecking and cannibalism in a caged layer flock. Br. Poult. Sci. 16, 441-451. Ambrose, N., and Morton, D. B. (1997). The effects of environmental enrichment on cage-cleaning aggression in male laboratory mice. B&K Sci. Now 6, 1-3.

Anderson, J. R., and Chamove, A. S. (1984). Allowing captive primates to forage. In "Standards in Laboratory Animal Management, Symposium Proceedings, Part 2," pp. 253-256. Universities Federation for Animal Welfare, Potters Bar, United Kingdom. Animal Welfare Information Center (1992). "Environmental Enrichment Information Resources for Nonhuman Primates: 1987-1992." National Agricultural Library, U.S. Department of Agriculture, Beltsville, Maryland. Apple, J. K., and Craig, J. V. (1992). The influence of pen size on toy preference of growing pigs. Appl. Anim. Behav. Sci. 35, 149-155. Appleby, M. C. (1995). Perch length in cages for medium hybrid laying hens. Br. Poult. Sci. 36, 23-31. Appleby, M. C., and Wood-Gush, D. G. M. (1988). Effect of earth as an additional stimulus on the behaviour of confined pigs. Behav. Process. 17, 8391. Appleby, M. C., Hughes, B. O., and Elson, A. E. (1992a). "Poultry Production Systems. Behaviour, Management, and Welfare." CAB International, Wallingford, Oxon, United Kingdom. Appleby, M. C., Smith, S. E, and Hughes, B. O. (1992b). Individual perching behaviour of laying hens and its effects in cages. Br. Poult. Sci. 33, 227238. Appleby, M. C., Smith, S. E, and Hughes, B. O. (1993). Nesting, dust bathing, and perching by laying hens in cages: Effects of design on behaviour and welfare. Br. Poult. Sci. 34, 835-847. Arey, D. S. (1993). The effect of bedding on the behavior and welfare of pigs. Anim. Well 2, 235-246. Arey, D. S., Petchey, A. M., and Fowler, V. R. (1991). The preparturient behaviour of sows in enriched pens and the effect of pre-formed nests. Appl. Anim. Behav. Sci. 31, 61-68.

32. LABORATORY ANIMAL BEHAVIOR

Arnold, C. E., and Estep, D. Q. (1990). Effects of housing on social preferences and behavior in male golden hamsters. Appl. Anim. Behav. Sci. 27, 253261. Arnold, C. E., and Estep, D. Q. (1994). Laboratory caging preferences in golden hamsters (Mesocricetus auratus). Lab. Anim. 28, 232-238. Askew, H. R. (1996). "Treatment of Behavior Problems in Dogs and Cats: A Guide for the Small Animal Veterinarian." Blackwell Science, Oxford. Baer, H. (1991). Long-term isolation stress and its effects on drug responses in rodents. Lab. Anim. Sci. 21, 341-349. Baker, K. C. (1996). Chimpanzees in single cages and small social groups: Effects of housing on behavior. Contemp. Top. Lab. Anim. Sci. 35, 71-74. Bantin, G. C., and Saunders, P. D. (1989). Animal caging: Is bigger necessarily better? Anim. Technol. 40, 45-54. Barnett, S. (1967). Rats. Sci. Am. 216, 201-213. Batchelor, G. R. (1991). Group housing on floor pens and environmental enrichment in sandy lop rabbits. Anim. Technol. 42, 109-120. Batchelor, G. (1993). An enriched commune housing system for laboratory rats--a preliminary review. Anim. Technol. 44, 201-213. Batchelor, G. (1994). The rest/activity rhythm of the laboratory rat housed under different systems. Anim. Technol. 45, 181-187. Baxter, S. H., Baxter, M. R., and McCormack, J. A. C., eds. (1983). Farm animal housing and welfare. Curr. Top. Vet. Med. Anim. Sci. 24, 310-317. Bayne, K. A. L. (1989). Nylon balls re-visited. Lab. Primate News. 28, 5-6. Bayne, K. (1996). Normal and abnormal behaviors of laboratory animals: What do they mean? Lab. Anim. 25, 21-24. Bayne, K., and McCully, C. (1989). The effect of cage size on the behavior of individually housed rhesus monkeys. Lab. Anim. 18, 25-28. Bayne, K., and Novak, M. (1998). Behavioral disorders. In "Nonhuman Primates in Biomedical Research: Diseases" (B. T. Bennett, C. R. Abee, and R. Henrickson, eds.), pp. 485-500. Academic Press, New York. Bayne, K., Suomi, S., and Brown, B. (1989). A new monkey swing. Lab. Primate News. 28, 16-17. Bayne, K., Mainzer, H., Dexter, S., Campbell, G., Yamada, E, and Suomi, S. (1991). The reduction of abnormal behaviors in individually housed rhesus monkeys (Macaca mulatta) with a foraging/grooming board. Amer. J. Primatol. 23, 23-35. Bayne, K., Hurst, J., and Dexter, S. (1992a). Evaluation of the preference to and behavioral effects of an enriched environment on male rhesus monkeys. Lab. Anim. Sci. 42, 38-45. Bayne, K., Dexter, S., and Suomi, S. (1992b). A preliminary survey of the incidence of abnormal behavior in rhesus monkeys (Macaca mulatta) relative to housing condition. Lab. Anim. 21, 38-46. Bayne, K., Dexter, S., Mainzer, H., McCully, C., Campbell, G., and Yamada, E (1992c). The use of artificial turf as a foraging substrate for individually housed rhesus monkeys (Macaca mulatta). Anim. Welf 1, 39-53. Bayne, K., Dexter, S., and Strange, G. (1993). The effect of food treat provisioning and human interaction on the behavioral well-being of rhesus monkeys (Macaca mulatta). Contemp. Top. Lab. Anim. Sci. 32, 6-9. Bayne, K. A. L., Strange, G. M., and Dexter, S. L. (1994). Influence of food enrichment on cage side preference. Lab. Anim. Sci. 44, 624-629. Bayne, K., Haines, M., Dexter, S., Woodman, D., and Evans, C. (1995). Nonhuman primate wounding prevalence: A retrospective analysis. Lab. Anim. 24, 40-44. Beaver, B. V. (1981). Behavioral considerations for laboratory dogs and cats. Compend. Contin. Educ. 2, 212- 215. Beaver, B. V. (1989). Environmental enrichment for laboratory animals ILAR News 31, 5-11. Beaver, B. V. (1992). "Feline Behavior: A Guide for Veterinarians." Saunders, Philadelphia. Beaver, B. V. (1999). "Canine Behavior: A Guide for Veterinarians." Saunders, Philadelphia. Bebak, J., and Beck, A. M. (1993). The effect of cage size on play and aggression between dogs in purpose-bred beagles. Lab. Anim. Sci. 43, 457-459.

1257 Benn, D. M. (1995). Innovations in research animal care. J. Am. Vet. Med. Assoc. 206, 465-468. Bernstein, I. S., and Mason, W. A. (1962). Group formation by rhesus monkeys. Anim. Behav. 11, 28- 31. Bishop, M. L., and Chevins, P. E D. (1989). Territory formation by mice under laboratory conditions: Welfare considerations. In "Laboratory Animal Welfare Research: Rodents" (T. B. Poole, ed.), pp. 25-48. UFAW, Wheathampstead, United Kingdom. Blanchard, R., Flannelly, K., and Blanchard, D. (1975). Conspecific aggression in the laboratory rat. J. Comp. Physiol. Psychol. 89, 1204-1209. Blanchard, R., Flannelly, K., and Blanchard, D. (1988). Life span studies of dominance and aggression in established colonies of laboratory rats. Physiol. Behav. 43, 1-7. Blokhuis, H. J. (1986). Feather pecking in poultry: Its relation with ground pecking. Appl. Anim. Behav. Sci. 16, 63-67. Blokhuis, H. J. (1989). The development and causation of feather pecking in the domestic fowl. Thesis, Agricultural Univ. of Wageningen, The Netherlands. Blom, H. J. M. (1993). "Evaluation of Housing Conditions for Laboratory Rats and Mice: The Use of Preference Tests for Studying Choice Behaviour." Febodruk, Enschede, The Netherlands. Blom, H. J. M., Baumans, V., van Vorstenbosch, C. J. A. H. V., van Zutphen, L. E M., and Beynen, A. C. (1993). Preference tests with rodents to assess housing conditions. Anim. Well 2, 1-87. Blom, H. J. M., van Tintelen, G., Baumans V., van den Broek, J., and Beynen, A. C. (1995). Development and application of a preference test system to evaluate housing conditions for laboratory rats. Appl. Anim. Behav. Sci. 43, 279-290. Blom, H. J. M., van Tintelen, G., van Vorstenbosch, C. J. A. H., Baumans, V., and Beynen, A. C. (1996). Preferences of mice and rats for types of bedding material. Lab. Anim. 30, 234-244. Bloom, K. R., and Cook, M. (1989). Environmental enrichment: Behavioral responses of rhesus to puzzle feeders. Lab. Anim. 18, 25-31. Bloomsmith, M. A., Finlay, T. W., Merhalski, J. J. and Maple, T. L. (1990). Rigid plastic balls as enrichment for captive chimpanzees. Lab. Anim. Sci. 40, 319-322. Bloomsmith, M. (1992). Environmental enrichment research to promote the well-being of chimpanzees. In "Chimpanzee Conservation and Public Health: Environments for the Future" (J. Erwin and J. G. Landon, eds.), pp. 155-163. Diagnon/Bioqual, Rockville, Maryland. Boccia, M. L. (1989). Preliminary report on the use of a natural foraging task to reduce aggression and stereotypies in socially housed pigtail macaques. Lab. Primate. News. 28, 3-4. Boice, R. (1977). Burrows of wild and albino rats: Effects of domestication, outdoor raising age, experience, and maternal state. J. Comp. Physiol. Psychol. 91, 649-661. Boot, R., Leussink, A. B., and Vlug, R. F. (1985). Influence of housing conditions on pregnancy outcome in cynomolgus monkeys (Macaca fascicularis). Lab. Anim. Sci. 19, 42-47. Bradshaw, A., and Poling, A. (1991). Choice by rats for enriched versus standard home cages: Plastic pipes, wood platforms, wood chips, and paper towels as enrichment items. J. Exp. Anal. Behav. 55, 245-250. Brain, P. E (1990). Stress in agonistic contexts in rodents. In "Stress in Domestic Animals" (R. Dantzer and R. Zayan, eds.), pp. 73-85. Kluwer Academic Publ., Dordrecht, The Netherlands. Brain, P. E (1992). Understanding the behaviours of feral species may facilitate design of optimal living conditions for common laboratory rodents. Anim. Technol. 43, 99-105. Brain, P. E (1997). Aggression in laboratory rodents as sources of pain and distress. In "Harmonisation of Laboratory Animal Husbandry," (P. N. O'Donoghue ed.), Proceedings of the Sixth FELASA Symposium, pp. 10-14. Royal Society of Medicine Press, Ltd., London, United Kingdom.

1258

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Brain, E E, and Benton, D. (1979). The interpretation of physiological correlates of differential housing in laboratory rats. Life Sci. 24, 99-116. Brain, E E, McAllister, K. H., and Walmesley, S. V. (1989). Drug effects on social behaviour: Methods in ethopharmacology. In "Neuromethods: Psychopharmcaology" (A. A. Boulton, G. B. Baker, and A. J. Greenshaw eds.), pp. 689-739. Humana Press, Totowa, New Jersey. Brastaad, B. O. (1990). Effects on behaviour and plumage of a key-stimuli floor and a perch in triple cages for laying hens. Appl. Anita. Behav. Sci. 27, 127139. Broom, D. M. (1969). Effects of visual complexity during rearing on chicksm reactions to environmental change. Anim. Behav. 17, 773-780. Broom, D. M. (1986). Stereotypies and responsiveness as welfare indicators in stall-housed sows. Anita. Prod. 42, 438-439. Broom, D. M., and Johnson K. G. (1993). "Stress and Animal Welfare." Chapman and Hall, London. Bryant, C. E., Rupniak, N. M. J., and Iversen, S. D. (1988). Effects of different environmental enrichment devices on cage stereotypies and autoaggression in captive cynomolgus monkeys. J. Med. Primatol. 17, 257-269. Bubier, N. E. (1996). The behavioral priorities of laying hens: The effects of two methods of environmental enrichment on time budgets. Behav. Process. 37, 239-249. Buhot, M. C. (1989). Exploration and choice by mice among nest boxes differing in size influence of the inner and outer dimensions. Q. J. Exp. Psychol. 41B, 49-64. Campbell, S. A., Hughes, H. C., Griffin, H. E., Landi, M. S., and Mallon, E M. (1988). Some effects of limited exercise on purpose-bred beagles. Am. J. Vet. Res. 49, 1298-1301. Capel, I., Jenner, M., Pinnock, M., Dorrell, H., and Williams, D. (1980a). The effect of overcrowding stress on carcinogen metabolising enzymes of the rat. J. Environ. Pathol. Toxicol. 4, 337-344. Capel, I., Jenner, M., Pinnock, M., Dorrell, H., and Williams, D. (1980b). The effect of isolation stress on some hepatic drug and carcinogen metabolising enzymes in rats. Environ. Res. 23, 162-169. Capitanio, J. (1998). Social experience and immune system measures in laboratory-housed macaques: Implications for management and research. ILAR J. 39, 12-20. Carder, B., and Berkowitz, K. (1970). Ratsmpreference for earned in comparison with free food. Science 167, 1273-1274. Carlstead, K. (1986). Predictability of feeding: Its effect on agonistic behaviour and growth in grower pigs. Appl. Anim. Behav. Sci. 16, 25-38. Chamove, A. S. (1989). Cage design reduces emotionality in mice. Lab. Anim. 23, 215-219. Chamove, A. S., and Moodie, E. M. (1990). Are alarming events good for captive monkeys. Appl. Anita. Behav. Sci. 27, 169-176. Chance, M. R. A. (1957). Mammalian behaviour studies in medical research. Lancet (October 5), 270, 678-690. Chmiel, D. J., and Noonan, M. (1996). Preference of laboratory rats for potentially enriching stimulus objects. Lab. Anim. 30, 97-101. Claassen, V. (1994). Neglected factors in pharmacology and neuroscience research: Biopharmaceutics, animal characteristics, maintenance, testing conditions. In "Techniques in the Behavioural and Neural Sciences," Vol. 12. Elsevier, Amsterdam. Clark, J. D., Calpin, J. E, and Armstrong, R. B. (1991). Influence of type of enclosure on exercise fitness of dogs. Am. J. Vet. Res. 52, 1024-1028. Clough, G. (1982). Environmental effects on animals used in biomedical research. Biol. Rev. 57, 487-523. Coe, C. L. (1991). Is social housing of primates always the optimal choice? In "Through the Looking Glass: Issues of Psychological Well-Being in Captive Nonhuman Primates" (M. A. Novak and A. J. Petto, eds.), pp. 78-92. American Psychological Assoc. Washington, D.C. Coe, C. L., and Rosenblum, L. A. (1984). Male dominance in the bonnet macaque: A malleable relationship. In "Social Cohesion. Essays toward a Sociophysiological Perspective" (E R. Barchas and S. E Mendoza, eds.), pp. 31-64. Greenwood Press, Westport, Connecticut.

Coelho, A. M., Carey, K. D., and Shade, R. E. (1991). Assessing the effects of social environment on blood pressure and heart rates of baboons. Am. J. Primatol. 23, 257-267. Collinge, N. (1989). Mirror reactions in a zoo colony of cebus monkeys. Zoo Biol. 8, 89-98. Cooper, S. J., and Hendrie, C. A. (1994). "Ethology and Psychopharmacology." Wiley and Sons, New York. Cooper, J. J., Emmans, G. C., and Friggens, N. C. (1994). Effect of diet on behaviour of individually penned lambs. Anim. Prod. 58, 441. Cowan, P. E. (1983). Exploration in small mammals: Ethology and ecology. In "Exploration in Animals and Humans" (J. Archer and L. Birke, eds.), pp. 147-175. Van Nostrand Reinhold, Wakingham, Berkshire, United Kingdom. Craig, W. (1918). Appetites and aversions as constituents of instincts. Biol. Bull. 34, 91-107. Crockett, C. M. (1998). Psychological well-being of captive nonhuman primates: Lessons from laboratory studies. In "Second Nature: Environmental Enrichment for Captive Animals" (D. J. Shepherson, J. D. Mellen, and M. Hutchins, eds.), pp. 129-152. Smithsonian Institution Press, Washington, D.C. Crockett, C. M., Bielitzky, J., Carey, A., and Velez, A. (1989). Kong toys as enrichment devices for singly-caged macaques. Lab. Primate News. 28, 21-22. Crockett, C. M., Bowers, D. M., Bowden, D. M., and Sackett, G. P. (1994). Sex differences in compatibility of pair-housed adult longtailed macaques. Am. J. Primatol. 32, 73-94. Crockett, C., Bellanca, R. U., Bowers, C. L., and Bowden, D. M. (1997). Grooming-contact bars provide social contact for individually caged laboratory macaques. Contemp. Top. Lab. Anim. Sci. 36, 53-60. Cronin, G. M., Simpson, G. J., and Hemsworth, E H. (1996). The effects of the gestation and farrowing environment on sow and piglet behaviour and piglet survival and growth in early lactation. Appl. Anita. Behav. Sci. 46, 175-192. Curtis, S. E. (1983). "Environmental Management in Animal Agriculture." Iowa State Univ. Press, Ames. Dailey, J. W., and McGlone, J. J. (1997). Oral/nasal/facial and other behaviors of sows kept individually outdoors on pasture, soil or indoors in gestation crates. Appl. Anita. Behav. Sci. 52, 25-43. Damon, E. G., Eidson, A. E, Hobbs, C. H., and Hahn E E (1986). Effect of acclimation on nephrotoxic response of rats to uranium. Lab. Anita. Sci. 36, 24-27. Dantzer, R., and Kelley, K. W. (1989). Stress and immunity: An integrated view of relationships between the brain and the immune system. Life Sci. 44, 1995 - 2008. Dawkins, M. S. (1989). Time budgets in red junglefowl as a baseline for the assessment of welfare in domestic fowl. Appl. Anita. Behav. Sci. 24, 77-80. Dawkins, M. S. (1990). From an animal's point of view: Motivation, fitness, and animal welfare. Behav. Brain Sci. 13, 1-61. Dawkins, M. S. (1992). "Animal Suffering: The Science of Animal Welfare," 2nd ed. Chapman and Hall, London. DeLuca, A. M., and Kranda, K. C. (1992). Environmental enrichment. Lab. Anim. 21, 38-44. Denny, M. (1975). The rat's long-term preference for complexity in its environment. Anita. Learn. Behav. 3, 245-249. DePass, L. R., Weil, C. S., Ballantyne, B., Lewis, S. C. Losco, E E. Reid, J. B., and Simon, G. S. (1986). Influence of housing conditions for mice on the results of a dermal oncogenicity assay. Fundam. Appl. Toxicol. 7, 601-608. de Passill6, A. M. B., Christopherson, R., and Rushen, J. (1993). Nonnutritive sucking by the calf and postprandial secretion of insulin, CCK, and gastrin. Physiol. Behav. 54, 1069-1073. Duncan, I. J. H. (1978). The interpretation of preference tests in animal behaviour. Appl. Anim. Ethol. 4, 197-200. Duncan, I. J. H. (1992). The effect of the researcher on the behaviour of poultry.

32. LABORATORY ANIMAL BEHAVIOR In "The Inevitable Bond" (H. Davis and D. Balfour, eds.), pp. 285-294. Cambridge Univ. Press, Cambridge. Duncan, I. J. H., and Kite, V. G. (1989). Nest site selection and nest building in domestic fowl. Anim. Behav. 37, 215-231. Duncan, I. J. H., and Wood-Gush, D. G. M. (1972). Thwarting of feeding behaviour in the domestic fowl. Anim. Behav. 20, 444- 451. Duncan, I. J. H., Appleby, M. C., and Hughes, B. O. (1992). Effects of perches in laying cages on welfare and production of hens. Br. Poult. Sci. 33, 25-36. Duncan, I. J. H., Rushen, J., and Lawrence, A. B. (1993). Conclusions and implications for animal welfare. In "Stereotypic Animal Behaviour. Fundamentals and Applications to Welfare" (A. B. Lawrence and J. Rushen, eds.), pp. 193-206. CAB International, Wallingford, Oxon, United Kingdom. Durschlag, M., and Stauffacher, M. (1996). Effects of environmental enrichment on agonistic interactions and endocrine states in male Zur :ICR mice. In "Second World Congress on Alternatives and Animal Use in the Life Sciences," ATLA Special Issue, p. 217. Frame, Nottingham, England. Eaton, G. G., Kelley, S. T., Axthelm, M. K., and Iliff-Sizemore, S. A. (1994). Psychological well-being in paired adult female rhesus (Macaca mulatta). Am. J. Primatol. 33, 89-99. Erwin, J. (1979). Aggression in captive macaques: Interaction of social and spatial factors. In "Captivity and Behavior: Primates in Laboratories, Breeding Colonies, and Zoos" (J. Erwin, T. Maple, and G. Mitchell, eds.), pp. 139171. Van Nostrand Reinhold, New York. Erwin, J., and Deni, R. (1979). Strangers in a strange land: Abnormal behavior or abnormal environments? In "Captivity and Behavior: Primates in Laboratories, Breeding Colonies, and Zoos" (J. Erwin, T. Maple, and G. Mitchell, eds.), pp. 1-28. Van Nostrand Reinhold, New York. Erwin, J., and Landon, J. G. (1992). Spacious biocontainment suites for chimpanzees in infectious disease research. In "Chimpanzee Conservation and Public Health: Environments for the Future" (J. Erwin and J. G. Landon, eds.), pp. 65-69. Diagnon/Bioqual, Rockville, Maryland. European Directive, Annex II, Guidelines for Accommodation and Care of Animals (1986). Council Directive 86/609/EEC on the approximation of laws, regulations, and administrative provisions of the member states regarding the protection of animals used for other scientific purposes. Official J. Eur. Communities L. 358 29, 1-29. Fara, J., and Catlett, R. (1971). Cardiac response and social behaviour in the guinea-pig. Anim. Behav. 19, 514-523. Faure, J. M., and Jones, R. B. (1982a). Effects of sex, strain, and type of perch on perching behaviour in the domestic fowl. Appl. Anim. Ethol. 8, 291293. Faure, J. M., and Jones, R. B. (1982b). Effects of age, access, and time of day on perching behaviour in the domestic fowl.Appl. Anim. Ethol. 8, 357-365. Federation of Animal Science Societies (FASS) (1999). "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching." Savoy, Illinois. Flannelly, K., and Lore, R. (1977). Observations of the subterranean activity of domesticated and wild rats (Rattus norvegicus): A descriptive study. Physiol. Rec. 27, 315-329. Fox, M. W. (1965). Environmental factors influencing stereotyped and allelomimetic behavior in animals. Lab. Anim. Care 15, 363-370. Fox, M. W. (1968). Introduction: The concepts of normal and abnormal behavior. In "Abnormal Behavior in Animals" (M. Fox, ed.), pp. 1-5. Saunders, London. Fox, M. W., and Bekoff, M. (1975). The behaviour of dogs. In "The Behaviour of Domestic Animals" (E. S. E. Hafez, ed.), p. 370. Williams and Wilkins, Balltimore. Fragaszy, D. M., and Adams-Curtis, L. E. (1991). Environmental challenges in groups of capuchins. In "Primate Responses to Environmental Change" (H. O. Box, ed.), pp. 239-264. Chapman and Hall, London. Fragaszy, D. M., and Mason, W. A. (1978). Response to novelty in Saimiri and Callicebus: Influence of social context. Primates 19, 311-331.

1259 Fraser, D. (1975). The effect of straw on the behaviour of sows in tether stalls. Anim. Prod. 21, 59-68. Fraser, D. (1978). Observations on the behavioural development of suckling and early weaned piglets during the first six weeks after birth. Anim. Behav. 26, 22-30. Fraser, D. (1987). Attraction to blood as a factor in tail-biting by pigs. Appl. Anim. Behav. Sci. 17, 61-68. Fraser, D. (1996). Preference and motivational testing to improve animal wellbeing. Lab. Anim. 25, 27-31. Fraser, A. F., and Broom, D. M. (1997). "Farm Animal Behaviour and Welfare," 3rd ed. CAB International, Wallingford, Oxon, United Kingdom. Fraser, D., Phillips, P. A., Thompson, B. K., and Tennessen, T. (1991). Effect of straw on the behavior of growing pigs. Appl. Anim. Behav. Sci. 30, 307318. Gamello, A., Villanua, A., Trancho, G., and Fraile, A. (1986). Stress adaptation and adrenal activity in isolated and crowded rats. Physiol. Behav. 36, 217221. Gao, W., Feddes, J. J. R., Robinson, E E., and Cook, H. (1994). Effects of stocking density on the incidence of usage of enrichment devices by White Leghorn hens. J. Appl. Poult. Res. 3, 336-341. Gardiner, S., and Bennett, T. (1977). The effects of short term isolation on systolic blood pressure and heart rate in rats. Med. Biol. 55, 325-329. Gentle, M. J., and Hunter, L. N. (1990). Physiological and behavioural responses associated with feather removal in Gallus gallus var. domesticus. Res. Vet. Sci. 50, 95-101. Gomez-Sanchez, E. P., and Gomez-Sanchez, C. E. (1991). Nordeoxycorticosterone, aldosterone, and corticosterone excretion in sequential urine samples from male and female rats. Steroids 56, 451-454. Gonyou, H. W., and Keeling, L. J., eds. (2001). "Social Behaviour in Domestic Animals." CAB International, Wallingford, Oxon, United Kingdom. Gonzalez, C. A., Coe, C. L., and Levine, S. (1982). Cortisol responses under different housing conditions in female squirrel monkeys. Psychoneuroendocrinology 7, 209-216. Grandin, T., ed. (1993). "Livestock Handling and Transport." CAB International, Wallingford, Oxon, United Kingdom. Grandin, T., ed. (1998). "Genetics and the Behavior of Domestic Animals." Academic Press, San Diego. Gunn, D., and Morton, D. B. (1995). Inventory of the behavior of New Zealand White rabbits in laboratory cages. Appl. Anim. Behav. Sci. 45, 277-292. Gust, D. A., Swenson, R. B., Smith, M. B., and Sikes, J. (1988). An apparatus and procedure for studying the choice component of foraging in a captive group of chimpanzees. Primates 29, 139-143. Gvaryahu, G., Arafat, E., Asaf, E., Lev, M., Weller, J. I., Robinzon, B., and Snapir, N. (1994). An enrichment object that reduces aggressiveness and mortality in caged laying hens. Physiol. Behav. 55, 313-316. Haemisch, A., Voss, T., and Gartner, K. (1994). Effects of environmental enrichment on aggressive behaviour, dominance hierarchies, and endocrine state in male DBA/2J mice. Physiol. Behav. 56, 1041-1048. Harlow, H. F., and Harlow, M. K. (1962). The effects of rearing conditions on behavior. Bull. Menninger Clin. 26, 213-224. Hart, L. A. (1994). Opportunities for environmental enrichment in the laboratory. Lab. Anim. 23, 24-27. Hatch, A., Wiberg, G., Balazs, T., and Grice, H. (1963). Long term isolation stress in rats. Science 142, 507. Hauschka, T. S. (1952). Whisker eating mice. J. Hered. 43, 77-80. Hemsworth, P. B., and Coleman, G. J. (1998). "Human-Livestock Interactions. The Stockperson and the Productivity and Welfare of Intensively Farmed Animals." CAB International, Wallingford, Oxon, United Kingdom. Herbers, J. M. (1981). Time resources and laziness in animals. Oecologia 49, 252-262. Her Majesty's Stationery Office (1997). Statistics of Scientific Procedures on Living Animals in Great Britainm1996. Cm. 3012. Her Majesty's Stationery Office, London. Herskin, M. S., Jensen, K. H., and Thodberg, K. (1998). Influence of environ-

1260

KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

mental stimuli on maternal behaviour related to bonding, reactivity, and crushing of piglets in domestic sows. Appl. Anim. Behav. Sci. 58, 241-254. Hessler, J. R., and Moreland, A. E (1984). Design and management of animal facilities. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 505-526. Academic Press, New York. Hetts, S. (1991). Psychologic well-being: Conceptual issues, behavioral measures, and implications for dogs. Vet. Clin. North Am. Small Anim. Pract. 21, 369-387. Hill, J. D., McGlone, J. J., Fullwood, S. D., and Miller, M. E (1998). Environmental enrichment influences on pig behavior, performance, and meat quality. Appl. Anim. Behav. Sci. 57, 51-68. Hite, M., Hanson, H. M., Bohidar, N. R., Conti, P. A., and Mattis, P. A. (1977). Effect of cage size on patterns of activity and health of beagle dogs. Lab. Anim. Sci. 27, 60-64. Hobbs, B. A., Kozubal, W., and Nebiar, E E (1997). Evaluation of objects for environmental enrichment of mice. Contemp. Top. 36, 69-71. Holson, R., Scallet, A., Ali, S., and Turner, B. (1991). Isolation stress revisited: Isolation-rearing effects depend on animal care methods. Physiol. Behav. 49, 1107-1118. Houpt, K. A. (1998). "Domestic Animal Behavior for Veterinarians and Animal Scientists," 3rd ed. Iowa State Univ. Press, Ames. Hubrecht, R. C. (1993). A comparison of social and environmental enrichment methods for laboratory housed dogs. Appl. Anim. Behav. Sci. 37, 345-361. Hubrecht, R. C., Serpell, J. A., and Poole, T. B. (1992). Correlates of pen size and housing conditions on the behaviour of kennelled dogs. Appl. Anim. Behav. Sci. 34, 365-383. Hughes, R. (1968). Behaviour of male and female rats with free choice of two environments differing in novelty. Anim. Behav. 16, 92-96. Hughes, B. O., and Appleby, M. C. (1989). Increase in bone strength of spent laying hens housed in modified cages with perches. Vet. Rec. 124, 483484. Hughes, B. O., and Duncan, I. J. H. (1972). The influence of strain and environmental factors upon feather pecking and cannibalism in fowl. Br. Poult. Sci. 13, 525-547. Hughes, B. O., and Elson, H. A. (1977). The use of perches by broilers in floor pens. Br. Poult. Sci. 18, 715-722. Hughes, H. C., Campbell, S., and Kenney, C. (1989a). The effects of cage size and pair housing on exercise of beagle dogs. Lab. Anim. Sci. 39, 302-305. Hughes, B. O., Duncan, I. J. H., and Brown, M. E (1989b). The performance of nest building by domestic hens: Is it more important than the construction of a nest? Anim. Behav. 37, 210-214. Hurst, J. L., Fang, J., and Barnard, C. J. (1993). The role of substrate odours in maintaining social tolerance between male house mice, Mus musculus domesticus. Anim. Behav. 45, 997-1006. Hurst, J. L., Barnard, C. J., Nevison, C. M., and West, C. D. (1998). Housing and welfare in laboratory rats: The welfare implications of social isolation and social contact among females. Anim. Welf. 7, 121-136. Jennings, M., Batchelor, G., Brain, P. F., Dick, A., Elliot, H., Francis, R. J., Hubrecht, R. C., Hurst, J. L., Morton, D. B., Peters, A. G., Raymond, R., Sales, G. D., Sherwin, C., and West, C. (1998). Refining rodent husbandry: The mouse. Lab. Anim. 32, 233-259. Jensen, G. D., Blanton, E L., and Gribble, D. H. (1980). Older monkeys' (Macaca radiata) response to new group formation: Behavior, reproduction, and mortality. Exp. Gerontol. 15, 399-406. Jerome, C. P., and Szostak, L. (1987). Environmental enrichment for adult, female baboons (Papio anubis). Lab. Anim. Sci. 37, 508-509. Jones, R. B. (1996). Fear and adaptability in poultry: Insights, implications, and imperatives. World Poult. Sci. J. 52, 131-174. Jones, S. E., and Brain, P. E (1987). Performance of inbred and outbred laboratory mice in putative tests of aggression. Behav. Genet. 17, 87-96. Jones, R. B., Harvey, S., Hughes, B. O., and Chadwick, A. (1980). Growth and the plasma concentrations of growth hormone and prolactin in chicks: Effects of "environmental enrichment," sex, and strain. Br. Poult. Sci. 21, 457-462.

Juarbe-Dfaz, S. V. (1997). Assessment and treatment of excessive barking in the domestic dog. In "Progress in Companion Animal Behavior, the Veterinary Clinics of North America: Small Animal Practice," pp. 27, 515-532. Saunders, Philadelphia. Kannan, G., and Mench, J. A. (1996). Influence of different handling methods and crating periods on plasma corticosterone concentrations in broilers. Br. Poult. Sci. 37, 21-31. Kaplan, J. R., Manning, P., and Zucker, E. (1980). Reduction of mortality due to fighting in a colony of rhesus monkeys (Macaca mulatta). Lab. Anim. Sci. 30, 565-570. Keeling, L. J. (1994). Feather peckingmwho in the group does it, how often, and under what circumstances? Proc. 9th Eur. Poult. Conf. 288-289. Keeling, L. J., and Gonyou, H. W., eds. (2001). "Social Behaviour in Domestic Animals." CAB International, Wallingford, Oxon, United Kingdom. Kempermann, G., Kuhn, H. G., and Gage, F. H. (1997). More hippocampal neurons in adult mice living in an enriched environment. Nature 386, 493495. Kessel-Davenport, A. L., and Brent, L. (1994). Effects of an enriched exercise cage on the behaviors of singly housed baboons (Papio sp.). Am. J. Primatol. 33, 220. Kessler, M. R., and Turner, D. C. (1997). Stress and adaptation of cats (Felis silvestris catus) housed singly, in paris, and in groups in boarding catteries. Anim. Welf. 6, 243-254. Kingston, S. G., and Hoffman-Goetz, L. (1996). Effect of environmental enrichment and housing density on immune system reactivity to acute exercise stress. Physiol. Behav. 60, 145-150. Kitchen, A. M., and Martin, A. A. (1996). The effects of cage size and complexity on the behaviour of captive common marmosets, Callithrixjacchus jacchus. Lab. Anim. 30, 317-326. Knowles, T. G., and Broom, D. M. (1990). Limb bone strength and movement in laying hens from different housing systems. Vet. Rec. 126, 354-356. Knowles, T. G., and Wilkins, L. J. (1998). The problem of broken bones during the handling of laying hens--a review. Poult. Sci. 77, 1798-1793. Kohn, D. E, and Barthold, S. W. (1984). Biology and diseases of rats. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 91-122. Academic Press, New York. Kostal, L., Savory, C. J., and Hughes, B. O. (1992). Diurnal and individual variation in behaviour of restricted-fed broiler breeders. Appl. Anim. Behav. Sci. 32, 361-374. Kyriazakis, I., and Savory, C. J. (1997). Hunger and thirst. In "Animal Welfare" (M. C. Appleby and B. O. Hughes, eds.), pp. 49-62. CAB International, Wallingford, Oxon, United Kingdom. Ladewig, J., de Passill6, A. M., Rushen, J., Schouten, W., Terlouw, E. M. C., and Von Borell, E. (1993). Stress and the physiological correlates of stereotypic behaviour. In "Stereotypic Animal Behaviour. Fundamentals and Applications to Welfare" (A. B. Lawrence and J. Rushen, eds.), pp. 97-118. CAB International, Wallingford, Oxon, United Kingdom. Lam, K., Rupniak, N. M. J., and Iversen, S. D. (1991). Use of a grooming and foraging substrate to reduce cage stereotypies in macaques. J. Med. Primatol. 20, 104-109. Lambeth, S. P., and Bloomsmith, M. A. (1992). Mirror as enrichment for captive chimpanzees (Pan troglodytes). Lab. Anim. Sci. 42, 261-266. Latane, B. (1969). Gregariousness and fear in laboratory rats. J. Exp. Soc. Psychol. 5, 61-69. Latane, B., Joy, V., Meltzer, J., Lubell, B., and Cappell, H. (1972). Stimulus determinants of social attraction in rats. J. Comp. Physiol. Psychol. 79, 13-21. Lawlor, M. M. (1984). Behavioural approaches to rodent management. In "Standards in Laboratory Animal Management," Proceedings of a LASA/UFAW Symposium, pp. 40-49. Universities Federation for Animal Welfare, Potters Bar, United Kingdom. Lawrence, A. B., and Rushen, J., eds. (1993). "Stereotypic Animal Behaviour. Fundamentals and Applications to Welfare." CAB International, Wallingford, Oxon, United Kingdom.

32. LABORATORY ANIMAL BEHAVIOR

Lindberg, A. C., and Nicol, C. J. (1994). An evaluation of the effect of operant feeders on welfare of hens maintained on litter. AppL Anim. Behav. Sci. 41, 211-227. Line, S. W., and Houghton, P. (1987). Influence of an environmental enrichment device on general behavior and appetite in rhesus macaques. Lab. Anim. Sci. 37, 508. Line, S. W., and Morgan, K. N. (1991). The effects of two novel objects on the behavior of singly caged adult rhesus macaques. Lab. Anim. Sci. 41, 365369. Line, S. W., Morgan, K. N., Markowitz, H., Roberts, J., and Riddell, M. (1990a). Behavioral responses of female long-tailed macaques (Macaca fascicularis) to pair formation. Lab. Primate News. 29, 1-5. Line, S. W., Morgan, K. N., Roberts, J. A., and Markowitz, H. (1990b). Preliminary comments on resocialization of aged macaques. Lab. Primate News. 29, 8-12. Line, S. W., Markowitz, H., Morgan, K. N., and Strong, S. (199 l a). Effects of cage size and environmental enrichment on behavioral and physiological responses of rhesus macaques to the stress of daily events. In "Through the Looking Glass: Issues of Psychological Well-Being in Captive Nonhuman Primates" (M. A. Novak and A. J. Petto, eds.), pp. 160-179. American Psychological Assoc. Washington, D. C. Line, S. W., Morgan, K. N., and Markowitz, H. (1991b). Simple toys do not alter the behavior of aged rhesus monkeys. Zoo Biol. 10, 473-484. Litterst, C. I. (1974). Mechanically self-induced muzzle alopecia in mice. Lab. Anim. Sci. 24, 806-809. Long, S. Y. (1972). Hair-nibbling and whisker-trimming as indicators of social hierarchy in mice. Anim. Behav. 20, 10-12. Lore, R. K., and Schultz, L. A. (1989). The ecology of wild rats: Applications in the laboratory. In "Ethoexperimental Approaches to the Study of Behavior" (R. J. Blanchard, P. F. Brain, D. C. Blanchard, and S. Parmigiani, eds.), pp. 607-622. Kluwer Academic Publ., Dordrecht, The Netherlands. Love, J. A. (1994). Group housing: Meeting the physical and social needs of the laboratory rabbit. Lab. Anim. Sci. 44, 5-11. Love, J. A. (1995). Implementation of innovative housing for laboratory animals. In "Proceedings of the Animals in Science Conference: Perspectives on Their Use, Care, and Welfare" (N. E. Johnston, ed.), p. 168. Monash Univ. Press, Melbourne, Australia. Lynch, J. J., Hinch, G. N., and Adams, D. B. (1992). "The Behaviour of Sheep." CAB International, Wallingford, Oxon, United Kingdom. Maki, S., and Bloomsmith, M. A. (1989). Uprooted trees facilitate the psychological well-being of captive chimpanzees. Zoo Biol. 8, 79-87. Malik, I., and Southwick, C. H. (1988). Feeding behavior and activity patterns of rhesus monkeys (Macaca mulatta) at Tughlaqabad, India. In "Ecology and Behavior of Food-Enhanced Primate Groups" (J. E. Fa and C. H. Southwick, eds.), pp. 95-111. Alan R. Liss, New York. Mandrell, T. D. (1996). Personnel considerations: Hiring, training, attitudes. In "The Human/Research Animal Relationship" (L. Krulisch, S. Mayer, and R. C. Simmonds, eds.), pp. 99-104. Scientists Center for Animal Welfare, Greenbelt, Maryland. Manning, P. J., Wagner, J. E., and Harkness, J. E. (1984). Biology and diseases of guinea pigs. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and F. M. Loew, eds.), pp. 149-181. Academic Press, New York. Manser, C. E. (1992). "The Assessment of Stress in Laboratory Animals." RSPCA, Horsham, United Kingdom. Manser, C. E., Morris, T. H., and Broom, D. M. (1995). An investigation into the effects of solid or grid cage flooring on the welfare of laboratory rats. Lab. Anim. 29, 353-365. Manser, C. E., Elliott, H., Morris, T. H., and Broom, D. M. (1996). The use of a novel operant test to determine the strength of preference for flooring in laboratory rats. Lab. Anim. 30, 1-6. Manser, C. E., Broom, D. M., Overend, P., and Morris, T. H. (1998a). Investigations into the preferences of laboratory rats for nest-boxes and nesting materials. Lab. Anim. 32, 23-35. Manser, C. E., Broom, D. M., Overend, P., and Morris, T. H. (1998b). Operant

1261 studies to determine the strength of preference in laboratory rats for nestboxes and nesting materials. Lab. Anim. 32, 36-41. Markowitz, H., and Gavazzi, A. (1995). Eleven principles for improving the quality of captive animal life. Lab. Anim. 24, 30-33. Marriott, B. M. (1988). Time budgets of rhesus monkeys (Macaca mulatta) in a forest habitat in Nepal and on Cayo Santiago. In "Ecology and Behavior of Food-Enhanced Primate Groups" (J. E. Fa and C. H. Southwick, eds.), pp. 125-149. Alan R. Liss, New York. Mason, G., McFarland, D. M., and Garner, J. (1998). A demanding task using economic techniques to assess animal priorities. Anim. Behav. 55, 10711075. Mason, G. J. (1991). Stereotypies: A critical review. Anim. Behav. 41, 10151037. McCully, C. L., Godwin, K. S., Brown, P., Mandrell, T., Bayne, K., Southers, J., and Poplack, D. G. (1992). A social housing strategy for rhesus monkeys used frequently in biomedical research. Contemp. Top. Lab. Anim. Sci. 31, 33-34. Mclntyre, D. T., and Petto, A. J. (1993). Effect of group size on behavior of group-housed female rhesus macaques (Macaca mulatta). Lab. Primate News. 32, 1-4. Mench, J. A. (1988). The development of aggressive behavior in male broiler chicks: A comparison with laying-type males and the effects of feed restriction. Appl. Anim. Behav. Sci. 21, 233-242. Mench, J. (1994). Environmental enrichment and exploration. Lab. Anim. 23, 38-41. Mench, J. A. (1998a). Why it is important to understand animal behavior. ILAR J. 39, 20-26. Mench, J. A. (1998b). Environmental enrichment and the importance of exploratory behavior. In "Second Nature: Environmental Enrichment for Captive Animals" (D. J. Shepherdson, J. D. Mellen, and M. Hutchins, eds.), pp. 30-46. Smithsonian Institution Press, Washington, D.C. Mench, J. A. (1999). Ethics, animal welfare, and transgenic farm animals. In "Transgenic Animals in Agriculture" (J. D. Murray, G. B. Anderson, A. M. Oberbauer, and M. M. McGloughlin, eds.), pp. 251-268. CAB International, Wallingford, Oxon, United Kingdom. Mench, J. A., and Keeling, L. J. (2001). Social behaviour of domestic birds. In "Social Behaviour in Domestic Animals" (L. J. Keeling and H. W. Gonyou, eds.), pp. 177-210. CAB International, Wallingford, Oxon, United Kingdom. Mench, J. A., and Mason, G. J. (1997). Behaviour. In "Animal Welfare" (M. C. Appleby and B. O. Hughes, eds.), pp. 127-143. CAB International, Wallingford, Oxon, United Kingdom. Meyer, D. (1995). Environmental enrichment: Creating a happy environment in the animal house. In "Animals in Science" (N. Johnston, ed.), pp. 257-262. Monash Univ. Press, Melbourne, Australia. Millar, S. K., Evans, S., and Chamove, A. S. (1988). Older offspring contact novel objects soonest in callitrichid families. Biol. Behav. 13, 82-96. Milton, K. (1980). "The Foraging Strategies of Howler Monkeys: A Study in Primate Economics." Columbia Univ. Press, New York. Mitchell, G. (1968). Persistent behavior pathology in rhesus monkeys following early social isolation. Folia Primatol. 8, 132-147. Morrow-Tesch, J. (1997). Environmental enrichment for dairy calves and pigs. AWIC Newsl. 7, 3 - 12. Morton, D. B., Jennings, M., Batchelor, G. R., Bell, D., Birke, L., Davies, K., Eveleigh, J. R., Gunn, D, Heath, M., Howard, B., Koder, P., Phillips, J., Poole, T., Sainsbury, A. W., Sales, G. D., Smith, D. J. A., Stauffacher, M., and Turner, R. J. (1993). Refinements in rabbit husbandry. Second report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Lab. Anim. 27, 301-329. Muiruri, H. K., Harrison, P. C., and Gonyou, H. W. (1990). Preferences of hens for shape and size of roosts. Appl. Anim. Behav. Sci. 27, 141-147. Murphy, L. B. (1977). Responses of domestic fowl to novel food and objects. Appl. Anim. Ethol. 3, 335-349. Nagel, R., and Stauffacher, M. (1994). Ethologische Grundlagen zur Beurtei-

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

lung der Tiergerechtheit der Haltung von Ratten in Vollgitterk~ifigen. Tierlaboratorium 17, 119-132. National Institutes of Health (NIH) (1991). "Nonhuman Primate Intramural Management Plan." National Institutes of Health, Bethesda, Maryland. National Research Council (NRC) (1994). "Dogs." National Academy Press, Washington, D.C. National Research Council (NRC) (1996). "Guide for the Care and Use of Laboratory Animals." National Academy Press, Washington, D.C. Neamand, J., Sweeny, W. T., Creamer, A. A., and Conti, P. A. (1975). Cage activity in the laboratory beagle: A preliminary study to evaluate a method of comparing cage size to physical activity. Lab. Anim. Sci. 25, 180-183. Newberry, R. C. (1995). Environmental enrichment: Increasing the biological relevance of captive environments. Appl. Anim. Behav. Sci. 44, 229-243. Newberry, R. C. (1999). Why chickens cross the road: Exploratory behaviour in young domestic fowl. Appl. Anim. Behav. Sci. 63, 311-321. Newberry, R. C., and Shackelton, D. M. (1997). Use of visual cover by domestic fowl: A Venetian blind effect? Anim. Behav. 54, 387-395. Newton, W. M. (1972). An evaluation of the effects of various degrees of longterm confinement on adult beagle dogs. Lab. Anim. Sci. 22, 860-864. Nicol, C. J. (1992). Effects of environmental enrichment and gentle handling on behavior and fear responses of transported broilers. Appl. Anim. Behav. Sci. 33, 367-380. Nicol, C. J., and Guilford, T. (1991). Exploratory activity as a measure of motivation in deprived hens. Anim. Behav. 41, 333-341. Noonan, D. (1994). The guinea-pig (Cavia porcellus). ANZCCART News 7, Insert. Ncrgaard-Nielsen, G., Vestergaard, K., and Simonsen, H. B. (1993). Effects of rearing experience and stimulus enrichment on feather damage in laying hens. Appl. Anim. Behav. Sci. 38, 345-352. Novak, M. A., and Drewson, K. H. (1989). Enriching the lives of captive primates: Issues and problems. In "Housing, Care, and Psychological Wellbeing of Captive and Laboratory Primates" (E. Segal, ed.), pp. 161-182. Noyes Publications, Park Ridge, New Jersey. NRAES-84 (1995). "Animal Behavior and the Design of Livestock and Poultry Systems." Northeast Regional Agricultural Engineering Service, Ithaca, New York. Odberg, F. O. (1987). The influence of cage size and environmental enrichment on the development of stereotypies in bank voles (Clethrionomys glareolus). Behav. Process. 14, 155-173. O'Neill, P. (1988). Developing effective social and environment enrichment strategies for macaques in captive groups. Lab. Anim. 17, 23-26. O'Neill, P. (1989). A room with a view for captive primates: Issues, goals, related research, and strategies. In "Housing, Care, and Psychological Wellbeing of Captive and Laboratory Primates" (E. Segal, ed.), pp. 135-160. Noyes Publications, Park Ridge, New Jersey. O'Neill-Wagner, P. L., Wright, A. C., and Weed, J. L. (1997). Curious response of three monkey species to mirrors. AZAA Regional Conf. Proc., pp. 95101. Orok-Edem, E., and Key, D. (1994). Response of rats (Rattus norvegicus) to enrichment objects. Anim. Technol. 45, 25-30. Paquette, D., and Prescott, J. (1988). Use of novel objects to enhance environments of captive chimpanzees. Zoo Biol. 7, 15-23. Paulk, H. H., Dienske, H., and Ribbens, L. G. (1977). Abnormal behavior in relation to cage size in rhesus monkeys. J. Abnorm. Psychol. 86, 87-92. Pearce, G. P., and Paterson, A. M. (1993). The effect of space restriction and provision of toys during rearing on the behaviour, productivity, and physiology of male pigs. Appl. Anim. Behav. Sci. 36, 11-28. Pearce, G. P., Paterson, A. M., and Pearce, A. N. (1989). The influence of pleasant and unpleasant handling and the provision of toys on the growth and behavior of male pigs. Appl. Anim. Behav. Sci. 23, 27-37. Petersen, V., Simonsen, H. B., and Lawson, L. G. (1995). The effect of environmental stimulation on the development of behavior in pigs. Appl. Anim. Behav. Sci. 45, 215-224.

Petherick, J. C., and Duncan, I. J. H. (1989). Behaviour of young domestic fowl directed towards different substrates. Br. Poult. Sci. 30, 229-238. Phillips, C. J. C. (1993). "Cattle Behaviour." Farming Press, Ipswich, United Kingdom. Podberscek, A. L., Blackshaw, J. K., and Beattie, A. W. (1991). The behaviour of group penned and individually caged laboratory rabbits. Appl. Anim. Behav. Sci. 28, 353-363. Poole, T. B. (1991). Criteria for the provision of captive environments. In "Primate Responses to Environmental Change" (H. O. Box, ed.), pp. 357-374. Chapman and Hall, London. Poole, T. B. (1992). The nature and evolution of behavioural needs in mammals. Anita. Welf. 1, 203-220. Poole, T. B. (1998). Meeting a mammal's psychological needs: Basic principles. In "Second Nature: Environmental Enrichment for Captive Animals" (D. J. Shepherson, J. D. Mellen, and M. Hutchins, eds.), pp. 1-12. Smithsonian Institution Press, Washington, D.C. Potter, M. P., and Borkowski, G. L. (1998). Apparent psychogenic polydipsia and secondary polyuria in laboratory-housed New Zealand White rabbits. Contemp. Top. Lab. Anim. Sci. 37, 87-89. Price, E. O. (1987). Farm animal behavior. In "The Veterinary Clinics of North America: Food Animal Practice Series." Saunders, Philadelphia. Price, E. O. (1998). Behavioral genetics and the process of animal domestication. In "Genetics and the Behavior of Domestic Animals" (T. Grandin, ed.), pp. 31-65. Academic Press, San Diego. Pyle, D. A., Bennett, A. L., Zarcone, T. J., and Turkkan, J. S. (1996). Use of two food foraging devices by singly housed baboons. Lab. Primate News. 35, 10-15. Raje, S., and Stewart, K. L. (1997). Group housing for male New Zealand White rabbits. Lab. Anim. 36, 36-38. Reed, H. J., Wilkins, L. J., Austin, S. D., and Gregory, N. G. (1993). The effect of environmental enrichment during rearing on fear reactions and depopulation trauma in adult caged hens. Appl. Anim. Behav. Sci. 36, 39-46. Reinhardt, V. (1988). Preliminary comments on pairing unfamiliar adult rhesus monkeys for the purpose of environmental enrichment. Lab. Primate News. 27, 1-3. Reinhardt, V. (1994a). Comparing the effectiveness of PVC swings versus PVC perches as environmental enrichment objects for caged female rhesus macaques. Lab. Primate News. 30, 5-6. Reinhardt, V. (1994b). Continuous pair-housing of caged Macaca mulatta: Risk evaluation. Lab. Primate News. 33, 1-4. Reinhardt, V. (1994c). Social enrichment for previously single-caged stumptail macaques. Anim. Technol. 5, 37- 41. Reinhardt, V., and Reinhardt, A. (1991). Impact of a privacy panel on the behavior of caged female rhesus monkeys living in pairs. J. Exp. Anim. Sci. 34, 55-58. Reinhardt, V., and Reinhardt, A. (1998). "Environmental Enrichment for Nonhuman Primates: An Annotated Bibliography for Animal Care Personnel," 2nd ed. Animal Welfare Institute, Washington, D.C. Reinhardt, V., Cowley, D., Eisele, S., Vertein, R., and Houser, W. D. (1988). Pairing compatible female rhesus monkeys for the purpose of cage enrichment has no negative impact on body weight. Lab. Primate News. 27, 13-15. Reinhardt, V., Pape, R., and Zweifel, D. (1991a). Multifunctional cage for macaques housed in pairs or in small groups. AALAS Bull. 30, 14-15. Reinhardt, V., Cowley, D., and Eisele, S. (1991 b). Serum cortisol concentrations of single-housed and isosexually pair-housed adult rhesus macaques. J. Exper. Anim. Sci. 34, 73-76. Renner, M. J., and Rosenzweig, M. R. (1987). "Enriched and Impoverished Environments. Effects on Brain and Behavior." Springer-Verlag, New York. Riley, V. (1981). Psychoneuroendocrine influences on immunocompetence and neoplasia. Science 212, 1100-1109. Roberts, J. A. (1989). Environmental enrichment, providing psychological wellbeing for people and primates. Am. J. Primatol. Suppl. 1, 25-30.

32. LABORATORY ANIMAL BEHAVIOR Roper, T. J. (1973). Nesting material as a reinforcer for female mice. Anim. Behav. 21, 733-740. Rose, M. (1993). Social behaviour of rats and mice m individuals and groups. ANZCCART News 6, 1O- 11. Rose, M. A. (1994). Environmental factors likely to impact on an animal's wellbeing: An overview. In "Improving the Well-Being of Animals in the Research Environment," Proceedings conference in Sydney, October, pp. 99116. ANZCCART. Rosenblum, L. A., and Coe, C. L. (1977). The influence of social structure on squirrel monkey socialization. In "Primate Bio-Social Development: Biological, Social, and Ecological Determinants" (S. Chevalier-Skolnikoff and E E. Poirier, eds.), pp. 479-499. Garland Publ. New York. Rosenblum, L. A., and Smiley, J. (1984). Therapeutic effects of an imposed foraging task in disturbed monkeys. J. Child Psychol. Psychiatry 25, 485497. Rumbaugh, D. M., Washburn, D., and Savage-Rumbaugh, E. S. (1991). On the care of captive chimpanzees: Methods of enrichment. In "Housing, Care, and Psychological Wellbeing of Captive and Laboratory Primates" (E. Segal, ed.), pp. 357-375. Noyes Publications, Park Ridge, New Jersey. Sachser, N. (1986). The effects of long term isolation on physiology and behaviour in male guinea-pigs. Physiol. Behav. 38, 31-39. Sachser, N., and Lick, C. (1991). Social experience, behaviour, and stress in guinea-pigs. Physiol. Behav. 50, 83-90. Sackett, G. P. (1965). Effects of rearing conditions upon monkeys (M. mulatta). Child Dev. 36, 855-868. Sambrook, T. D., and Buchanan-Smith, H. M. (1996). What makes novel objects enriching? A comparison of the qualities of control and complexity. Lab. Primate News. 35, 1-4. Sanotra, G. S., Vestergaard, K. S., Agger, J. F., and Lawson, L. G. (1995). The relative preferences for feathers, straw, wood-shavings, and sand for dustbathing, pecking, and scratching in domestic chicks. Appl. Anim. Behav. Sci. 43, 263-277. Sarna, J. R., Dyck, R. H., and Whishaw, I. Q. (2000). The Dalila effect: C57BL6 mice barber whiskers by plucking. Behav. Brain Res. 1t)8, 39-45. Savory, C. J. (1995). Feather pecking and cannibalism. World Poult. Sci. J. 51, 215-219. Schaefer, A. L., Salomons, M. O., Tong, A. K. W., Sather, A. P., and LePage, P. (1990). The effect of environmental enrichment on aggression in newly weaned pigs. Appl. Anim. Behav. Sci. 27, 41-52. Scharmann, W. (1991). Improved housing of mice, rats, and guinea-pigs: A contribution to the refinement of animal experiments. ATLA 19, 108-114. Schwartz, R., Sackler, A., and Weltman, A. (1974). Adrenal relationships and aggressiveness in isolated female mice. Experientia 30, 199-200. Scott, L. (1991). Environmental enrichment for single housed common marmosets. In "Primate Responses to Environmental Change" (H. O. Box, ed.), pp. 265-274. Chapman and Hall, London. Shepherdson, D. J. (1998). Tracing the path of environmental enrichment in zoos. In "Second Nature: Environmental Enrichment for Captive Animals" (D. J. Shepherson, J. D. Mellen, and M. Hutchins, eds.), pp. 1-12. Smithsonian Institution Press, Washington, D. C. Sherwin, C. M. (1993). The pecking behaviour of laying hens provided with a simple motorized pecking device. Br. Poult. Sci. 34, 235-240. Sherwin, C. M. (1996a). Laboratory mice persist in gaining access to resources: A method of assessing the importance of environmental features. Appl. Anim. Behav. Sci. 48, 203-214. Sherwin, C. M. (1996b). Preferences of individually housed TO strain laboratory mice for loose substrate or tubes for sleeping. Lab. Anim. 30, 245-251. Sherwin, C., and Nicol, C. (1995). Changes in meal patterning by mice measure the cost imposed by natural obstacles. Appl. Anim. Behav. Sci. 43, 291300. Shettleworth, S., and Jordan, V. (1986). Rats prefer handling food to waiting for it. Anim. Behav. 34, 925-927. Silverman, A. P. (1978). "Animal Behaviour in the Laboratory." Chapman and Hall, London.

1263 Singh, D. (1970). Preference for bar pressing to obtain reward over freeloading in rats and children. J. Comp. Physiol. Psychol. 73, 320-327. Smith, A., Lindburg, D. G., and Vehrencamp, S. (1989). Effect of food preparation on feeding behavior of lion-tailed macaques. Zoo Biol. 8, 57-65. Snowdon, C. T., and Savage, A. (1991). Psychological well-being of captive primates: General considerations and examples from callitrichids. In "Housing, Care, and Psychological Wellbeing of Captive and Laboratory Primates" (E. Segal, ed.), pp. 75-88. Noyes Publications, Park Ridge, New Jersey. Spoolder, H. A. M., Burbridge, J. A., Edwards, S. A., Simmins, P. H., and Lawrence, A. B. (1995). Provision of straw as a foraging substrate reduces the development of excessive chain and bar manipulation in food restricted sows. Appl. Anim. Behav. Sci. 43, 249-262. Stanton, M. E., Patterson, J. M., and Levine, S. (1985). Social influences on conditioned cortisol secretion in the squirrel monkey. Psychoneuroendocrinology 10, 125-134. Stauffacher, M. (1997a). Comparative studies on housing conditions. In "Harmonisation of Laboratory Animal Husbandry" (P. N. O'Donoghue, ed.), Proceedings of the Sixth FELASA Symposium, pp. 5-9. Royal Society of Medicine Press, Ltd., London. Stauffacher, M. (1997b). Housing requirements: What ethology can tell us. In "Animal Alternatives, Welfare and Ethics" (L. E M. Van Zutphen and M. Balls, eds.), pp. 179-186. Elsevier, Amsterdam. Stermann, M. B., Knauss, T., Lehmann, D., and Clemente, C. D. (1965). Circadian sleep and waking patterns in the laboratory cat. Electroencephalogr. Clin. Neurophysiol. 19, 509-517. Stolba, A., and Wood-Gush, D. G. M. (1989). The behaviour of pigs in a seminatural environment. Anim. Prod. 48, 419-425. Strier, K. B. (1987). Activity budgets of woolly spider monkeys or muriquis (Brachyteles arachnoides). Am. J. Primatol. 13, 385-395. Sutherland, S., and Festing, M. (1987). The guinea-pig. In "Handbook on the Care and Management of Laboratory Animals" (T. B. Poole, ed.), 6th ed., pp. 309-330. Longman Science and Technical, Harlow, United Kingdom. Tauson, R. (1984). Effects of a perch in conventional cages for laying hens. Acta Agric. Scand. 34, 193-209. Tauson, R. (1985). Mortality in laying hens caused by differences in cage design. Acta Agric. Scand. 35, 165-174. Tauson, R. (1986). Avoiding excessive growth of claws in caged laying hens. Acta Agric. Scand. 36, 95-106. Tauson, R., and Svensson, S. A. (1980). Influence of plumage condition on the hen's feed requirement. Swedish J. Agric. Res. 10, 35-39. Taylor, I. A. (1995). Designing equipment around behavior. In "Animal Behavior and the Design of Livestock and Poultry Systems." Northeast Regional Agricultural Engineering Service, Ithaca, New York. Terlouw, E. M. C., Lawrence, A. B., and Illius, A. W. (1991). Influences of feeding level and physical restriction on development of stereotypies in sows. Anim. Behav. 42, 981- 991. Thorington, R. W., Jr. (1968). Observations of squirrel monkeys in a Colombian forest. In "The Squirrel Monkey" (L. A. Rosenblum and R. W. Cooper, eds.), pp. 69-85. Academic Press, New York. Thornberg, L. P., Stowe, H. D., and Pick, J. E (1973). The pathogenesis of the alopecia due to hair chewing in mice. Lab. Anim. Sci. 23, 843-850. Tipton, C. M., Carey, R. A., Eastin, W. C., and Erickson, H. H. (1974). A submaximal test for dogs: Evaluation of effects of training, detraining, and cage confinement. J. Appl. Physiol. 37, 271-275. Tuli, J. S. (1993). Stress and parasitic infection in laboratory mice. Ph.D. thesis, University of Birmingham. Turner, R. J., Held, S. D. E., Hirst, J. E., Billinghurst, G., and Wootton, R. J. (1997). An immunological assessment of group-housed rabbits. Lab. Anim. 31, 362-372. U.S. Department of Agriculture (1991). Title 9, CFR Part 3, Animal Welfare; Standards; Final Rule. Fed. Reg., Vol. 56, No. 32, February 15. Van de Weerd, H. A. (1996). "Environmental Enrichment for Laboratory Mice:

1264

KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

Preferences and Consequences." Print Partners Ipskamp, Enschede, The Netherlands. Van de Weerd, H. A., Baumans, V., Koolhaus, J., and van Zutphen, L. (1994). Strain specific behavioural response to environmental enrichment in the mouse. J. Exp. Anim. Sci. 36, 117-127. Van de Weerd, H. A., van den Broek, E A. R., and Baumans, V. (1996). Preference for different types of flooring in two rat strains. AppL Anim. Behav. Sci. 46, 251-261. Van de Weerd, H. A., van Loo, P. L. P., van Zutphen, L. E M., Koolhaas, J. M., and Baumans, V. (1997). Preferences for nesting material as environmental enrichment for laboratory mice. Lab. Anim. 31, 133-143. van Liere, D. W., and Bokma, S. (1987). Short-term feather maintenance as a function of dust-bathing in laying hens. Appl. Anim. Behav. Sci. 18, 197204. van Liere, D. W., Kooijman, J., and Wiepkema, P. R. (1990). Dustbathing behaviour of laying hens as related to quality of dustbathing material. AppL Anim. Behav. Sci. 26, 127-141. van Putten, G., and Dammers, J. (1976). A comparative study of the well-being of piglets reared conventionally and in cages. Appl. Anim. Ethol. 2, 339356. van Putten, G., and van de Burgwal, J. A. (1990). Vulva biting in group housed sows: Preliminary report. Appl. Anim. Behav. Sci. 26, 181-186. Vestergaard, K. S., and Lisborg, L. (1993). A model of feather pecking development which relates to dustbathing in the fowl. Behaviour 126, 89-106. Vom Saal, E S. (1991). Prenatal gonadal influences on mouse sociosexual behaviours. In "Heterotypical Behaviour in Man and Animals" (M. Haug, P. E Brain, and C. Aron, eds.), pp. 42-70. Chapman and Hall, London. Wadham, J. J. B. (1996). Recognition and reduction of adverse effects in research on rodents. Ph.D. thesis, University of Birmingham. Wadham, J. J. B., and Morton, D. B. (In press). Measurement of benzodiazepine receptors in the brains of rats kept in enriched and conventional environments. Lab. Anim. Ward, G., and DeMille, D. (1991). Environmental enrichment for laboratory mice. Anim. Technol. 42, 149-156. Watson, D. S. B. (1991). A built-in perch for primate squeeze cages. Lab. Anim. Sci. 41, 378-379. Watson, S. L., and Shively, C. A. (1996). Effects of cage configuration on behavior in cynomolgus macaques. International Primatological Society/ American Society of Primatologist Congress Abstracts. Webb, N. G., and Nilsson, C. (1983). Flooring and injuryman overview. In "Farm Animal Housing and Welfare" (S. H. Baxter, M. R. Baxter, and J. A. C. McCormack, eds.). Curr. Top. Vet. Med. Anim. Sci. 24, 226-261. Wechsler, B., and Huber-Eicher, B. (1988). The effect of foraging material and perch height on feather pecking and feather damage in laying hens. Appl. Anim. Behav. Sci. 58, 131-142. Weihe, W. (1987). The laboratory rat. In "Handbook on the Care and Management of Laboratory Animals" (T. B. Poole, ed.), 6th ed., pp. 309-330. Longman Science and Technical, Harlow, United Kingdom. Weld, K., Metz, B., and Erwin, J. (1991). Environmental enrichment for Macaca fascicularis: Effects of shape and substance of manipulable objects. Am. J. Primatol. 24, 139.

Wemelsfelder, E (1990). Boredom and laboratory animal welfare. In "The Experimental Animal in Biomedical Research" (B. E. Rollin and M. L. Kesel, eds.), Vol. 1, pp. 243-272. CRC Press, Boca Raton, Florida. Wemelsfelder, F. (1993). The concept of animal boredom and its relationship to stereotyped behaviour. In "Stereotypic Animal Behaviour. Fundamentals and Applications to Welfare" (A. B. Lawrence and J. Rushen, eds.), pp. 6595. CAB International, Wallingford, Oxon, United Kingdom. Wemelsfelder, E, and Birke, L. (1997). Environmental challenge. In "Animal Welfare" (M. C. Appleby and B. O. Hughes, eds.), pp. 35-47. CAB International, Wallingford, Oxon, United Kingdom. Whary, M., Peper, R., Borkowski, G., Lawrence, W., and Ferguson, E (1993). The effects of group housing on the research use of the laboratory rabbit. Lab. Anim. 27, 330-341. White, W. (1990). The effect of cage space and environmental factors. In "Guidelines for the Well-Being of Rodents in Research" (H. N. Guttman, ed.), pp. 29-44. Scientists Center for Animal Welfare, Greenbelt, Maryland. White, W., Balk, M., and Lang, M. (1989). The use of cage space by guinea pigs. Lab. Anim. 23, 208-214. Widowski, T. M., and Curtis, S. E. (1990). The influence of straw, cloth tassel, or both on the prepartum behavior of sows. Appl. Anim. Behav. Sci. 27, 53-71. Wiepkema, P. R, Broom, D. M., Duncan, I. J. H., and van Putten, G. (1983). "Abnormal Behaviours in Farm Animals." Commission of the European Communities, Brussels. Williams, L. E., Abee, C. R., Barnes, S. R., and Ricker, R. B. (1988). Cage design and configuration for an arboreal species of primate. Lab. Anim. Sci. 38, 289-291. Wolff, A., and Ruppert, G. (1991). A practical assessment of a non-human primate exercise program. Lab Anim. 20, 36-39. Wolfle, T. (1996). How different species affect the relationship. In "The Human/Research Animal Relationship" (L. Krulisch, S. Mayer, and R. C. Simmonds, eds.), pp. 85-90. Scientists Center for Animal Welfare, Greenbelt, Maryland. Wood-Gush, D. G. M. (1972). Strain differences in response to suboptimal stimuli in domestic fowl. Anim. Behav. 20, 72-76. Wood-Gush, D. G. M., and Vestergaard, K. (1991). The seeking of novelty and its relation to play. Anim. Behav. 42, 599-606. Wood-Gush, D. G. M., Vestergaard, K., and Petersen, V. (1990). The significance of motivation and environment in the development of exploration in pigs. BioL Behav. 15, 39-52. Wiirbel, H., Stauffacher, M., and Von Hoist, D. (1996). Stereotypies in laboratory mice mquantitative and qualitative description of the ontogeny of "wire chewing" and "jumping" in Zur:ICR and Zur:ICR nu-mice. Ethology 102, 371-385. Wtirbel, H., Chapman, R., and Rutland, C. (1998). Effects of feed and environmental enrichment on development of stereotypic wire-gnawing in laboratory mice. Appl. Anim. Behav. Sci. 60, 69-81. Young, R. J., Carruthers, J., and Lawrence, A. B. (1994). The effect of a foraging device (the "Edinburgh foodbalr') on the behaviour of pigs. Appl. Anim. Behav. Sci. 39, 237-247.

Index

Abomasal disorder, 599-600 Abortion bovine, 541-542, 561 brucellosis, 539-540 campylobacteriosis, 540-542 caprine, 583 chlamydial, 583 enzootic, 583 listeriosis, 553 mycoplasmosis, 562 neosporosis, 588-589 ovine, 540-541,553, 583 query fever, 564 ruminant, 562 yersiniosis, 562 Abscesses caseous lymphadenitis, 547 ferret, 497 foot, 551 hepatic, 733 ruminant, 551, 611 sole, 611 turtle, 849 yersiniosis, 733 Absidia corymbifera, 505 ABSL, 1048 Acanthocephalosis grasshopper mouse, 265 guinea pig, 225 primate, 762 reptile, 852 Acanthocheilonema viteae, 276, 282 Acaropsis docta, 1096 Acepromazine clostridia infection, 742 tetanus, 544

tranquilizer dog, 973,974 gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 719, 720, 726 rabbit, 968 rat, 963 rodent, 960 ruminant, 987, 988 swine, 981 Acetaminophen hamster, 964 mouse, 962 primate, 726 rabbit, 972 rodent, 961 Acetylsalicylic acid. See Aspirin Achatina sp., 1013 Acidosis, lactic, 601,602 Acinal cell atrophy, mouse, 109 Ackertia marmotae, 319, 323 Acral lick granuloma, 434-435, 1247 Acriflavine, 820 Actinobacillosis, ruminants, 537 Actinobacillus sp. Actinobacillus lignieresii, 537 Actinobacillus pleuropneumoniae, 637, 640 Actinomyces sp. Actinomyces bovis, 555 Actinomyces pyogenes, 218 bite wound, 497 Actinomycosis, ruminants, 538 Acyclovir, 1069 Addisonian adrenal necrosis, 173 Adenocarcinoma cotton rat, 272

1265

1266 Adenocarcinoma (cont.) endometrial, 331 frog, 817-818 gastric, 277, 357 gastric tubular, 272 gerbil, 277 ground squirrel, 254 hamster, 187, 193, 197 Helicobacter pylori, 492 intestinal, 187 kangaroo rat, 261 Lucke's renal, 817-818, 823 mammary gland, 197, 261,281,356 mouse, 113 pancreatic, 193, 357 pulmonary, 113 rabbit model, 331 renal, 817-818, 823 snake, 857 uterine, 193, 263, 356 vole, 281 wood rat, 263 Adenoma adrenal, 187 bile duct, 357 bronchial, 237 chromophobe, 155 cortical, 154, 155 hepatic, 197 islet cell, 154 liver, 154 mammary gland, 154 papillary, 237 parathyroid, 187 pars distalis, 154 pituitary, 173 pulmonary, 113 thyroid, 187, 356 turtle, 857 Adenomatosis, pulmonary, 577-578 Adenovirus canine, 405-406 cotton rat, 270 goat, 582 guinea pig, 222 hamster, 185 mouse, 63-64 primate, 751 rat, 147 reptile, 850 ruminant, 565 waterborne human, 369 Adiaspiromycosis, 267 Adrenal cortical nodules, mouse, 110 Adrenal gland diseases cortical hyperplasia, 510 cysts, 189 ferret, 508-510 hamster model, 173 mouse, 110 neoplasms, 154, 155, 357, 508-510 rat, 154, 155 Adrenalectomy, 1028-1029, 1147

INDEX Advanced intercross line, 37, 39 Adynamic ileus, 154 Aequorin, 865-866 Aeromonas sp., 497 Aeromonas caviae, 898 Aeromonas hydrophila, 814-815, 878, 898 Aeromonas salmonicida, 898 Aeromonas schubertii, 898 Aeromonas sobria, 898 Aeromonas veronii, 898 nitrification cycle, .869 Afipia felis, 1080 Aflatoxicosis, guinea pig, 230 African clawed frog, 795 African green monkey, 750, 751, 753 Age-related diseases cotton rat, 272 degu, 285 dog, 449-451 ferret, 512-513 gerbil, 279 guinea pig, 239-240 mouse, 109-113 multimammate rat, 284-285 woodchuck, 325-326 Aggression, in confinement, 1242, 1243 Aging, genetic research, 1193-1195 AIDS. See HIV/AIDS; Simian AIDS Airborne transmission, 367 Airway hyperresponsiveness, 207 Albendazole, 100, 323,416, 643 Albinism, white-tailed rat, 275 Aleutian disease, 499-500 Aleutian disease virus, 510 Alfentanil, 960, 982 Algae, 874 Allegheny wood rat, 261 Alligator, 829, 830, 844 Allodermanyssus sanguinus, 1096 Allograft, 1033 Allopurinol, 854 Allotransplantation, 1107-1108 Alopecia chinchilla, 289 dog, 425,427 ferret, 508-510 guinea pig, 211,236-237 hamster, 186 mouse, 107, 110 prairie dog, 257 rabbit, 350-351 rat, 158 Alphaloxone-alphadolone gerbil, 965 hamster, 964 mouse, 962 primate, 991,992, 993 rat, 963 rodent, 959 Althesin, 959 Alveolar histiocytosis, 158 Alveolar hyperplasia, 108 Alytes obstetricans, 808

1267

INDEX Alzheimer's disease, 170, 1194-1195 Amami rabbit, 330 Amantadine, 641 Ambylomma americanum, 1096 Ambyostoma sp. Ambyostoma mexicanum, 794, 795 Ambyostoma tigrinum, 794, 806 American Association for Laboratory Animal Science (AALAS), 5, 7, 10-12, 1198 American College of Laboratory Animal Medicine (ACLAM), 1231-1234 animal models information, 1198 animal selection, 1231 education and training, 1231-1234 grants and awards, 1236 history, 13, 1203 American Physiological Society (APS), 8-10 American Rabbit Breeders Association, 330 American Society of Laboratory Animal Practitioners (ASLAP), 14 American Veterinary Medical Association (AVMA), 1-2, 13, 897 Amicarbolide, 586 Amikacin colibacillosis, 547, 674 klebsiellosis, 735 mycobacteriosis, 816 reptile dosing, 849 salmonellosis, 561 shell fracture, 856 Amino acid deficiency, 235 Aminocaproic acid, 907 Aminoglycosides, 815 Amitraz demodectic mange, 657 demodicosis, 425, 504 louse, 657 sarcoptic mange, 425 Ammonia, 869-870 Ammonium chloride, 406 Ammonium hydroxide, 502 Ammonium molybdenate, 606 Amoebiasis amphibian, 819-822 mouse, 99 primate, 758 rat, 149 reptile, 851 zoonotic, 1089-1090 AmoxiciUin. See also Penicillins aspiration injury, 440 bordetellosis, 735 greasy pig disease, 655 helicobacteriosis dog, 413 ferret, 494 human, 1085 mouse, 90 primate, 731 rat, 140 lumpy jaw, 538 Lyme disease, 412, 554 navel ill, 559 primate dose, 726 skin wound, 433 streptococcal meningitis, 633, 634

Amphibian acquisition and sources, 796 anatomy and physiology, 801-803 behavior, 806-807 clinical chemistry data, 803, 806 diseases bacterial, 814-817 fungal, 822 metabolic and nutritional, 822-823 neoplasms, 823 parasitic, 819-822 traumatic, 823 viral and chlamydial, 817-819 handling and restraint, 799-800 hematology data, 805 housing, 796-797 husbandry, 796-801,808 longevity, 803 metamorphosis, 808 nutrition and diet, 803-804, 806 physical examination, 813-814 reproduction, 807-808 research uses, 795-796 taxonomy, 793-795 temperature, 798-799 water quality, 797-798 Ampicillin. See also Penicillins actinobacillosis, 537 balantidiasis, 1090 clostridia infection, 648 colibacillosis, 731 gastric dilatation, 777 leptospirosis, 1083 listeriosis, 554 lumpy jaw, 538 navel ill, 559 pasteurellosis, 559, 735 primate dose, 726 salmonellosis, 561 streptococcal meningitis, 633, 634, 735 Amprolium, 417, 587, 652 Amrinone, 975 Amyloid P, 188 Amyloidosis cat model, 461 degu, 285 guinea pig, 239 hamster, 188 hamster model, 170, 188 mouse, 105, 109, 110 ruminant, 611 Amyloodinium sp., 879 Analgesia animals, effect on, 1156-1157 dog, 974, 978-979 frog model, 795 gerbil, 965 guinea pig, 965-966 hamster, 964-965 mouse, 962-963 NSAID dog, 974, 979 primate, 991,997

1268 Analgesia, NSAID (cont.) rabbit, 972-973 rodent, 961 ruminant, 987, 989-990 swine, 981,985 primate, 726, 991,992, 997 rabbit dosage, 968-969 rat, 963- 964 rodents, 961-962 ruminant, 987, 989-990 swine, 981,985 Xenopus sp., 812-813 Anaphylaxis, 610, 1209 Anaplasma sp. Anaplasma marginale, 584 Anaplasma ovis, 584 Anaplasmosis, 584-585 Anatrichostoma cynomolgi, 766, 770 Ancylostoma sp., 478 Ancylostoma braziliense, 418-420, 1094 Ancylostoma caninum, 418-420, 1094 Ancylostoma duodenale, 1094 Androlaelaps fahrenholzi, 324 Anemia, 665 Anesthesia animals, effect on, 1156-1157 cardiac surgery, 977, 984, 995 dog, 974-978 epidural, 979, 985,997 fish, 896-897 gerbil, 965 guinea pig, 965-966 hamster, 964-965 hypnosis, 971-972 isoflurane-induced extramedullary hematopoiesis, 486 local, 979, 985, 989, 997 mouse, 962-963 neurosurgery, 984, 996 obstetric/gynecology, 984, 995-996 pediatric, 977, 984, 996 porcine stress syndrome, 662 pregnancy, 960-961 primate, 726, 990-996 rat, 963 - 964 regional, 979, 985,997 reptile, 845 rodent, 957-961 ruminant, 986-989 spinal, 971 stereotactic surgery, 961 swine, 980-984 thoracic surgery, 977 woodchuck, 312 Xenopus sp., 812-813 Anesthetics dog dosage, 974 gerbil dosage, 965 guinea pig dosage, 966 hamster dosage, 964 local, 960, 961,972 mouse dosage, 962 primate restraint, 719-720 rabbit dosage, 969-970 rat dosage, 963

INDEX swine dosage, 981

Xenopus sp., 812-813 Angiography, 1028 Animal and Plant Health Inspection Service (APHIS), 21, 25 Animal biosafety level (ABSL), 1048 Animal Care Panel, 11-12 Animal experimentation, 1, 2-7 abnormal behavior, 1248, 1253 amphibian, 795-796 animal models database, 250 animal resources, 1234 animal selection, 1230-1231 age, 1145 circadian rhythm, 1146 endocrine factors, 1146-1147 genetic, 1144-1145 husbandry, 1147-1153 immune and nutritional status, 1146 sex, 1145-1146 baboon, 711 cat, 460-462 crisis in, 1203 diagnostic laboratory support, 1235 dog, 395-396 ferret, 484 fish, 886 genetically defined animals, 1118-1119 genetics, 1144-1145 grants and awards, 1233, 1235-1236 ground squirrel, 252 guinea pig, 203 hamster, 169 history, 1200-1201, 1206-1214 information support, 1235 marmoset, 685-686 mouse, 35 origin and history, 2-3 owl monkey, 692 paradigm shifts, 1201-1202 rabbit, 330-331 rat, 114, 152-153 research design, 1230 research laboratory support, 1234 results reporting, 1230-1231 serendipity, 1201 squirrel monkey, 697-698 swine, 617-618 tamarin, 685-686 technological breakthroughs, 1201-1202 woodchuck, 310 zebrafish, 862 Animal facilities. See Facility design; Facility management Animal model defined, 1185-1190 induced, 1186-1189, 1198 journals, 1198-1199 mutagenesis, 1190 source and supply, 1197-1200, 1234 spontaneous, 1186-1189, 1198 validation, 1189-1190 Animal Models and Genetic Stocks Information Program, 1197, 1234 Animal Welfare Act (1970), 14, 20-25,397, 717 Animal Welfare Information Center, 23 Animal Welfare Regulations (1985), 1240

1269

INDEX

Anisocytosis, 334 Anole, 844 Anorexia, 236, 848 Ant, fluke vector, 595 Antacids, 601 Anthrax, 538-539 Antibiotics, 726, 1157-1158 Antibodies, 1209-1210 Antibody assay, 375-377, 380-382 Antibody production, 331,374-375 Antiemetic, 795 Antigen assay, 375-377 Antigens, swine leukocyte, 627,628 Antivenom, 262, 1055 Antivivisectionism, 7-9 Aotus sp. Aotus lemurinus griseimembra, 687 Aotus lemurinus lemurinus, 687 Aotus nancymaae, 688 Aotus vociferans, 687, 688 taxonomy, 686-687 Aphthovirus sp., 574 Apramycin, 547 Aquarium, 872 Arachidonic acid, 107 Arbovirus, 276 Arbovirus sp., 569 Arcanobacterium sp. Arcanobacterium bovis, 537-538 Arcanobacterium pyogenes, 537-538, 549, 557-558 Arenavirus, 223, 262 Argas persicus, 1096 Argentinian hemorrhagic fever, 1096 Arginine vasotocin, 808, 854 Argyrol, 598 Armadillo, 1012 Armed Forces Institute of Pathology, 1198 Arsenic, 423 Arterivirus sp., 660-662 Arthritis caprine arthritis encephalitis virus, 555,569-570 mouse, 91, 92 mycoplasmal, 639-640 polyarthritis, 583-584, 631 reptile, 854 ruminant, 551-552 swine, 639-640 thromboembolic meningoencephalitis, 551-552 Artificial insemination campylobacteriosis control, 542 dog, 404-405 ferret, 490 guinea pig, 211-212 method, 1025 rat, 133 ruminant, 533-534 Arvey Ordinance, 8 Ascariasis cat, 478 dog, 417-418 guinea pig, 225-228 hamster, 186 mouse, 102-105 rat, 151

swine, 642-643 Ascaris sp. Ascaris lumbricoides, 766, 769, 1094 Ascaris suum, 642-643, 652 Ascending pyelitis, 110 Aspergillosis, 352 Aspergillus sp., 230, 853 Aspergillus flavus, 352 Aspergillus fumigatus, 151-152, 352 Aspicularis tetraptera, 101-102, 149 Aspiration lung injury, 439-440, 986-987 Aspirin dog, 974 primate, 726, 991,997 rabbit, 972 swine, 981 Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International), 14, 25-26, 28, 397 Associations, 7-14, 28 Asthma, 207, 1213 Ataxia, enzootic, 605-606 Atherosclerosis, 286, 331,617-618, 692 Atipamezole dog, 974 medetomidine reversal, 959 primate, 992 rabbit, 969 rat, 963 ruminant, 987, 988 Atrial thrombosis, 108 Atrophic rhinitis, 635-637 Atropine dog, 973, 974 gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 726, 990, 991,992 rabbit, 968 rat, 963 rodent, 960 swine, 982 Atropine esterase, 355 Aujeszky's disease, 578-579 Austrobilharzia variglandis, 276 Autoclaving, 369 Autograft, 1033 Autoimmune disease, 282, 284 Avermectin, 597, 643, 657 Avertin, 958 Axolotl, 794, 795, 802, 804 Azaperone, 981 Azathioprine, 1211 Azurophil, 841 B

B-cell lymphoma, 111 B virus infection (cercopithecine), 1068-1069 Babesia sp. Babesia bigemina, 585 Babesia bovis, 585 Babesia divergens, 275 Babesia microti, 281

1270 Babesia sp. (cont.) Babesia pitheci, 761 Babesiosis, 585-586 Baboon, 678 blood sample collection, 1007 body weight, 711 clinical chemistry data, 710 diseases enteric, 730-734 mycotic, 744-746 nutritional, 773-774 respiratory and nervous system, 734-738 tuberculosis and mycobacterial, 738-742 viral, 746 - 757 drug dosages, 726 hematology data, 709 housing, 708, 710 husbandry, 708, 710 natural history, 706-707 nutrition and diet, 710-711 reproduction, 707-708 research uses, 711 taxonomy, 706, 707 vaccination, 725 xenotransplantation, 628 Bacillary hemoglobinuria, 544-545 Bacillus sp. Bacillus anthracis, 538-539 cilia-associated respiratory bacillus infection, 83, 140-142 mastitis, 555 Bacitracin, 274 Bacterial diseases animals, effect on, 1161-1163 diagnosis, 373-374 multimammate rat model, 282 zoonotic, 1077-1088 Bacteroides sp., 551 Bacteroides distasonis, 46 Bacteroides melaninogenicus, 550 reptile, 850 Balanoposthitis, 572-573 Balantidiasis, 651,759, 1090 Balantidium sp. Balantidium caviae, 224 Balantidium coli, 223, 644, 651,759, 1090 Bandaging, 433 Bang's disease, 539-540 Barbering, 52, 108, 236, 1241-1242 Barber's pole worm, 592 Barbiturates, 957-958 Barrier, 917 Barth's syndrome, 461 Bartonella henselae, 479, 1079-1080 Basal cell tumor, 356, 357 Bat, 1070 Batrachochytrium dendrobatidis, 1188 Baylisascaris sp., 255, 319 Baylisascaris columnaris, 323 Baylisascaris laevis, 323 Baylisascaris procyonis, 225, 289, 323, 1094 Bayou virus, 268 Baytril, 726 Beagle clinical chemistry data, 400-401

INDEX congenital disorders, 449 growth data, 398 hematology data, 399 juvenile polyarteritis syndrome, 451-453 neoplasms, 442 normative data, 402 research uses, 396 thyroid carcinoma, 448-449 Bedding disposal, 933 fungus in, 152 guinea pig, 205 hamster, 178 mouse, 40-41 rat, 153 reptile, 831 woodchuck, 311 Beechey ground squirrel, 250-254 Behavior alligator, 844 amphibian, 806-807 birds, 1253 cat, 464-465,466, 1246-1248 cattle, 535-536 chinchilla, 290 crocodile, 844 dog, 405, 1246-1248 frog, 806-807 goat, 535-537 guinea pig, 209, 241, 1244 hamster, 1245 history of study, 1240-1241 isolation stress phenomenon, 1164 marmoset, 684 mouse, 51-52, 1241-1242 owl monkey, 688 primate, 1248-1252 rabbit, 336-337, 1245 rat, 130, 133-134, 1243 reptile, 844 rhesus monkey, 701 rodents, 1241-1245 salamander, 806-807 sheep, 535-536 snake, 844 squirrel monkey, 695 stereotypy, 1240-1242, 1247, 1252 stress, 1240-1241 study methods, 1240 swine, 623, 665, 1252-1253 tamarin, 684 turtle, 844 woodchuck, 319 Behavioral disorders acral lick granuloma, 434-435 dog, 434-435 mouse, 108 Belladonna alkaloids, 355 Benign epidermal monkeypox, 1061 Benign prostatic hyperplasia, 450-451 Benzalkonium chloride, 822 Benzimidazoles, 100, 101,588, 592, 643 Bezoar, 260 Bicarbonate, 601

1271

INDEX Bighead, 544-545,609 Bile duct manipulation, 1017-1018 Bile duct neoplasms, 357 Bile sampling, 1017-1018 Bio MedNet Mouse Knockout database, 1199 Biofilms, 370 Biofiltration, 869, 888-889, 893 Biohazards containment, 1048-1053 facility design, 917- 918 legislation and guidelines, 30-31 management and control, 1047-1056 needles and syringes, 1053 risk assessment, 1048 safety cabinets, 943,945 security and controlled access, 1053 warning sign, 1054 waste disposal, 1053 Bioinformatics, 1202-1203 Biological filtration, 869, 888-889 Biological safety cabinet, 943, 945, 1048-1053 Biopsy liver, 1018-1019 method, 442 reptile, 847, 848 skin, 728-729 testicular, 1024 Biosecurity, 366-372, 720-723 Biotransformation, 1153 Birds behavior, 1253 blood collection, 1012-1013 cannulation technique, 1014 psittacosis vector, 1077 Birnavirus, 905 Birth complication, 507 Bismuth, 90, 140, 731 Bismuth subsalicylate, 413, 494, 1085 Bite wound dog, 433 ferret, 497 primate, 775-776 reptile, 855-856 ruminant, 610 swine, 665 Bites and scratches, 721, 1077-1081 Bittner virus, 112 Black disease, 544-545 Black mouse, 107 Black-tailed prairie dog, 254-257 Blackleg, 544-545 Bladder worm, 595 Blastomyces dermatitidis, 230 Blepharitis, 241 9Blindness, 110 Bloat, 600-601,776-777, 987 Blood pressure technique, 1027 Blood sample collection amphibian, 814 baboon, 1008 birds, 1012-1013 carotid-jugular shunt, 1027 cat, 1008, 1011-1012 chinchilla, 1011

dog, 1008, 1011-1012 ferret, 1011 gerbil, 1009 guinea pig, 1008, 1010-1011 hamster, 1008 marmoset, 1008 mink, 1011 mouse, 1007, 1008 primate, 727-728, 729, 1008, 1012 rabbit, 1008, 1011 rat, 1008, 1010 reptile, 846 rodents, 1008-1011 swine, 617-623 woodchuck, 312 Blowpipe, 719 Bluetongue, 565-566 Bolivian squirrel monkey, 693-694, 695 Bolomys sp., 268 Bone marrow biopsy, 729 Boophilus sp. Boophilus annulatus, 585 Boophilus decoloratus, 585 Boophilus microplus, 585 Border disease, 573 Bordetella sp.

Bordetella bronchiseptica dog, 405-406 ferret, 496-497 guinea pig, 212-213 isolation and culture, 373 kennel cough, 405-406 pneumonia, 321,407-408, 496-497 primate, 735,737 swine, 635-637 vole, 281 woodchuck, 321 zoonotic, 1088 feline upper respiratory tract infection, 476 Bordetellosis guinea pig, 212-213 primate, 735,737 zoonotic, 1088 Boric acid, 598 Borrelia sp. Borrelia burgdorferi, 1096 grasshopper mouse, 265 rat vector, 262 tick vector, 411-412, 554-555 zoonotic, 1098 Borrelia recurrentis, 1096 Borreliosis. See Lyme disease Bos sp. Bos indicus, 520 Bos taurus, 520 Botfly, 597 Bothridium sp., 852 Botriocephalus sp., 852 BovGbase, 1200 Brachydanio rerio, 862 Brachylagus idahoensis, 330 Brachyspira sp. Brachyspira hyodysenteriae, 643-645

Brachyspira pilosicoli, 645

1272 Brain, 1030-1031 Breeding systems cat management, 466- 474 colony management, 1121-1122 defined, 37 genetic contamination, 1119-1120 heterozygosity, residual, 1120 inbreeding, incomplete, 1120 mouse, 36-37 mutagenicity, 864 mutant alleles, 1120 transgenic/knockout mouse, 1135-1137 zebrafish, 864 Bretylium tosylate, 982 British Veterinary Association, 15, 895 Bronchial hyperplasia, 108 Bronchitis cat, 476 hamster model, 172 swine, 642-643 verminous, 642- 643 Bronchoalveolar lavage, 1023 Bronchoscopy, 1023 Brown Pierce carcinoma, 357 Brucella sp. Brucella abortus, 218, 539-540 Brucella canis, 1081-1082 Brucella melitensis, 218, 539-540 Brucella neotomae, 261 Brucella ovis, 539-540 Brucella suis, 218, 657-658 Brucellosis guinea pig, 218 ruminant, 539-540 swine, 657-658 wood rat, 261 zoonotic, 1081-1082 Brugia sp. Brugia malayi, 276, 282 Brugia pahangi, 276, 282 Brush rabbit, 344 Bubonic plague, 1082-1083 Bufo sp. Bufo marinus, 800 reproduction, 807 Bunamidine, 772 Bunolagus monticularis, 330 Bunostomum trigonocephalum, 593 Bunyoro rabbit, 330 Buphthalmia, 353-354 Bupivacaine, 960, 974, 997 Buprenorphine dog, 974 gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 726, 991,992, 997 rabbit, 972 rodent, 961 ruminant, 987 swine, 981 Burns, 440, 856-857 Bushman rabbit, 330

INDEX Bushy-tailed wood rat, 261 Butenafine, 229 Butorphanol dog, 974 hamster, 964 mouse, 962 primate, 726, 992 rabbit, 972 reptile, 845 rodent, 961 swine, 981 Xenopus sp., 813 C Caecilian, 793-794 Cage paralysis, 773 Cages animal experimentation, 1152-1153 caging systems, 937-939, 944 germ-free isolator, 945 identification, 1006-1007 isolation unit, 941 primate, 716-717, 940, 1249 restraint, 718 rodent, 939-943 sanitation and sterilization, 931-935 Caiman, 850-851 Calcification. See Mineralization, soft tissue Calcium, 662, 869 Calcium borogluconate, 604 Calcium chloride, 975 Calcium deficiency amphibian, 822-823 primate, 774 reptile, 853 ruminant, 603-604 Calcium excess, 443 Calcium imaging, 865-866 Calcium supplements, 853,854 Calcivirus feline, 476-477 rabbit, 330, 345 vaccination, 476 California ground squirrel, 250-254 California vole, 279 Callithrix sp. Callithrixjacchus, 681-682, 750 taxonomy, 682 Callitrichid primate. See also Marmoset; Tamarin defined, 678 diseases nutritional, 773-775 respiratory and nervous system, 735 tuberculosis and mycobacterial, 738-742 Camallanus sp., 903 Campylobacter sp., 218, 331 Campylobacter coli, 732, 1084 Campylobacterfetus subsp, intestinalis, 540-541 Campylobacterfetus subsp, jejuni, 180, 491 Campylobacterfetus subsp, venearealis, 541-542 Campylobacter helveticus, 1084 Campylobacterjejuni, 408-409, 491-492, 540-541,732, 1084 Campylobacter upsalensis, 1084

1273

INDEX isolation and culture, 374 proliferative bowel disease, 494 Campylobacteriosis dog, 408-409 ferret, 491-492 primate, 731,732 ruminant, 540 zoonotic, 1084 Canadian Council on Animal Care (CCAC), 15 Cancer eye, 611 Candida albicans, 98, 230, 745-746, 853, 899 Candidiasis, 230, 745-746 Cane mouse biology and physiology, 269 description, distribution, habitat, 269 diseases, 270 husbandry, 269-270 nutrition and diet, 269 research uses, 269 taxonomy, 268 Canis sp. Canis familiaris, 395 Canis latrans, 395 Canis lupus, 395 Canis lupus chanco, 395 Canis lupus pallipes, 395 Canis rufus, 395 Capillaria sp., 878 Capillaria hepatica, 253,282, 323 Capillaria philippinensis, 276 Capillaria tamiasstriate, 323 fish, 903 Capnocytophagia sp., 479 Capra sp. Capra falconeri, 520 Capra hircus, 520 Capra ibex, 520 Capra pyrenaica, 520 Caprine arthritis encephalitis, 532 Caprolagus hispidus, 330 Captan, 598 Capture myopathy, 324 Carassius auratus, 880 Carbadox, 630, 645 Carbamates, 227 Carbon dioxide anesthesia fish, 897 gerbil, 965 hamster, 964 mouse, 962 rat, 963 rodent, 959-960 Carbon tetrachloride, 762 Carcinogens, 442 Carcinoma adrenal, 187 bile duct, 357 bovine, 611 Brown Pierce, 357 dog, 448-449 ground squirrel, 253-254 hamster, 197 hepatocellular, 253, 257, 310, 321-322, 325 islet cell, 154, 155

liver, 112-113, 154, 253-254 mammary gland, 112, 154 mucoepidermoid, 357 pars distalis, 154, 155 prairie dog, 257 renal, 187 sebaceous gland, 357 squamous cell ground squirrel, 254 hamster, 197 rabbit, 356, 357 reptile, 857 ruminant, 611 thyroid, 448-449 turtle, 857 uterine, 187 VX-2, 331,357 woodchuck, 310, 321-322, 325 Carcinosarcoma, 112 Cardiac puncture, 1008-1009 Cardiomyopathy cat model, 461 cotton rat, 272 ferret, 512 hamster model, 169 nutritional, 774 primate, 774 rat, 157-158 woodchuck, 326 Cardiovascular diseases aortic rupture, 326 atherosclerosis, 331 atrial thrombosis, 108 congenital lesions, 156 dog, 421-423 fibrinopurulent pericarditis, 215 guinea pig, 215 heartworm, 421-423 juvenile polyarteritis syndrome, 451-453 mouse, 108 myocardial and epicardial mineralization, 105-106, 108 periarteritis, 108 rat, 156, 157-158 septal congenital, gerbil, 279 swine model, 617-618 woodchuck, 326 Carotene, 487 Carotid-jugular shunt, 1027 Carprofen dog, 974 rodent, 961 ruminant, 987, 989 swine, 981 Caseous lymphadenitis, 547 Castration, 451,535, 1025 Cat acquisition and sources, 462-463 behavior, 464-465,466, 1246-1248 blood sample collection, 1007, 1011-1012 breeding colony management, 466-474 cat scratch disease vector, 1080 diseases congenital, 460 feline lower urinary tract infection, 475

1274 Cat, disease (cont.) feline spongiform encephalopathy, 461 model, 460-462 parasitic, 478 prevention, 475-476 upper respiratory tract infection, 476-477 environmental enrichment, 1246-1247 estrous cycle, 466-467 helicobacteriosis vector, 1085 housing, 463-466, 1246 identification, 472-474 infertility, 470-471 microbiological quality control, 462-463 noise control, 1247 nutrition and diet, 474-475 reproduction, 466-474 research uses, 460-462 social interaction, 1246-1247 stress, 1247 toxoplasmosis vector, 590-591 Cat scratch disease, 479, 1079-1080, 1081 Cataracts chinchilla, 291 degu, 285-286 dog, 449-450 guinea pig, 241 mouse, 110 Catarrhine primate, 679-680 Catheter indwelling, 436-437, 610-611,664, 729 infection, 664 intestinal cannulation, 1019 intracerebral implantation, 1030 neuromuscular damage, 610-611 spinal, 1031 urinary, 728 urine sample collection, 1020-1021 vascular cannulation technique, 1013-1015 Cattle acquisition and sources, 522-524 analgesia, 987, 989-990 anesthesia, 986-989 behavior, 535-536 cannulation technique, 1014 clinical chemistry data, 528 databases, 1200 development, 532-533 digestive system, 525 diseases bacterial, 537-562 genetic, 599 iatrogenic, 610-611 management-related, 608-610 metabolic, 599-605 mycoplasmal, 562-563 neoplasms, 611 nutritional, 607-608 protozoal, 584-592 rickettsial, 563-564 traumatic, 610 viral, 565-583 embryo transfer, 534-535 estrous cycle, 530, 534

INDEX health screening, 523 hematology data, 527 histocompatibility complex, 525 husbandry, 524, 531 identification, 1007 mastitis, 555-556 metabolic disease model, 522 normative data, 526 nutrition and diet, 526-529 physiology, 525-526 postoperative recovery, 988 preanesthesia, 986 reproduction, 529-531 research uses, 521,522 tail docking, 535 taxonomy, 520 Cattle grub, 597 Cattle stomach worm, 592-593 Cavia porcellus, 203-204 Cecal mucosal hyperplasia, 182 Cecal volvulus, 109 Cecotrophy, 332 Cefoxitin, 255 Ceftazidime, 849, 856 Ceftiofur atrophic rhinitis, 636 navel ill, 559 pasteurellosis, 559, 637 pleuropneumonia, 637 salmonellosis, 630 streptococcal meningitis, 634 Ceftriaxone, 735 Cellular blue nevi, 187 Cellular immunity, 1210-1211 Centers for Disease Control and Prevention (CDC), 30 Cephalexin, 726 Cephalohematoma, 775 Cephalosporins colibacillosis, 547 greasy pig disease, 655 heel wart, 551 Lyme disease, 554 polyarthritis, 631 primate dose, 726 sepsis, 439 skin wound, 433 Cephalothin, 735 Cephapirin, 726 Cercopithecine herpesvirus, 746, 747, 748, 1068-1069 Cerebral larvae migrants, 225 Cerebromedullar exposure, 1030 Cerebrospinal fluid collection, 728, 1031-1032 Cerebrovascular disease, 193, 275, 326 Cestodiasis amphibian, 821 dog, 423-424 dwarf, 100 gerbil, 277 gerbil model, 276 grasshopper mouse, 265 guinea pig, 225 hamster, 186 mouse, 100

1275

INDEX

prairie dog, 257 primate, 772 rat, 150-151 reptile, 852 ruminant, 594-595 wood rat, 263 woodchuck, 324 zoonotic, 1094 Chabertia ovis, 593 Chagas' disease, 261,286 Chamaleo jacksonii, 853 Chameleon, 853 Charles River Laboratories, 1199 Checkered-cross rabbit, 355 Chediak-Higashi syndrome, 275, 461 Cheek pouch, 170, 190, 679 Chemical agents containment, 918 disinfectant, 369- 370 hazardous substances, 31 radioactive agents and equipment, 31 reptile toxicity, 857 restraint, 719 xenobiotics, 1153-1156 Chemical burn, 440 Cherry eye, 453-454 Cheyletiella mite, 1095 Cheytiella sp., 1096 Cheytiella johnsoni, 349-350 Cheytiella ochotonae, 349-350 Cheytiella parasitovorax, 349-350 Cheytiella takahashii, 349-350 Chicken blood collection, 1013 cannulation technique, 1014 databases, 1200 housing, 1254 ChickGBase, 1200 Chimpanzee breeding program, 712 clinical chemistry data, 715 diseases enteric, 730-734 mycotic, 744-746 nutritional, 773-774 respiratory and nervous system, 734-738 tuberculosis and mycobacterial, 738-742 viral, 746-757 drug dosages, 726 hematology data, 714 housing, 713-714 husbandry, 713-714 natural history, 713 nutrition and diet, 714 reproduction, 713 research uses, 714-715 taxonomy, 711-712 vaccination, 725 Chinchilla behavior, 290 biology and physiology, 286 blood sample collection, 1011 description, distribution, habitat, 286

diseases age-related, 291 bacterial, mycoplasmal, and rickettsial, 287-288 genetic and congenital, 291 neoplasms, 290-291 parasitic, 288-289 traumatic, 290 viral and chlamydial, 288 dustbath, 291 hematology data, 256 husbandry, 286-287 reproduction, 286, 290 research uses, 286 Chinchilla laniger, 256, 286 Chinese hamster ovary (CHO) cell, 190 Chirodiscoides caviae, 225, 226-228 Chlamydia sp. Chlamydia laevis, 800 Chlamydia pneumoniae, 800 Chlamydia psittaci, 220-221,583, 819 Chlamydia trachomatis, 96, 180 feline upper respiratory tract infection, 476 Chlamydiosis amphibian, 819 guinea pig, 220-221 mouse, 96 zoonotic, 1076-1077 Chlamydophyla psittaci, 1077, 1096 Chloral hydrate, 959, 963 Chloralose dog, 974, 976 guinea pig, 966 hamster, 964 rabbit, 969 rat, 963 rodent, 959, 961 swine, 981 Chloramines, 797 Chloramphenicol amoebiasis, 758 bordetellosis, 735 campylobacteriosis, 409 clostridia infection, 217 colibacillosis, 343 ehrlichiosis, 410 eye infections, 241 kennel cough, 406 lymphadenitis, 215 pasteurellosis, 735 primate dose, 726 proliferative bowel disease, 494 pseudomoniasis, 816 red leg, 815 streptococcal meningitis, 735 yersiniosis, 731 Chlorhexidine catheter infection, 437 dermatomycosis, 428 dermatophilosis, 548-549 disinfectant, 406 skin preparation and disinfection, 432 Chlorine, 906 Chlorine dioxide, 352, 437

1276 Chloroleukemia, 111 Chloroquine, 758, 761 Chlorous acid, 352 Chlorpromazine lactogenesis, 665 rodent, 960 tetanus, 544 tranquilizer, 960 Chlortetracycline campylobacteriosis, 541 erysipelas, 632 foothill abortion, 561 leptospirosis, 553, 659 pleuropneumonia, 637 proliferative enteropathy, 646 CHO cell, 190 Choke, esophageal, 289 Cholangiofibrosis, 190 Cholangioma, 187 Cholera, 172 Cholesteatoma, 279 Cholesterol, 331 Cholestyramine, 342 Choline deficiency, 107, 231,235 Chondrodysplasia, 599 Chordoma, 512 Chorionic gonadotropin, 506-507 Chorioptes sp. Chorioptes bovis, 596 Chorioptes caprae, 596 Chorioptes ovis, 596 Chorioptic mange, 596 Chromomycosis, 822 Chromophobe adenoma, 155 Chronic ulcerative dermatitis, 107 Chylomicronemia, 461 Cilia-associated respiratory (CAR) bacillus infection, 83, 140-142, 144 Cimetidine, 442, 494 Ciprofloxacin, 476 Circadian rhythm, 1146 Circling disease, 553-554 Cirrhosis, 190 Cisapride, 848 Citellina sp. Citellina bifurcaturn, 323 Citellina triradiata, 323 Citrobacter sp., 240 Citrobacter freundii, 218 Citrobacter koseri diversus, 320 Citrobacter rodentium, 85-86, 90, 94, 277, 1161-1162 Citrullinemia, 599 Clara cell, 113 Claviceps purpurea, 240 Clavulanate, 433,440 Climazolam, 981 Clinical chemistry data amphibian, 803, 806 baboon, 710 beagle, 400-401 cattle, 528 chimpanzee, 715 crocodile, 842 cynomolgus monkey (macaque), 705

INDEX

degu, 284-285 ferret, 487 frog, 803, 806 goat, 526, 528 guinea pig, 208 lizard, 842 marmoset, 686 mouse, 44 owl monkey, 691 rabbit, 334-335 Rana sp., 806 rat, 128 reptile, 842 rhesus monkey, 703 sheep, 528 snake, 842 squirrel monkey, 698 swine, 624-625 Syrian (golden) hamster, 175 tamarin, 686 turtle, 842 woodchuck, 314, 316 Cloacal prolapse, 854 Clofazimine, 741 Cloning, 1192 Clostridia infection. See also Tyzzer's disease antibiotic-associated, 217-218 chinchilla, 288 enteritis, 644, 648-649 guinea pig, 217-218 mouse, 95 primate, 742, 776 reptile, 850 ruminant, 542-546 swine, 648-649 Clostridium sp., 850. See also Tyzzer's disease Clostridium chauvoei, 544-545 Clostridium difficile, 182, 189, 217-218, 255, 327 Clostridium novyi, 544-545, 595 Clostridium perfringens, 599, 644, 645, 776 Clostridium perfringens type A, 288, 342, 490-491,542, 648 Clostridium perfringens type B, 542 Clostridium perfringens type C, 542-543, 648 Clostridium perfringens type D, 95, 542, 543-544 Clostridium perfringens type E, 542 Clostridium piliforme animals, effect on, 1162 guinea pig, 218 hamster, 183 isolation and culture, 374 mouse, 83-85, 90 rat, 135-137 Clostridium septicum, 545-546 Clostridium tetani, 543-544, 742 Clostridium welchii, 342 Coccidioides immitis, 230, 265, 744 Coccidioidomycosis, 744 Coccidiosis. See also Cryptosporidiosis cat, 478 diagnosis, 373 dog, 417 ferret, 502 grasshopper mouse, 265

1277

INDEX guinea pig, 223-224 mouse, 99 prairie dog, 255 primate, 758 rabbit, 346-347 reptile, 851 ruminant, 586-587 swine, 644, 651-652

Cochliomyia hominovorax, 281,597 Cod liver oil, 903 Codeine, 966 Coelomocyte, 1013

Coenuris cerebralis, 595 Coenurosis, 772 Coisogenic inbred strain, 37 Colibacillosis mouse, 94-95 primate, 731,732-733 rabbit, 342-343 ruminant, 546-547 swine, 644, 646-648 Colitis, 496 Colony health, 319-320 Colostrum, 608, 627 Common field vole, 279 Common vole, 279 Comparative medicine, 2 Comparative Medicine program, NCRR, 1236 Comp!ement fixation test, 375,381 Computational fluid dynamics, 920-921 Conductivity, water, 869 Congenic inbred strain, 37 Congestive heart failure, 169, 326 Conjunctivitis cattle, 556-557 goat, 563, 584 guinea pig, 241 sheep, 563,584 Constipation, 289 Contact transmission, 367-368 Convention on International Trade in Endangered Species (CITES, 1973), 30 Cooperia sp. Cooperia curticei, 593 Cooperia oncophora, 593 Cooperia pectinata, 593 Cooperia punctata, 593 Copper deficiency, 599, 605-606 Copper sulfate clostridia infection, 342 dermatomycosis, 352 foot rot, 550 heel wart, 551 Ichthyobodo necatrix, 901 protozoal infection, 820, 901 Copper toxicosis, 606-607,797 Copra itch, 278 Corneal mineralization, 156 Corneal opacity, 108 Corneal ulcers, 435-436 Coronavirus canine, 414 cattle, 582-583 feline enteric, 477-478

goat, 582 guinea pig, 223 mouse, 74-79, 90 rabbit, 345-346 rat, 144-146 sheep, 582 waterborne human, 369 Coronavirus-like particle, 223 Cortical adenoma, 154 Corticosteroids acral lick granuloma, 435 arthritis, 854 flea infestation, 428 gout, 854 guinea pig resistance, 206 hamster sensitivity, 176 juvenile polyarteritis syndrome, 453 malignant catarrhal fever, 576 perivascular injection injury, 441 stress release of, 1164 subluxation with spinal edema, 353 Cortisol, 692 Cortisone, 777 Corucia zebrata, 842 Corynebacteriosis, 92-93 Corynebacterium sp., 497 Corynebacterium bovis, 92-93 Corynebacterium cystitidis, 547-548 Corynebacterium kutscheri,92, 135, 182, 218, 373 Corynebacterium paulometabulum, 182 Corynebacterium pilosum, 547-548 Corynebacterium pseudotuberculosis, 547, 555

Corynebacterium pyogenes, 218 Corynebacterium renale, 547-548 Corynebacterium ulcerans, 252 Cotton rat aging, 272 biology and physiology, 271 congenital disorders, 272 description, distribution, habitat, 270 diseases, 271-272 husbandry, 271 nutrition and diet, 271 research uses, 270-271 Cotton-top tamarin, 731,734 Cottontail rabbit, 330, 345 Coumaphos, 596, 597, 657 Council for International Organizations of Medical Sciences (CIOMS), 29 Cowpox, 277, 281 Coxiella burnetii, 265, 564, 1076 Crayfish, fluke vector, 424 Creeping eruption, 1094 Creutzfeldt-Jakob disease, 170, 698 Cricetulus sp. Cricetulus griseus, 168, 190 Cricetulus migratorius, 168, 193 Cricetus sp. Cricetus barabensis, 192 Cricetus cricetus, 168, 194-195 CRISP database, 1236 Crocodile anatomy and physiology, 837-841 anesthesia, 845

1278 Crocodile (cont.) behavior, 844 blood collection, 846 clinical chemistry data, 842 handling and restraint, 834, 845 hematology data, 842 nutrition and diet, 841 sexing, 843-844 taxonomy, 827, 828 viral diseases, 850 Crocodylus porosus, 842 Crotoxyphos, 597 Cryopreservation history, 1192 method, 1026-1027 mouse embryo, 1139-1140 rat, 133 Cryptobranchus sp., 794 Cryptobranchus alleganiensis, 805 Cryptocaryon irritans, 900 Cryptococcosis, 746 Cryptococcus neoformans, 229-230, 278,746 Cryptosporidiosis. See also Coccidiosis ferret, 502-503 guinea pig, 224 mouse, 99 primate, 759, 760 rabbit, 347 ruminant, 587-588 zoonotic, 1090-1091 Cryptosporidium sp., 289, 502-503 Cryptosporidium baileyi, 587 Cryptosporidium cuniculus, 347 Cryptosporidium meleagridis, 587 Cryptosporidium muris, 99, 587 Cryptosporidium parvum, 99, 347, 587, 651, 1090 Cryptosporidium wrairi, 224 reptile, 851 Ctenocephalides sp., 504 Ctenocephalides canis, 427, 1097 Ctenocephalides felis, 228, 427-428, 1097 Culture medium, 373-375,637 Cutaneous biopsy, 728-729 Cutaneous streptothricosis, 548-549 Cuterebra sp., 291 Cybermouse project, 1199 Cyclophosphamide, 445 Cyclosporin, 628 Cynomolgus monkey (macaque) clinical chemistry data, 705 diseases enteric, 730-734 mycotic, 744-746 nutritional, 773-774 respiratory and nervous system, 734-738 tuberculosis and mycobacterial, 738-742 viral, 746-757 drug dosages, 726 hematology data, 705 housing, 704 natural history, 704 nutrition and diet, 704 reproduction, 704

INDEX research uses, 705 taxonomy, 678, 700, 704 vaccination, 725 Cynomys sp. Cynomys leucurus, 257 Cynomys ludovicianus, 254, 256 Cynomys mexicanus, 257 Cynops pyrrhogaster, 805 Cystic dilatation of cortical sinusoids, 110 Cystic endometrial hyperplasia, 110 Cystic prostatic hypertrophy, 169 Cysticercosis, 595, 771,772 Cysticercus fasciolaris, 151 Cystitis, 234-235,265 Cystocentesis, 728 Cystoisospora sp. Cystoisospora burrowsi, 417 Cystoisospora canis, 417 Cystoisospora neorivolta, 417 Cystoisospora ohioensis, 417 Cysts chinchilla, 288 cotton rat, 271 dog, 454 gerbil, 278 guinea pig, 239, 240 hamster, 188-189, 195 ovarian, 278 prairie dog, 257 ruminant, 595 Cytokines, 53, 54, 627 Cytomegalovirus grasshopper mouse, 265 mouse, 59-61 primate, 747 swine, 641 xenotransplant transmission, 1108, 1110-1111 Cytomegalovirus-inducing insulitis, 285

Dactylogyridiasis, 902 Damalina sp. Damalina bovis, 596 Damalina caprae, 596 Damalina crassipes, 596 Damalina limbatus, 596 Damalina ovis, 596 Danio rerio, 862 Dantrolene, 662, 982 Dapsone, 741 Dasypus novemcinctus, 1012 Databases AGRICOLA, 1198 Animal Models and Genetic Stocks Information Program, 1197 animal models database, 250 Bio MedNet Mouse Knockout database, 1199 BovGbase, 1200 ChickGBase, 1200 CRISP database, 1236 Dysmorphic Human-Mouse Homology database, 1199 European Collaborative Interspecific Mouse Backcross database, 1199 Gene Expression database, 1199

1279

INDEX Gene Knockouts, 1199 International Laboratory Code Registry, 1197 Jackson Laboratory, 1199 Japan Animal Genome, 1199 Mouse Atlas Project, 1199 Mouse Genome Database, 36, 38, 1199 MutaMouse, 1199 Oak Ridge National Laboratory, 1199 Pigbase, 1200 Sheepbase, 1200 Taconic Farms, Inc., 1199 TBASE, 1199 transgenic mouse, 1199 Transgenic Systems for Mutation Analysis-Big Blue, 1199 Debridement, 433 Dechlorination, 797 Decontamination. See Disinfection Decoquinate, 587 Decubital ulcer, 433-434 Deer mouse, 265-267, 412 Degu biology and physiology, 284-285 clinical chemistry data, 284-285 description, distribution, habitat, 284 diseases, 285-286 hematology data, 284-285 husbandry, 285 research uses, 284 Dehydration amphibian, 823 mouse, 108 rat, 153 reptile, 854-855 Demodectic mange, 186, 192, 656 Demodex sp., 253,265, 503 Demodex aurati, 186 Demodex canis, 424-425 Demodex caviae, 225, 226 Demodex criceti, 186 Demodex cricetuli, 194 Demodex leucogasteri, 265 Demodex musculi, 102 Demodex ovis, 596 Demodex phylloides, 656 Demodex sinocricetuli, 192 Demodicosis, 186, 424 - 425, 503-504 Dengue, 752, 1063 Dental disease. See also Malocclusion caries, 170-171,273 periodontitis, 193, 273, 278, 281 primate, 777 rabbit, 354, 355,356 root canal, 1016-1017 sheep, 611 squamous cell carcinoma, 356 tooth extraction, 1016 wear, 611 woodchuck, 327 Dentostomella translucida, 186, 277 Department of Agriculture. See USDA Department of Health and Human Services, 30 Department of the Interior, 30 Dermacentor sp., 228

Dermacentor andersoni, 426, 561,585, 1096 Dermacentor occidentalis, 585, 1096 Dermacentor variabilis, 426, 1096 Dermanyssus gallinae, 1096 Dermatitis acral lick granuloma, 434-435, 1247 bacterial folliculitis, 319 bighead, 544-545,608 caprine staphylococcal, 542 chronic ulcerative, 107 contagious pustular, 573-574 creeping eruption, 1094 dog, 424-425 facial, gerbil, 277 gangrenous, 252 goat, 608 reptile, 853 ruminant, 551,596 swimmer's itch, 276 wart, 551 woodchuck, 319 Dermatomycosis, 598 cat, 479 chinchilla, 289 dog, 428-429 ferret, 505 guinea pig, 228-229 human risk factor, 479 mouse, 98 prairie dog, 257 primate, 744 rabbit, 351-352 rat, 152 ruminant, 598 zoonotic, 1088-1089 Dermatophagoides scheremtewskyi, 1096 Dermatophilosis, 548-549 Dermatophilus congolensis, 548-549 Dermatophytosis. See Dermatomycosis Dermatosis human, 1095 ovine viral, 576-577 ulcerative, 572-573 Dermorphin, 801 Desert gerbil, 275 Desflurane, 959, 983 Desulfovibrio sp., 494 Desulfovibrio desulfuricans, 645-646 Detomidine, 987, 988 Dexamethasone dehydration, 823 parturition induction, 603 polioencephalomalacia, 608 primate dose, 726 Dexon suture, 433 Dextrose, 603,776 Diabetes mellitus cataract, 449 chinchilla, 289 degu, 285 ground squirrel, 253 guinea pig, 236 hamster model, 173, 192-193

1280 Diabetes mellitus (cont.) mouse, 110 mouse model, 1187 periodontitis, 193 rabbit model, 331 rat model, 152 vole model, 280 white-tailed rat model, 273, 274 Diagnosis antibody assay, 380-382 bacteriology, 373-374 facility design, 914 microbial isolation, 373-375 parasitology, 373 pathology, 373 polymerase chain reaction (PCR), 377-380 results interpretation, 385-387 serology, 375-377, 380-382 specimen examination, 372-373 virology, 374-375 Diaphragmatic hernia, 325 Diarrhea antibiotic-associated, 255 bovine viral, 567-569 coccidiosis, 586-587 nutritional, 609 prairie dog model, 255 primate, 730-734 rabbit model, 1019 rotavirus, mouse, 73-74 ruminant, 582-583, 586-587 swine, 643-654 transmissible murine colonic hyperplasia, 85-86 woodchuck, 319 Diastema, 249 Diazepam clostridia infection, 742 polioencephalomalacia, 608 seizure control, 44 1-442 streptococcal meningitis, 735 tranquilizer dog, 973-975 hamster, 964 mouse, 962 primate, 720, 726, 991,992-993 rabbit, 968 rat, 963 reptile, 845 rodent, 960 ruminant, 987, 988 swine, 981 Diazinon, 657 Diazoxide, 508 Dichelobacter nodosus, 549-550, 551 Dichlorvos, 597, 643, 653,766 Dicrocoelium dendriticum, 324, 595 Dictyocaulus sp. Dictyocaulus filaria, 594 Dictyocaulus viviparus, 594 Dieldrin, 1188 Diet. See Nutrition and diet Diethylcarbamazepine, 423 Difloxacin, 646 Dihydrostreptomycin, 408, 632, 659

INDEX Diiodohydroxyquin, 651,758, 759, 1090 Diltiazem, 982 Dimethyl sulfoxide, 435, 441,578 Dimetradizole, 98, 99, 149 Diminazene diaceturate, 586 Dipetalonema viteae, 282 Dipodomys sp. Dipodomys merrami, 259 Dipodomys spectabilis, 259 Diprenorphine, 981 Dipylidium caninum, 228, 423-424, 478 Dirofilaria immitis, 421-423, 505-506 Dirofilariasis, 421-423 Disinfection. See also Sanitation aquatic facility, 890 canine parvovirus, 413 canine tracheobronchitis, 406 coccidiosis, 502 Klein-DeForest scheme, 370 methods, 368-370 mycobacteriosis, aquatic, 876-877 primate housing, 722-723 pseudorabies, 579 scrapie, 581 virus, 369-370 Distemper, 414-415,498-499 DNA hybridization, 378 Dobutamine, 975, 991 Dog. See also Beagle acquisition and sources, 396-397 analgesia, 974, 978-979 anatomy, 401-403 anesthesia, 974-978 behavior, 405, 1246-1248 blood sample collection, 1007, 1011-1012 cannulation technique, 1014 diseases age-related, 449-451 bacterial, mycoplasmal, and rickettsial, 405-413 congenital, 449, 450 fungal, 428-429 hip dysplasia, 450 iatrogenic, 436-442 metabolic and nutritional, 429-432 neoplasms, 442-449 obesity, 431-432 parasitic, 416-428 traumatic, 432-436 viral and chlamydial, 405-406, 413-416 environmental enrichment, 1246-1247 estrous cycle, 402, 403 euthanasia, 979 exercise, 24 helicobacteriosis vector, 1085 housing, 919, 1246 husbandry, 397 identification, 1007 intraoperative monitoring and support, 978 neosporosis vector, 589 noise control, 1247 nutrition and diet, 397-400 postoperative recovery, 978 preanesthesia, 973-975 pulpectomy/pulpotomy, 1016 - 1017

INDEX

1281

reproduction, 401-405 research uses, 395-396 social interaction, 1246-1247 stress, 1247 taxonomy, 395 Dopamine, 975, 991 Doramectin, 643, 657 Doxapram, 726 Doxorubicin, 441 Doxycycline campylobacteriosis, 409 chlamydiosis, 819 ehrlichiosis, 410 leptospirosis, 408, 1083 Lyme disease, 412 mycobacteriosis, 899 mycoplasmal pneumonia, 640 pasteurellosis, 637 primate dose, 726 Droperidol-fentanyl (Innovar-Vet), 312, 961, 981 Drymarchon corais, 842 Dull eye, 241 Dunnifilaria meningica, 263 Duodenitis, 284 Duragesic, 974 Dusky-footed wood rat, 261 Dwarf tapeworm, 100 Dysecdysis, 855 Dysentery, 583, 643-645 Dysmorphic Human-Mouse Homology database, 1199 Dystocia, 237, 854 E

Ear diseases chinchilla model, 286 ferret, 504-505 mouse, 110 otitis externa, 320 otitis interna, 339 otitis media, 339-340 rabbit, 339-340 woodchuck, 320 Earthworm, blood collection, 1013 Eastern meadow vole, 279 Ebola virus, 751,753, 1064-1065 Echidnophaga gallinacea, 427, 1097 Echinococcus sp. Echinococcus granulosus, 276, 423,595,772 Echinococcus multiocularis, 263,271,276, 281 Echinolaelaps sp., 281 Ecthyma, 573-574 Ectromelia virus, 55-59 Eczema, 277 Edema, malignant, 545-546 EDIM virus, 73-74 Education and training, 13-14 American College of Laboratory Animal Medicine (ACLAM), 1231-1234 facility management, 946-947 Internet sites, 1231 laboratory animal medicine, 13-14, 1231, 1231-1234 personnel, 13-14, 946-947, 1049-1050, 1051, 1231-1234 veterinarians, 1231-1234 Edwardseilla tarda, 836, 878, 899

Ehlers-Danlos syndrome, type II, 461 Ehrlichia sp. Ehrlichia canis, 409-411 Ehrlichia phagocytophilia, 262 Ehrlichia platys, 411 Ehrlichiosis dog, 409-411 monocytic, 409-411 rat vector, 262 thrombocytic, 411 Eimeria sp., 253, 255, 258, 651 Eimeria alabamensis, 586 Eimeria arloingi, 586 Eimeria ashata, 586 Eimeria auburnensis, 586 Eimeria bovis, 586 Eimeria caviae, 223 Eimeria chinchillae, 289 Eimeria christenseni, 586 Eimeria crandalis, 586 Eimeria falciformis, 99 Eimeria flavescens, 347 Eimeria furonis, 502 Eimeria ictidea, 502 Eimeria intestinalis, 347 Eimeria irresidua, 347 Eimeria magna, 347 Eimeria media, 347 Eimeria monacis, 323 Eimeria neoleporis, 347 Eimeria ninakohlyakimoviae, 586 Eimeria onychomysis, 265 Eimeria os, 323 Eimeria ovinoidalis, 586 Eimeria perforans, 347 Eimeria perforoides, 323 Eimeria piriformis, 347 Eimeria roperi, 271 Eimeria sigmodontis, 271 Eimeria steidae, 346, 357 Eimeria tuscarorensis, 323 Eimeria tuskegeesis, 271 Eimeria webbae, 271 Eimeria zuernii, 586 Elbow hygroma, 435 Electromagnetic radiation, 369 ELISA, 381-382 Embryo transfer, 133, 534-535, 1026-1027 Embryology, 1188 Embryonic development, 862, 863 Embryonic nephroma, 664 Embryonic stem cell-mediated homologous integration, 1130-1131, 1191 Emetine, 1090 EMLA cream, 972 Emmonsia sp., 281 Emmonsia crescens, 253 EMTU, 958, 963, 968 Encephalitis caprine arthritis, 532, 569-570 listeriosis, 553 Powassan virus, 322-323 rabbit, 322-323, 347-349 thromboembolic meningoencephalitis, 551-552 Encephalitis virus, 265, 276

1282 Encephalitozoon cunicuii guinea pig, 224 mouse, 99-100 primate, 762 rabbit, 347-349 rat, 147 Encephalitozoonosis, 347-349 guinea pig, 224 mouse, 99-100 primate, 762, 763 rat, 147 Encephalomyelitis virus, 79-80, 147, 253 Encephalomyocarditis, 757 Encephalopathy cat model, 461 cattle model, 599 hamster model, 170 hepatic, 441-442 myeloencephalopathy, 599 spongiform, 461,580 Encyclopedia of the Mouse Genome, 1199 Endangered species, 30 Endeleinellus marmotae, 324 Endemic typhus, 1074-1075 Endocardial fibroelastosis, 461 Endocrine system, 1146-1147 Endocrine system diseases. See also Adrenal gland diseases dog, 429-431 ferret, 506-507 hyperestrogenism, 506-507 hypothyroidism, 429-431 mouse, 110 rabbit, 357 Endometrial adenocarcinoma, 331 Endometrial stromal polyp, 154 Endoscopy, 847 Endotracheal intubation, 845, 847, 986-987, 1021-1022 Enflurane, 662, 959 Enilconazole, 428 Enrichment, environmental, 1241 Enrofloxacin aspiration injury, 440 bite wound, 856 bordetellosis, 476 campylobacteriosis, 409 colibacillosis, 547, 674 ehrlichiosis, 410 mastitis, 498 mycobacteriosis, 816 mycoplasmosis, 850 pasteurellosis, 87, 340, 637 pleuropneumonia, 637 proliferative enteritis, 181 reptile dosing, 849 salmonellosis, 561 sepsis, 439 septicemia, 878 shell fracture, 856 shigellosis, 731, 1088 streptococcal meningitis, 634 Entamoeba sp. See also Amoebiasis Entamoeba histolytica, 282, 758, 1089-1090 Entamoeba invadens, 851 Entamoeba muris, 99, 148, 149, 323

INDEX

Entamoeba ranarum, 819 Entamoebiasis. See Amoebiasis Enteric helicobacteriosis, 1084-1085 Enteritis antibiotic-associated, 182, 274 guinea pig model, 230 hamster, 182, 197 primate, 730-734 proliferative, 77, 180-182 rabbit model, 331 swine, 643-654 Enterobacter aerogenes, 555 Enterobius infection. See Pinworm Enterobius sp. Enterobius anthropopitheci, 765 Enterobius vermicularis, 765, 1094 Enterococcosis, 134-135 Enterococcus sp. Enterococcus faecalis, 135 Enterococcusfaecium-durans 2, 135 Enterococcus hirae, 135 navel ill, 558 Enterocolitis, 189, 649 Enteropathy proliferative, 344, 644, 645-646 rabbit, 344 swine, 644, 645-646 Enterotoxemia, 342, 542-543 Entopolypoides macacai, 761 Entropion, 598 Environmental disorders, 107-108 Environmental enrichment cat, 1246-1247 dog, 1246-1247 farm animals, 1254-1255 guinea pig, 1244-1245 mouse, 1242-1243 primate, 1248-1250, 1251-1252 rabbit, 1245 rat, 1243-1244 Environmental Protection Agency (EPA), 27, 30, 1188 Enzootic ataxia, 605-606 Enzootic pneumonia, 640-641 Enzyme immunoassay, 376-380 Eosinophilic granulomatous pulmonary inflammation, 156 Ependymoma, 357 Eperythrozoon sp. Eperythrozoon coccoides, 95~96, 367 Eperythrozoon ovis, 564 Eperythrozoon suis, 654 Eperythrozoon tegnodes, 564 Eperythrozoon tuomii, 564 Eperythrozoon wenyoni, 564 Eperythrozoonosis, 563-564, 654 Ephedrine, 975 Epidermitis, 654-655 Epidermophyton sp., 1089 Epidermophyton floccosum, 598 Epilepsy, 275. See also Seizures Epinephrine, 610, 726 Epizootic diarrhea of infant mouse, 73-74 Epstein-Barr virus, 748-749, 1108-1109 Equine encephalitis virus, 265 Equipment

INDEX aquatic health, 871-873 bedding dispensers and disposal, 933-934 biological safety cabinet, 943,945, 1048-1053 cage sanitation and sterilization, 931-933,934 chemical restraint devices, 719-720 osmotic minipump, 1014-1015 primary containment, 721,722 protective, 718-719, 721,722, 1052-1053 pumps and compressors, 887-888 robotics, 934, 935 rodent anesthesia, 959 sterilization, 934- 935 ventilation, 922-923 water bottle sterilization, 936 water filters, 888-889 Ergot poisoning, 240 Erysipelas, 548, 632-633 Erysipelothrix rhusiopathiae, 218, 548, 632, 898 Erythroblastosis fetalis, 627 Erythrocebus patas, 751 Erythroleukemia, 111 Erythromycin anthrax, 539 campylobacteriosis, 492, 731, 1084 foot rot, 550 greasy pig disease, 655 leptospirosis, 659 listeriosis, 554 navel ill, 559 primate dose, 726 proliferative enteropathy, 646 Erythropoietic porphyria, 599 Escherichia coli dog, 407 ferret, 496-497 guinea pig, 218, 234 hamster, 180-182 hemolytic, 497 mouse, 94-95 navel ill, 558 primate, 732-733 rabbit, 342-343, 344, 345 ruminant, 546-547 sheep, 555 swine, 644, 646-648 woodchuck, 320, 321 Esfenvalerate, 1188 Esmolol, 975 Esophageal choke, 289 Essential fatty acids deficiency, 107 Ethambutol, 1088 Ethanol, 349 Ethylmalonyl urea, 958, 963,968 Ethylnitrosourea, 331 Etomidate dog, 974, 976 primate, 991,993 rodent, 959 swine, 981,983 Etorphine, 981 Eulaelaps stabularis, 102, 1096 European brown hare virus, 345 European Collaborative Interspecific Mouse Backcross database, 1199 European rabbit, 330

1283 European Union (EU), 29 Eustrongyloides sp., 899, 903 Euthanasia amphibian, 814 animals, effect on, 1157 dog, 979 fish, 897 guidelines, 26 guinea pig, 966 primate, 997 reptile, 848 rodent, 966 swine, 986 Eutrombicula sp., 1096 Exocrine pancreatic insufficiency, 109 Exophthalmos, 241,254, 272 Experimental technique, 846. See also Anesthesia; Blood sample collection adrenalectomy, 1028-1029 artificial insemination, 1025 bandaging, 433 bile sampling, 1017-1018 biopsy dog, 442 reptile, 847, 848 skin, 728-729 blood pressure, 1027 bone marrow biopsy, 729 bronchoalveolar lavage, 1023 bronchoscopy, 1023 cardiovascular system, 1027-1028 carotid-jugular shunt, 1027 castration, 1025 cerebromedullary exposure, 1030 cerebrospinal fluid sampling, 728, 1031-1032 cryopreservation, 1026-1027 debridement, 433 dental procedures, 730 digestive system, 1015-1019 electrode implantation, 1030-1031 embryo transfer, 1026-1027 embryological, 864 endocrine system, 1028-1029 endoscopy, 847 endotracheal intubation, 1021-1022 fecal examination, 814, 846-847 gastric intubation, 1016 gavaging, 814 genetic, 864 heart surgery, 1028 hepatectomy/biopsy, 1018-1019 hypophysectomy, 1028 identification, 1006-1008 imaging, 1033-1034 indwelling catheter, 729 injections amphibian, 814 reptile, 846 swine, 617 intestinal cannulation, 1019 intestinal loop isolation, 1019 mtracerebral implantation, 1030 mtraosseous infusion, 730 lntraperitoneal injection, 1015 intratracheal injection, 1022

1284 Experimental technique, 846. See also Anesthesia; Blood sample collection (cont.) intrauterine fetal surgery, 1027 laparoscopy, 1024 lumbar sympathectomy, 1029-1030 lymph node biopsy, 729 magnetic resonance imaging, 1034 microangiography, 1028 molecular, 865 nasogastric intubation, 730 neurosurgery, 1029-1032 oral gavage, 1015-1016 orthopedic methods, 1029 pancreatic function, 1019 parathyroidectomy, 1029 pharyngeal sampling, 1021 pinealectomy, 1028 pregnancy diagnosis, 1025-1026 pulmonary arterial blood pressure, 1027 pulpectomy/pulpotomy, 1016-1017 radiology, 729, 1034 radiotelemetry, 1034 rectal prolapse repair, 730 reproductive system, 1023-1027 respiratory system, 1021-1023 semen collection, 1025 skin preparation and disinfection, 432-433 skin sampling and biopsy, 728-729 skin suture, 433 spinal catheter, 1031 spinal laminectomy, 1030 stereotactic surgery, 1030-1031 subcutaneous injection, 1015 testicular biopsy, 1024 thyroidectomy, 1029 tooth extraction, 1016 toxicology, 865 tracheal culture/wash, 847, 1022 tracheal pouch formation, 1022-1023 tracheostomy, 1023 tumor transplantation, 1032-1033 ultrasonography, 729, 1034 urine sample collection, 728, 1019-1021 vascular cannulation, 1013-1015 Experimentation. See Animal experimentation Exportation, 29-30 Extramedullary hematopoiesis, 109, 485-486 Extravasation injury, 440-441 Eye diseases cattle, 556-557 chinchilla, 291 dog, 435-436, 449-450, 453-454 entropion, 598 guinea pig, 241 mouse, 110 woodchuck, 319

F1 hybrid, 36, 37 Facility design cage sanitation and sterilization, 931-933 caging systems, 937-946

INDEX

circulation pattern, 912 cleaning, 915-916 commissioning and validation, 946 communications, 930 cubicles, 912-913 doors, 929 energy conservation, 925-926 environmental monitoring, 931 ergonomics, 887-888 fish, 886-891 floor plans, 912 goals and objectives, 910-911 heating, ventilation, air conditioning, 919- 926 interior surfaces, 927-928 location and arrangement, 911-912 noise control, 930 plumbing and drainage, 927 power and lighting, 926-927 retrofit, 890 security and controlled access, 930-931 shipping and receiving, 916 storage, 915 - 916 support area, 913- 919 tanks and housing, 892-893 temperature and humidity control, 925 vacuum cleaner, 946 vermin control, 929-930 waste storage and removal, 916 water management, 935-937 Facility management administrative suite, 913- 914 aquatic health, 866 biohazards, 1048-1053 biosecurity, 366-372 cat disease control, 462-463,466 crisis management/disaster planning, 947 design and, 911 disinfection, 890 education and training, 946-947 environmental, 1148-1150 fish, 870-874 heating, ventilation, air conditioning, 1148-1150 housing, 1152-1153 humidity, 1148 lighting, 1151-1152 microbiological surveillance, 372-387 noise control, 1150-1151 nutrition and diet, 1153-1155 rabbit disease control, 365-387 radiation control, 1152 rodent disease control, 365-387 stress management, 1164-1165 temperature management, 1147-1148 transgenic/knockout mouse, 1134-1135 water management, 1155-1156 woodchuck colony health, 319-320 xenobiotics, 1153-1156 Fainting, 599 Famotidine, 413 Farm animals behavior, 1252-1256 environmental enrichment, 1254-1255 feeding, 1254

INDEX housing, 1253-1254 manipulanda, 1254-1255 personnel interaction, 1256 social behavior, 1255-1256 Fasciola sp. Fasciola gigantica, 225 Fasciola hepatica, 225,595 Fascioliasis, 595-596 Fascioloides magna, 595 Fat cow syndrome, 600 Fate mapping, 864-865 Fatty acids, essential, 107 Fecal examination, 814, 846-847 Fecal flotation, 373 Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA, 1947), 27 Federation of American Societies for Experimental Biology (FASEB), 9, 10 Federation of Animal Science Societies (FAAS), 28 Federation of European Laboratory Animal Science Associations (FELASA), 383 Feline immunodeficiency virus, 460-461,477 Feline infectious peritonitis, 477-478 Feline leukemia virus, 460, 477, 510 Feline lower urinary tract infection, 475 Feline spongiform encephalopathy, 461 Felis cattus, 460 Fenbendazole Anatrichostoma cynomolgi, 766 ascariasis, 418, 643 cestodiasis, 423 giardiasis, 323, 416, 478 lungworm, 594 nematodiasis, 821,878, 903 pinworms, 351 primate dose, 726 strongyloidiasis, 420 trematodiasis, 424 trichuriasis, 653 whipworm, 420 Fentanyl analgesia dog, 974 rabbit, 972 swine, 981 anesthesia gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 991,992, 993-994 rabbit, 968, 969 rat, 963 rodent, 958, 961 swine, 982 tranquilizer rodent, 960 swine, 981 Fenthion, 423 Fenvalerate, 657 Ferret acquisition and sources, 484 anatomy, 485-486 blood sample collection, 1011 cannulation technique, 1014

1285

clinical chemistry data, 487 development, 490 diseases age-related, 512-513 bacterial, 490-498 congenital, 512 fungal, 504 iatrogenic, 507 metabolic and nutritional, 506-507 nematodiasis, 505 neoplasms, 507-512 parasitic, 502-504 pneumonia, 496-497 traumatic, 507 viral, 498-502 estrous cycle, 488, 506-507 hematology data, 486 housing, 484-485 husbandry, 484-485,489 normative data, 486 nutrition and diet, 485,486-487, 506, 508 reproduction, 487-490 research uses, 484 taxonomy, 483-484 urinalysis data, 487 Fertility, 173 Fetal surgery, 1027 FETAX solution, 811-812 FETAX system, 795, 1188-1189 Fialuridine (FIAU), 325 Fibrinopurulent pericarditis, 215 Fibroadenoma, 154, 238 Fibroelastosis, 461 Fibromatosis, 344, 754 Fibropapillomatosis, 850, 857 Fibrosarcoma dog, 443-444 guinea pig, 237, 238 hamster, 187 prairie dog, 257 rabbit, 356, 357 snake, 857 turtle, 857 Field vole, 256, 279 Filariasis dog, 421- 423 ferret, 505-506 gerbil model, 276 snake, 852 Filoviruses, 751,753, 1064-1065 Filtration air supply, 369, 941-943,944, 1050 water management, 369, 888-889 Finasteride, 451 Fish. Se'e also Zebrafish acquisition and sources, 875 anesthesia, 896-897 cannulation technique, 1014 clinical signs and diagnosis, 875-876 diseases fluke infestation, 902-903 fungal (water mold), 879-880 infectious pancreatic necrosis, 905-907

1286 Fish. See also Zebrafish, diseases (cont.) lymphocystis, 904-905 microsporidiosis, 903-904 mycobacteriosis, 876-877, 899-900 myxosporidiosis, 904 nematodiasis, 878, 903 parasitic, 902-903 protozoal, 901- 902 septicemia, 878 trichodinosis, 881 velvet disease, 878-879 viral, 882 white spot (ich), 880-881,900-901 euthanasia, 897 facility design, 870-874, 886-893 health screening, 894-895 husbandry, 866-874, 893-894 lighting, 870-871 nutrition and diet, 874-875, 894 pain perception, 895 research uses, 886 Streptococcus iniae vector, 1080 tanks and housing, 871-873, 892-893 taxonomy, 886 temperature management, 891-892 water quality, 866-869 Fish and Wildlife Service (FWS), 30 Fish tank granuloma, 1078 Fish tuberculosis, 876-877 Flank gland, hamster, 171,172 Flaviviruses, 751,752 Flavobacterium infection, 816-817 Flavobacterium sp., 878 Flavobacterium indologenes, 816 Flavobacterium meningosepticum, 816 Flavobacterium oderans, 816 Flea cat infestation, 478 cestode vector, 423 cotton rat infestation, 272 dog infestation, 427-428 ferret infestation, 504 grasshopper mouse infestation, 265 guinea pig infestation, 228 hosts, 1097 photograph, 1095 plague vector, 1082, 1098 typhus vector, 1075, 1098 woodchuck infestation, 324 Yersinia pestis transmission, 252 Flexibacter sp., 144 Flexibacter columnaris, 878 Florfenicol, 559, 637 Fluanisone gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 992 rabbit, 968 rat, 963 Flubendazole, 766 Fluconazole, 429

INDEX Fluke dog, 424 fish, 902- 903 gerbil, 276 reptile, 852 ruminant, 595-596 woodchuck, 324 Flumazenil, 969, 974 Flunixin acral lick granuloma, 435 analgesia primate, 726 rabbit, 972 ruminant, 987, 989 Xenopus sp., 813 Fluoroquinolones. See also Cephalosporins anthrax, 539 bordetellosis, 213 clostridia infection, 217 eye infections, 241 guinea pig toxicity, 240 lymphadenitis, 215 mycoplasmosis, 562 polyarthritis, 631 Flutamide, 451 Fly, 597-598 Foa-Kurloff cell, 206 Fog fever, 594 Foleyella sp., 820-821 Folic acid, 762 Folic acid deficiency, 107, 697, 774 Follicle-stimulating hormone, 1132 Follicular center cell lymphoma, 111 Folliculitis, 319, 320 Fomite transmission, 367, 368-370 Food and Drug Administration (FDA), 15, 26-27 Food Animal Residue Avoidance Bank, 537 Food Security Act (1985), 20 Foot-and-mouth disease, 574-575 Foot rot, 549-550 Foot scald, 549-550 Foot wart, 551 Foothill abortion, 561 Formalin encephalitozoonosis, 349 fluke infestation, 902 foot rot, 550 heel wart, 551 Ichthyobodo necatrix, 901,902 infectious pancreatic necrosis, 906 protozoal infection, 901,902 trematodiasis, 903 trichodinosis, 881 velvet disease, 879 water mold, 880 white spot (ich), 901 Formica fusca, 595 Foundation Center, 1236 Fractures chinchilla, 290 orthopedic methods, 1029 rabbit, 352-353 reptile, 853, 857

INDEX

1287

shell, 856 Francisella tularensis, 1096 Free-gas bloat, 600-601 Freemartin, 533, 683-684 Frog. See also Xenopus sp. anatomy and physiology, 801-803 behavior, 806-807 clinical chemistry data, 803, 806 diseases bacterial, 814-817 fungal, 822 metabolic and nutritional, 822-823 parasitic, 819-822 viral and chlamydial, 817-819 global decline, 1187-1189 handling and restraint, 799-800 hematology data, 805 housing, 796-797 husbandry, 796-801 larvae, 804, 806 longevity, 803 metamorphosis, 808 nutrition and diet, 803-804, 806 reproduction, 807-808 research uses, 795-796 taxonomy, 794-795 Frog embryo teratogenesis assay: Xenopus (FETAX), 795, 811-812, 11881189 Frog erythrocytic virus, 818-819 Frothy bloat, 600-601 Fungal diseases, effect of, 1163 Fur chewing, 289-290 Fur ring, 290 Fur slip, 289-290 Furazolidone, 416, 652, 758, 1092 Furosemide, 726 Furunculosis, 93 Fusarium sp., 853 Fusobacterium sp., 551 Fusobacterium necrophorum, 549-551,586, 601, 611 Fuzzies, 573 G Gait abnormality, 325 Galapagos tortoise, 846 Gamma radiation, 369 Gangicyclovir, 1069 Gangliosidosis, 461 Gangrenous dermatitis, 252 Gas bubble disease, 823, 868 Gastric dilatation, 600-601,776-777 Gastric helicobacteriosis, 1085-1086 Gastric intubation, 1016 Gastric lesions and ulcers, 109, 240 Gastrodiscoides hominis, 772 Gastroenteritis eosinophilic, 512, 513 ferret, 512, 513 swine, 644, 649-650 Gavaging, 814, 1015-1016 Gecko, 840 Gene Expression database, 1199

Gene Knockouts database, 1199 Genetic drift, 152 Genetic mapping cat, 460, 461 human genome project, 1193 Mouse Genome Database, 36 overview, 1193 zebrafish, 866 Genetics aging research, 1193-1195 animal models, 1187 animal research, 1144 - 1145 behavior, 1122-1123 biochemical monitoring, 1123-1124, 1125 congenic strains, 1120-1121 contamination, 1119-1120 DNA analysis techniques, 1124-1125 genetic drift, 1120 genetic purity, 1144-1145 heterozygosity, residual, 1120 history, 1207-1208 immunologic monitoring, 1123 inbreeding, incomplete, 1120 karyotypes, 1124 knockout mouse, 864, 1129-1140 molecular methods, 865 monitoring, 1117-1126 mouse nomenclature, 39-40 mutagenicity, 862, 863-864 mutant alleles, 1120 obesity, 1204-1206 rat, 152-153 transgenic mouse, 39, 1129-1140 Genital tract diseases, 110, 446-447, 572-573 Gentamicin anthrax, 539 clostridia infection, 217 colibacillosis, 674, 731 kennel cough, 406 klebsiellosis, 735 lymphadenitis, 215 mastitis, 557 mycoplasmal pneumonia, 563 plague, 1083 primate dose, 726 pseudomoniasis, 139, 744, 816 red leg, 815 salmonellosis, 561,630 yersiniosis, 731 Geochelone elephantopus, 846 Geomys sp., 257 Gerbil anesthesia and analgesia, 965 biology and physiology, 276 blood sample collection, 1009 description, distribution, habitat, 275 diseases age-related, 279 bacterial, mycoplasmal, and rickettsial, 277 congenital, 279 fungal, 278 metabolic and nutritional, 278 model, 275-276

1288 Gerbil (cont.) neoplasms, 278-279 parasitic, 277-278 toxicity, antibiotic-induced, 278 traumatic and iatrogenic, 278 viral and chlamydial, 277 drug dosages, 965 hematology data, 256 husbandry, 276-277 research uses, 275-276 sentinel animal, 383 Gerstmann-Staussler syndrome, 170 Giardia sp., 288-289 degu, 285 Giardia duodenalis, 148, 323,416 Giardia intestinalis, 644, 652 Giardia lamblia, 275,588, 1091 Giardia muris, 98, 99, 147, 148 Giardiasis chinchilla, 288-289 dog, 416-417 kitten, 478 mouse, 98 primate, 758 rat, 148-149 ruminant, 588 swine, 644, 652 zoonotic, 1091-1092 Gid, 595 Glasser's disease, 631 Glaucoma, 353-354 Gliricola porcelli, 228 Globocephalus urosublatus, 652 Globoid cell leukodystrophy, 461 Glomerulonephritis, 109, 284, 326-327, 627 Glomerulonephropathy, chronic, 109 Gloves, 718, 1052-1053 Glucose, 776 Glugea sp., 903 Glycogenosis, 461 Glycopyrrolate dog, 973, 974 primate, 991 rabbit, 968 rodent, 960 ruminant, 987, 988 Glygyphagus cadaverum, 1096 Gnotobiology, 15 Goat acquisition and sources, 522-524 analgesia, 987, 989-990 anesthesia, 986-989 behavior, 535-537 castration, 535 clinical chemistry data, 526, 528 development, 532-533 digestive system, 525 diseases bacterial, 537-562 chlamydial, 583-584 genetic, 598-599 iatrogenic, 610-611 management-related, 608-610 metabolic, 599-605

INDEX mycoplasmal, 562-563 neoplasms, 611 nutritional, 605-608 protozoal, 584-592 rickettsial, 563-564 traumatic, 610 viral, 565-583 estrous cycle, 530, 534 health screening, 523 hematology data, 526, 527 histocompatibility complex, 525 husbandry, 524, 531 lidocaine sensitivity, 989 normative data, 526 nutrition and diet, 526-529 physiology, 525-526 postoperative recovery, 988 preanesthesia, 986 regurgitation prevention, 986 reproduction, 529-531 research uses, 521-522 taxonomy, 520 Goiter, 599 Gold dust disease, 878-879 Goldfish, 880 Gonadectomy, 1147 Gonadotropin-releasing hormone, 506-507 Good Laboratory Practice (GLP), 15, 26-27 Gopher, pocket, 257-259 Gout, 853-854 Government. See Laws, regulations, and guidelines Granulocyte-macrophage colony stimulating factor, 54 Granuloma acral lick, 434-435 acral lick granuloma, 1247 adjuvant-induced pulmonary, 236 fish tank, 1078 fungal, 853 pulmonary, 193, 236 sterile pyogranuloma complex, 454 swimming pool, 1078 Granulomatosus encephalitis, 348 Grapevine gerbil, 275 Grasshopper mouse biology and physiology, 264 description, distribution, habitat, 263-264 diseases bacterial, 265 fungal, 265 parasitic, 264, 265 seizure, 265 viral, 265 husbandry, 264 research uses, 264 taxonomy, 263 Gray squirrel, 256 Greasy pig disease, 654-655 Great ape, 678 Greene melanoma, 357 Griseofulvin, 229, 351-352, 429, 505,598 Ground squirrel anatomy, 254-255 biology and physiology, 252 dentition, 254

1289

INDEX description, distribution, habitat, 250-251 diseases bacterial, 252 diabetes mellitus, 253 fungal, 253 neoplasms, 253-254 parasitic, 253 viral, 252-253 husbandry, 252 monkeypox vector, 1060 nutrition and diet, 252 research uses, 252 Guanarito virus, 269, 270 Guelph system (artificial insemination), 534 Guidelines. See Laws, regulations, and guidelines Guinea pig Abyssinian, 204, 205 acquisition and sources, 204 aging, 239-240 anatomy, 206-207,209, 241 anesthesia and analgesia, 965-966 artificial insemination, 211 behavior, 209, 241, 1244 blood sample collection, 1007, 1010-1011 breeding systems, 210 caging systems, 939-943 cannulation technique, 1014 cardiovascular system, 207, 208 clinical chemistry data, 208 dentition, 207 development, 211 diseases age-related, 239-240 bacterial, mycoplasmal, and rickettsial, 212-220 chlamydial, 220-221 eye, 241 iatrogenic and management, 236-237 9metabolic and nutritional, 230-236 mycoses, 228-230 neoplasms, 237-238 parasitic, 223-228 plant toxicosis, 240 toxemia of pregnancy, 232-234 urolithiasis and cystitis, 234-235 viral, 221-223 ear, 207 English, 204 environmental enrichment, 1244-1245 euthanasia, 966 gastrointestinal tract, 207 Hartley, 204 hematology, 206 hematology data, 208 housing, 204-205, 939-943, 1244-1245 husbandry, 205-206, 209-212 hypovitaminosis, 231 lymphoreticular system, 206-207 normative data, 208, 210 nutrition and diet, 208-209, 231-232, 235, 240-241 Peruvian, 204, 206 physiology, 206-207 pituitary gland, 207 reproduction, 209-212 respiratory system, 207, 208

sexing, 211 taxonomy, 203-204 Guyanese squirrel monkey, 693-694 Gyrate atrophy, choroid and retina, 461 Gyrodactylidiasis, 902-903 Gyrodactylus sp., 821,902-903 Gyropus ovalis, 228

H-1 virus, 1161

Haematopinus sp. Haematopinus eurysternus, 596 Haematopinus quadripeTtusus, 596 Haematopinus suis, 657 Haemobartonella sp. Haemobartonella bovis, 564 Haemobartonella microti, 281 Haemobartonella muris, 95, 95-96, 143, 151 Haemogamasus pontiger, 102, 1096 Haemolaelaps sp. Haemolaelaps casalis, 102, 1096 Haemolaelaps glasgowi, 102 Haemonchus sp. Haemonchus contortus, 275, 592 Haemonchus placei, 592 Haemophilus sp. Haemophilus aphrophilus, 218 Haemophilus parainfluenzae, 218 HaemophiIus paraphilus, 218 Haemophilus parasuis, 631,635-637 Haemophilus somnus, 551-552 Hageman trait bleeding disorder, 461 Hair shedding, 110 Hair worm, 593 Hairy foot wart, 551 Hairy shaker disease, 573 Haloperidol, 355 Halothane anesthesia dog, 974 gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 720, 726, 991,994 rat, 963 rodent, 959 swine, 983 porcine stress syndrome, 662 Hamster anesthesia and analgesia, 964-965 Armenian (gray), 168, 193-194 background, 168 behavior, 1244 blood sample collection, 1007 Chinese, 168 anatomy, 190-191 hematologic data, 191 husbandry, 190-191, 193 infectious diseases, 191-192 metabolic/genetic diseases, 192-193 neoplasms, 193 nutrition and diet, 190

INDEX

1290

Hamster, Chinese (cont.) reproduction data, 191 traumatic disease, 193 Djungarian, 168 diseases, 196-197 husbandry, 196 neoplasms, 197 nutrition and diet, 196 reproduction, 196 drug dosages, 964 European, 168 anatomy, 195 hematologic data, 195 reproduction, 194-195 handling and restraint, 179 housing, 1244 identification, 1007 Siberian, 168 Syrian (golden), 168 anatomy, 170-173 breeding, 177 clinical chemistry data, 175 dentition, 170-171 development and physiology, 173-174 disease models, 169 diseases bacterial, 180-183 neoplasms, 186-187 parasitic, 185-186 viral, 183-185 endocrine system, 173 estrous cycle, 177 gastrointestinal tract, 172-173 genetics, 174 Harderian gland, 173 hematologic data, 175 hibernation, 170 housing, 175-176, 178-179 husbandry, 176, 178-179 immunology, 170 normative data, 175 nutrition and diet, 171,174-176, 177 origin and history, 169 pancreas, gallbladder, and biliary tract, 172 physiology, 170-171 reproduction, 169, 176-178 respiratory system, 171-172 sexing, 173, 174 strains, 169 urinary system, 173 taxonomy, 168 Hansen's disease, 741 Hantavirus infection prairie dog, 262 rat, 147 rice rat, 268 rodent vector, 1065 vole, 281 zoonotic, 1065-1066 Hard udder, 555 Hardbag, 555 Harderian gland, 173, 254, 277 Hardness, water, 869

Hardware disease, 602 Hare, 330 Haverhill fever, 91 Hazardous substances, 30-31. See also Biohazards Head grub, 597 Headplate, 1031 Health Research Extension Act (1985), 25-26 Health surveillance. See Surveillance Heartgard Plus, 418, 423. See also Ivermectin; Pyrantel pamoate Heartworm, 421-423, 505-506 Heat stress, 236 Heel wart, 551 Helicobacter sp. animals, effect on, 1162 Helicobacter aurati, 172 Helicobacter bilis, 88-90, 139-140, 412-413, 1162 Helicobacter bizzozeroni, 139-140, 412-413, 1085-1086 Helicobacter canis, 412-413 Helicobacter cholecystus, 190 Helicobacter cinaedi, 1084-1085 Helicobacter felis, 412-413, 461-462, 1085 Helicobacter heilmannii, 139-140, 412-413 Helicobacter hepaticus, 88-90, 139-140, 190, 372, 1162 Helicobacter muridarum, 88-90, 139-140 Helicobacter mustelae, 492-494, 510 Helicobacter pylori ferret, 492, 494 gerbil, 277 guinea pig, 218 primate, 734 rat, 139-140 zoonotic, 1085-1086 Helicobacter rappini, 88-90, 412-413 Helicobacter rodentium, 88-90 Helicobacter trogontum, 139-140 Helicobacter typhlonicus, 88-90 isolation and culture, 373, 374 Helicobacteriosis cat model, 461-462 dog, 412-413 enteric, 1084-1085 ferret, 492-494 gastric, 1085-1086 mouse, 88-90, 140 primate, 731,734 rat, 139-140 Heligmosomoides kurilensis, 276 Hellbender, 794, 805 Hemagglutination inhibition (HA1) test, 375, 376, 381, 1026 Hemagglutinin/neuraminidase virus, 641 Hemangioma, 187, 357 Hemangiosarcoma, 445-446 Hematology data amphibian, 805 baboon, 709 beagle, 399 black-tailed prairie dog, 256 cattle, 527 chimpanzee, 714 chinchilla, 256 Chinese hamster, 130 crocodile, 842 cynomolgus monkey (macaque), 705

1291

INDEX degu, 284-285 European hamster, 130 ferret, 486 frog, 805 gerbil, 256 goat, 526, 527 gray squirrel, 256 guinea pig, 208 lizard, 841,842 marmoset, 685 mouse, 43 multimammate rat, 256 owl monkey, 691 prairie dog, 256 rabbit, 334 Rana sp., 805 rat, 127-129, 130 reptile, 841,842 rhesus monkey, 702 sheep, 525-526, 527 snake, 841,842 squirrel monkey, 697 swine, 624 Syrian (golden) hamster, 130 tamarin, 685 tree squirrel, 256 turtle, 841,842 vole, 256 woodchuck, 314, 315 Hematoma, 775 Hematopoietic diseases dog model, 396 ferret, 485-486 mouse, 110-111 rabbit, 356-357 Hemipene, 843,844 Hemitrichodina sp., 881 Hemobartonella muris, 367 Hemobartonellosis, 143 Hemolymphopoietic neoplasms, 237 Hemophilia, 461 Hemorrhagic enteritis, 643-645 Hemorrhagic enterotoxemia, 543 Hemorrhagic fever Argentinian, 1096 canine, 409- 411, 415 multimammate rat model, 282 rabbit, 330, 345 rat model, 273 rat vector, 147 with renal syndrome, 147, 1065-1066 simian, 751-753, 751-753 zoonotic, 1063-1066 Hemorrhagic septicemia, 559-560 Hemosiderin, 109 HEPA filtration, 369, 941-943,944, 1050 Hepatic encephalopathy, 441-442 Hepatic lipidosis, 324-325,600 Hepatic neoplasia, 90 Hepatitis ground squirrel, 252-253 hamster, 190 mouse, 72, 74-79

mouse hepatitis virus, 369, 1159-1160 primate, 757 ruminant, 553 woodchuck, 310, 321-322 zoonotic, 1070-1072 Hepatocellular necrosis, 763 Hepatocellular neoplasms, 154, 310, 321-322, 325 Hepatocystosis, 761 Hepatoma, 193 Hernia, 325, 358, 665 Herpesviruses alpha, 748 ateline, 748 bovine, 567 bovine rhinotracheitis, 570-571 canine, 405-406, 415 cercopithecine, 746, 747, 748 feline, 476-477 gamma, 747, 748-749 guinea pig, 221-222 Herpes simplex virus, 1108 Herpesvirus cuniculi, 345 Herpesvirus sylvilagus, 345 human, 746, 747, 748 human risk factor, 718 inclusion body rhinitis, 641 malignant catarrhal fever, 575 of marmots, 322 mouse, 59- 61 porcine, 578 pseudorabies, 634-635 rabbit, 345 reptile, 850 saimirine, 746, 747, 748 woodchuck, 322 Heterodoxus spiniger, 426 Hexamita, 185, 270 Hibernation, 170, 195, 310, 313-314 High-efficiency particulate air filtration (HEPA), 369, 941-943,944, 1050 Hip dysplasia, 450 Hispid hare, 330 Histamine, 176, 1209 Histiocytic lymphoma, 111, 154 Histiocytic sarcoma, 156 Histiocytoma, 357, 444 Histiocytosis, 158 Histocompatibility complex, 36 Histophilus ovis, 555 Histoplasma capsulatum, 230, 289, 744-745 Histoplasmosis, 169, 230, 289, 744-745 History, of animal medicine and experimentation, 2-8 HIV/AIDS cat feline immunodeficiency virus, 460-461 origin, 1072 rhesus monkey model, 701 simian, 754, 756 Hoof diseases, 549-550, 608-609 Hookworm cat, 478 dog, 418-420 reptile, 852 ruminant, 593

1292

INDEX

Hoplopsyllus anomalus, 252 Host-pathogen-environment interaction, 866, 867 Housing amphibian, 796-797 baboon, 708, 710 caging systems, 937-946 cat, 463-466, 1246 chicken, 1254 chimpanzee, 713-714 cynomolgus monkey (macaque), 704 dog, 919, 1246 facility design, 917-919 farm animals, 1253-1254 ferret, 484-485 fish, 871-873, 892-893 frog, 796-797 guinea pig, 204-205,939-943, 1244-1245 hamster, 1245 heating, ventilation, air conditioning, 919-926 isolation unit, 941 marmoset, 684 mouse, 40-41, 53, 55,939-943 owl monkey, 690 primate, 715-717, 721-723, 918-919, 940, 1249 rabbit, 338,938, 1245-1246 rat, 122-123, 134, 153, 939-943, 1243-1244 reptile, 829-834 rhesus monkey, 700-701 salamander, 796-797 snake, 829-834 squirrel monkey, 696-697 stress, 1164-1165 swine, 616, 622 tamarin, 684 woodchuck, 311,317, 319-320 Xenopus sp., 796-797, 809-810 zebrafish, 871-873 Human. See Personnel Human Genome Project, 1193, 1202 Hurler syndrome, 461 Hyalosis, 291 Hyaluronidase, 441 Hybridization assay, 378 Hydatid cyst disease, 595 Hydatidosis, 595,772 Hydrocephalus, 156, 331,336, 353 Hydrometra, 357-358 Hydronephrosis, 156, 507 Hydrophthalmia, 353-354 Hydroxyflutamide, 451 Hygroma, 435 Hygromycin, 643 Hyla sp., 795 Hyla gratiosa, 795, 805 Hyla septentrionalis, 805 reproduction, 807 Hymenolepis sp. Hymenolepis citelli, 265 Hymenolepis diminuta, 100, 150-151, 186, 277

Hymenolepis microstroma, 100 Hymenolepis nana (dwarf tapeworm) gerbil, 277 hamster, 186

hosts, 1094 mouse, 100 primate, 772 rat, 150-151

Hyostrongylus rubidus, 652 Hyperammonemia, 507 Hyperestrogenism, 506-507 Hyperinsulinemia, 508 Hyperkeratosis, 92-93 Hyperkeratotic dermatitis, 92 Hyperlipidemia, 331 Hyperostosis, 775 Hyperplasia adrenocortical, 510 benign prostatic, 450-451 cecal mucosal, 182 cystic endometrial, 110 epididymal, 279 nictitating membrane gland, 453-454 nodular, 193 seminiferous tubule, 279 stomach, 272 subcapsular spindle cell, 110 transmissible murine colonic, 85-86 Hypertension, 152 Hyperthermia, 776 Hypertrophic cardiomyopathy, 461 Hyperuricemia, 853-854 Hypnorm guinea pig, 966 hamster, 964 mouse, 962 primate, 992 rat, 963 rodent, 958 Hypnosis, 971-972 Hypoderma sp. Hypoderma bovis, 505,597 Hypoderma lineatum, 597 Hypoglycemia, 508, 665,776 Hypophysectomy, 1028, 1147 Hypothalamic-pituitary axis, 1164 Hypothermia anesthesia, 961 mouse, 108 primate, 776 rodent, 961 Hypothyroidism, 429-431 Hypovitaminosis. See specific vitamin deficiencies Hysterectomy, 507 Hystricognath rodent, 249-250 Hystricomorph rodent, 249 I

I-cell disease, 461 Ibuprofen, 972

Ichthyobodo necatrix, 901-902 Ichthyophthiirius multifiliis, 880-881,900-901 Idaho pygmy rabbit, 330 Identification methods, 1006-1008, 1138 Iguana iguana, 842 Ileitis, 180-182

INDEX Ileus, 107, 154 Imaging anesthesia, 961,977-978, 984, 996 facility design, 914 method, 1032-1033 microangiography, 1028 Imidazoles, 592 Imidocarbdiprionate, 586 Immobilon, 981 Immunity, passive, 608 Immunoassay, 381-382 Immunodeficiency genetic, 109 human, 1072 mouse model, 53, 55 rhesus monkey model, 701 Immunofluorescence, 376 Immunoglobulins hamster, 170 hepatitis, 1071-1072d mouse, 52-53 passive immunity, 608 rabies, 1070 swine, 627 Immunology animal selection, 1146 cellular immunity, 1210-1211 genetic monitoring, 1123 hamster model, 170 history, 1208-1214 immunosuppressive agents, 1211-1212 swine, 623-625, 627-629 transplantation, 1211-1213 vaccination, 1213 Importation, 29-30 Inactin, 958, 963 Inbred strain, 37, 109, 1120-1126 Inclusion body rhinitis, 641 Indian desert gerbil, 275 Indirect immunofluorescence assay (IFA), 380-381 Indwelling catheter, 436-437 Infectious diseases adventitious, chain of, 366-367 animals, effect on, 1158-1163 aquatic health, 866 biosafety levels, 722 cat model, 460- 462 containment and eradication, 371-372 hamster model, 169-170 host-pathogen-environment interaction, 866, 867 prevention, 365-387, 475-476 primate, 720-721 risk factors, 367-368, 720-721 sampling, 383-384 surveillance program, 382-385 transmission, 366, 1108 xenotransplantation, 1107-1113 Infectious pancreatic necrosis, 905-907 Inflammatory bowel disease, 88-90, 327, 331 Influenza ferret, 499-500 ferret model, 500 parainfluenza, 571- 572, 753

1293 ruminant, 571-572 swine, 641 zoonotic, 1074 Inguinal hernia, 665 Injection amphibian, 814 anesthesia, 957 intraperitoneal, 1015 intratracheal, 1022 oral gavage, 1015-1016 perivascular injection injury, 440-441 reptile, 846 subcutaneous, 1015 swine, 617 Innovar-Vet, 312, 958 Institute for Animal Research, 250 Institute of Laboratory Animal Research (ILAR), 12-13, 28, 397, 1197, 1203 Institutional Animal Care and Use Committee (IACUC), 21-22, 25-26 Insulin-like growth factors, 207 Insulinoma. See Islet cell neoplasms Insulitis, 285 Intellectual property rights, 1140, 1237 Interagency Research Animal Committee, 25, 26 Interdigital cyst, 454 Interdigital necrosis of cattle, 550 Interferons, 54 Intedeukins, 54, 628 International Council on Laboratory Animal Science (ICLA), 12 International Laboratory Code Registry, 1197 Internet sites American Association for Laboratory Animal Science (AALAS), 1198 American College of Laboratory Animal Medicine (ACLAM), 1231, 1232 animal model supply, 1199-1200 animal models database, 250 animal welfare regulations, 32, 38 Bio MedNet Mouse Knockout database, 1199 BovGbase, 1200 Charles River Laboratories, 1199 ChickGBase, 1200 CRISP database, 1236 Cybermouse project, 1199 Dysmorphic Human-Mouse Homology database, 1199 education and training, 1231 Encyclopedia of the Mouse Genome, 1199 European Collaborative Interspecific Mouse Backcross database, 1199 Foundation Center, 1236 Gene Expression database, 1199 Gene Knockouts database, 1199 gene maps, 1197 genetics and genomics, 38 grants and awards, 1236 Jackson Laboratory, 1199 Japan Animal Genome, 1199 Japanese Association of Laboratory Animal Science, 1198 Laboratory Animal Science Association of the United Kingdom, 1198 mouse, 38 Mouse Atlas Project, 1199 Mouse Genome Database, 36, 38, 1199 MutaMouse, 1199 National Agriculture Library, 1198 National Center for Research Resources, 1233, 1234 National Institutes of Health (NIH), 1231 National Research Service Awards, 1233

1294 Internet sites (cont.) Nature America, Inc, 1198 NIH Comparative Medicine, 1233 Oak Ridge National Laboratory, 1199 Pigbase, 1200 Portable Dictionary of the Mouse Genome, 1199 rodent jaw anatomy, 249 Sheepbase, 1200 Special Emphasis Research Career Awards, 1234 Taconic Farms, Inc., 1199 Transgenic Systems for Mutation Analysis-Big Blue, 1199 Whitehead Institute, 1199 Interstitial cell tumor, 154, 356 Interstitial nephritis, 109 Intestinal access port, 437, 438 Intestinal diseases antibiotic-associated, 182, 189 cat, 478 chinchilla, 287-288, 289 coccidiosis, 347, 502 cysts, 189 degu, 285 dog, 408-409, 412-414, 416-417, 418-420, 423-424 ferret, 490-494, 496, 501-503 gerbil, 277 giardiasis, 98, 416-417 ground squirrel, 253 guinea pig, 216, 218, 223-225 hamster, 180-183 helicobacteriosis, 88-90, 412-413 hepatic encephalopathy, 441-442 inflammatory bowel disease, 88-90, 327 mouse, 109 multimammate rat, 282, 284 parasitic, mouse, 98-105 prairie dog, 255 primate, 730-734, 758-760 proliferative enteritis, 77, 180-182 rabbit, 342-343, 344, 345-346, 347, 357 rat, 135, 147-151 salmonellosis, 90- 91 spironucleosis, 98-99 streptococcal enteropathy, 135 transmissible murine colonic hyperplasia, 85-86 Tyzzer's disease, 83-85, 135-137 ulcers, 240 volvulus, 438 woodchuck, 319, 323-324, 327 yersiniosis, 287 Intracerebral implantation, 1030 Intraosseous infusion, 730 Intravenous injection, 729 Iodine, 559, 906 Iodine deficiency, 107 Iodophors, 598, 906 Iospora sp., 271 Iridovirus, 818, 904 Iron deficiency, 665 Iron dextran, 620, 654 Ischial callosities, 679 Islet cell neoplasms, 154, 155, 507-508, 509 Isoflurane anesthesia

INDEX dog, 974, 976 gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 720, 726, 991,994 rabbit, 970 rat, 963 reptile, 845, 846 rodent, 959 swine, 983 extramedullary hematopoiesis, 486 porcine stress syndrome, 662 Isolation stress phenomenon, 1164 Isoniazid, 1088 Isospora sp. Isospora laidlawi, 502 lsospora suis, 644, 651 primate, 758 Itraconazole, 229, 429, 853 Ivermectin. See also Heartgard Plus acanthocephalosis, 852 ascariasis, 103, 151,227, 643 botfly, 597 Cheytiella sp., 350 demodectic mange, 657 demodicosis, 425 ear mite, 478 heartworm, 423, 506 louse, 657 lungworm, 594 mite, 596, 822, 852 nematodiasis, 101,149, 592, 820-821,878 oxyuriasis, 149, 351 primate dose, 726 psoroptic mange, 349 Pterygodermatites sp., 766 sarcoptic mange, 350, 425, 762 strongyloidiasis, 653,765 Trichosomoides crassicauda, 150 lxodes sp., 262, 426, 1096 lxodes cookei, 323,324 Ixodes ricinus, 554 Ixodes scapularis, 412, 1096

J

Jaagsiekte, 577-578 Jackrabbit, 330 Jackson Laboratory, 1199 Jamestown Canyon virus, 262 Japan Animal Genome, 1199 Japanese Association of Laboratory Animal Science, 1198 Jird, 275-279 Johne's disease, 557-558 Joint diseases arthritis, 91, 92 hip dysplasia, 450 joint ill, 557-558 polyarthritis, 583-584 Journals, 1198-1199 Juvenile polyarteritis syndrome, 451-453

INDEX

K virus, 65 Kalicephalus sp., 852 Kanamycin, 406, 735 Kangaroo rat biology and physiology, 260 description, distribution, habitat, 259 diseases central auditory system, 260 metabolic and nutritional, 260 neoplasms, 261 parasitic, 260 husbandry, 260 research uses, 259-260 taxonomy, 259 Karyotype, 687-688 Ked, 598 Kennel cough complex, 405-406 Keratin, 837 Keratoconjunctivitis, 556-557, 563, 584 Keratopathy, lipid, 823 Ketamine anesthesia crocodile, 845 dog, 974 gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 719-720, 726, 777, 991,992-993 rabbit, 968, 969 rat, 963 rodent, 958-959 ruminant, 987, 988 swine, 981 wood rat, 262 woodchuck, 312 sedation, 777 Ketoconazole, 229, 428, 429, 853 Ketoprofen, 961 Ketorolac, 991,997 Ketorolac tromethamine, 973, 974 Ketosis, of pregnancy, 232-234, 506, 602-603 Kidney diseases amyloidosis with nephrotic syndrome, 188 calcium-induced, 336 chronic infarct, 436 coccidiosis, 99 cysts, 189 hydronephrosis, 507 immune-mediated glomerulonephritis, 326-327 mouse, 109 nephrocalcinosis, 157 nephropathy, chronic progressive, 157 nephrosclerosis, 193 pulpy kidney disease, 543-544 pyelonephritis, 95 rat, 157 renal amyloidosis, 106 Reye's-like syndrome, 106 woodchuck, 326-327 Kilham's rat virus, 146, 1161

1295 Kimmel, Charles, 862 Kindling, 338 Klebsiella sp. Klebsiella oxytoca, 95 Klebsiella pneumoniae degu, 285 dog, 407 ferret, 496-497 fish, 899 goat, 555 guinea pig, 218-219 mouse, 95 primate, 735, 736-737 Klebsiellosis, 95,735, 736-737 Klein-DeForest scheme, 369, 370 Klinefelter's syndrome, 461 Klossiella cobayae, 224-225 Klossiellosis, 224-225 Knockout mouse breeding systems, 1135-1137 colony management, 1131-1134, 1138-1139 databases, 1199 embryo cryopreservation, 1138-1139 gene expression control, 1191 genotype analysis, 1137, 1138 husbandry, 1131-1134 nomenclature, 1138-1139 property rights, 1140 strain selection, 1196 Koisella muris, 99 Krabbe's disease, 461 Kurloff cell, 206 Kyasanur Forest disease, 752 Kyphosis, 268

Laboratory animal care, 1 Laboratory animal medicine comparative medicine, 1228-1229 defined, 1-2 education and training, 13-14, 1231-1234 grants and awards, 1233 history, 1228 laws, regulations, and guidelines, 14-15 organizations, 7-14 research design, 1230 research foundation, 1229 Laboratory animal science, 2 Laboratory Animal Science Association of the United Kingdom, 1198 Laboratory Animal Welfare Act (1966), 14, 20 Laboratory testing. See Diagnosis; Surveillance Lacey Act (1900), 30 Lactate dehydrogenase-elevating virus (LDV) animals, effect on, 1160 diagnosis, 375 mouse, 65-66 transmission, 367 Lactic acidosis, 601,602 Lactobacillus sp. lactic acidosis, 601 Lactobacillus acidophilus, 46 Lactobacillus animalis, 46

1296 Lactobacillus sp. (cont.) Lactobacillus lactis, 46 Lactobacillus murinis, 46 Lactobacillus salivarius, 46 Lactulose, 442

Laelaps echidninus, 102, 1096 Lagomorph. See Rabbit Laminectomy, spinal, 1030 Laminitis, 608-609 Langerhans giant cell, 740 Laparoscopy, 1024 Large granular lymphocytic leukemia, 154 Large-mouth bowel worm, 593 Large stomach worm, 592 Larvae, 804, 806 Lasalocid, 587 Laws, regulations, and guidelines animal welfare, 20-29, 397 Canada, 15 Europe, 29 hazardous substances, 30-31, 1047 importation and exportation, 29-30 interagency cooperation, 27 international, 28-29 Internet sites, 32, 38 personnel health risks, 720-721 primate space requirements, 716 recombinant DNA research, 31 reptile study, 829 United Kingdom, 15 United States, 14-15, 20-32

Lawsonia intracellularis cynomolgus monkey (macaque), 731 ferret, 494 hamster, 180-181 rabbit, 344 rhesus monkey, 734 swine, 644, 645-646 Leiomyoma, 237,239, 356 Leiomyosarcoma, 356, 357 Leishmania sp., 169 Leishmania braziliensis, 273 Leishmania donovani, 271,273, 282 Leishmania major, 282, 1146 Leishmania mexicana, 262, 273 Leishmaniasis, 169, 273,692 Lemur catta, 680 Lentiviruses, 756 Lentivirus sp., 569 Leporid herpesvirus, 345 Leprosy, 741 Leptopsylla segnis, 1097, 1098 Leptospira sp., 281 Leptospira australis, 659 Leptospira autumnalis, 1083 Leptospira ballum, 1083 Lepwspira brattislava, 658-659, 1083 Leptospira canicola, 658, 1083 Leptospira grippotyphosa, 552, 658 Lepwspira hardjo, 552, 658, 1083 Leptospira icterohaemorrhagiae, 219, 552, 658-659, 899, 1083 Leptospira interrogans, 552, 658, 1083 Leptospira interrogans sensu lato, 407-408

INDEX

Leptospira interrogans serovar ballum, 95 Leptospira pomona, 552, 658-659, 1083 Leptospira sejroe, 658 Leptospira tarassovi, 658 Leptospirosis dog, 407-408 mouse, 95 primate, 742 ruminant, 552-553 swine, 658-659 zoonotic, 1083-1084 Lepus sp., 330 Lesser ape, 678 Leukemia bovine lymphosarcoma, 566-567 cat feline leukemia model, 460 chloroleukemia, 111 erythroleukemia, 111 feline leukemia virus, 460 ferret, 510 guinea pig, 237 large granular lymphocytic, 154 lymphocytic, 187 mouse, 111 myelocytic, 187 myelogenous, 111 myeloid, 357 myelomonocytic, 156 sheep, 611 Leukocyte adhesion deficiency, 599 Leukosis, 566-567 Levamisole ascariasis, 643 heartworm, 423 lungworm, 594 nematodiasis, 592, 820-821 Oesophagostomum sp., 765 strongyloidiasis, 653, 765 trichuriasis, 653,766 Levothyroxine, 430 Libyan jird, 275 Lidocaine dog, 975 perivascular injection injury, 441 primate, 991,997 rabbit, 972 rodent, 960 swine, 982 ventricular dysrhythmia, 975 Life-support system, aquatic, 871-873 Lime sulfur, 425, 596, 598 Lincomycin erysipelas, 632 greasy pig disease, 655 heel wart, 551 mycoplasmal pneumonia, 640 mycoplasmosis, 639 swine dysentery, 645 Lindane, 596 Linognathus sp. Linognathus africanus, 596 Linognathus ovilus, 596 Linognathus pedalis, 596

INDEX Linognathus setosus, 426 Linognathus stenopis, 596 Linognathus vituli, 596 Linoleic acid, 107 Lipid keratopathy, 823 Lipidosis cat model, 461 hepatic, 324-325,600 woodchuck, 324-325 Lipofuscin, 110 Lipofuscinosis, 110, 461 Lipoma, 257, 356, 444 Liponyssoides sanguineus, 102, 277 Listeria sp. Listeria ivanovii, 553 Listeria monocytogenes, 219, 288, 366, 553-554 Listeriosis, 219, 288, 553-554 Litomosoides sp. Litomosoides carinii, 265, 271,282 Litomosoides thomomydis, 258 Litomosoides westi, 258 Liver diseases. See also Hepatitis coccidiosis, 346-347 coronavirus, mouse, 74-79 dog, 441-442 Helicobacteriosis, 88-90 hepatectomy/biopsy, 1018-1019 hepatic encephalopathy, 441-442 lipidosis, 324-325 liver fluke, 595-596 lobe torsion, 358 mouse, 109, 112-113 neoplasms, 112-113 rabbit, 346-347 Reye's-like syndrome, 106 woodchuck, 324-325 Lizard anatomy and physiology, 837-841 blood collection, 846 clinical chemistry data, 842 diseases dysecdysis, 855 dystocia, 854 metabolic and nutritional, 853-855 parasitic, 851-852 viral, 850 handling and restraint, 834 hematology data, 841,842 nutrition and diet, 843 sexing, 843 taxonomy, 827, 828 Loa loa, 276 Lockjaw, 543-544 Longistriata adunca, 272 Lordosis, 467, 468 Louse cestode vector, 423 cotton rat infestation, 272 dog infestation, 426 guinea pig infestation, 228 mouse, 96 parasitic vector, 367 prairie dog infestation, 259

1297 rat infestation, 151 rice rat infestation, 268 rickettsia transmission, 95, 143, 151 ruminant, 596-597 spiny rat, 143, 268 swine infestation, 657 tapeworm vector, 426 woodchuck infestation, 324 Lucke tumor herpesvirus, 817- 818 Lumbar sympathectomy, 1029-1030 Lumpy jaw, 537-538 Lumpy wool, 548-549 Lung diseases. See also Pneumonia dog, 439-440 fluke, 424 ground squirrel, 253 mouse, 108-109, 113 neoplasms, 113 rat, 140-143 Lungworm, 594, 852 Luteinizing hormone, 1132 Lyme disease dog, 411-412 grasshopper mouse, 265 ruminant, 554-555 white-footed mouse, 267 wood rat, 262 zoonotic, 1098 Lymph nodes, 109, 619, 624, 729 Lymphadenitis, 214-215, 547 Lymphoblastic lymphoma, 111, 331 Lymphocystis, 904-905 Lymphocytes, 1210-1211 Lymphocytic choriomeningitis virus (LCMV) animals, effect on, 1160-1161 diagnosis, 374 hamster, 183-184 immunity test, 375 mouse, 66-69, 366, 367 zoonotic, 1066-1068 Lymphocytic leukemia, 187 Lymphohistiocytosis, 147 Lymphokines, 627 Lymphoma canine, 442- 443 ferret, 510, 511 follicular center cell, 111 Helicobacter mustelae, 510 histiocytic, 111,154 lymphoblastic, 111, 331 malignant, 356 SIV-induced, 756 viral, 187 Lymphoreticular disorders guinea pig, 213-215 mouse, 109, 110-111 neoplasms, 155-156 rat, 154, 155-156 Lymphosarcoma bovine, 566-567, 611 hamster, 187 rabbit, 356-357 sheep, 611

1298

INDEX

Lymphosarcoma (cont.) snake, 857 swine, 664 turtle, 857 Lysol, 349 Lyssavirus sp., 579-580

M

Macaca sp. Macacafasicularis, 700, 703 Macaca mulatta, 680, 698-699 Macaque. See Cynomolgus monkey (macaque) Macdonaldius oschei, 852 Macracanthorhynchus hirudinaceus, 652 Mad cow disease, 580 Mad itch, 578-579 Maedi pneumonia, 576 Mafenide, 440 Magainins, 801 Maggot, 253 Magnesium, 475, 869 Magnesium antacids, 601 Magnesium deficiency, 107 Magnet, 602 Magnetic resonance imaging anesthesia, 961,977-978, 984, 996 method, 1034 Malachite green, 853, 880 Malaria, 692, 697-698 Malassezia sp., 505 Malathion, 596, 657, 1188 Malignant catarrhal fever, 575-576 Malignant edema, 545-546 Malocclusion chinchilla, 289 guinea pig, 238-239 mouse, 108 prairie dog, 259 rabbit, 355 rat, 124 vole, 281 Mammary gland diseases cattle, 555-556 dog, 447-448 ferret, 497-498 goat, 555 guinea pig, 238 kangaroo rat, 261 mammillitis, 567 mastitis, 497-498, 555-556, 665 mouse, 111-112 neoplasms, 111-112, 154, 238, 447-448 rat, 154 sheep, 555 swine, 665 Mandibular prognathia, 355 Manganese deficiency, 107 Mange chorioptic, 596 demodectic, 186, 192, 656 psorergatic, 283, 596

psoroptic, 349 sarcoptic, 350, 425-426, 596, 656-657, 762 Mannosidosis cat model, 461 cattle model, 599 goat, 598-599 goat model, 522 Marburg virus, 753, 1063-1064 Markers, biochemical biochemical markers, 1123-1124, 1125 Marmoset. See also Callitrichid primate behavior, 684 blood sample collection, 1007 clinical chemistry data, 686 diseases enteric, 730-734 mycotic, 744-746 nutritional, 773-774 respiratory and nervous system, 734-738 viral, 746-757 distinguishing features, 680, 680-681 hematology data, 685 housing, 684-685 husbandry, 684 natural history, 681-683 normative data, 685 nutrition and diet, 685 reproduction, 683-684 research uses, 685-686 taxonomy, 678, 681,682 vaccination, 725 Marmota monax, 253, 309- 310 Maroteaux-Lamy syndrome, 461 Marten, 483 Martes sp., 483 Masseter muscle, rodent, 249 Mast cell tumors, 111,444-445, 510-512 Mastitis cattle, 555-556 ferret, 497-498 goat, 555 sheep, 555 swine, 665 Mastomys natalensis, 256, 281-284 Mastophorus muris, 265 Mayo Clinic, 3-4, 6 Meadow mouse, 279-281 Meadow vole, 280 Measles, 753,754, 755, 1073 Mebendazole, 100 ascariasis, 766 cestodiasis, 423 enterobiasis, 765 Oesophagostomum sp., 765 Pterygodermatites sp., 766 strongyloidiasis, 765 trichuriasis, 766 Medetomidine anesthesia guinea pig, 966 hamster, 964 mouse, 962 primate, 992

1299

INDEX rat, 963 rodent, 958-959, 960 ruminant, 987, 988 tranquilizer dog, 974 gerbil, 965 primate, 991 rabbit, 968 Medium stomach worm, 592 Megaesophagus, 512 Melanin, 110, 196 Melanocortin, 1205 Melanoma, 187, 357, 664 Melarsomine, 423 Melophagus ovinus, 598 Meningitis, 633-634, 734-738 Meningoencephalitis, 94 Meperidine, 972, 974, 981,987 Mercaptopurine, 904, 1211 Mercury poisoning, 331 Meriones sp. Meriones crassus, 275,276 Meriones hurruanae, 275, 276 Meriones libycus, 275, 276 Meriones unguiculatus, 256, 265,275 Meriones vinagradovi, 275, 276 Mesocestoides sp., 423, 852 Mesocricetus auratus, 168, 169 Mesothelioma, 357 Metastrongylus sp. Metastrongylus elongatus, 642 Metastrongylus pudendotectus, 642 Metastrongylus salmi, 642 Methemoglobinemia, 461 6-Methocybenzoxazolinone, 280 Methohexital dog, 976 hamster, 964 mouse, 962 rabbit, 968 rat, 963 Methoxychlor, 657 Methoxyflurane, 662 Methoxyprogesterone, 133 Metomidate, 897, 959, 981 Metronidazole amoebiasis, 758, 820, 1090 balantidiasis, 651,759, 1090 clostridia infection, 218 gastrointestinal bleeding, 442 giardiasis, 1092 cat, 478 dog, 416 primate, 758 rat, 149 ruminant, 588 swine, 652 Syrian hamster, 185 woodchuck, 323 helicobacteriosis dog, 413 ferret, 494 human, 1085

mouse, 90 primate, 731 rat, 140 primate dose, 726 proliferative bowel disease, 494 protozoal infection, 851 sepsis, 439 trichomoniasis, 758 Mexican wood rat, 261 Miconazole, 428, 1089 Micrococcus sp., 555 Microfiltration, 369 Microhyla ornata, 1188 Microsporidiosis, 903-904 Microsporum sp. Microsporum canis cat, 479 chinchilla, 289 dog, 428 ferret, 505 goat, 598 guinea pig, 228-229 primate, 744 rabbit, 351 zoonotic, 1089 Microsporum gypseum, 289, 428, 598 Microtus sp. Microtus arvalis, 279 Microtus californicus, 256, 279 Microtus montanus, 279 Microtus ochrogaster, 279 Microtus oeconomus, 279 Microtus pennsylvanicus, 279 Microtus pinetorum, 279 Midazolam anesthesia primate, 726, 993 swine, 981 tranquilizer dog, 974 gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 rabbit, 968 rat, 963 rodent, 960 Milbemycin ascariasis, 418 demodicosis, 425 heartworm, 423 hookworms, 418 sarcoptic mange, 425 whipworm, 420 Milk fever, 603-604 Mineral deficiency, 107 Mineral oil, 352 Mineralization, soft tissue chinchilla, 290 cotton rat, 272 guinea pig, 235 mouse, 105-106, 108, 110 rabbit, 336

1300 Mineralization, soft tissue (cont.) rat, 156-157 Minerals, deficiency and excess, 235 Mink, 483, 1011 Minute virus of mice, 61-63, 1161 Mite amphibian infestation, 822 cane mouse infestation, 270 cat infestation, 478 diagnosis, 373 dog infestation, 424-426 ferret infestation, 503-504 fur, 102, 104, 105 gerbil infestation, 277-278 grasshopper mouse infestation, 264 ground squirrel infestation, 253 guinea pig infestation, 225-228 hair follicle, 102, 103, 105 hamster infestation, 186, 194 hosts, 1096 house mouse, 102 mouse infestation, 102-105 multimammate rat, 283 northern fowl, 102 photograph, 1095 rabbit infestation, 349-350 rat infestation, 151 reptile infestation, 852-853 ruminant infestation, 596 snake, 852-853 spiny rat, 102 tapeworm vector, 594 tropical rat, 102 vole, 281 woodchuck infestation, 324 zoonotic, 1096, 1098 MMTV-S virus, 112 Mogula manhattensis, 234 Molineus sp. Molineus elegans, 765 Molineus torulosis, 765 Molluscum contagiosum, 750 Molybdenum, 606 Monensin, 587, 590, 591,601 Monezia sp. Monezia benedini, 594 Monezia expansa, 594 Mongolian gerbil, 265, 275-279 Moniliformis clarki, 265, 281 Monitoring, genetic, 1117-1126 Monkey, 678 Monkeypox, 750, 1060-1061 Monodelphis domestica, 1012 Mononuclear cells, 109 Monopsyllus exilis, 265 Montane vole, 279 Moraxella sp., 736 Moraxella bovis, 556-557 Moraxella catarrhalis, 738 Morbillivirus sp., 414-415, 498-499, 754 Morganella morganii, 320 Moroccan leather appearance, 593 Morphine dog, 974

INDEX guinea pig, 966 hamster, 176, 964 mouse, 962 primate, 991,992, 997 rabbit, 972 rodent, 961 swine, 981 Xenopus sp., 813 Morro Bay virus, 262 Mortellaro's disease, 551 Mosquito, 421 Mouse. See also Cane mouse; Grasshopper mouse; Vole; White-footed mouse aggression, 1242 aging, 108-113 anatomy, 43-49 anesthesia and analgesia, 962-963 barbering, 52, 108, 1241-1242 behavior, 51-52, 108, 1241-1242 blood and reticuloendothelial system, 47 blood sample collection, 1007 breeding systems, 36-37, 39 caging systems, 939-943 cannulation technique, 1014 cardiovascular system, 48, 49 cellular immunology, 53 clinical chemistry data, 44 cytokines, 53, 54 diabetes mellitus model, 1187, 1205 diseases age-related, 108-109 bacterial, 80-95 congenital, 108 environmental, behavioral, and traumatic, 107-108 metabolic and nutritional, 105-107 mycotic, 96-98 neoplasms, 110-111 parasitic, 98-105, 277 plague, 265 postpartum ileus, 107 rickettsial and chlamydial, 95-96 skin trauma, 108 viral, 55- 80 drug dosages, 962 embryo cryopreservation, 1027, 1138-1139 environmental enrichment, 1242-1243 estrous cycle, 50 gastrointestinal tract, 46-47 genetics, 36 genital system, 48 genome databases, 1199 gestation, 51 growth, 42-43 hematology data, 43 housing, 40-41, 53, 55, 939-943 husbandry, 41 identification, 1007 immunodeficiency model, 53, 55, 109 immunology, 52-53 Internet sites, 38 lymphoreticular system, 47 mating, 51 musculoskeletal system, 48 nervous system, 48 normative data, 43

1301

INDEX

nutrition and diet, 41, 42 obesity model, 1205 origin and history, 35-36 pheromones, 50, 51 physiology and anatomy, 41-48 postnatal development and weaning, 51 reproduction, 48-51 respiratory system, 43, 45 sexual maturation, 49 strains A, 56, 105, 111 129/SvEv, 1130-1131 lpr, 55 A/J, 88, 110 A/JCr, 88, 90 AEJ /GnL-ae/A w-J,40 AKR, 56, 65, 81, 111 AKR.B6-H2b, 40 ASF series, 46-47 athymic, 94 B10.BR, 1146 B6; 129-Cftr tm/vn, 40 B6.129-Myf5My~ 40 B6C3F1, 40, 88, 90, 110, 112 B6.CBA-D4Mit25-D4Mit80, 40 B6.Cg -m +/+ Lepr ab, 40 B6.Cg -m Leprdb/++, 40 B6EiC3-Ts65Dn, 40 B.A-Chr 1, 40 BALB/c, 51, 56, 81, 93, 98, 105, 106, 109, 111, 1146 BALB/cByJ, 106 BALB.K, 1146 beige, 55 black, 107 breeding systems, 36-37, 39 BXD- 1/Ty, 40 C3H, 56, 81, 93, 105, 106, 109, 110, 111,112 C3H/He, 98, 110 C3H/HeNCr, 88 C3H/HeSn-ash/+, 40 C57BL, 81,105, 111 C57BL/10, 98, 107 C57BL/6, 42, 56, 65, 69, 84, 88, 93, 95, 98, 107, 110, 1130-1131 C57BL/6J-mtBALB/c,40 C57BL/6J-Tyrc2J/+, 40 C58, 65 CBA, 81, 110 CBA/J, 1146 CcS 1, 40 CcS 1(N4), 40 CD1, 42 CE, 1147 CF1, 42 cytokine KO, 55 DBA, 93, 106, 1147 DBA/1, 56 DBA/2, 56, 69, 81, 84, 111 DBA/21, 40 DBA/ZN, 42 development, 35-36 fighting, 108 FVB/N, 1130-1131 FVB /N-m rgl/zzz, 40 FVB/N-TgN(MBP) 1Xxx, 40

gld, 55 Hsd:ICR, 40 IL 10% 90 immunodeficiency model, 53, 55, 109 inbred, 1122-1123, 1125-1126 knockout, 1129-1140 moth-eaten, 55 NOD, 110 nomenclature, 39-40 nude, 55, 1211 NZB x NZW, 109 Pri:B6,D2-G#, 40 Rag- 1, 55 Rag-2, 55 receptor KO, 55 SCID, 55, 90, 109, 1211 SJL, 81,105 SJL/NCr, 88 SWR, 81 table, 40 transgenic, 39, 1129-1140 XID, 55 temperature and water regulation, 42-43 urinalysis data, 44 urinary system, 45-46 vaginal plug, 51 Mouse antibody production (MAP) test, 374-375 Mouse Atlas Project, 1199 Mouse Genome Database, 36, 38, 1199 Mouse hepatitis virus, 369, 1159-1160 Mouse parvovirus, 1161 Mousepox, 55-59 MS222, 812-813 Mucoepidermoid carcinoma, 357 Mucometra, 110 Mucopoly saccharoidosis, 461 Mucor sp., 853 Mucormycosis, 505,853 Mucosal disease, 568 Mudpuppy, 794, 795, 804 Muellerius capillaris, 594 Multiceps sp., 423,772 Multimammate rat, 256 biology and physiology, 283 description, distribution, habitat, 282 diseases, 284-285 hematology data, 256 husbandry, 283 normative data, 283 nutrition and diet, 283 research uses, 282 taxonomy, 281-282 Murine respiratory mycoplasmosis, 142-143 Murine typus, 1074-1075 Mus sp. Mus musculus, 35,265 Mus musculus castaneus, 35, 36 Mus spretus, 35, 36 Muscular dystrophy, models, 169, 396, 461 Mustela sp. Mustela eversmanii, 484 Mustela furo, 484 Mustela grammogale, 483 Mustela lutreola, 483

1302 Mustela sp. (cont.) Mustela putorius, 483,484 Mustela putoriusfuro, 483-484 Mustela vision, 483 Mutagenicity, 862, 863-864, 1188, 1190 MutaMouse, 1199 Mycobacteriosis amphibian, 816 atypical, 1078 ferret, 494-496 fish, 876-877, 899 hamster, 169 mouse, 95 primate, 738-742 reptile, 836, 849 zoonotic, 1088 Mycobacterium sp., 219 Mycobacterium abscessus, 1078 Mycobacterium africanum, 738 Mycobacterium avium, 92, 494-496, 557, 1088 Mycobacterium avium-intracellulare, 95, 740-741 Mycobacterium bovis, 494-496, 557, 738, 1088

Mycobacterium chelonae amphibian, 816 fish, 876-877, 898, 899 reptile, 836, 849 zoonotic, 1078

Mycobacteriumfortuitum amphibian, 800, 816 fish, 876-877, 898, 899 zoonotic, 1078 Mycobacterium kansasii, 741-742 Mycobacterium leprae, 741,899 Mycobacterium lepraemurium, 95

INDEX

Mycoplasma ovipneumoniae, 562-563 Mycoplasma pneumoniae, 169 Mycoplasma pulmonis animals, effect on, 1162 guinea pig, 220 mouse, 80-82, 83, 87 rat, 140, 142-143, 144, 146 Mycoplasmosis guinea pig, 220 mouse, 80-83 pneumonia of swine, 640-641 rat, 142-143 reptile, 850 ruminant, 562-563 swine, 639-641 Mycoptes musculinus, 225, 226 Mycosporum gypseum, 257 Myelocytic leukemia, 187 Myeloencephalopathy, 599 Myelogenous leukemia, 111 Myeloid leukemia, 357 Myelomonocytic leukemia, 156 Myiasis, 253, 505 Myobia musculi, 102, 104-105, 151 Myocoptes musculinus, 102, 104 Myodegeneration, nutritional, 607 Myoglobinuria, 829 Myomorph rodent, 249 Myopathy, 324 Myotonia congenita, 522, 599 Mystromys albicaudatus, 272-273 Myxomatosis, 344 Myxosporidiosis, 904

Mycobacterium marinum amphibian, 800, 816 fish, 876, 898, 899-900 reptile, 849

Mycobacterium paratuberculosis, 557-558 Mycobacterium thamnopheos, 849 Mycobacterium tuberculosis ferret, 494-496 fish, 899 hamster, 170 primate, 738 ruminant, 557 zoonotic, 1088

Mycobacterium ulcerans, 282 Mycobacterium xenopi, 800, 816 snail vector, 874

Mycoplasma sp., 182 feline upper respiratory tract infection, 476 isolation and culture, 374 mastitis, 555-556 Mycoplasma agassizii, 850 Mycoplasma arthritidis, 80, 81, 82 Mycoplasma bovigenitalium, 562 Mycoplasma bovis, 562 Mycoplasma caviae, 220 Mycoplasma collis, 82-83 Mycoplasma conjunctivae, 563 Mycoplasma hyorhinis, 639 Mycoplasma mycoides, 555, 563 Mycoplasma neurolyticum, 83

Nalbuphine, 961,972 Naloxone dog, 974 primate, 991,992, 994 rabbit, 969 rodent, 960 woodchuck, 312 Naltrexone, 434 Nanofabrication, 1195-1197 Naproxen, 726 Nasal bot, 597 Nasal myiasis, 597 Nasogastric intubation, 730 Nasolacrimal duct occlusion, 358 Nasopsyllusfasciatus, 1097, 1098 National Academy of Sciences (NAS), 12 National Agriculture Library, 23, 1198 National Antivivisection Society, 8 National Association for Biomedical Research (NABR), 10 National Center for Research Resources, 1203, 1233, 1234 National Institute for Occupational Safety and Health (NIOSH), 31 National Institutes of Health (NIH) animal models database, 1197-1198 CRISP database, 1236 education and training, 1231 grants and awards, 1203, 1233, 1236 laws, regulations, and guidelines, 14-15 Office of Animal Welfare (OLAW), 25

INDEX National Research Service Awards, 1233 National Society for Medical Research (NSMR), 8-10 Nature America, Inc, 1198 Navel ill, 538, 557-558 Necator americanus, 1094 Necrohemorrhagic typhlitis, 182 Necropsy, 914 Necturus sp., 794, 795 Nematodiasis amphibian, 820-821 chinchilla, 289 cotton rat, 271,272 dog, 417-423 fish, 878, 903 gerbil, 277 gerbil model, 275-276 grasshopper mouse, 265 guinea pig, 225 hamster, 186 hosts, 1094 mouse, 100-102 multimammate rat model, 282 prairie dog, 258 primate, 765-766 rat, 149-150 reptile, 852 ruminant, 592-594 swine, 642-643, 644, 652-654 woodchuck, 319, 323-324 zoonotic, 1093-1094, 1098 Nematodiris sp. Nematodiris battus, 593 Nematodiris spathiger, 593 Nematospiroides dubius, 275 Nematotaenia sp., 821 Neobellieria citellivora, 253 Neomycin, 343,409, 442, 630 Neonate anesthesia, 961,977, 984, 996 Neoplasms benign, 187 biopsy, 442 chinchilla, 290-291 cotton rat, 272 degu, 285 dog, 442-449 gerbil, 278-279 ground squirrel, 253-254 guinea pig, 206, 237-238 hamster, 186-187, 195 hamster model, 169-170, 172, 173, 194, 197 liver, 112-113, 154 lymphatic and hematopoietic, 110-111, 154 mammary gland, 111-112, 154 mouse, 110-112 mouse model, 110-112 multimammate rat, 283 multimammate rat model, 282 ovarian, 154 pituitary, 154, 155 rabbit, 355-357 rabbit model, 331,357 rat, 154-156 testicular, 154, 155 uterine, 154

1303 vole, 281 white-tailed rat, 274 woodchuck, 325 Neospora caninum, 588 Neosporosis, 588-589 Neostigmine, 975 Neotenic, 794 Neotoma sp. Neotoma albigula, 261 Neotoma cinerea, 261,262 Neotoma floridana, 261,262 Neotoma fuscipes, 261 Neotoma lepida, 261 Neotoma mexicana, 261 Neotoma micropus, 261-262 Neotropical primate, 678, 680 Nephritis, interstitial, 109, 279, 281. See also Glomerulonephritis Nephroblastoma, 331 Nephrocalcinosis, 157 Nephrolithiasis, 234 Nephroma, 356, 664 Nephromyces sp., 234 Nephropathy, chronic progressive, 157, 272 Nephrosclerosis, 193, 239 Nephrosis, 290 Nephrotic syndrome, 188 Nerium oleander, 240 Nervous system diseases gerbil, 278 hamster model, 170 hydrocephalus, 156, 331,336, 353 kangaroo rat, 260 meningoencephalitis, 94 mouse, 110 prairie dog, 255,257 woodchuck, 319 Nesolagus netscheri, 330 Nets, 719 Neuroaxonal dystrophy, 461 Neurofibrosarcoma, 356, 357 Neuronal ceriod lipofuscinosis, 461 Neuropathy, peripheral, 110 Neuropeptide Y, 1205 Neutralization test, 375 New World monkey, 678, 680, 726 New Zealand White rabbit, 334 Newcastle disease virus, 1074 Newt, 803 Niacin deficiency, 231,235 Niclosamide, 186, 772 Nictitating membrane, 453-454 Niemann-Pick disease, 461 Nigg agent, 96 Nippostrongylus brasiliensis, 282 Nitrate, 869-870 Nitrification cycle, 869-870, 889 Nitrite, 869-870 Nitroglycerin, 975, 982 Nitroprusside sodium, 975 N-nitroso-N-ethylurea, 864 Nitrosomonas sp., 869, 870 Nitrous oxide dog, 977 primate, 994

1304

INDEX

Nitrous oxide (cont.) rodent, 959 swine, 983 Nocardia sp., 736, 738

Nocardia asteroides, 898 Nocardia kampachi, 898 Nocardiosis, 736, 738 Nodular hyperplasia, 193 Norepinephrine, 975, 991 Norway rat, 113, 123 Nosema sp., 903 Nosopsyllus fasciatus, 228 Notoedres sp., 186 Notoedres cati, 350, 1096 Notoedres muris, 225 Notoedres musculi, 102

Notophthamlus viridescens, 807 Nuclear Regulatory Commission (NRC), 31 Nuclear sclerosis, 449-450 Nutrition and diet, 1146, 1153-1155 Nutritional muscular dystrophy, 607 Nystatin, 853 O Oak Ridge National Laboratory, 1199 Obeliscoides cuniculi, 323-324 Obesity dog, 431-432 hamster model, 169 rat model, 152 research program, 1204-1206 Occupational Safety and Health Administration (OSHA), 30-31,720-721, 1047 Ochotona sp., 330 Octodon degus, 284 Odontoma, 257 Oesophagostomum sp. hosts, 1094 Oesophagostomum columbianum, 593 Oesophagostomum venulosum, 593 primate, 765, 769 swine, 652 Oestrus ovis, 597 Office of Laboratory Animal Welfare (OLAW), 25, 26 Old World monkey, 678, 680, 733 Old World rabbit, 330 Omeprazole, 413,494 Omphalitis, 538, 557-558 Omphalophlebitis, 538 Onchocerca volvulus, 276 Onchomys sp. Onychomys leucogaster, 263 Onychomys torridus, 263 Onychomycosis, 745 Oocytes, 173 Oodinium sp., 819 Ophionyssus sp., 852 Ophionyssus natricis, 1096 Ophiotaenia sp., 852 Opossum, 1012 Oral dosing, 814, 1015-1016 Oral examination, 1015 Off, 573-574, 1061-1062

Organ transplantation, 627- 629 Organizations animal model supply, 1197-1198 Internet sites, 38 laboratory animal science, 7-14 professional and scientific organizations, 28, 1203 Organophosphate insecticides, 151 flea infestation, 428 fluke infestation, 902 mite infestation, 852 sarcoptic mange, 425 trematodiasis, 903 Ornithodorus sp., 1096 Ornithodorus coriaceus, 561 Ornithonyssus sp. Ornithonyssus bacoti, 1098 grasshopper mouse, 264 hamster, 186 hosts, 1096 mouse, 102 rat, 270 Ornithonyssus bursa, 1096 Ornithonyssus sylviarum, 102, 1096 Ornithosis, 1076-1077 Oropsylla montanus, 252 Orphans of the Storm, 8 Oryctolagus sp., 344 Oryctolagus cuniculus, 330 Oryzomys sp. Oryzomys capito, 267 Oryzomys eliurus, 268 Oryzomys laticeps, 268 Oryzomys palustris, 267-268 Oryzomys subflavus, 268 OSHA. See Occupational Safety and Health Administration (OSHA) Osteoarthritis, 109, 283-284 Osteoarthrosis, 239 Osteodystrophy, 109 Osteomalacia, 774 Osteoporosis, 109 Osteosarcoma, 356, 357 Ostertagia sp. Ostertagia bisonis, 592 Ostertagia circumcinata, 275, 592 Ostertagia leptospicularis, 592 Ostertagia ostertagi, 592-593 Otitis externa, 320, 478 Otitis interna, 339 Otitis media, 339-340 Otodectes cynotis, 478, 504, 505 Outbred strain, 37 Ovarian atrophy, 110 Ovarian cysts, 239, 240 Ovariohysterectomy, 507 Ovis sp. Ovis aries, 520 Ovis nivicola, 520 Ovulation induction, 1132-1133, 1147 Owl monkey behavior, 688 body weight, 692 clinical chemistry data, 691 diseases enteric, 730-734

1305

INDEX mycotic, 744-746 nutritional, 773-774 respiratory and nervous system, 734-738 tuberculosis and mycobacterial, 738-742 viral, 746-757 hematology data, 691 housing, 690 husbandry, 690 karyotypes, 687-688 natural history, 687-688 nutrition and diet, 690-691 reproduction, 689-690 research uses, 692 taxonomy, 687-688 urinalysis data, 692 vaccination, 725 Oxfendazole, 423,643 Oxibendazole, 420 Oxygen, dissolved, water management, 867-868 Oxymorphone dog, 974 primate, 991,992, 997 swine, 981 Oxytetracycline anaplasmosis, 585 anthrax, 539 atrophic rhinitis, 636 chlamydial abortion, 583 chlamydial conjunctivitis, 584 chlamydiosis, 819 Citrobacter rodentium, 277 dermatophilosis, 548-549 eperythrozoonosis, 564 erysipelas, 632 foot rot, 550 klebsiellosis, 735 leptospirosis, 553,659 listeriosis, 554 Lyme disease, 554 mastitis, 557 mycoplasmal pneumonia, 563, 640 pasteurellosis, 559, 637 pleuropneumonia, 563, 637 polyarthritis, 584 query fever, 564 rickettsial eperyzoonosis, 654 septicemia, 878 thromboembolic meningoencephalitis, 552 Tyzzer's disease, 342 Oxytocin dystocia, 854 lactogenesis, 665 mastitis, 555 parturition induction, 404 primate dose, 726 uterine prolapse, 609 Oxyuriasis, 149, 350-351 Oxyuris ambigua, 350-351 Ozone, 890, 906 P

Pack rat, 261-263 Pain assessment. See also Analgesia

dog, 978 fish, 895, 895-896 rabbit, 972 rodent, 961 swine, 985 Pan sp., 711-712 Panacur, 903 Pancreatic diseases age-related, 109, 240 cysts, 189 fatty infiltration, 240 infectious pancreatic necrosis, 905-907 mouse, 109 neoplasms, 154, 155, 173, 507-508 rat, 154, 155 Pancreatic function, 1019 Pancreatic necrosis virus, 882 Pancuronium, 975,982, 991 Pancytopenia, 409-411 Panleukopenia, 476 Panophthalmitis, 241 Pantothenic acid deficiency, 107, 231,235 Papilloma, 187, 197, 237, 331 Papillomatosis digital dermatitis, 551 rabbit, 345 ruminant, 551,578, 611 turtle, 850, 857 Papillomavirus, 282, 283, 345, 578, 751 Papio anubis, 706 Papovavirus, 64-65 Paragonimus sp. Paragonimus heterotremus, 276 Paragonimus kellicotti, 424 Paragonimus westermanii, 424, 772 Parainfluenza virus, 147, 405-406 Parakeratosis, 601 Paralysis, 79-80, 169, 821 Paramphistoma sp. Paramphistoma cervi, 596 Paramphistoma microbothroides, 596 Paramyxovirus parainfluenza 1, 69, 143 - 144, 184 Paramyxoviruses, 753,754, 851 Paraovarian cyst, 110 Paraphimosis, 290 Parapoxvirus, 577 Parasitic diseases, effect of, 1163 Parasitology, 373 Paraspidodera uncinata, 225 Parathyroidectomy, 1029 Paratrichodina sp., 881 Paratuberculosis, 557-558 Parker's rat coronavirus, 144 Parkinson disease model, 1187 Paromomycin, 758, 820, 1090, 1091 Parrot fever, 1076-1077 Pars distalis neoplasms, 154, 155 Parturient paresis, 603-604 Parvovirus Aleutian disease virus, 499-500 canine, 413- 414 cattle, 582-583 hemagglutination inhibition (HAI) test, 376 mouse, 61-63, 1161

1306 Parvovirus (cont.) porcine, 659-660 primate, 751 rat, 146, 1161 Passalurus ambiguus, 350-351 Pasteurella sp., 321,497 Pasteurella haemolytica, 555, 559 Pasteurella multocida animals, effect on, 1162 cat, 479 dog, 407, 433 guinea pig, 219 owl monkey, 737-738 rabbit, 339-340 ruminant, 559-560 squirrel monkey, 737-738 swine, 635-637, 640 woodchuck, 320 zoonotic, 1080 PasteureUa pneumotropica, 87-88, 137-138, 144, 182 Pasteurella tularensis, 561 zoonotic, 1080 Pasteurellosis cat, 479 guinea pig, 219 human risk factor, 479 primate, 735, 737-738 rabbit, 339-340 rat, 137-138 ruminant, 559-560 swine, 637 zoonotic, 1080 Patas monkey, 751 Patents, transgenics, 1140 Pathogenicity, biohazard, 1048 Pathology, 373 PDS suture, 433 Pediculosis dog, 426 rat, 151 ruminant, 596-597 swine, 657 Pelger-Hu~t syndrome, 331,334 Pelvic organ prolapse, 698 D-Penicillamine, 606 Penicillins. See also Amoxicillin; Ampicillin; Procaine penicillin actinobacillosis, 537 anthrax, 539 bighead, 545 campylobacteriosis, 541 dermatophilosis, 548-549 entropion, 598 erysipelas, 548, 632 foot rot, 550 greasy pig disease, 655 heel wart, 551 lactic acidosis, 601 leptospiremia, 408 leptospirosis, 659, 1083 listeriosis, 554 lumpy jaw, 538 Lyme disease, 554 malignant edema, 546

INDEX mastitis, 557 Moraxella sp., 736 navel ill, 559 pasteurellosis, 559, 637, 735 pleuropneumonia, 637 polyarthritis, 631 primate dose, 726 proliferative enteropathy, 646 pyelonephritis, 547-548 rat bite fever, 1079 sepsis, 439 streptococcal meningitis, 633, 634, 735 tetanus, 544 treponematosis, 344 Penis, 290 Pentalagus furnessi, 330 Pentastomiasis, 225, 852 Pentazocine, 972, 981 Pentobarbital, 312 anesthesia dog, 974, 976 gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 726, 991,993 rabbit, 968, 970 rat, 963 rodent, 958, 961 swine, 981,982 euthanasia dog, 979 primate, 997 swine, 986 Periarteritis, 108 Pericarditis, 215 Peripheral neuropathy, 110 Peritonitis, 477-478, 602 Peritonitis virus, 477-478 Perivascular injection injury, 440-44 1 Perivascular lymphoid nodules, 239 Permethrin, 227, 657 Peromyscus sp. Peromyscus leucopus, 264, 265,412 Peromyscus maniculatus, 265 Personnel biohazard control, 1047-1056 education and training, 13-14, 946-947, 1049-1050, 1051, 1231-1234 ergonomics, 887-888 facility design, 912 farm animal interaction, 1256 health risks allergies, 1054 amoebiasis, 1089-1090 amphibian, 800 anthrax, 539 atypical mycobacteriosis, 1078 bacterial, 720, 898-899, 1077-1088 balantidiasis, 1090 benign epidermal monkeypox, 1061 bites and scratches, 1077-1081 bordetellosis, 1088 brucellosis, 540, 1081-1082

1307

INDEX campylobacteriosis, 1084 cat disease transmission, 478-480, 1055 cat scratch disease, 1079-1080, 1081 cercopithecine herpesvirus, 1068-1069 chlamydiosis, 1076-1077 cryptosporidiosis, 1090-1091 dengue, 1063 Ebola virus, 1064-1065 enteric helicobacteriosis, 1084-1085 fish, 898-899 fungal diseases, 899, 1088-1089 gastric helicobacteriosis, 1085-1086 giardiasis, 1091-1092 hantavirus infection, 1065-1066 hemorrhagic fever, 1063-1066 hepatitis, 1070-1072 herpesviruses, 718 influenza virus, 1074 leptospirosis, 1083-1084 lymphocytic choriomeningitis virus, 1066-1068 Marburg virus disease, 1063-1064 monkeypox, 1060-1061 murine typus, 1074-1075 mycobacteriosis, 1088 nematodiasis, 1093-1094, 1098 Newcastle disease virus, 1074 off virus, 1061-1062 OSHA guidelines, 720-721 parasitic diseases, 720, 899 pasteurellosis, 1080 plague, 1082-1083 poxvirus, 1060-1062 primate handling, 720-721, 1055 protozoal infection, 1089-1093 Q fever, 1076 rabies, 1055, 1069-1070 rat bite fever, 1078-1079 reptile, 836-837, 845 respiratory infections, 1088 rickettsial pox, 1075-1076 salmonellosis, 1086-1087 shigellosis, 1087-1088 simian foamy virus, 1073 simian immunodeficiency virus, 1072-1073 spongiform encephalopathy, 720 streptococcosis, 1080-1081 toxins, 899 toxoplasmosis, 1092-1093 tuberculosis, 496, 1088 venom, 836-837, 1055-1056 viral, 720, 899 xenotransplantation, 1107-1113 yellow fever, 1063 zoonoses, 1055 hygiene facility, 915 immunization guidelines, 1055 occupational health program, 727 primate interaction, 1252 protection biohazards, 30-31 protective equipment, 718-719, 721,722, 1052-1053 safety, 887-888 Peruvian squirrel monkey, 693-694

Pesticides, 1188 Pet Theft Act (1990), 20 Pharyngeal sampling, 1021 Phenmidine diisethionate, 586 Phenobarbital, 442 Phenothiazine, 351 Phenotyping, 1195-1197 Phenylbutazone, 981,987, 989 Phenylephrine, 975, 991 Pheochromocytoma, 154 Pheromones, 1158-1159 Philometra sp., 899 Phodopus sp., 196-197 Phodopus campbelli, 168 Phodopus sungorus, 168, 196-197 Phosmet, 657 Photoperiodicity, 1151-1152 Photosensitization, 544-545 Phycomycosis, 822 Pigbase, 1199 Pigeon, 1014, 1072 Pine vole, 279 Pineal gland, 196 Pinealectomy, 1028 Pineapple juice, 352 Pink eye, 556-557 Pinworm cane mouse, 270 diagnosis, 373 egg, 768 gerbil, 277 guinea pig, 225 hamster, 186 mouse, 100-102 primate, 765-766 rabbit, 350-351 rat, 101, 149 Pipa pipa, 807-808 Piperazine ascariasis, 643 nematodiasis, 101, 186, 903 oxyuriasis, 351 Piroxicam, 972 Pisinoodinium sp, 878-879 Pituitary neoplasms, 154, 155, 357 Pituophis sp., 837-841 Placenta, 290 Placentitis, 553 Plagiorchis sp.

Plagiorchis javensis, 151 Plagiorchis muris, 151 Plagiorchis philippinensis, 151 Plague, 262, 265, 282, 1082-1083. See also Yersiniosis

Planorbella sp., 874 Plant toxicoses, 240 Plasma cell tumors, 111, 187 Plasmodium infection, 761 Plasmodium sp. Plasmodium berghei, 282 Plasmodium brazilianum, 761 Plasmodium cynomolgi, 761 Plasmodium eylesi, 761 Plasmodiumfalciparum, 687, 697-698

1308 Plasmodium sp. (cont.) Plasmodium fieldi fragile, 761 Plasmodium hylobati, 761 Plasmodium inui, 761 Plasmodium knowlesi, 761 Plasmodium pitheci, 761 Plasmodium rodhairi, 761 Plasmodium schwetzi, 761 Plasmodium vivax, 687, 697-698 Plasmodium youngi, 761 Platyrrhine primate, 679, 679-680 Pleistophora sp., 903 Plethodon cinereus, 806-807 Pleuropneumonia, 563, 637, 743 Plistophora sp., 819 Pneumocystis carinii animals, effect on, 1163 ferret, 504-505 mouse, 96-98, 109, 152 primate, 744 rabbit, 352 rat, 135, 152 Pneumocystosis mouse, 96-98 primate, 744 Pneumonia aspiration injury, 440 caprine, 563 cotton rat model, 270 dog, 406-407, 440 enzootic, 559-560, 640-641 ferret, 496-497 fungal, 853 giant cell, 1073 hamster, 182, 184-185 listeriosis, 553 mouse, 96-98 mycoplasmal, 81-82, 640-641 ovine mycoplasmal, 562-563 ovine progressive, 576 pleuropneumonia, 563, 637, 743 Pneumocystis carinii, 96-98, 152 primate, 734-735, 736-738, 743 rabbit, 339-340 rat, 152 reptile, 853 respiratory syncytial virus, 572 ruminant, 553, 559-560, 572 Sendai viral, 69-71 swine, 637, 640-641,642-643 verminous, 642-643 virus of mice, 71-72, 146 woodchuck, 320-321 Pneumoviruses, 754 Pocket gopher biology and physiology, 258 description, distribution, habitat, 257-258 diseases, 258-259 husbandry, 258 research uses, 258 taxonomy, 257-258 Pododermatitis, 153, 219-220, 353 Podophyllin, 578 Poelagus marjorita, 330

INDEX

Pole-and-collar restraint, 719 Polecat, 484 Policies. See Laws, regulations, and guidelines Polioencephalomalacia, 607-608 Poliomyelitis, 65, 369 Poliovirus, 222-223,757 Pollution effects, 1188 Poloxalene, 601 Polyarteritis, juvenile, 451-453 Polyarthritis, 583-584, 631 Polyclonal antibody, 331 Polycystic kidney disease, 110, 461 Polydaetyly, 599 Polymerase chain reaction (PCR), 376-380 Polymyxin, 274, 744 Polyomavirus, 64-65, 185 Polyomavirus macacae, 751 Polyplax sp. Polyplax serrata, 96 Polyplax spinulosa, 143, 151,268 Polyps, 187 Polyserositis, 631,639-640 Polystoma sp., 821 Polyvinylpyrrolidone-iodine, 906 Porcupine, 249 Porphyria, 461,599 Porphyrin, 173, 277 Portable Dictionary of the Mouse Genome, 1199 Positron-emission tomography, 984 Posthitis, 547-548 Postpartum ileus, 107 Povidone-iodine, 432, 440, 548-549 Powassan virus, 322-323 Poxviruses marmoset, 750 primate, 747, 750 rabbit, 344-345 reptile, 850-851 ruminant, 576-577 zoonotic, 1060-1062 Prairie dog biology and physiology, 255 clinical chemistry, 255 description, distribution, habitat, 254 diseases, 255, 257 hematology data, 256 husbandry, 255 research uses, 254-255 Prairie vole, 279 Praziquantel, 100 cestodiasis, 423, 772, 821,852 fluke infestation, 902 trematodiasis, 424, 772, 821,852, 903 Prednisolone, 428, 445,454 Prednisone, 428, 453, 508 Preeclampsia, 232-234 Pregnancy diagnosis, 1025-1026 hypocalcemia, 603-604 intrauterine fetal surgery, 1027 personnel health risks, 1055 rodent anesthesia, 960-961 toxemia, 232-234, 506, 602-603 Pregnant mare serum, 133

INDEX Prehensile tail, 678-679 Pressure sore, 433-434 Preventive medicine. See also Surveillance infectious diseases cat, 475-476 rabbit, 365-387 rodent, 365-387 primate management, 724-727 ruminant screening, 523 Prilocaine, 972 Primaquine, 761 Primates analgesia, 991,992, 997 anesthesia, 990-996 behavior, 1248-1252 blood sample collection, 727-728, 1007, 1012 bone marrow biopsy, 729 cannulation technique, 1014 cercopithecine herpesvirus vector, 1068-1069 characteristics, 677 clinical techniques, 727-730 dental procedures, 730 diseases acute gastric dilatation, 776-777 bacterial, 730-744 dentition, 777 enteric, 730-734 hyperthermia/hypothermia, 776 hypoglycemia, 776 metabolic, 776 mycotic, 744-746 nematodiasis, 765-766 nutritional, 757,773-775 parasitic, 757, 758-772 protozoal, 761-762 traumatic, 775-776 tuberculosis and mycobacterial, 738-742 viral, 746-757 drug dosages, 726 environmental enrichment, 717, 1248-1252 euthanasia, 997 feeding, 1250 geographic distribution, 677-678 handling and restraint, 718-720 hepatitis vector, 1071, 1072 housing, 715-717, 721-723, 918-919, 940, 1249-1250 husbandry, 1249-1250 identification, 1007 intraoperative monitoring and support, 994-995 intraosseous infusion, 729-730 lymph node biopsy, 729 manipulanda, 1250 monkeypox vector, 1060 nasogastric intubation, 730 nutrition and diet, 725, 727 Parkinson disease model, 1187 personnel health risks, 720-723 personnel interaction, 1252 physical examination, 725 postoperative recovery, 996-997 preanesthesia, 990 preventive medicine, 724-727 psychological welfare, 24 radiology technique, 729

1309 records and documentation, 722-723 shigellosis vector, 1087 simian immunodeficiency virus vector, 1072 skin sampling and biopsy, 728-729 social behavior, 1250-1251 space requirements, 716 taxonomy and nomenclature, 677-680 terminology, 678-679 toys, 1250 ultrasound technique, 729 vaccination, 724-725 Yaba virus vector, 1061 Prion disease, 170 Probenecid, 854 Procaine penicillin. See also Penicillins erysipelas, 632 foot rot, 550 otitis media, 340 pasteurellosis, 340 pseudomoniasis, 744 tetanus, 544 Progressive degenerative myeloencephalopathy, 599 Progressive retinal atrophy, 461 Prolapse cloacal, 854 pelvic, 698 primate, 730 rectal, 610, 730 reptile, 854 ruminant, 609-610 squirrel monkey model, 698 uterine, 609-610 vaginal, 609- 610 Proliferative bowel disease, 494, 496 Proliferative enteropathy rabbit, 344 swine, 494, 644, 645-646 Proliferative stomatitis, 577 Promazine, 960 Pronotagus sp., 330 Pronuclear microinjection, 1130-1131, 1190-1191 Propiolactone, 906 Propofol dog, 974, 975-976 mouse, 962 primate, 991,992, 993 rabbit, 968, 969, 970 rat, 963 reptile, 845 rodent, 958 swine, 981,982 Propylene glycol, 603 Prosimian primate, 678, 679-680 Prostaglandins, 603, 808 Prostate, 450 - 451 Prosthenorchis sp. Prosthenorchis elegans, 762, 764 Prosthenorchis spirula, 762 Protein energy malnutrition, 602-603 Proteus infections, 95, 558 Proteus sp., 320 navel ill, 558 Proteus mirabilis, 95, 555 Protospirura muris, 272

1310 Protostrongylus rufescens, 594 Protozoal disease amphibian, 819-820 dog, 416-417 ferret, 502-503 gerbil, 275 guinea pig, 223-225 hamster, 185 mouse, 98-100 multimammate rat model, 282 primate, 758-760 rat, 147-149 ruminant, 584-592 woodchuck, 323 Pseudocapillaroides xenopi, 820-821 Pseudocowpox, 577 Pseudomonas sp. amphibian, 816 degu, 285 dog, 407 fish, 878 goat, 555 nitrification cycle, 869 Pseudomonas aeruginosa animals, effect on, 1162-1163 biofilms, 370 chinchilla, 287-288 ferret, 496-497 goat, 555 guinea pig, 219 mouse, 86-87, 95 rat, 138-139 Pseudomoniasis amphibian, 816 chinchilla, 287-288 guinea pig, 219 mouse, 86-87 primate, 743-744 rat, 138-139 Pseudopregnancy dog, 405 ferret, 485 hamster, 177 rabbit, 337-338 Pseudoprehensile tail, 679 Pseudorabies, 578-579, 634-635 Pseudotuberculosis, 135, 216-217 Psittacosis, 1076-1077 Psorergates sp. Psorergates ovis, 596 Psorergates simplex, 102, 103, 105, 281 Psorergatic mange, 283, 596 Psoroptes sp. Psoroptes bovis, 596 Psoroptes cuniculi, 349, 596 Psoroptes ovis, 596 Psoroptic mange, 349 Psychodermatosis, 434-435 Pterygodermatites sp. Pterygodermatites alphi, 766 Pterygodermatites nycticebi, 766 Public Health Service, 25-26, 30 Public opinion, biohazard, 1053 Pulex irritans, 427, 1097

INDEX

Pulmonary adenomatosis, 577-578 Pulmonary histiocytosis, 108 Pulpectomy/pulpotomy, 1016 Pulpy kidney disease, 543-544 Pyelitis, ascending, 110 Pyelonephritis, 547-548 Pyrantel pamoate. See also Heartgard Plus ascariasis, 418, 478, 643,766 enterobiasis, 765 hookworms, 420 strongyloidiasis, 765 Pyrethrins, 151,428, 504 Pyrethroids, 428, 597 Pyridoxine deficiency, 231,235 Pyrimethamine, 762, 1093 Pyruvate kinase deficiency, 461 Python regius, 842

Q Q fever, 564, 1076 Quality control cat disease control, 462-463,466 genetic monitoring, 1117-1126 rabbit disease control, 365-387 rodent disease control, 365-387 woodchuck colony health, 319-320 Quarantine amphibian, 800 facility design, 917 fish, 873, 875, 895 foot-and-mouth disease, 575 postshipment, 368 primate, 724 reptile, 836 time requirement, 30 Query fever, 564, 1076 Quinacrine, 416, 417, 1092 Quinaldine, 897 Quinine hydrochloride, 879 Quinine sulfate, 820 Quinolones guinea pig toxicity, 240 kennel cough, 406 Lyme disease, 412 mycoplasmal pneumonia, 563 R

Rabbit analgesia, 969-970, 972-973 anesthesia, 967-970, 971-972 behavior, 336-337, 1245 bile sampling, 1018 blood sample collection, 1007, 1011 breeds, 330 cannulation technique, 1014 cardiovascular system, 332-333 clinical chemistry data, 334-335 dentition, 331 digestive system, 331-332 diseases bacterial, 213, 339-344 congenital, 353-355

INDEX fungal, 351-352 neoplasms, 355-357 protozoal, 346-349 traumatic, 352-353 viral, 344-346 endotracheal intubation, 1021-1022 environmental enrichment, 1245 fecal circadian rhythm, 332 hematology data, 334 housing, 338, 938, 1245-1246 husbandry, 338, 341-342 identification, 1007 intraoperative monitoring and support, 971 metabolism, 333-334 microbiological quality control, 365-387 normative data, 335 nutrition and diet, 335-336 physiology, 331-334 population control, 329 preanesthesia, 967 reproduction, 337-338 research uses, 330 respiratory system, 332 taxonomy, 330 urogenital system, 333 Rabies dog, 415-416 ferret, 500-501 ground squirrel, 253 personnel health risk, 1055 primate, 756-757 ruminant, 579-580 wood rat, 263 zoonotic, 1069-1070 Raccoon, 225 Radifordia sp. Radifordia affinis, 102, 105, 151 Radifordia ensifera, 151 Radioactive agents and equipment, 31 Radiology pregnancy diagnosis, 1026 radioisotope containment, 918 reptile, 847-848 technique, 729, 1034 Radioresistance, 170 Radiotelemetry, 1034 Raillietina sp, 272 Rana sp. clinical chemistry data, 806 hematology data, 805 Rana catesbeiana, 805, 1188 Rana pipiens, 805, 817-818, 1188 Rana sylvatica, 1188 Rana temporaria, 1188 taxonomy, 795 Random bred stock, 37 Ranitidine, 442 Rat, 1243. See also Cotton rat; Degu; Kangaroo rat; Rice rat; White-tailed rat; Wood rat acquisition and sources, 122 aggression, 1243 aging, 157-158 anatomy, 123-126 anesthesia and analgesia, 963-964

1311 artificial insemination, 133 behavior, 130, 133-134 blood sample collection, 1007, 1010 caging systems, 939-943 cannulation technique, 1014 cardiovascular system, 126 central nervous system, 126 clinical chemistry data, 128 cryopreservation, 133 development, 132 digestive system, 124 diseases age-related, 157-158 bacterial, mycoplasmal, and rickettsial, 134 - 143 environmental, 153 fungal, 151-152 genetic and congenital, 156 metabolic and nutritional, 152-153 neoplasms, 154-156 parasitic, 147-151,349-351 traumatic and iatrogenic, 153-154 viral, 143-147 drug dosages, 963 embryo transfer, 133 estrous cycle, 127-129, 133 genitourinary system, 125-126 hearing frequency range, 123, 153 hematology data, 127-129 history, 113-114 hormone normative ranges, 129 housing, 122-123, 134, 153, 939-943, 1243-1244 husbandry, 122-123, 131-132, 153 identification, 1007 leukocyte parameters, 130 morphophysiology, 123-126 multimammate, 281-284 normative data, 127 nutrition and diet, 126-127, 131, 153, 154, 176 parturition, 132 plague vector, 1082 pregnancy, 131-132 rat bite fever vector, 1078 reproduction, 127-133 respiratory system, 124-125 rickettsial pox vector, 1075 sensory organs, 123 sexing, 132 skeleton, 123-124 strains ACI, 35 BB/Wor, 122 BDIX, 157 BN-Bi, 156 BN (Brown Norway), 122, 133, 152, 156, 158 Brattleboro, 122 BUF (Buffalo), 122 CD, 133 CDF(F-344)CdBR, 130 COP (Copenhagen), 122 Crl:CD(SD)BR, 130 Crl:(WI)BR, 130 Dahl, 152, 1144 F-344 (Fischer 344), 122, 123, 133, 154, 155, 157 F-344/NHsd, 130

1312

Rat, 1243. See also Cotton rat; Degu; Kangaroo rat; Rice rat; White-tailed rat; Wood rat, strains (cont.) fawn-hooded, 152 Gunn, 122, 156 hematologic data, 130 Hsd:SD, 130 Hsd:WI, 130 leukocyte parameters, 130 LEW (Lewis), 122, 133, 143-144, 152 LOU/C, 122 Norway, 123 nude, 122 obese SHR, 122 SD, 133 SHR, 122, 152, 1144 Sprague-Dawley, 123, 133, 154, 155, 156, 157 SS/Jr, 1144 table, 122 Tac:N(SD)fBR, 130 WF (Wistar-Furth), 122 Wistar, 123, 151,155 Wistar Kyoto, 1144 Wistar:Han, 133 Wistar:WU, 133 Zucker, 122, 152 taxonomy, 113 typhus vector, 1075 urinalysis data, 127 weaning, 132-133 Rat antibody production (RAP) test, 374-375 Rat bite fever, 91, 1078-1079 Rat parvovirus, 146, 1161 Rattus sp. Rattus norvegicus, 113 Rattus rattus, 113 Recombinant congenic strain, 39 Recombinant DNA research, 31, 1053-1054, 1129-1140 Recombinant inbred strain, 37, 39 Record keeping primate, 724 standard operating procedures, 947 transgenic/knockout mouse, 1138 USDA standards, 23-24 Rectal prolapse, 610, 730 Red leg, 814-815 Red-tailed jird, 275 Red water, 544-545 Regulations. See Laws, regulations, and guidelines Regurgitation prevention, 986 Relative humidity, 1151 Reovirus bluetongue, 565-566 canine, 405-406 feline upper respiratory tract infection, 476 mouse, 72-74 type 1, 72-73, 369 type 2, 72-73, 369 type 3, 72-73, 147, 369 Reproductive and respiratory syndrome, 660-662 Reptile acquisition and sources, 829 anatomy and physiology, 837-841 behavior, 844 blood collection, 846

INDEX clinical chemistry data, 842 diseases bacterial and mycoplasmal, 848-850 burn injury, 856-857 fungal, 853 metabolic and nutritional, 853-855 neoplasms, 857 parasitic, 851-853 traumatic, 855-856 viral, 850 experimental techniques, 846-848 handling and restraint, 834-835, 845 hematology data, 841,842 housing, 829-834 husbandry, 829-837 lighting, 832-833 nutrition and diet, 841,843, 848 physical examination, 845-846 reproduction, 843-844, 854 research uses, 828-829 taxonomy, 827-828 Research. See Animal experimentation Research cycle, 1229-1230 Respiratory tract diseases. See also Influenza; Lung diseases; Pneumonia adenovirus, 222 alveolar histiocytosis, 158 anaphylaxis, 610 aspergillosis, 352 bordetellosis, 212-213 cotton rat model, 270 dog, 405-406 eosinophilic granulomatous pulmonary inflammation, 156 ferret, 494-496, 499-500 guinea pig, 215-216, 222 hamster model, 169, 172 kennel cough, 405-406 lymphohistiocytosis, 147 mouse, 108-109 prairie dog, 257 primate, 734-738 rabbit, 339-340, 352 rat, 140-146, 151-152, 156, 158 respiratory distress syndrome, 331 respiratory syncytial virus, 572 ruminant, 551-552 thromboembolic meningoencephalitis, 551-552 tuberculosis, 494-496 upper respiratory infection, 476-477 woodchuck, 319 zoonotic, 1088 Restraint chemical, 719-720 physical, 718-719 pole-and-collar, 719 primate, 718-720 protective equipment, 718-719 Reticulitis, 602 Reticulum cell sarcoma, 111, 187 Retinal degeneration, 110, 153, 156, 461 Retroperitoneal fibromatosis, 754, 756 Retrovirus cat, 477 primate, 753-754, 756 rat, 147

INDEX ruminant, 569 transmission, transplantation, 1110-1111 zoonotic, 1072-1073 Reverse osmosis, 369 Revivon, 981 Reye's-like syndrome, 106 Rhabdias sp., 820-821,852 Rhabdomyomatosis, 239 Rhabdomyosarcoma, 356, 357 Rheobatrachus sp., 808 Rhesus monkey behavior, 701 bite and scratch safety procedures, 721 clinical chemistry data, 703 diseases mycotic, 744-746 nutritional, 773-774 respiratory and nervous system, 734-738 tuberculosis and mycobacterial, 738-742 viral, 746-757 hematology data, 702 housing, 700-701 husbandry, 700-701 natural history, 699 nutrition and diet, 701 reproduction, 699 research uses, 701 serum electrolyte values, 704 taxonomy, 699, 700 Rhinitis, 635-637, 641 Rhinotracheitis, 476-477, 501-502, 570-571 Rhipicephalus sanguineus, 410, 426, 1096, 1098 Ribeiroia sp., 821, 1188 Riboflavin deficiency, 231,235 Rice rat chromosome polymorphism, 268 description, distribution, habitat, 268 diseases parasitic, 268 viral, 268 husbandry, 268 research uses, 268 taxonomy, 268 Richardson's ground squirrel, 250-254 Rickets, 605,773. See also Vitamin D deficiency Rickettsia sp. Rickettsia akari, 277, 1075-1076, 1096 Rickettsia mooseri, 1096 Rickettsia rickettsii, 1096 Rickettsia tsutsumagushi, 271 Rickettsia typhi, 261, 1074-1075 Rickettsial infection guinea pig, 220 mouse, 95-96 pox, 1075-1076 rat, 143 ruminant, 563-564 swine, 654 wood rat, 261 zoonotic, 1074-1076 Rifampin, 741,899, 1088 Rifle, 719 Rift Valley Fever virus, 276 Ringer's solution, 777

1313 Ringtail cotton rat, 272 mouse, 108 rat, 153 white-footed mouse, 267 white-tailed rat, 274 woodchuck, 325-326 Ringworm. See Dermatomycosis Rock hare, 330 Rocky Mountain spotted fever, 324 Rodents analgesia, 961-962 anesthesia, 957-961 behavior, 1241-1245 blood collection, 1008 breeding, commercial and academic, 15 dentition, 248-249 endotracheal intubation, 1021 euthanasia, 966 gnotobiology, 15 intraoperative monitoring and support, 960 leptospirosis vector, 1083 microbiological quality control, 365-387 preanesthesia, 956-957 sources, commercial, 250 swine dysentery vector, 645 taxonomy, 249-250 urine sample collection, 1020 Rolling disease, 83 Romerolagus diazzi, 330 Root canal, 1016-1017 Rotavirus cattle, 582-583 ferret, 501 goat, 582 mouse, 73-74 porcine, 650-651 rabbit, 345 sheep, 582 swine, 644 waterborne human, 369 Rotavirus-like agent infection, 147 Rotenone, 349, 504 Roundworm. See Ascariasis Rubeola, 1073 Rumen disorder, 600-602 Rumen fluke, 596 Rumenitis, 601-602 Ruminant, orf virus vector, 1061-1062. See also Cattle; Goat; Sheep Ruptured follicle, 849 S Saffan, 959 Saguinus sp. Saguinus nigricollis, 681 Saguinus oedipus, 681,734 taxonomy, 682 Saimiri sp., 680, 692 Saimiri bolivensis, 692, 693 Saimiri bolivensis bolivensis, 693, 695 Saimiri oerstedii, 693 Saimiri sciureus, 693 Saimiri ustus, 693

1314 Salamander anatomy and physiology, 801-803 behavior, 806-807 diseases bacterial, 814- 817 fungal, 822 metabolic and nutritional, 822-823 parasitic, 819-822 viral and chlamydial, 817-819 handling and restraint, 799 housing, 796-797 husbandry, 796-801 larvae, 804, 806 metamorphosis, 808 nutrition and diet, 803-804, 806 reproduction, 807-808 taxonomy, 794 Salmon, cannulation technique, 1014 Salmonella sp., 496 antigen assay, 375 hemorrhagic enteritis, 643-645 mastitis, 555-556 Salmonella anatum, 560, 733 Salmonella arizona, 288 Salmonella choleraesuis amphibian, 817 cat, 496 primate, 733 swine, 630-631,644, 649 zoonotic, 1086-1087 Salmonella derby, 733 Salmonella dublin, 560 Salmonella enteritidis, 90-91, 138, 288, 496, 560 Salmonella hadar, 496 Salmonella kentucky, 496 Salmonella montevideo, 560 Salmonella newport, 496, 560 Salmonella oranienburg, 733 Salmonella stanley, 733 Salmonella typhimurium ferret, 496 hamster, 216 mouse, 90-91 primate, 733 ruminant, 560-561 swine, 630, 644, 645, 649 Salmonella typhisuis, 630 taxonomy, 560 Salmonellosis amphibian, 817 chinchilla, 288 ferret, 496 guinea pig, 216 hamster, 182-183 mouse, 90-91 primate, 731,733 rat, 138 reptile, 836, 849 ruminant, 560-561 swine, 629-631,644 zoonotic, 1086-1087 Salt bath fluke infestation, 902

INDEX fungal infection, 822 protozoal infection, 820 trematodiasis, 903 trichodinosis, 881 velvet disease, 879 water mold, 880 white spot (ich), 880 Salt poisoning, 662-663 Sampling, 383-384, 386 San Miguel sea lion virus, 899 Sanitation, 721-723, 1053. See also Disinfection Saprolegnia sp., 879-880 Saprolegnia ferax, 1188 Saprolegniosis, 822 Sarcocystis sp., 323 Sarcocystis campestris, 253 Sarcocystis capricanus, 589 Sarcocystis cruzi, 589 Sarcocystis hirsuta, 589 Sarcocystis hominis, 589 Sarcocystis ovicanus, 589 Sarcocystis sigmoidontis, 271 Sarcocystis tenella, 589 Sarcocystosis, 289, 589-590 Sarcoma guinea pig, 237 hemangiosarcoma, 445-446 histiocytic, 156 neurofibrosarcoma, 356 osteosarcoma, 356, 357 reticulum cell, 111, 187 rhabdomyosarcoma, 356, 357 Sarcoptes sp. Sarcoptes rupicaprae, 596 Sarcoptes scabiei dog, 425 ferret, 503 guinea pig, 225, 226 hosts, 1096 photograph, 764 primate, 762, 764 rabbit, 350 ruminant, 596 swine, 656-657 Sarcoptic mange, 350, 425-426, 596, 656-657, 762 Satellite phenomenon, 637 Savaging, 623 Scales, 158 Schaedler flora, 46-47 Schistosoma sp. Schistosoma haematobium, 276, 772 Schistosoma japonicum, 276 Schistosoma mansoni, 282, 772 Schistosoma matheei, 772 Schistosomiasis, 276, 772 Schistosomus reflexus, 291 Scientists' Center for Animal Welfare (SCAW), 10 Sciurognath rodent, 249-250 Sciuromorph rodent, 249 Sciurus carolinensis, 256 Scorbutus, 230-232 Scrapie, 170, 580-581 Screwworm, 281,597

1315

INDEX Scrub typhus, 271 Scurvy, 230-232, 773. See also Vitamin C deficiency Sea squirt, 234-235 Sebum, 158 Segregating inbred strain, 37 Seizures. See also Epilepsy dog, 441-442 gerbil, 278 grasshopper mouse, 265 hepatic encephalopathy, 441-442 rat, 156 Selenium, 607 Selenium deficiency, 235, 324, 607 Selenium excess, 607 Self-mutilation, 355, 775 Semen collection, 535, 1025 Sendai virus animals, effect on, 1160 hamster, 184-185 mouse, 69-71, 87 rat, 143-144 Sensitivity, test, 385-386 Sentinel animal, 383,873 Sepsis, 437-439 Septicemia amphibian, 815 fish, 878 mouse, 86-87 ruminant, 553 swine, 630 Serology, 375-377 Serpulina hyodysenteriae, 644 Serratia marscesens, 555 Sevoflurane dog, 974, 977 primate, 994 rodent, 959 swine, 983 Shedding, 855 Sheep acquisition and sources, 522-524 analgesia, 987, 989-990 anesthesia, 986-989 artificial insemination, 534 behavior, 535-536 cannulation technique, 1014 castration, 535 clinical chemistry data, 528 databases, 1200 development, 532-533 digestive system, 525 diseases bacterial, 537-562 chlamydial, 583-584 genetic, 598-599 iatrogenic, 610-611 management-related, 608-610 metabolic, 599-605 mycoplasmal, 562-563 neoplasms, 611 nutritional, 605-608 protozoal, 584-592 rickettsial, 563-564

traumatic, 610 viral, 565-583 embryo transfer, 534 estrous cycle, 534 health screening, 523 hematology data, 525-526, 527 histocompatibility complex, 525 husbandry, 524, 531 lipogenesis, 525 normative data, 526 nutrition and diet, 526-529 physiology, 525-526 postoperative recovery, 988 preanesthesia, 986 regurgitation prevention, 986 reproduction, 529-531 research uses, 521 semen collection, 535 taxonomy, 520 Sheep ked, 598 Sheepbase, 1200 Shell fracture, 856 Shigella sp., 1087 Shigellosis, 730-732, 1087-1088 Shipping containers, 368 Shipping fever, 559-560 Shope papilloma, 331 Shope virus, 344, 345 Sialodacryoadenitis, 144-146 Sialodacryoadenitis virus, 144, 369, 1161 Sigmodon sp. Sigmodon fulviventer, 270 Sigmodon hispidus, 147, 270 Silage disease, 553-554 Silvadene, 856 Simian AIDS, 754, 756 Simian foamy virus, 1073 Simian immunodeficiency virus, 756, 1072-1073 Simian primate, 678 Sin nombre virus, 262 Skin diseases. See also Dermatomycosis; Ringtail actinobacillosis, 537 alopecia, 107 bacterial folliculitis, 320 burn injury, 856-857 cattle, 537 chronic ulcerative dermatitis, 107 dermatomycosis (ringworm), 98 dog, 424-435 dysecdysis, 855 ectromelia virus, 55-59 ferret, 505, 510-512 furunculosis, 94 gangrenous dermatitis, 252 gerbil, 277 ground squirrel, 252 hair shedding, 110 hyperkeratosis, 92-93 hyperkeratotic dermatitis, 92-93 lesions, 252 mousepox, 55-59 neoplasms, 237, 357, 510-512 papillomavirus, 345

1316 Skin diseases. See also Dermatomycosis; Ringtail (cont.) parasitic, 324 pododermatitis, 219-220 rabbit, 345, 349-351,357 rat, 153-154, 158 ruminant, 548-549 sebum accumulation, 158 sheep, 537 suppurative dermatitis, 93 trauma, mouse, 108 ulcerative dermatitis, 93 woodchuck, 320, 324 Skin, sampling and biopsy, 728-729 Skink, 849 Slider, 829, 843 Small intestinal worm, 593 Smallpox, 1060 Smoke inhalation, 169, 172 Snail, 424, 595, 874, 1013 Snake anatomy and physiology, 837-841 behavior, 844 blood collection, 846 clinical chemistry data, 842 diseases dysecdysis, 855 dystocia, 854 neoplasms, 857 paramyxoviral, 851 parasitic, 851-852 viral, 850 handling and restraint, 834, 845 hematology data, 841,842 housing, 829-834 nutrition and diet, 841,843 sexing, 843-844 taxonomy, 827, 828 Snake hookworm, 852 Snapping turtle, 828 Society for the Prevention of Cruelty to Animals (SPCA), 3 Sodium excess, 662-663 Sodium hydroxide, 581 Sodium hypochlorite, 598 Sodium ion toxicosis, 662-663 Sodium levothyroxine, 430 Sodium molybdenate, 606 Sodium thiosulfate, 606 Soft tissue mineralization. See Mineralization, soft tissue Solenoptes capillatus, 596 Somatomedins, 207 Sore hock, 353 Sore mouth, 573-574 Special Emphasis Research Career Awards, 1234 Specific pathogen-free (SPF), 366 Specificity, test, 385-386 Spectinomycin, 551,559, 674 Sperm granuloma, 110 Spermophilus sp. Spermophilus armatus, 250-254 Spermophilus beecheyi, 250-254 Spermophilus elegans, 253 Spermophilus franklinii, 253 Spermophilus fulvus, 253

INDEX

Spermophilus lateralis, 250-254 Spermophilus richardsonii, 250-254 Spermophilus tridecemlineatus, 250-254 Sphingomyelin lipidosis, 461 Spider lamb syndrome, 599 Spinal anesthesia, 971 Spinal catheter, 1031 Spinal edema, 353 Spinal laminectomy, 1030 Spindle cell, 187 Spindly leg, 823 Spirillosis, 1079-1080 Spirillum minus, 1078-1079 Spirometra sp., 423 Spironucleosis, 98-99 Spironucleus muris, 98-99, 147, 148 Splay leg, 354-355 Splendore-Hoeppli phenomenon, 513 Splenic disease, 189 Splenomegaly, 512 Spleorodens clethrionomys, 186 Spondylosis, 193 Spongiform encephalopathy, 461,580 Spontaneous neoplasia, 195 Sprague-Dawley rat, 123 Spraguea sp., 903 Squamous cell carcinoma, 197, 356, 357 Squirrel. See Ground squirrel Squirrel monkey availability, 692 behavior, 695 birth weight, 696 body weight, 698 clinical chemistry data, 698 diseases enteric, 730-734 mycotic, 744-746 nutritional, 773-774 respiratory and nervous system, 734-738 tuberculosis and mycobacterial, 738-742 viral, 746-757 hematology data, 697 housing, 696-697 husbandry, 696-697 metabolic rate, 686-687 natural history, 693-695 nutrition and diet, 697 reproduction, 692, 695-696 research uses, 692, 697-698 taxonomy, 693 vaccination, 725 St. Louis equine encephalitis virus, 265 St. Thomas Hospital'rabbit strain, 331 Stanozolol, 726 Staphylococcosis caprine staphylococcal dermatitis, 542 guinea pig, 219-220 mouse, 93-94 primate, 742-743 reptile, 850 Staphylococcus sp., 234, 290, 321,497 dog, 407 fish, 898

INDEX navel ill, 558 skin infection, 433

Staphylococcus aureus cattle, 555 gerbil, 277 goat, 542 guinea pig, 219-220 mouse, 93 rat, 153-154 sheep, 555 squirrel, 252 woodchuck, 320

Staphylococcus epidermidis, 93, 555 Staphylococcus hyicus, 654-655 Staphylococcus intermedius, 542 Staphylococcus xylosus, 277 Stem-cell factor, xenotransplantation, 628 Stereotactic surgery, 961, 1030 Stereotypy defined, 1240-1241 dog, 1247 farm animals, 1252 mouse, 1241-1242 Sterile pyogranuloma complex, 454 Sterne vaccine, 539 Sticker tumor, 446-447 Stiff lamb disease, 607 Stomatitis, 577, 581-582 Strawberry foot rot, 548-549 Streptobacillary fever, 1079-1080 Streptobacillosis, 91-92, 139, 220 Streptobacillus moniliformis, 91-92, 139, 220, 1078-1079 Streptococcosis dog, 406-407 guinea pig, 213-215, 215-216 mouse, 94 primate, 734-736 rat, 134-135 reptile, 850 swine, 633-634 zoonotic, 1080-1081 Streptococcus sp., 320, 321,497 fish, 898 lactic acidosis, 601 Streptococcus agalactiae, 555 Streptococcus dysgalactiae, 555 Streptococcus equi subsp, zooepidemicus, 213-215 Streptococcus equisimilis, 94 Streptococcus iniae, 878, 1080-1081 Streptococcus pneumoniae, 134-135, 182, 215-216, 237, 734-736 Streptococcus pyogenes, 220 Streptococcus suis, 633 Streptococcus uberis, 555 Streptococcus zooepidemicus, 406-407, 496-497, 555 Streptomycin, 636, 1079, 1083 Streptothricosis, 548-549 Streptozotocin, 173 Stress animals, effect on, 1163-1165 cat, 1247 disease resistance, 476 disease susceptibility, 1165 dog, 1247

1317 isolation stress phenomenon, 1164 porcine stress syndrome, 662 shipping, 1164 Strongyloides sp., 272, 323 egg, 767, 768 Strongyloides cebus, 765, 767-768 Strongyloidesfulleborni, 765, 1094 Strongyloides papillosus, 549, 593 Strongyloides ransomi, 644, 653-654 Strongyloides stercoralis, 275,420, 1094 Strongyloides sterocoralis, 765 Strongyloidiasis, 420, 593, 653-654 Struck, 542-543 Styphylodora sp., 852 Subcapsular spindle cell hyperplasia, 110 Subluxation, 353 Succinylcholine, 662, 982 Sucralfate, 413 Sucrose, 776 Sufentanil, 981,982 Suipoxvirus sp., 655-656 Sulfa drugs, 406, 537, 631. See also Trimethoprim-sulfa Sulfadiazine, 440, 762 Sulfadimethoxine, 417, 502 Sulfamerazine, 83, 347 Sulfamethoxine, 758 Sulfaquinoxaline, 347 Sulfathalidine, 496 Sulfonamides. See also Trimethoprim-sulfa atrophic rhinitis, 636 chlamydiosis, 221 coccidiosis, 224, 587, 652 Pasteurella pneurnotropica, 88 pasteurellosis, 559 toxoplasmosis, 1093 Sulfonomethazine, 550 Sulfur, 608 Sumatra short-haired rabbit, 330 Suppurative dermatitis, 93 Surgical facility, 914 Surveillance. See also Preventive medicine aquatic systems, 893 environmental, 931 genetic, 1117-1126 implementation, 384-385 infectious agent selection, 383 primate preventive medicine, 725 results interpretation, 385-387 sampling, 383-384, 386 sentinel animal, 383, 873 test schedule, 384-385 Susliks, 250-254 Sutures, 433 Swayback, 605-606 Swimmer's itch, 276 Swimming pool granuloma, 1078 Swine acquisition and sources, 615-616 analgesia, 981,985 anatomy and physiology, 618-620 anesthesia, 980-984 balantidiasis vector, 1090 behavior, 623, 665, 1252-1253

INDEX

1318 Swine (cont.) blood collection, 617-621 breeds, 616 cannulation technique, 1014 clinical chemistry data, 624-625 databases, 1200 diseases bacterial, 180, 629-641,644, 646-648, 654-659 circulatory, 654 gastrointestinal, 643-654 iatrogenic, 664 metabolic and nutritional, 662-664 mycoplasmal, 639-641 neoplasms, 664 parasitic, 642-643,652-654 polysystemic, 629-635 proliferative enteropathy, 494 reproductive, 657-662 respiratory, 634-635 skin, 654 viral, 634-635, 641-644, 650-651,655-656, 659-662 estrous cycle, 621 euthanasia, 986 hematology data, 624 hepatitis vector, 1072 housing, 616, 622 husbandry, 616-617 immunology, 623-625, 627 intraoperative monitoring and support, 983-984 nutrition and diet, 620-621,626, 663 postoperative recovery, 984-985 preanesthesia, 979-980 pseudorabies vector, 579 reproduction, 621-623 research uses, 617-618 taxonomy, 615 xenotransplantation, 627- 629 Swine flu, 641 Swordtail, 886 Sylvilagus bachmani, 344 Sylvulagus sp., 330 Sympathectomy, 1029-1030 Syndactyly, 599 Syphacia sp., 270 Syphacia muris (rat pinworm), 101,149, 186 Syphacia obvelata (mouse pinworm), 100-102, 149, 186, 277 Syphacia sigmoidontis, 272 Syphilis, 343 T Taconic Farms, Inc., 1199 Taenia sp., 257 Taenia crassiceps, 276, 319, 324 Taenia hydatigena, 595 Taenia multiceps, 595 Taenia mustelae, 257, 324 Taenia pisiformis, 423 Taenia polyacantha, 276 Taenia taeniaformis, 100, 151 Taeniidae, 772 Tail docking, 535,665

injury, reptile, 857 prehensile, 678-679 pseudoprehensile, 679 tail-cuff blood pressure, 1027 Tamarin. See also Callitrichid primate behavior, 684 clinical chemistry data, 686 diseases enteric, 730-734 measles, 755 mycotic, 744-746 nutritional, 773-774 parasitic, 764 viral, 746-757 distinguishing features, 680-681 hematology data, 685 housing, 684-685 husbandry, 684 natural history, 681-683 normative data, 685 nutrition and diet, 685 reproduction, 683-684 research uses, 685-686 taxonomy, 678, 681,682 Tanapox, 750 Tapeworm. See Cestodiasis Tarsier, 677, 678 Telazol. See Tiletamine Temperature-related disorders, 108 Teratology FETAX system, 795 hamster model, 173 Xenopus sp., 795 Teratoma, 237-238, 356, 357 Ternidens deminutus, 1094 Terrapene carolina, 842, 849 Testicular atrophy, 110 Testicular teratoma, 356 Testosterone, 1147 Testudi sp. Testudo hermanni, 842 Testudo radiata, 842 Tetanus, 543-544, 742 Tether and vest restraint, 719 Tetracyclines. See also Chlortetracycline; Oxytetracycline actinobacillosis, 537 amoebiasis, 758, 820 atrophic rhinitis, 636 balantidiasis, 651,759, 1090 bighead, 545 bordetellosis, 735 conjunctivitis, 563 ehrlichiosis, 410 heel wart, 551 helicobacteriosis, 140, 1085 kennel cough, 406 klebsiellosis, 735 lactic acidosis, 601 Lyme disease, 412 mycoplasmal pneumonia, 640 mycoplasmosis, 82 pleuropneumonia, 637 polyarthritis, 631

1319

INDEX primate dose, 726 proliferative enteritis, 181 rat bite fever, 1079 red leg, 815 Tyzzer's disease, 342 yersiniosis, 562 Thecoma, 187 Theiler's murine encephalomyelitis virus (TMEV), 369 Thermal burn, 440 Thermoneutral zone, 1147-1148 Thiabendazole, 100 dermatomycosis, 229, 598 nematodiasis, 820 Oesophagostomum sp., 765 strongyloidiasis, 653,765 Trypanoxyuris sp., 765 Thiacetarsamide, 423,441,506 Thiamin deficiency, 107, 231,235, 607-608 Thiamine hydrochloride, 608 Thiamylal, 968, 981,987, 988 Thiopental sodium dog, 974 dog, 976 mouse, 962 primate, 726, 991,993 rabbit, 968 rat, 963 rodent, 958 ruminant, 987, 988-989 swine, 981,982 Thiosemicarbazide, 1188 Thirteen-lined ground squirrel, 250-254 Thomomys sp., 257 Thomsen's disease, 522 Thorny-headed worm, 764 Thread-necked worm, 593 Threadworm, 644, 653-654 Thromboembolic meningoencephalitis, 551-552 Thyanosoma actinoides, 594 Thymic lymphoma, 111 Thymic virus, 60-61 Thymoma, 195, 611 Thyroid carcinoma, 448-449 Thyroid cysts, 110 Thyroid disease, 599 Thyroid function tests, 430 Thyroid-stimulating hormone (TSH), 430 Thyroidectomy, 1029 Thyroiditis, 282, 284, 429 Thyrotropic hormone deficiencies, 110 Thyroxine, 430 Tiamulin, 645, 646 Tick Ackertia marmotae vector, 323 anaplasmosis vector, 585 babesiosis vector, 426, 585-586 Borrelia burgdorferi vector, 324 cotton rat infestation, 272 ehrlichiosis vector, 410, 411,426 ferret infestation, 505 foothill abortion vector, 561 guinea pig, 228 hemobartonellosis vector, 426

hepatozoonosis vector, 426 hosts, 1096 Lyme disease vector, 262, 324, 412, 426, 554, 1098 Powassan virus vector, 323, 324 removal, 427 reptile infestation, 853 Rocky Mountain spotted fever vector, 324, 426, 1098 ruminant infestation, 597 sheep ked, 598 tularemia vector, 561 typhus vector, 1098 woodchuck infestation, 324 zoonotic, 1098 Tick-bite paralysis, 427 Tiger salamander, 794 Tiletamine dog, 974 hamster, 964 primate, 719-720, 726, 991,992, 993 rodent, 958 swine, 981 Tilmicosin atrophic rhinitis, 636 pasteurellosis, 340, 559 proliferative enteropathy, 646 Tilorone, 906 Toad, 795, 807-808 Toll gene, 1202 Tolnaftate, 229 Toolan's H- 1 rat virus, 146 Tooth extraction, 1016 Torticollis, 339 Torulopsis pintolopesii, 230 Toxascaris leonina, 417, 478 Toxemia of pregnancy ferret, 506 guinea pig, 232-234 ruminant, 602-603 Toxic Substances Control Act (TSCA, 1976), 27, 31 Toxins fish, 869-870 frog, 800, 801 venom, 836-837 Toxocara sp. Toxocara canis, 417-418, 1094 Toxocara cati, 478, 1094 Toxocara leonina, 1094 Toxoplasma gondii cat, 479 chinchilla, 288 guinea pig, 224 mouse, 100 Neospora caninum vs., 588 primate, 762, 763 rat, 148 ruminant, 590-591 vole, 281 woodchuck, 323 zoonotic, 1092-1093 Toxoplasmosis cat, 479 chinchilla, 288 guinea pig, 224

1320 Toxoplasmosis, (cont.) human risk factor, 479 mouse, 100 primate, 762, 763 rat, 148 ruminant, 590-591 woodchuck, 323 zoonotic, 1092-1093 Tracheal culture/wash, 847, 1022 Tracheal pouch formation, 1022-1023 Trachemys scripta, 842 Tracheobronchitis, 405-406 Tracheostomy, 1023 Tracker dog disease, 409-411 Training and education, 13-14, 23 Tranexamic acid, 907 Tranquilizers animals, effect on, 1156-1157 gerbil dosage, 965 guinea pig dosage, 966 hamster dosage, 964 mouse dosage, 962 primate restraint, 719-720 rabbit, 968-969 rat dosage, 963 rodent, 960 Transgenic mouse, 1129-1140 Alzheimer's disease, 1194-1195 breeding systems, 1135-1137 colony management, 1134-1135, 1138-1139 databases, 1199 embryo cryopreservation, 1138-1139 gene expression control, 1191 genotype analysis, 1137 husbandry, 1131-1134, 1138 nomenclature, 1138-1139 property rights, 1140 Transgenic Systems for Mutation Analysis-Big Blue, 1199 Transmissible gastroenteritis, 649-650 Transmissible murine colonic hyperplasia, 85-86 Transmissible venereal tumor, 446-447 Transmission, biohazard, 1048 Transplantation allotransplantation, 1107-1108 history, 1211-1213 infectious diseases, 1107-1113 organ, 627-629 tumor, 1032-1033 xenotransplantation, 627-629, 1107-1113 Tree frog, 795 Tree squirrel, 256 Trematodiasis amphibian, 821 dog, 424 fish, 902-903 gerbil model, 276 guinea pig, 225 multimammate rat model, 282 primate, 772 rat, 151 reptile, 852 ruminant, 595 woodchuck, 324 Treponema sp.

INDEX

Treponema cuniculi, 344 Treponema palladium, 170, 343-344, 345-346 Treponema paraluis, 343 - 344 Treponematosis, 343- 344 Triamcinolone, 445 Tribosphenomys minutus, 249 Tribromoethanol, 958, 960-961,962, 965 Tricaine methane sulfonate (MS222), 812-813, 896-897 Trichinella spiralis, 272 Trichlorfonmebendazole, 878 Trichobezoar, 260, 289, 352, 610 Trichodectes canis, 426 Trichodina sp., 819, 881 Trichodinella sp., 881 Trichodinosis, 881 Trichoecius romboutsi, 102 Trichoepithelioma, 357 Trichofolliculoma, 237 Trichomonas sp., 192 Trichomonas vaginalis, 522 Trichomoniasis cattle model, 522 mouse, 99 primate, 758 ruminant, 591-592 Trichophyton sp.

Trichophyton mentagrophytes cat, 479 chinchilla, 289 dog, 428 ferret, 505 guinea pig, 228-229 mouse, 98 primate, 744 rabbit, 351 rat, 152 ruminant, 598 zoonotic, 1089

Trichophyton schoenleinii, 598 Trichophyton verrucosum, 598, 1089 Trichophyton violaceum, 744 Trichosomoides sp. Trichosomoides axe.i, 323 Trichosomoides crassicauda, 149-150 Trichospirura leptosoma, 766 Trichostrongylus sp. Trichostrongylus axei, 593, 1094 Trichostrongylus colubriformis, 275, 593, 1094 Trichostrongylus vitrinus, 593 Trichuris sp. primate, 766 ruminant, 594

Trichuris bradleyi, 285 Trichuris suis, 644, 645, 652-653 Trichuris trichiura, 770, 1094 Trichuris vulpis, 420 Trimethoprim-sulfa bordetellosis, 213 clostridia infection, 217 coccidiosis, 417, 652 colibacillosis, 547, 731 eye infections, 241 flavobacterium infection, 817 greasy pig disease, 655

1321

INDEX klebsiellosis, 735 lymphadenitis, 215 mucormycosis, 505 Pasteurella pneumotropica, 88 Pneumocystis carinii, 505 primate dose, 726 proliferative enteritis, 181 salmonellosis, 561,630 septicemia, 878 shigellosis, 1087 skin wound, 433 yersiniosis, 731 Tripartiella sp., 881 Trisetum flavescens, 608 Tritrichomonas sp., 522 Tritrichomonas caviae, 277 Tritrichomonas fetus, 591 Tritrichomonas muris, 99, 185 Triturus sp., 807 Trixacarus caviae, 225-228, 1096 Tropical pancytopenia, 409-411 Tropical rat mite, 1095 Trumpet-tailed rat, 284-286 Trypanosoma sp. amphibian, 819 Trypanosoma brucei rhodensiense, 282 Trypanosoma cruzi, 261, 761 Trypanosoma kansasensis, 262 Trypanosoma microti, 281 Trypanosoma neotomae, 262-263 Trypanosomiasis primate, 761 vole model, 280 Trypanoxuris sp., 765 Tuberculin test, 725,740, 1209 Tuberculosis ferret, 494-496 fish, 876-877 primate, 738-742 primate testing, 725 ruminant, 557 zoonotic, 1088 Tularemia, 255, 561 Tumor growth factor, 54 Tumor necrosis factor, 54 Tumor transplantation, 1032-1033 Tundra vole, 279 Turkey, 1252 Turtle anatomy and physiology, 837-841 behavior, 844 blood collection, 846 clinical chemistry data, 842 diseases abscess, 849 dysecdysis, 855 dystocia, 854 Herpesvirus sp., 850 metabolic and nutritional, 853-855 neoplasms, 857 parasitic, 852 shell fracture, 856 handling and restraint, 834, 845 hematology data, 841,842

nutrition and diet, 843 research uses, 829 sexing, 843 taxonomy, 827-828 28-Hour Law, 20 Tylosin erysipelas, 632 greasy pig disease, 655 leptospirosis, 659 mycoplasmal pneumonia, 563 mycoplasmosis, 639 pasteurellosis, 559 pleuropneumonia, 563 polyarthritis, 631 Tylosin phosphate, 646 Tympanites, 600-601 Type C oncovirus, 185 Type C reticulum cell tumors, 111 Typhlitis, hemorrhagic, 182, 274 Typhlocolitis, 76, 77 Typhus canine, 409-411 cotton rat, 271 murine, 1074-1075 wood rat, 261 Tyrophagus castellani, 278 Tyzzer's disease. See also Clostridium piliforme gerbil, 277, 383 guinea pig, 218 hamster, 182, 183, 191 mouse, 83-85 rabbit, 340-342 rat, 135-137 sentinel animal, 383 white-footed mouse, 267 white-tailed rat, 274 U Uinta ground squirrel, 250-254 Ulcerative dermatitis, 93, 153 Ulcerative dermatosis, 572-573 Ulcerative mammillitis, 567 Ulcers abomasal, 599-600 corneal, 435-436 decubital, 433-434 duodenal, 284, 492, 599-600 gastric, 492, 663-664 multimammate rat, 282, 284 tongue, 747 vulvovaginal, 547-548 Ultrafiltration, 369 Ultrasonography, 729, 847-848, 1026, 1034 Ultraviolet radiation aquatic disinfection, 890, 906 frog deformity, 1188 housing disinfection, 369 infectious pancreatic necrosis, 906 reptile housing, 832-833, 853 vitamin D deficiency, 774 Umbilical hernia, 665 Uncinaria sp., 478 Uncinaria stenocephala, 418-420, 1094

INDEX

1322

University of Chicago, 7 Urethane guinea pig, 966 hamster, 964 rabbit, 969, 970 rat, 963 rodent, 959, 961 Urinalysis data ferret, 487 mouse, 44 owl monkey, 692 rat, 127 Urinary tract obstruction, 110 Urine sample collection, 728, 1019-1020 Urolithiasis cat, 475 guinea pig, 234-235 rabbit, 358 ruminant, 604-605 USDA AGRICOLA database, 1197-1198 Animal Welfare Regulations (1985), 1240 animal welfare regulations and enforcement, 20-25, 397 importation and exportation, 29-30 Uterine prolapse, 609-610

Vaccination anaphylaxis, 610 anaplasmosis, 585 anthrax, 539 border disease, 573 bovine rhinotracheitis virus, 571 bovine viral diarrhea, 568-569 brucellosis, 540 calcivirus, 476 campylobacteriosis, 542 canine distemper, 414-415,498-499 canine parvovirus, 413 clostridia infection, 648 contagious pustular dermatitis, 574 dog, 396-397 erysipelas, 632 feline panleukopenia, 476 ferret, 498-499, 500 foot-and-mouth disease, 575 history, 1207, 1213 infectious disease prevention, 372 kennel cough, 406 leptospirosis, 408, 553 malaria, 697-698 measles, 1074 papillomavirus, 578 parainfluenza 3, 571-572 parvovirus, 660 of personnel, 1055 pleuropneumonia, 637 primate, 724-725 Q fever, 1076 rabies, 416, 500, 580 respiratory syncytial virus, 572 rhinotracheitis, 476

rodent, 372 salmonellosis, 561,630 smallpox, 1061 swine, 616 tetanus, 544 trichomoniasis, 591-592 tuberculosis, 1088 vaccinia virus, 59 Vaccinia virus, 59 Vachomia sp., 881 Vaginal prolapse, 609-610 Vapona strip, 852 Varicella, 746, 749 Varicella zoster virus, 1108 Vascular adhesion molecule- 1 (VCAM- 1), 340 Vector transmission, 370-371 Vecuronium, 969, 975, 982, 991 Velvet disease, 878-879 Venereal disease balanoposthitis, 572-573 ovine viral dermatosis, 576-577 sheep, 572-573 trichomoniasis, 591-592 Venereal granuloma, 446-447 Venezuelan hemorrhagic fever, 269, 270 Venipuncture. See Blood sample collection Venom, 836-837, 1055-1056 Ventilation, 919- 926 Verrucae, 578 Vervet monkey disease, 1063-1064 Vesicular stomatitis, 581-582 Vestibular syndrome, 110 Veterinarians animal welfare, 23 early, 3-7 education and training, 1231-1234 financial support, 1233-1234 grants and awards, 1233 Vibration, 1153 Vibrio sp. See also Campylobacter sp. Vibrio cholerae, 898 Vibrio parahaemolyticus, 898 Vibrio vulnificus, 898 Vibriosis. See also Campylobacteriosis Vicryl suture, 433 Vinblastine, 441 Vincristine, 445,447 Viral diseases. See also Hepatitis hamster, 183-185 mouse, 111 neoplasms, 111-112, 169-170 primate, 746-757 rat, 143-147 woodchuck, 310 zoonotic, 1060-1074 Virazole, 906 Virginiamycin, 646 Virulent foot rot, 549-550 Viruses. See also Herpesviruses Aleutian disease, 510 bovine lymphosarcoma, 566-567 bovine rhinotracheitis, 570-571 bovine viral diarrhea, 567-569, 582-583

INDEX cache valley, 569 caprine arthritis encephalitis virus, 555, 569-570 disinfection, 369-370 Ebola, 751 feline immunodeficiency virus, 460-461,477 feline leukemia virus, 460, 477, 510 frog erythrocytic virus, 818-819 H1N1 swine, 641 isolation and culture, 373, 374-375 Lucke tumor herpesvirus, 817- 818 oncogenic, 747 pancreatic necrosis virus, 882 parainfluenza 3, 571-572 pseudocowpox, 577 respiratory syncytial virus, 572 San Miguel sea lion virus, 899 simian hemorrhagic fever, 751-753 simian leukemia, 753 simian virus 40, 751 swine influenza, 641 swine pox, 655-656 transmissible gastroenteritis, 644, 649-650 transmission, transplantation, 1108 tumor, 110, 111-112 vesicular stomatitis, 581-582 waterborne human, 369 Yaba, 750 Visna pneumonia, 576 Vitamin A, 588 Vitamin A deficiency guinea pig, 231,235 mouse, 106 primate, 773 rabbit hydrocephalus model, 331,336, 353 rat, 153 reptile, 853 Vitamin A excess guinea pig, 231,235 primate, 773 rabbit model, 331 Vitamin B complex, 608 Vitamin B complex deficiency, 106-107, 231,235 Vitamin B12, 603 Vitamin B12 deficiency, 773 Vitamin C, 107, 208-209 Vitamin C deficiency guinea pig, 205, 219, 230-232, 241 primate, 773,775 Vitamin D deficiency guinea pig, 231,235 primate, 773 ruminant, 605 Vitamin D excess, 608 Vitamin E, 487, 607 Vitamin E deficiency guinea pig, 231,235 mouse, 106 primate, 774 rabbit, 336 rat, 153 ruminant, 607 Syrian hamster, 175 woodchuck, 324

1323

Vitamin K deficiency, 153, 231,235 Volcano rabbit, 330 Vole description, distribution, habitat, 279-280 diseases, 281 hematology data, 256 husbandry, 281 nutrition and diet, 280-281 physiology, 280 reproduction, 281 research uses, 280 Vomeronasal organ, 280 Vulvovaginitis, 547-548

W

Waardenburg's disease, 461 Wart. See Papillomatosis Wasting disease, 183, 223, 577-578 Watanabe heritable hyperlipidemic rabbit strain, 331 Water belly, 604-605 Water management aeration, 868 air supply, 888, 889-890 algae control, 874 ammonia, nitrates, nitrites, 869-870 amphibian, 797-798 biofiltration, 869 change procedure, 893-894 circulation, 888, 893-894 conductivity, 869 dechlorination, 797, 798 drinking water, 1155-1156 facility design, 871-873 filtration, 888-889 hardness, 869 nitrogen cycle, 869-870 oxygen, dissolved, 867-868 pH, 868-869 plumbing, 870, 887-888 source and supply, 873, 888-891,935-936 Water mold, 879-880 Watsonius sp., 772 Weasel, 483 Western blot, 382 Whipworm degu, 285 dog, 420 ruminant, 594 swine, 652-653 White-footed mouse biology and physiology, 266-267 cross-breeding, 264 dentition, 266 description, 265-266 diseases, 267 husbandry, 266-267 Lyme disease, 265 nutrition and diet, 266 research uses, 266 White muscle disease, 324, 607 White spot disease (ich), 880-881,900

1324 White-tailed rat clinical chemistry, 274 description, distribution, habitat, 272-273 diseases, 274-275 husbandry, 273 normative data, 274 physiology, 273-275 reproduction, 274 research uses, 273 White-throated wood rat, 261 Whitehead Institute, 1199 Whitewater Arroyo virus, 262 Whooping cough, 1088 Winter coccidiosis, 586 Winter dysentery, 583 Wistar Institute, 114 Wolf, 395 Wood rat biology and physiology, 262 description, distribution, habitat, 261 diseases, 262-263 husbandry, 262 nutrition and diet, 262 research uses, 261-262 taxonomy, 261 Woodchuck acquisition and sources, 311 anesthesia, 312 annual metabolic cycle, 310, 312-313 behavior, 319 clinical chemistry data, 314, 316 clinical techniques, 312 description, distribution, habitat, 309-310 diseases age-related, 325-326 bacterial, 320-321 cardiovascular, 326 congenital, 325 dermatitis, 319 diarrhea, 319 gastrointestinal, 327 hepatitis, 310, 311,319-320 hepatocellular carcinoma, 310, 319-320 iatrogenic, 325 metabolic and nutritional, 324-325 nematodiasis, 319 neoplasms, 310, 319-320, 325 parasitic, 323-324 rabies, 253, 310 traumatic, 319, 325 urogenital, 326-327 viral, 321-323 estrous cycle, 316, 318 hematology data, 314, 315 housing, 311, 317, 319-320 husbandry, 311- 312 longevity, 314 neonatal development, 318 nutrition and diet, 314, 319 physiology, 312- 314 reproduction, 314- 319 taxonomy, 309-310 Wooden tongue, 537

INDEX Wound care, 432- 433

Wuchereria bancrofli, 276, 282

Xenobiotics bacterial, 1161-1163 biotransformation, 1153 fungal, 1163 hormones, 1147 microbial, 1159-1163 nutrition and diet, 1153-1155 parasitic, 1163 pharmaceuticals, 1156-1158 pheromones, 1156-1158 stressors, 1163 viral, 1159-1161 water supply, 1155-1156 Xenograft, 1033 Xenopsylla cheopis, 1097, 1098 Xenopus sp. anatomy and physiology, 801-803, 809 animal model, 1187-1188 diseases bacterial, 814-817 nematodiasis, 821 parasitic, 819-822 viral and chlamydial, 817-819 embryo collection, 811-812 housing, 796-797, 809-810 husbandry, 796-801,809-810 lateral line system, 804 magainin, 801 metamorphosis, 812 natural history, 809 nutrition and diet, 810-811 oocyte harvest, 812-813 pollution deformity, 1188 reproduction, 807, 811 research uses, 795, 808-809 tadpole biology, 812 taxonomy, 795 Xenopus laevis, 795, 806, 808-809 Xenopus tropicalis, 808-809 Xenotransplantation, 627-629, 1107-1113 Xiphophorus sp., 886 Xylazine anesthesia gerbil, 965 guinea pig, 966 hamster, 964 mouse, 962 primate, 720, 726, 991,992 rabbit, 969 rat, 963 rodent, 958 ruminant, 987, 988 swine, 981 woodchuck, 312 Xenopus sp., 813 tranquilizer rabbit, 968 swine, 981

INDEX

Yaba virus, 750, 1061 Yellow fever, 752, 1063 Yersinia sp. Yersinia enterocolitica, 287, 562, 733 Yersinia pestis chinchilla, 287 mouse, 265 multimammate rat, 282 squirrel, 252 wood rat, 262 zoonotic, 1082-1083 Yersinia pseudotuberculosis cane mouse, 270 chinchilla, 287 guinea pig, 216-217 primate, 733 ruminant, 562 swine, 645 vole, 281 Yersinia ruckeri, 899 Yersiniosis. See also Plague chinchilla, 287 primate, 731,733, 733-734 ruminant, 562 Yohimbine, 959, 969, 987, 988

Zebrafish biomedical model, 863-864 diseases fungal (water mold), 880 mycobacteriosis, 876-877 nematodiasis, 878 septicemia, 878 trichodinosis, 881 velvet disease, 878-879 viral, 882 embryonic development, 863 experimental techniques, 864-866 housing, 871-873 husbandry, 866-874 natural history, 862 nutrition and diet, 874-875 research uses, 862, 886 water quality, 866-869 Zeolite, 797 Zinc deficiency, 107 Zinc excess, 408, 507 Zinc sulfate, 550 Zolazepam dog, 974 hamster, 964 primate, 719-720, 991,992, 993 swine, 981

1325

Zollinger Ellison syndrome, 282 Zoonoses. See also Personnel, health risks amoebiasis, 1089-1090 atypical mycobacteriosis, 1078 bacterial, 898-899, 1077-1088 balantidiasis, 1090 benign epidermal monkeypox, 1061 bordetellosis, 1088 brucellosis, 1081-1082 campylobacteriosis, 1084 cat scratch disease, 1079-1080, 1081 cercopithecine herpesvirus, 1068-1069 chlamydiosis, 1076-1077 cryptosporidiosis, 1090-1091 dengue, 1063 Ebola virus, 1064-1065 enteric diseases, 1084 enteric helicobacteriosis, 1084-1085 fish, 898-899 fungal diseases, 1088-1089 gastric helicobacteriosis, 1085-1086 giardiasis, 1091-1092 helminth infections, 1093-1094, 1098 hemorrhagic fever, 1063-1066 hemorrhagic fever, renal syndrome, 1065-1066 hepatitis, 1070-1072 influenza, 1074 leptospirosis, 1083-1084 lymphocytic choriomeningitis virus, 1066-1068 Marburg virus, 1063-1064 monkeypox, 1060-1061 murine typus, 1074-1075 nematodiasis, 1093-1094, 1098 Newcastle disease virus, 1074 off virus, 1061-1062 pasteurellosis, 1080 plague, 1082-1083 protozoal infection, 1089-1093 Q fever, 1076 rabies, 1069-1070 rat bite fever, 1078-1079 respiratory infections, 1088 retroviral, 1072-1073 rickettsial diseases, 1074-1076 rickettsial pox, 1075-1076 salmonellosis, 1086-1087 shigellosis, 1087-1088 simian foamy virus, 1073 simian immunodeficiency virus vector, 1072 streptococcosis, 1080-1081 toxoplasmosis, 1092-1093 transmission, transplantation, 1107-1113 tuberculosis, 1088 viral, 1060-1074 Yaba virus, 1061 yellow fever, 1063 Zygodontomys brevicauda, 268-269

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